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Photosynthetic polyol production

Savakis, P.E.

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PHOTOSYNTHETIC POLYOL PRODUCTION Keywords: cyanobacteria, biofuels, genetic engineering, microbial consortia Printed by: Ipskamp Drukkers B.V. Front & Back: Blueprint-style illustration of the topic of this thesis. Sunlight drives pho- tosynthesis in a cyanobacterium, which converts carbon dioxide into bu- tanediol or glycerol.

Chapters 1, 4, 7, 8, 9: © 2016 by P.E. Savakis Chapters 2, 3, 5: © 2014, 2013, 2014 by Elsevier B.V. (reprinted with permission) Chapter 6: © by the authors.

All rights reserved. No part of this publication may be reproduced in any form without prior written permission from the copyright holders.

The research reported in this thesis was carried out in the Molecular Microbial Physiology Group of the Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Amsterdam, The Netherlands and the Metabolic Engineering Group of the Qingdao Institute of Bioenergy and Bioprocess Technology, Qingdao, People’s Republic of China.

This work was carried out within the research programme of BioSolar Cells, co-financed by the Dutch Ministry of Economic Affairs.

The thesis layout is based on the TU Delft LaTeX template.

ISBN 978-94-028-0372-3 PHOTOSYNTHETIC POLYOL PRODUCTION

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam, op gezag van de Rector Magnificus prof. dr. ir. K.I.J. Maex, ten overstaan van een door het College voor Promotie ingestelde commissie, in het openbaar te verdedigen in de Agnietenkapel op dinsdag 1 november 2016, te 10:00 uur

door

Philipp Emmanuil SAVAKIS

geboren te Aken, Duitsland. Promotiecommissie: Promotor: prof. dr. K. J. Hellingwerf Copromotor: dr. F.Branco dos Santos

Overige leden: prof. dr. M. A. Haring Universiteit van Amsterdam dr. E. P. Hudson KTH Royal Institute of Technology, Stockholm, Zweden prof. dr. J. Hugenholtz Universiteit van Amsterdam prof. dr. R. Wever Universiteit van Amsterdam prof. dr. A. Wilde Albert-Ludwigs Universität Freiburg, Duitsland

Faculteit der Natuurwetenschappen, Wiskunde en Informatica SUMMARY

N recent decades, there has been an increasing awareness that the societal dependence I on fossil fuels has drastic ecological consequences. In this context, the use of "biofuels", combustible compounds that are derived from organic matter, has been proposed to mit- igate man-made carbon emissions. First generation biofuels are derived from food crops. Their use thus requires arable land and competes with food supply. Therefore, alternatives are currently being explored. Cyanobacteria are prokaryotic organisms that are able to convert light, water and car- bon dioxide into biomass, in a process referred to as oxygenic photosynthesis. Cyanobacte- ria perform photosynthesis more efficiently than plants, since the latter devote biomass to roots, stems and mitochondria, among other things. This thesis explores the use of genetically engineered Synechocystis sp. PCC6803 (Syne- chocystis) as cell factories for the production of organic molecules – the polyols glycerol and 2,3-butanediol, as well as butanone and s-butanol. The general approach for this is to in- troduce one or more enzymes into the cells. Thus, molecules from the central metabolism can be converted to products of interest. In Chapter1 , attributes of oxygenic photosynthesis and the biology of cyanobacteria are briefly introduced. Furthermore, 2,3-butanediol and glycerol, the two most important molecules this thesis revolves around, are presented. Finally, aspects of chemical reactions and molecular symmetry (i.e. chirality) are briefly touched upon. In Chapter2 , recent progress in the genetic engineering of cyanobacteria for the syn- thesis of fuels and chemical feedstocks is reviewed. Enzymatic pathways and final titres are presented. Additionally, challenges that this relatively recent research field faces, are laid out and speculations for future developments are made. Chapter3 addresses the conversion of light, carbon dioxide and water into 2,3-butane- diol. There, an enzymatic pathway for the synthesis of meso-2,3-butanediol, derived from chemotrophic bacteria, is introduced into Synechocystis. In the last step of this pathway, acetoin is reduced to butanediol, in an NADH-dependent reaction. It is demonstrated that an increase in the availability of NADH increases the titre of butanediol. The yield of butanediol is further increased, when an enzyme is selected that prefers NADPH over NADH as the cosubstrate. In Chapter4 , extracellular [butanediol]/[acetoin] ratios, measured in Chapter3 , are used to calculate intracellular [NAD(P)H]/[NAD(P)] ratios. It is found that the NADPH pool is more reduced than the NADH pool. In Chapter5 , the enzyme phosphoglycerol phosphatase from Saccharomyces cerevisiae is introduced into Synechocystis and so a mutant strain is generated that produces glycerol from light, water and carbon dioxide. The glycerol titres can further be increased through the addition of sodium chloride at low, but stressful concentrations. Interestingly, it was found that the wild-type strain of Syne- chocystis produces small amounts of glycerol under the same mild stress conditions.

v vi SUMMARY

This latter finding inspired the research in Chapter6 , where it is demonstrated that the glycerol produced by Synechocystis stems from the degradation of an endogenous osmo- protectant, glucosylglycerol. An enzyme could be identified that is involved in the re-assimilation of glucosylglycerol. This enzyme, Slr1670, previously had no previously assigned function. Proteins with a homologous sequence are widely distributed in the cyanobacteria, purple bacteria and even in archaea. With genome-scale modeling, an increase in growth rate is predicted, when exogenously supplied glucosylglycerol is used as a carbon source during photoheterotrophic growth. In Chapter7 , a co-culturing approach for the synthesis of optically active alcohols is presented. The compartmentalisation of oxidation and reduction reactions into Synechocys- tis and Escherichia coli or Bacillus subtilis, respectively, allows the construction of an enzy- matic pathway that would be difficult to realise in a single species. (S,S)-2,3-butanediol is utilised as a model system. Careful selection of the cosubstrate and enantiospecificity of two acetoin reductases allows the construction of a driving force to- wards this compound. This means that (S,S)-2,3-butanediol can be synthesised from any compound (or mixture of compounds) within this pathway. An extension of the pathway is demonstrated to be functional such that optically active 2,3-butanediol can be synthesised from light, carbon dioxide and water. A inter-organismal electron-carrier system is envi- sioned, which allows the transfer of redox equivalents from Synechocystis to a chemotrophic organism. This would allow the realisation of light-driven reduction reactions that would be difficult to realise in cyanobacteria. This approach would further allow the autotrophic growth of an organism like Escherichia coli. Chapter8 addresses radical-mediated dehydration reactions to convert the polyols 2,3- butanediol and glycerol into the corresponding carbonyl compounds, butanone and reuter- in. These are subsequently converted into the corresponding alcohols, s-butanol and 1,3- propanediol. The conversion of meso-butanediol to butanone could be demonstrated in Synechocystis, albeit at low yields and under special conditions. Dehydratase reactions are oxygen-sensitive and therefore difficult to realise in cyanboacte- ria, which are oxygen-producing organisms in the light. Therefore, the use of the chemo- trophic organisms Escherichia coli and Lactobacillus reuteri is explored to perform these reactions, instead of in Synechocystis. The conversion of meso-butanediol to butanone by recombinant Escherichia coli is demonstrated. Furthermore, the conversion of CO2, light and water to 1,3-propanediol is demonstrated in a consortium of Synechocystis and Lactobacillus reuteri. In Chapter9 , the findings of this thesis are put into ecological, societal and economic context. Strategies for the optimisation of product yield are discussed. Additionally, current challenges of this research field are examined and solutions are proposed. SAMENVATTING

N recente decennia is er een toenemende bewustwording van het feit dat de maatschap- I pelijke afhankelijkheid van fossiele brandstoffen drastische ecologische gevolgen heeft. In deze context is het gebruik van ‘biobrandstoffen’, brandbare stoffen afkomstig van bio- logisch materiaal, voorgesteld ter compensatie voor de koolstofemissies waarvoor de mens verantwoordelijk is. De eerste generatie biobrandstoffen zijn afkomstig van landbouwge- wassen waarvoor vruchtbare landbouwgrond nodig is en wat tevens concurreert met de voedselvoorziening. Om die reden worden alternatieven onderzocht. Cyanobacteriën zijn prokaryote organismen die in staat zijn licht, water en koolstofdioxide om te zetten in bi- omassa, een proces waaraan gerefereerd wordt als oxygene fotosynthese. Aangezien cy- anobacteriën geen wortels hebben, is hun fotosynthetische efficiëntie hoger dan dat van planten. Deze is hoger omdat planten wortels, een stam en andere onderdelen moeten ma- ken die niet direct bijdragen aan fotosynthese. Dit proefschrift verkent de mogelijkheden voor genetisch bewerkte Synechocystis sp. PCC6803 (Synechocystis) om ze in te zetten als zo- genaamde ‘cellulaire fabrieken’ voor de productie van organische moleculen – de polyolen glycerol en 2,3-butaandiol – alsook butanon en s-butanol. De algemene aanpak hiervoor is om één of meerdere enzymen te introduceren in de cellen. Hiermee kunnen moleculen uit het centrale metabolisme van de cellen worden omgezet in de gewenste producten. In Hoofdstuk1 worden de eigenschappen van de oxygene fotosynthese en de biologie van cyanobacteriën kort geïntroduceerd. Voorts worden 2,3-butaandiol en glycerol, de twee belangrijkste moleculen waar dit proefschrift om draait, gepresenteerd. Als laatste worden aspecten van chemische reacties en moleculaire symmetrie (chiraliteit) kort genoemd. In Hoofdstuk2 wordt een overzicht gegeven van de recente vooruitgang in de geneti- sche bewerking van Synechocystis voor de synthese van brandstoffen en grondstoffen voor de chemische industrie. Enzymatische routes en maximale concentraties worden gepre- senteerd. Daarnaast worden de uitdagingen waar dit relatief nieuwe onderzoeksgebied voor staat blootgelegd en wordt er gespeculeerd over toekomstige ontwikkelingen. Hoofdstuk3 bespreekt de omzetting van licht, koolstofdioxide en water in 2,3-butaan- diol. In dit Hoofdstuk wordt een enzymatische route, afgeleid van chemotrofe bacteriën, geïntroduceerd in Synechocystis. In de laatste stap van deze route wordt acetoïne geredu- ceerd tot butaandiol in een NADH-afhankelijke reactie. De relatie tussen de beschikbaar- heid van NADH en de maximale concentraties van butaandiol wordt gedemonstreerd. De opbrengst van butaandiol wordt verder verhoogd wanneer een enzym geselecteerd wordt dat NADPH prefereert boven NADH as co-substraat. In Hoofdstuk4 , worden extracellulaire [butanediol]/[acetoin] verhoudingen, bepaald in Hoofdstuk3 , gebruikt om intracellulaire [NAD(P)H]/[NAD(P)] verhoudingen te berekenen. In Synechocystis zijn de NADPH/NADP ratio’s groter is dan de NADH/NAD ratio’s. In Hoofdstuk5 wordt het enzym fosfoglycerolfosfatase van Saccharomyces cerevisiae geïntroduceerd in Synechocystis waarmee een mutant gegenereerd wordt dat glycerol pro- duceert uit licht, water en koolstofdioxide. De maximale concentraties van glycerol kun- nen verder worden verhoogd door toevoeging van natriumchloride in lage, doch stress-

vii viiiS AMENVATTING inducerende concentratie. Een interessante observatie is dat het wild type van Synecho- cystis onder deze condities ook kleine hoeveelheden glycerol produceert. Deze ontdekking inspireerde het onderzoek beschreven in Hoofdstuk6 , waarin gedemonstreerd wordt dat glycerolproductie in Synechocystis afhankelijk is van de afbraak van een Synechocystis-eigen molecuul dat beschermt tegen osmotische stress, namelijk glucosylglycerol. Een enzym dat betrokken is bij de her-assimilatie van glucosylglycerol kon worden geïdentificeerd. Dit en- zym, Slr1670, had geen eerder bekende functie. Eiwitten met homologe sequentie zijn wijd- verspreid onder cyanobacteriën, purperbacteriën en zelfs onder de Archea. Een toename in groeisnelheid werd voorspeld met behulp van een genoom-schaal stoichiometrisch model wanneer glucosylglycerol kunstmatig wordt toegevoegd als koolstofbron tijdens fotohete- rotrofe groei. In Hoofdstuk7 wordt een methode voor co-cultivatie voor de synthese van optisch actieve alcoholen gepresenteerd. Het onderverdelen van de oxidatie- en reductiereacties in compartimenten, respectievelijk in Synechocystis en Escherichia coli of Bacillus subtilis, maakt het mogelijk een enzymatische route te construeren die moeilijk te realiseren is in een enkel organisme. (S,S)-2,3-butaandiol is gebruikt als modelsysteem waarin zorgvuldige selectie van co-substraat en enantiospecificiteit van twee acetoïnereductases een drijvende kracht richting het gewenste product creëert. Dit betekent dat (S,S)-2,3-butaandiol gesyn- thetiseerd kan worden uit elke stof (of mengsel van stoffen) dat deel uitmaakt van deze en- zymatisch route. Een functionele extensie van de route dat optisch actief 2,3-butaandiol synthetiseert uit licht, koolstofdioxide en water is aangetoond. Een inter-soortelijke elek- tronentransportketen, waarin redoxequivalenten van Synechocystis naar een chemotroof organisme getransporteerd worden behoort hiermee tot de mogelijkheden. Dit maakt licht- gedreven reductiereacties mogelijk die moeilijk zijn te realiseren in cyanobacteriën. Daar- naast maakt deze aanpak ook autotrofe groei van een organisme zoals Escherichia coli mo- gelijk. In Hoofdstuk8 wordt de radicaal-gemedieerde dehydratatie van polyolen 2,3-butaan- diol en glycerol naar de corresponderende carbonylen, butanon en reuterine, besproken. Deze worden verder omgezet in de alcoholen s-butanol en 1,3-propaandiol. De omzetting van meso¬-butaandiol naar butanon kon worden aangetoond in Synechocystis, hoewel met lage opbrengst en onder speciale omstandigheden. Dehydratiereacties zijn zuurstofgevoe- lig en daarom moeilijk te realiseren in cyanobacteriën, welke zuurstof produceren wanneer blootgesteld aan licht. De mogelijkheid deze reacties te laten uitvoeren door chemotrofe or- ganismen Escherichia coli en Lactobacillus reuteri in plaats van Synechocystis is onderzocht. De omzetting van meso-butaandiol naar butanon door een gemodificeerde Escherichia coli is aangetoond. Verder is de omzetting van koolstofdioxide, licht en water in 1,3-propaandiol aangetoond in een consortium van Synechocystis en Lactobacillus reuteri. In Hoofdstuk9 worden de verkregen inzichten van dit proefschrift in ecologische, maat- schappelijke en economische context geplaatst. Strategieën voor de optimalisatie van pro- ductopbrengst worden besproken. Daarnaast worden de huidige uitdagingen van het on- derzoeksveld uitgelicht en oplossingen voorgesteld. ZUSAMMENFASSUNG

N den letzten Jahrzehnten hat sich immer deutlicher abgezeichnet, dass die gesellschaft- I liche Abhängigkeit von fossilen Brennstoffen drastische Konsequenzen für die Umwelt hat. In diesem Zusammenhang wurden Biokraftstoffe vorgeschlagen, Substanzen mit ho- hem Energiegehalt, die aus Biomasse gewonnen werden können. Biokraftstoffe der ersten Generation werden aus essbaren Pflanzen wie Mais oder Raps gewonnen und konkurrieren daher mit der Nahrungsmittelversorgung. Cyanobakterien sind prokaryotische Organismen, die Licht, Wasser und Kohlenstoffdioxid in Biomasse umwandeln können. Dieser Prozess heißt oxygene Photosynthese. Die cyano- bakterielle Photosynthese ist effizienter als die der Pflanzen, da die Letzteren unter ande- rem auch Biomasse für Wurzeln, Stämme und Stengel und Mitochondrien zur Verfügung stellen müssen. Diese Arbeit untersucht die Benutzung genetisch verbesserter Stämme des Cyanobakteri- ums Synechocystis sp. PCC6803 als Zellfabriken zur Synthese organischer Moleküle – der Polyole Glycerin und 2,3-Butandiol, sowie s-Butanol. Im Allgemeinen werden hierzu eines oder mehrere Fremdenzyme in die Zellen gebracht. So können Moleküle aus dem zentralen Stoffwechsel in wirtschaftlich interessante Stoffe überführt werden. In Kapitel1 werden Aspekte der Photosynthese und der Biologie der Cyanobakterien kurz erläutert. Danach werden Glycerin und 2,3-Butandiol, die zwei Moleküle, mit denen sich diese Arbeit im Wesentlichen befasst, vorgestellt. Zuletzt werden einige Attribute che- mischer Reaktionen und der Molekülsymmetrie (Chiralität) erläutert. In Kapitel2 wird der jüngste Fortschritt auf dem Gebiet der genetischen Veränderung von Cyanobakterien zur Chemikalien- und Kraftstoffsynthese referiert. Enzymatische Reak- tionswege und Ausbeuten werden präsentiert. Zusätzlich wird darüber spekuliert, welchen Herausforderungen dieses Forschungsgebiet entgegenblickt. In Kapitel3 wird die Umwandlung von Kohlenstoffdioxid, Licht und Wasser zu 2,3- Butandiol demonstriert. In diesem Zusammenhang wird ein Reaktionsweg zur Synthese von meso-2,3-Butandiol aus chemotrophen Bakterien in Synechocystis eingesetzt. Der letz- te Schritt dieses Reaktionsweges ist die NADH-abhängige Reduktion von Acetoin zu Butan- diol. Es wird gezeigt, dass eine Zunahme der NADH-Konzentration sich in einer Zunahme der Butandiolausbeute auswirkt. Die Ausbeute konnte weiter erhöht werden, wenn statt der NADH- eine NADPH abhängige Acetoinreduktase eingesetzt wurde. In Kapitel4 , werden extrazelluläre Verhältnisse von [Butandiol] zu [Acetoin], die bereits InKapitel3 gemessen wurden, in intrazelluläre [NAD(P)H]/[NAD(P)] Verhältnisse übersetzt. Es wird gezeigt, dass der NADPH pool weiter reduziert ist als der NADH pool. In Kapitel5 wird das Enzym Phosphoglycerinphosphatase aus Saccharomyces cerevisiae nach Synechocystis transferiert. Hierdurch wird eine Mutante generiert, die aus Licht, Koh- lenstoffdioxid und Wasser Glycerin produzieren kann. Die Glycerinausbeute kann weiter erhöht werden, wenn Natriumchlorid in kleinen, aber zum Erzeugen einer Stressantwort ausreichend hohen Mengen zugegeben wird. Interessanterweise stellte sich heraus, dass

ix xZ USAMMENFASSUNG der Wildtyp unter den gleichen Stressbedingungen kleine Mengen an Glycerin absondert. Dieses Forschungsergebnis war die Grundlage für Kapitel6 . Dort wird gezeigt, dass das Glycerin, das Synechocystis unter diesen Bedingungen produziert, aus dem Abbau von Glu- cosylglycerin, stammt. Die Synthese dieses Moleküls ist Teil der Salzstressantwort von Syn- echocystis. Es wurde ein Enzym identifiziert, das am Abbau von Glucosylglycerin beteiligt ist. Dieses Enzym, Slr1670, hatte keine zuvor bekannte Funktion. Proteine mit homologer Sequenz sind weitverbreitet unter den Cyanobakterien, finden sich aber auch in einigen Purpur- bakterien und sogar Archäen. Durch genomweite Modellierung wurde eine Steigerung der Wachstumsrate vorhergesagt, wenn Glucosylglycerin über diesen neu identifizierten Stoff- wechselweg abgebaut wird. In Kapitel7 wird ein Forschungsansatz verfolgt, in dem Kokulturen von rekombinan- ten Synechocystis-Stämmen und chemotrophen Bakterien, wie Escherichia coli oder Bacil- lus subtilis zur Synthese optisch aktiver Alkohole verwendet werden. Hier erlaubt räumliche Auftrennung einen Reaktionsweg, der in einer einzigen Zelle schwierig umzusetzen gewe- sen wäre. Als Modellsystem wird S,S-2,3-Butandiol untersucht. Durch sorgfältige Auswahl der Kofak- torspezifität der beteiligten Acetoinreduktasen lässt sich eine Triebkraft konstruieren, die den Reaktionsablauf in die gewünschte Richtung drängt. Das hat zur Folge, dass S,S-2,3- Butandiol aus jedem beliebigen Intermediat (oder Mischung von Intermediaten) im Re- aktionsweg mit dem gleichen Kokulturensystem hergestellt werden kann. Zusätzlich wird gezeigt, wie der Reaktionsweg erweitert werden kann, sodass Kohlenstoffdioxid zu S,S-2,3- Butandiol umgesetzt werden kann. Konzeptionell lässt sich dieser Ansatz so verstehen, dass Elektronen zwischen Synechocystis und dem chemotrophen Bakterium ausgetauscht wer- den. Es wäre daher möglich, Reduktionsreaktionen, die sich ohne weiteres nicht in Synecho- cystis durchführen ließen, dennoch lichtgetrieben zu realisieren. Des Weiteren wäre es möglich, Mutanten von Escherichia coli zu konstruieren, die selbst zur Kohlenstofffixierung befähigt sind. Kapitel8 behandelt die radikalvermittelte Dehydratisierung von Polyolen. Diese Reak- tion setzt Diole zu Carbonylverbindungen um. Die nachfolgende Reduktion ergibt dann die entsprechenden Alkohole. Es konnte gezeigt werden, dass rekombinante Synechocystis meso-2,3-Butandiol zu Butanon umsetzen konnten, wenn auch mit geringen Ausbeuten und unter besonderen Reaktionsbedingungen. Da Dehydratasen sauerstoffanfällig sind, ist eine funktionelle Produktion in Cyanobakterien, sauerstoffproduzierenden Organismen, schwierig. Deshalb wurde ein Ansatz verfolgt, in dem chemotrophe Organismen wie Esche- richia coli und Lactobacillus reuteri diese Reaktionen durchführen. Außerdem konnte ein Kokulturensystem gezeigt werden, in dem Synechocystis und Lactobacillus reuteri CO2, Licht und Wasser zu 1,3-Propandiol umwandeln. In Kapitel9 werden die Ergebnisse dieser Arbeit in einen ökologischen, gesellschaft- lichen und wirtschaftlichen Zusammenhang gesetzt. Strategien zur Optimierung der Pro- duktausbeute werden vorgestellt. Zusätzlich werden gegenwärtige Herausforderungen die- ses Forschungsgebietes diskutiert und Lösungsansätze vorgeschlagen. CONTENTS

Summary v

Samenvatting vii

Zusammenfassung ix

1 General Introduction1

2 Engineering cyanobacteria for direct biofuel production from CO2 17

3 Synthesis of meso-2,3-butanediol by Synechocystis sp. PCC6803 25

4 Redox cosubstrate levels in mutant Synechocystis strains 55

5 Photosynthetic production of glycerol by a recombinant cyanobacterium 65

6 Identification of a novel protein from Synechocystis sp. PCC 6803 involved in re- assimilation of the osmolyte glucosylglycerol 75

7 A coculturing approach for generation of optically active 2,3-butanediol isomers 95

8 On polyol dehydration 109

9 General discussion 121

References 135

Bibliography 135

Appendix 155

A Abbreviations 157

B Alterations of the genome-scale model of Synechocystis 161

Acknowledgements 165

List of Publications 167

xi

1 GENERAL INTRODUCTION

Philipp SAVAKIS

1 2 1.G ENERAL INTRODUCTION

1 RESOURCECYCLES -SCARCITYAND ABUNDANCE LANETARY escape1 aside, all elements are cycled between different chemical and phys- P ical states and locations. While there is considerable knowledge on some aspects of these cycles, their mutual interactions are understood, at best, poorly. It is understood very well, however, that practically all elemental cycles have been perturbed by human action over the last centuries [2]. For the sake of brevity, the section below will focus on carbon, nitrogen and phosphorus. Carbon. CO2 is the thermodynamic sink for the reaction of any organic molecule with ex- cess O2. As far as absolute numbers are concerned, inorganic carbon deposits and sedi- mentary carbonates outweigh the amount of carbon in the biosphere by far [2]. Temporal changes in the concentration of atmospheric carbon dioxide are a function of its emission and deposition (Fig. 1.1A). Atmospheric carbon is in rapid equilibrium with dis- solved oceanic and soil carbon. Oxygenic photosynthesis converts CO2 into organic mol- ecules, which, in time, are released into the soil. There, the decomposition of biomaterial leads to CO2 emission into the atmosphere. Similar cycles of photosynthetic fixation and respiratory oxidation occur in water bodies, where sedimentary processes remove carbon from both limnal and pelagic systems. While ice core data identified oscillations in the range of 100 parts per million by volume 2 (ppmv) over the last 100,000 years, in 2000 the atmospheric CO2 concentration was almost 100 ppmv higher than the record concentration in the 420,000 year timeframe analysed in [2]. Increases occured at a rate 10 to 100 times of pre-industrial revolution estimates [2].

A B man-made emissionsreactor-based plant photosynthesis equilibration photosynthesis man-made emissions

microbial respiration

sedimentation

marine fossil reservoirs photosynthesis & respiration

Figure 1.1: Simplified representation of the carbon cycle in a society based on fossil fuels (A) and a future society based on renewable fuels (B). There, human carbon emissions are matched by reactor- based synthesis. Reduction of atmospheric CO2 levels could occur if bioreactor-based products would be used for the synthesis of polymers.

1Gaseous molecules that surpass escape velocity can vanish into outer space. See [1] for an overview. 2 The year the study was published. Since then, atmospheric CO2 levels have risen further. In January 2016, the recent global monthly mean CO2 concentration was estimated at 402.59 ppmv [3]. In the year 2000, values around 370 ppmv were reported [3]. Pre-industrial values have been reported at 280 ppmv [2]. 3

The main anthropogenic contributors to the global carbon cycle are fossil fuel combustion and the expansion of agriculture. Utilising new land for agriculture may have a twofold 1 effect: More carbon dioxide is emitted, especially, when forests are cleared by burning, whereas less may be fixed, especially, when acres are ploughed every season. As the hu- man population level increases, so will the amount of land used for agricultural purposes. CO2 levels are therefore expected to rise further. Despite these dramatic increases, the atmospheric concentration of CO2 is still dwarfed by those of oxygen and nitrogen. The latter, however, do not absorb infrared (IR) light3. Solar radiation in the IR range that is reflected by the earth’s surface thus can be absorbed and reemitted by CO2 and other greenhouse gases, creating the atmospheric greenhouse effect. Measured data in the late 20th century are in good agreement with model predictions for greenhouse gas-induced rises in temperature [4]. To combat this, the use of alternative fuel sources is being explored, and in the case of first generation biofuels4, actively applied: In the US, 33.6% of the corn harvest was used for the production of fuel ethanol in the market year 2014/2015 [5]. The use of first genera- 5 tion biofuels indeed can mitigate the increases of atmospheric CO2 . The drawback is that the concomitant increase in agricultural activity heavily influences other resource cycles, as discussed in the following paragraphs. Phosphorus. All living organisms require phosphorus (P), chiefly as part of the backbone of their nucleic acids, of energy and redox equivalents such as ATP and NADPH and of phos- pholipids, which make up membranes. Virtually all inorganic phosphorus is present as phosphate6. With many cations, phosphate forms mineral salts of low solubility. Weathering of these minerals7 is the only natural pro- cess by which new phosphorus can enter the biosphere [6]. Riverine transport of phos- phate that has been washed out of soils represents the only source for phosphorus in ma- rine ecosystems. Sedimentation processes remove phosphorus from the sea. On geologi- cal timescales, subduction and uplifting move minerals such that they can be exposed to weathering again [6]. There is considerable human impact on the phosphorus cycle. An important plant nutrient, the main application for mined phosphorus is fertilizer [7]. Artificially increased phosphate levels in soils benefit plant growth, but also lead to increased efflux to lakes and seas. There, eutrophication can lead to algal and cyanobacterial blooms. Subsequent degradation of the biomass can lead to hypoxic conditions and put the entire ecosystem at risk [8]. Overuse of phosphate thus not only threatens biodiversity, but may lead to scarcity in the near future: Following increases in energy prices and in phosphorus demand, prices peaked in 2008 to unprecedented values, necessitating fertilizer rationing [9]. Increased efforts for recycling are therefore of paramount importance. Nitrogen. Nitrogen (N) is a key component of amino acids and nucleobases. Despite its

3A requirement for photoinduced vibrational and rotatianol transitions is that their dipole moment changes. While CO2 in its ground state has no dipole moment, some of its vibrational modes do. Consequently, CO2 ab- sorbs light in the IR range. 4First generation biofuels are those that come from the conversion of food crops. Examples include ethanol, which is generated by fermentation, and fatty acid esters, that are obtained through the transesterificiation of oils with methanol or ethanol. 5 Note that because these fuels are to be consumed, the amount of CO2 in the atmosphere can not decrease solely as a result of this technology, even when brought to perfect efficiency. 6 3– 2– Note that while phosphate is represented as PO4 , in aqueous systems, it is protonated to HPO4 . 7 – – – Mainly apatites: Ca5(PO4)3X; X OH ,F ,Cl . 4 1.G ENERAL INTRODUCTION

abundance in the atmosphere, nitrogen is often a limiting nutrient in the biosphere due 1 to the low reactivity in its elemental state. In the natural cycle of this element8, N enters the biosphere through the action of nitrogenases9, enzyme complexes that produce am- monia (NH3) in an energetically costly fashion [13]. Some cyanobacteria, among them the filamentous Anabaena and, crucially, the unicellular Trichodesmium [14] are capable of ni- trogen fixation. In terrestrial systems, a substantial part is fixed by symbiotic bacteria at 10 plant roots [15] and leaves . Another source of NH3 is the decay of biomass. Ammonia is 11 – oxidised to nitrite and, subsequently, nitrate, in a process termed nitrification [17] . NO3 is assimilated by plants, or converted back into elementary nitrogen by denitrifying bacteria. As a result of human activity, the amount of nitrogen that is made available to ecosystems in general has doubled [10]. Generation of NOx during the combustion of fossil fuels, in combination with ’acid rain’ is a substantial contributor. The nitrogen in artificial fertilisers is derived from ammonia, which in turn is synthesised from N2 and H2 in the Haber-Bosch process [18]. In highly industrialised regions, chemical nitrogen fixation is greater than bi- ological nitrogen fixation [10]. Agriculture has led to an increase in biological nitrogen fix- ation as well: Rhizobium occurs in symbiosys with legume roots and the total amount of nitrogen fixed by such systems is greater than if the same space were occupied by natural plant communties [15]. Nitrogen often is a limiting element and agriculture has artificially increased the amount that is available in the biosphere. Closing cycles. Alternatives to first-generation biofuels are being developed. The term "sec- ond generation biofuels" has been used for fuels derived from non-edible biomass. This can stem from microalgae [19]12 as well as non-food plant material [20]. An alternative ap- proach13 uses genetically manipulated cyanobacteria for the direct production of fuels and chemical feedstocks [21]. The synthesis of commodity chemicals and fuels by recombinant cyanobacteria (presented in Chapters2,3,5,7,8 of this thesis) can help to close the cycle and ultimately transition to an economy that is fully bio-based (Fig. 1.1B). While the cultivation of cyanobacteria in closed photobioreactor systems does produce wastewaters that contain nitrogen and phosphorus, these are easier to capture, and ulti- mately recycle. If realised on a sufficiently large scale, cultivation of genetically modified cyanobacteria can aid in closing the carbon cycle.

8See [10] for a comprehensive review of nitrogen cycles and the anthropogenic impact thereon. See [11] for an – organism that converts molecular nitrogen (N2) into nitrate (NO3 ). 9 At the high temperatures during lightning strikes, N2 and O2 can react to NOx [12]. 10See references in [16]. 11Recently, an organism was described that can perform all these steps [11]. 12Microalgae-derived fuels are sometimes given their own category: Third generation biofuels. 13Mostly put into the category "Fourth generation biofuels". 5

PHOTOSYNTHESIS 1 The term photosynthesis was suggested by Charles Reid Barnes in 1893 [22]14. Since then, the word has undergone changes in defintion (See [23] for an overview). The most widely accepted definition stems from Kamen [24]:

Photosynthesis is a series of processes in which electromagnetic energy is con- verted to chemical energy used for biosynthesis of organic cell material; [...]

Photosynthesis can either be chlorophyll- or proteorhodopsin based. Chlorophyll-based photosynthesis can be oxygenic or anoxygenic. Proteorhodopsin-based photosynthesis is anoxygenic15. There are different hypotheses as to which kind evolved first (see [25] for a short review). Green plants carry out oxygenic photosynthesis, while bacteria show more variability. Among the bacteria, there are five major classes that perform photosynthesis. These are the proteobacteria (purple bacteria)16, the heliobacteria, the chloroflexi (green non-sulfur bac- teria), the chlorobi (green sulfur bacteria) and the cyanobacteria [25]. The first four classes are anoxygenic. Cyanobacteria can perform oxygenic photosynthesis17. Chemiosmotic theory [27] They appear consistently throughout the geological The energy that is released as record; stromatolites, sedimented biofilms of cyanobac- electrons flow from a higher to terial origin, date back to 3.5 or even 3.8 Ga ago [29], sig- a lower potential is conserved nificantly predating the great oxygenation event (2.5 Ga in a proton gradient accross ago) [30]. a membrane. The increased Oxygenic photosynthesis. During oxygenic photosyn- concentration of protons on thesis, electrons are transported from H2O to NADP (and one side relative to the other ultimately to CO2) in a series of reactions that jointly constitutes an eletrochemical form the Z-scheme18. In the course of the electron flow potential, which is harnessed from water to NADP, protons are translocated from the to drive ATP synthesis. cytoplasm through the thylakoid membrane into the thy- lakoid lumen19. The chemiosmotic theory [27] explains ATP formation in photosynthesis. (Fig. 1.2C). Chlorophyll molecules can be excited by red light (Photosystem I (PS I): absorbs maximally at 700 nm, Photosystem II (PS II) at 680 nm). Excitation can occur directly at the special pair20 that is at the heart of each photosystem.

14Interestingly, Barnes himself preferred "photosyntax" over "photosynthesis", but the latter term caught on. 15For the remainder of the text of this thesis, proteorhodopsin-based photosynthesis will not be taken into con- sideration. 16Note that while there are photosynthetic proteobacteria, not all proteobacteria are photosynthetic. Initially, the photosynthetic proteobacteria were classified based on phenotype into purple sulfur- and purple non-sulfur bacteria. Based on 16S rRNA sequence data, this class was later merged into the proteobacteia, but there is no correlation between genotype and phenotype. Recently, it was proposed to resolve this conundrum with a com- bination of ring- and treelike evolution patterns [26]. Divergent gene flow from one ancestor is thus represented by continuous branching, while horizontal gene transfer and endosymbiotic events yield ring structures. 17Notably, some marine nitrogen fixers lack photosystem II and consequently do not produce oxygen [28]. 18Named so because of its N-shape. 19Notably, the cyanobacterium Gloeobacter violaceus lacks thylakoid membranes. Consequently, in this organism, the entire photosynthetic machinery is located in the cytoplasmic membrane [31]. This organism also contains a proteorhodopsin [32]. 20Two Mg-chlorophyll complexes oriented such that their ring planes are parallel to each other. 6 1.G ENERAL INTRODUCTION

The probability of photon absorption is greatly increased by the presence of antennae in the 1 form of phycobilisomes; when a photon is absorbed by an antenna complex, the resulting exciton21 can be relayed to a special pair. After excitation of PS II, the excited electron can be transported via chlorophyll and pheo- phytin molecules to plastoquinone, forming a radical anion. A second reduction step yields plastoquinol. Water is split at the oxygen evolving complex, essentially a Mn/Ca/O cluster that catalyses oxidation of two H2O into 4 H+,O2 and 4 e−, the latter of which fill the positively charged electron hole(s) at PS II. 2–electron oxidation of plastoquinol at the cytochrome b6f (cyt b6f) complex recycles plas- toquinone and formally transfers two (extra) protons from the cytoplasm into the thylakoid lumen. Electrons from cyt b6f are transferred to plastocyanin (PC). Also at PS I, chlorophyll molecules are excited by (red) light. The electrons are transported from the secondary acceptor of PS I via ferredoxin and ferredoxin:NADP oxidoreductase (FNR), to NADP, thus forming NADPH [33]. The resulting electron hole at PS I is filled by another electron derived from reduced PC. The proton gradient is used to fuel ATP synthesis [27]; as protons are cycled back from the thylakoid lumen to the cytosol, they pass through ATP synthase, a protein complex that re- sembles a rotary motor. Proton transport through this system propels the rotor part, which leads to structural changes in the nucleotide binding sites in the cytoplasmic domain of the enzyme (F1). Enforced proximity of ADP and phosphate in these binding pockets leads to generation of ATP,which is released into the cytoplasm by continued rotation of the shaft of the enzyme. With strictly linear electron transport, the NADPH/ATP ratio provided by the photosyn- thetic machinery does not exactly match the preferred ratio of the Calvin-Benson-Bassham cycle (CBB or Calvin cycle, Fig.1.2B). Cyclic electron transfer around PS I, for instance, can be used to ensure optimally adjusted output [34, 35]. Phototrophs harbour a wealth of alter- native routes of electron transfer, often referred to as alternative electron flow (AEF). Those systems are also relied on in stress situations, when specific energy demand is increased. An alternative use of AEF is to translate varying energy input (varying light quality and quan- tity) into comparatively constant output of chemical energy. Thus, alternative electron flow pathways have been proposed to increase photosynthetic robustness [36]. Light-independent reactions of photosynthesis. In the dark reactions of photosynthesis22, ATP and NADPH drive the Calvin cycle23 (See Fig. 1.2 B, [37,38]). The key enzyme of the Calvin cycle is Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). Reaction of CO2 with Ribulose-1,5-bisphosphate (RuBP) yields two molecules of 3-phosphoglycerate (3PG). ATP then drives the phosphorylation of 3PG to yield 1,3-bisphosphoglycerate, which in turn is reduced to glyceraldehyde-3-phosphate (GAP), in an NADPH dependent reaction. Three molecules of CO2 need to be fixed to generate one molecule of GAP,which can be used to build up biomass24. Recycling of RuBP starts with the conversion of GAP into dihydroxy

21electron/electron-hole pair. 22Note, that these reactions require ATP and NADPH and therefore happen in the light. No direct photon input is needed, however. 23Also referred to as the reductive pentose phosphate pathway. 24Although the description above (and Fig. 1.2 suggest(s) GAP to be the only molecule from which further biosyn- thesis originates, this is a simplification. Indeed, most CBB cycle intermediates are shared with other central metabolic pathways. 7 acetone phosphate (DHAP)25, followed by a series of carbon-carbon bond formations and backbone rearrangements catalysed by aldolase and transketolase. Aldolase catalyses the 1 reaction of an aldose phosphate and a ketose phosphate to a single ketose bisphosphate. In the CBB cycle, aldolase catalyses the reactions of GAP and dihydroxyacetone phosphate to fructose-1,6-bisphosphate and of erythrose-4-phosphate (E4P) and DHAP to sedoheptu- lose-1,7-bisphosphate. The bisphosphates are converted into the terminal monophos- phates by dedicated phosphatases. Transketolase catalyses the transfer of a C2 unit from a ketose onto an aldose, yielding an aldose with a carbon chain length diminished by two and a ketose with a chain length increased by two. Accordingly, transketolase catalyses the reaction of fructose-6-phosphate with GAP to xylulose-5-phosphate (Xu5P) and E4P as well as the reaction of sedoheptulose-7-phosphate (S7P) with GAP to ribose-5-phosphate (R5P) plus Xu5P. R5P and Xu5P are converted into ribulose-5-phosphate (Ru5P) by ribose phos- phate isomerase and phosphopentose epimerase, respectively. The phosphoribulo-kinase- catalysed phosphorylation of Ru5P recycles RuBP,closing the CBB cycle.

CYANOBACTERIA

Cyanobacteria are phototrophic prokaryotic organisms. They have simple growth require- ments and consequently have conquered many ecological niches, ranging from polar [39] to equatorial regions. Cyanobacteria dwell in seas [40], lakes [41], hot springs [42], even deserts [43] and, rather more recently, laboratories. Given that primordial cyanobacteria are thought to be the ancestors of plant chloroplasts ([44], [45] and references therein), their habitat formally extends to the inside of green plants. In short: wherever there is sunlight, you can expect to find cyanobacteria. When conditions are favourable, cyanobacteria are known to form blooms [46]. Some cya- nobacterial strains produce extremely potent toxins [47]. This does not only prohibit recre- ational use of lakes but may endanger existent ecosystems and put drinking water supply at risk [48]. In lakes, degradation of cyanobacterial biomass in the aftermath of a bloom can lead to anoxic conditions26, which poses a threat to fish populations. A better understand- ing of how such blooms come about could help to reduce risk for these ecosystems [49]. Structure and morphology. Cyanobacterial morphology is diverse; some species are uni- cellular, some are filamentous (linear, branched, helical), others form colonies or are part of microbial mats. Individual cells are spherical or rod-shaped [50]. In addition to vege- tative cells, filamentous, nitrogen-fixing strains like Anabaena possess heterocysts, whose microoxic [51] interior allows the operation of nitrogenase [52]. Altogether, cyanobacteria display a cell wall structure27 akin to Gram-negative bacteria, albeit there are some differences28. With one exception [31], the interior of a cyanobacterial cell is dominated by the presence of thylakoid membranes. Thylakoids differ in composition from the cytoplasmic membrane

25catalysed by triose phosphate isomerase. 26I.e. a disturbance in the oxygen cycle. 27Outside to inside: exopolysaccharide layer (some species)-S-layer-outer membrane-peptidoglycan layer- periplasmic space-cytosolic membrane. 28E.g. the peptidoglycan layer is thicker and shows a degree of crosslinking resembling that of Gram-positive bacteria. See [53] and references therein for further information. 8 1.G ENERAL INTRODUCTION

A FNR B Calvin-Benson-Bassham cycle 1 FD Q-pool NADPH tial FBP F6P E4P S1,7BP S7P e n

Pi

on po t DHAP

t r R5P Xu5P CO2 ele c

PSII Cyt b6f PC PSI GAP 1,3BPG 3PG Ru1,5BP Ru5P

Biomass

P, ADP ATP C i

H+

NADP NADPH H+ PSII

PQ FNR Cyt b FD 6f

PQH2

+ 2 H2O O2, 4 H H+ PSI PC ATPase

Figure 1.2: Schematic overview of Photosynthesis. (A) The Z-scheme; a qualitative representation of an electron’s potential energy as it travels from water to NADP. (B) The Calvin Benson Bassham cy- cle. Reaction of Ribulose-1,5-bisphosphate (Ru1,5BP) yields two molecules of 3-phosphoglycerate, which undergo phoosphorylation to 1,3-bisphosphoglycerate. NADPH-mediated reduction gives glyceraldehyde-3-phosphate (GAP), which can be isomerised to dihydroxyacetone phosphate (DHAP) or react with a number of ketoses in transketolase catalysed reactions: Reaction of GAP with DHAP gives fructose-1,6-bisphosphate, which is dephosporylated to Fructose-6-phosphate (F6P). Reaction of GAP with F6P gives ribose-5-phosphate (R5P) and erythrose-4-phosphate (E4P). E4P and DHAP react to sedoheptulose-1,7-bisphosphate, which first gets dephosphorylated (to yield sedoheptulose- 7-phosphate) and then reacts with GAP to give R5P and Xylulose-5-phosphate (Xu5P). R5P and Xu5P isomerise to Ribulose-5-phosphate, the phoshporylation of which gives Ru1,5BP,closing the cycle.(C) Schematic representation of electron and proton flow in oxygenic photosynthesis. Electrons originat- ing from water move through membrane proteins and molecules to NADP, forming NADPH. Abbre- viations: PS: Photosystem, Q-pool: plastoquinone pool, PQ: Plastoquinone, PQH2: Plastoquinol, Cyt: Cytochrome, PC: Plastocyanin, FD: Ferredoxin, FNR: Ferredoxin-NADP oxidoreductase 9 and are the site of both photosynthetic and respiratory electron flow [54]29. Cyanobacteria have evolved microcompartments, termed carboxysomes, which harbour 1 RuBisCO and carbonic anhydrase. Compared to O2, CO2 is a rather poor substrate for Ru- 30 BisCO . In the carboxysomes, the CO2 concentration is higher than in the cytoplasm, which allows more efficient operation of RuBisCO (see [57–59] for review articles on the subject). Other major intracellular structures include ribosomes, polyphosphate bodies, polyhydrox- yalkanoate granules, cyanophycin granules and lipid bodies [60]. Some species harbour cylindrical proteinaceous gas vesicles that take part in buoyancy regulation [61, 62]. As prokaryotes, cyanobacteria lack a nucleus and the DNA is therefore located in the cy- tosol. Cyanobacteria have circular chromosomes31 and may hold several additional plas- mids. Genome sizes range from 1.44 Mb (UCYN-A [64]) to 12.07 Mb (Scytonema hofmanni PCC7110 [65]) in length. The number of chromosome copies per cell depends on the species; experimentally determined gene copy numbers vary from 1 (Synechococcus WH8101 [66]) to a staggering 218 (exponentially growing Synechocystis [67]). Phylogeny. The Cyanobacteria are a monophyletic clade in the Bacteria. Details of cyano- bacterial phylogeny are the subject of extensive debate [68], the conclusion of which seems to be that this taxonomy needs to be revised [69]. Initially, cyanobacteria were considered to be plants32 by all but a few "shrewd and isolated pioneers" [70]. After techniques with high spatial resolution (electron microscopy) became available [70], cyanobacteria were re- classified by Rippka et al. under bacteriological criteria (morphology, reproduction) into five subsections [71]. This system, although not in accordance with nomenclatural rules (subsections instead of classes, orders), is convenient, and therefore, still widely used33 A blind- Subsections (Rippka et al. [71]) Classes (NCBI) [73] text to I Chroococcales h Gloeobacteria in- II Pleurocapsales h Nostocales clude III Oscillatoriales i h Oscillatoriophycideae the IV Nostocales box. h Pleurocapsales and V Stigonematales h Prochlorales an- other h Stigonematales one more. i This class is composed of the Chroococcales and the Oscillatoriales.

29 Light-harvesting phycobilisome antenna complexes are located on the cytoplasmic side of the thylakoid mem- brane [54] and seem to control the distance between the respective sheets [55]. Thylakoid shape and position are species-dependent; (See [56] for very clear schematic illustrations). 30Photosynthesis evolved in an oxygen-poor environment [29]. 31Cyanothece ATCC51142, apart from a circular chromosome and plasmids, harbours a linear chromosome, as well [63]. 32Hence, the historical term, "blue-green algae". 33Often, newly available molecular data is at odds with the current taxonomy; sequence analyses have shown that a number of morphological characteristics used for classification have evolved multiple times [72] and conse- quently cannot be reliable indicators of lineage. In light of newer approaches [26] that incorporate divergent as well as convergent evolution, it would be highly interesting to rethink this statement. 10 1.G ENERAL INTRODUCTION

Synechocystis sp. PCC6803 belongs into the Chroococcales. Although mentioned already 1 earlier in this section, it is the cyanobacterium this thesis revolves around and therefore de- serves a separate, short introduction. The PCC stands for Pasteur Culture Collection. The original isolate was the third that was deposited in the BCC (Berkeley Culture Collection) in 1968 and was consequently given the number 6803 [74]. In the older literature, Synechocys- tis is referred to as Aphanocapsa [74]. In 1996, Synechocystis sp. PCC6803 was the first cya- nobacterial strain for which a genome sequence was published [75]. Synechocystis is a nat- urally transformable [76], unicellular cyanobacterium that preferentially grows photoau- totrophically but can also use glucose. It is accessible to genetic modification through the integration of foreign DNA into the chromosome and into endogenous plasmids [77]. Fur- thermore, replicative plasmids, introduced by conjugation, were shown to be maintained [78]. Although initially isolated from a Californian freshwater lake [74], Synechocystis can toler- ate salt concentrations of up to 1.5 mol/L. For a recent review on cyanobacterial salt stress acclimation, see [79]. Salt stress response. As bacteria face osmotic stress (e.g. by an increased extracellular NaCl concentration), water diffuses out of the cells, while salt ions diffuse inwards [80,81], caus- ing the cells to shrink temporarily [80]34. Glucosylglycerol Simultaneously, compatible solutes [82]35 are accumulated OH [80]. These are small organic molecules that are characterised 36 O by high solubility in water, lack of net charge and by being HO preferentially excluded from the immediate vicinity of pro- HO glucose 37 HO teins [84] . O The nature of cyanobacterial compatible solutes is strain- OH

dependent; There is no correlation of the osmolyte accumu- glycerol OH lating and the habitat of a given strain38. The main osmolyte of Synechocystis is glucosylglycerol [88]. Figure 1.3: The cyanobac- Osmolyte synthesis is energetically costly and these com- terial osmolyte glucysolglyc- erol consists of a glucose- pounds are often of only temporary use. In Synechocystis, a and a glycerol moiety linked degradation pathway for glucosylglycerol was suspected [89], by a glycosidic bond. but could not be identified [90]. Applications of cyanobacteria. Historically, cyanobacteria have been used as sustenance

34Na+ influx is counteracted by the activation of efflux pumps [79]. 35In the literature, the terms osmolytes and osmoprotectant are also used. 36Interestingly, some picoplanktonic cyanobacteria have been shown to accumulate glucosylglycerate, with a net charge of –1 [83]. 37 Accumulation of compatible solutes has two effects; it raises the intracellular osmotic pressure and therefore balances the increased extracellular osmotic pressure, which allows the cells to retain the cytoplasmic water. Additionally, compatible solutes increase protein stability. An explanation for the latter can be found in the water exclusion hypothesis: The interaction of solute and protein is unfavourable (either due to unfavourable protein- solute interaction or due to a structural effect that excludes the solute from the protein-solvent interface [85]). Therefore, the system tends to minimise protein-solvent contact. Folded proteins have a smaller surface area than denatured ones. Consequently, the native state of the protein is preferred [86]. Thus, compatible solutes increase stability of proteins, not by stabilising the native state but by destabilising the denatured state. 38Instead, a correlation between osmolyte and maximum osmotolerance was found [87]: Strains that only tolerate low (<0.6 M NaCl) salt concentrations use sucrose, moderately halotolerant (<1.6 M NaCl) strains utilise gluco- sylglycerol (GG, Fig. 1.3) and highly salt resistant strains (>1.5 M NaCl) use glycin betaine [79]. As a comparison, the NaCl concentration of seawater is approximately 0.5 M (assuming 3% (w/v)). 11 for both livestock and humans alike [91]39. Nowadays, Arthrospira is sold as dietary supple- ment, food and feed. 1 Their relation to chloroplasts found in today’s green plants has made cyanobacteria impor- tant experimental model systems in efforts to shed light onto the details of photosynthe- sis. This is facilitated in particular by the fact that many cyanobacteria can grow (photo- )heterotrophically [71], a feat that allows the construction of mutant strains deficient in some aspects of photosynthesis. Such strains are serviceable tools in the quest to assign functions to individual parts of the photosynthetic apparatus. In the course of these funda- mental research projects, a number of genetic tools for the manipulation of cyanobacteria was developed and continuously expanded [92]. In recent years, cyanobacteria have attracted considerable interest for biotechnological ap- plications. While some wild-type strains produce attractive molecules on their own [93], the majority of research efforts has focused on the addition of desirable traits, through ge- netic engineering (see Chapter2 for a short overview on progress during the past few years). Their ability to convert CO2 into organic molecules using little more than sunlight and water is extremely attractive from an ecological as well as an economic standpoint.

POLYOLS Polyols are carbon- based molecules with more than one hydroxy group. Applications of these compounds are numerous. Polyols are used among other things as antifreeze agents, tooth-friendly sweeteners and raw materials for polymer production. This thesis will focus on two very simple polyols: Glycerol and 2,3-butanediol. Glycerol is a C3 compound (C3H8O3) some derivatives of which are central to the metabo- lism of all living organisms. The first industrial application of glycerol was the synthesis of nitroglycerin by Nobel. Industrially important derivatives include esters of inorganic (nitric acid in particular) and organic acids and polyglycerols (See [94] for a more complete list). Glycerol is used in the food, cosmetics, tobacco and pharmaceutical industry as a moistur- izer, preservative and solvent [94]. As glycerol is the backbone of lipid molecules, splitting their ester bonds through hydrolysis or transesterification reactions yields glycerol as a by- product. At this point, the biodiesel industry has to be emphasised as a major source of crude glycerol; 100 kg of glycerol are formed per ton of biodiesel [94]. An alternative syn- thetic route of industrial importance is the synthesis from propene (via allyl chloride and epichlorohydrin) [94]. Although glycerol is on the list of compounds for which biosynthetic routes should be researched, there is no fermentative route of economic significance for its synthesis [94]. In 1995 supply of glycerol surpassed demand, a fact that is expected to be exacerbated by continuing use of first generation biofuels and, particularly, biodiesel[94]. 2,3-Butanediol (C4H10O2) has three stereoisomers: R,R-, S,S-, and R,S-(meso) (Fig. 1.4). Technical synthesis of 2,3-butanediol is accomplished by hydrolysis of 2,3-butene oxide [95]. Natural producers of 2,3-butanediol include Klebsiella pneumoniae and Enterobacter aerogenes. Most species produce R,R- or R,S-2,3-butanediol. Biological production of 2,3- butanediol peaked in World War II [96]. Double elimination of water from 2,3-butanediol yields 1,3-butadiene40, the precursor of synthetic rubber. 2,3-butanediol can be used as

39Strains of the genus Arthrospira (also referred to as Spirulina) were known to the Aztecs of central America as techuitlatl and are to this day harvested and processed to Dihé by the Kanembu of Chad ([91] and references therein). 40Industrially, this was achieved through pyrolysis of the diacetate [95]. 12 1.G ENERAL INTRODUCTION

a glycerol substitute, cryoprotectant, a lubricant, humectant, an additive in cosmetics, a 1 cross-linker in polyurethanes and a starting point for chemical synthesis (of insecticides, for the pharmacetical industry) [95, 97]. It was shown to increase the octane number of fu- els [97]. Furthermore, 2,3-butanediol can be used as a precursor of 2-butanol, a promising fuel candidate.

ASYMMETRY If a molecule cannot be overlayed with its mirror image, it is called chiral41. Two molecules that are mirror images of each other are called enantiomers. An equal mixture of two enan- tiomers is called a racemic mixture, or racemate42. In a given non-racemic mixture of two enantiomers, the enantiomeric excess (%ee) is calculated as follows:

n1 n2 %ee | − | 100% = n1 n2 · (1.1) + n amount of isomer i i = When n n , i.e. in a racemic mixture, the ee is zero. 1 = 2 Achiral molecules that can be converted into chiral molecules in one step are called prochi- ral. Examples of such molecules in this thesis are glycerol43, meso-2,3-butanediol and bu- tanone. Stereoisomeric molecules that differ in the absolute configuration of at least one, but not all stereocentres, are called diastereomers. Chirality comes in many flavours, among them helical, axial and planar. As far as mole- cules in nature are concerned, the most commonly observed form of chirality is central chirality44. This term refers to a central atom with at least four different substituents, whose positions are sufficiently fixed. If inversion barriers are low, molecular vibration will convert one enantiomer into the other (as seen in e.g. substituted amines). A ray of linearly polarised light can be seen as a superposition of two rays of opposite cir- cular polarisation. Chiral entities interact differently with enantiomers of other chiral en- tities45. Since one ray interacts more strongly than the other, it is retarded along the light axis. The combination of the two rays again gives linearly polarised light, but now the plane of polarisation is rotated. Although not recommended, the D/L nomenclature is still widely used in the literature, par- ticularly for sugars and amino acids (historically, this method has been attributed to Emil Fischer; see Fig. 1.4A for an illustration). To determine the absolute configuration with this method, the molecule is rotated such that the carbon atom with the highest oxidation num- ber is at the top. The carbon chain extends from top to bottom (Fischer-projection). Now, the carbon atom that has a substituent other than hydrogen and that is furthest away from the top needs to be identified. If this substituent is on the right side of the chain, you are looking at the D-form. If it is on left, you are looking at the L-form.

41derived from the χει%, (Greek: hand) [98]; chiral molecules differ like the left hand differs from the right. 42After an antiquated name of tartaric acid (racemic acid, from the French acide racemique [99]). 43While glycerol is achiral, enzymes can distinguish the two primary alcohols. Glycerol kinase, thus converts glyc- erol into the R isomer, exclusively. 44Helical chirality of e.g. DNA or amylose is a consequence of central chirality in the sugar moieties. The direction of α-helices in proteins similarly is determined by the central chirality of the amino acids. 45For hands-on experience, try uncorking a bottle with your left. 13

A Fischer-projection 1 O O O O ! = = = H D OH HO H OH OH

B CIP-nomenclature HO HO HO

2 3

4 1 OH H OH OH

HO HO OH 4 H 1 2 3 2 OH OH R S 4 3 H 1OH R,R-2,3-butanediol S,S-2,3-butanediol R,S-2,3-butanediol

Figure 1.4: Illustration of nomenclature for entire molecules according to Fischer (A), and for indi- vidual stereocentres in molecules according to Cahn, Ingold and Prelog (B) [100]. Arrows in the first line denote rotations of molecules or rotations around binding axes. Arrows in the bottom row indi- cate the stereoconfiguration of the respective sereocentre. The grey plane denotes a plane of mirror symmetry, which renders the molecule achiral.

The recommended nomenclature for stereocentres is the (R/S)- or CIP (Cahn-Ingold-Prelog)- system [100]. Priorities are assigned to substituents of a stereocentre. In the simplest case, atoms with the higher atomic number gain the higher priority. The stereocentre is then ro- tated such that the substituent of lowest priority faces away from the observer. Now, if draw- ing a circle from the first via the second to the third priority results is a clockwise motion, the stereocentre has R-configuration (rectus, lat.: straight). If the motion is counterclock- wise, the molecule has S-configuration (sinister, lat.: left). See Fig. 1.4B for an illustration. If a molecule with more than one stereocentre has a plane of mirror symmetry, the molecule is achiral. An example in this thesis is meso-2,3-butanediol.

DRIVINGFORCES Given a kinetic pathway between two states, the composition of a system will move towards lower Gibbs energy. A crucial task of metabolic engineering is thus to survey the thermody- namics of production pathways and to apply insights of these studies to the engineering of 14 1.G ENERAL INTRODUCTION

the pathway at hand. 1 In the biosynthetic pathway to n-butanol for instance, the first step, i.e. the synthesis of acetoacteyl-CoA from two acetyl-CoA is endergonic. In the natural host, e.g. Clostridium species, the reaction is driven by the high amount of acetyl-CoA, which is derived from sub- strate catabolism. Furthermore, high amounts of NADH, also from substrate catabolism, drive reduction reactions further downstream, altogether creating a thermodynamically favourable pathway. Fatty acid biosynthesis similarly begins with acetoacetyl-CoA. To avoid the endergonic reac- tion of two acetyl-CoA to acetoacetyl-CoA, the direct reaction is replaced with two exergonic reactions: the reaction of acetyl-CoA to malonyl-CoA, in which hydrolysis of ATP provides the driving force, and the condensation of malonyl-CoA and acetyl-CoA to acetoacetyl-CoA, which is driven forward by the liberation of CO2. Lan and Liao demonstrated that the re- placement of the direct reaction to acetoacetyl-CoA with the two steps derived from fatty acid biosynthesis substantially increases yields when the butanol pathway is expressed in transgenic Synechococcus elongatus PCC7942 [101]. The butanediol biosynthetic pathway that is presented in chapter3 of this thesis contains two decarboxylation reactions, which provide a driving force for the respective reactions. In the same chapter, it is demonstrated that the substitution of an NADH dependent acetoin reductase with an NADPH dependent homologue provides a driving force for the synthesis of 2,3-butanediol. Moreover, in chapter7, the use of acetoin reductases with different co- substrate specificity allows the construction of a driving force towards S,S-2,3-butanediol. 15

SCOPE 1 This thesis focuses on the engineering of the cyanobacterium Synechocystis sp. PCC6803. Heterologous reactions are introduced into the metabolism of Synechocystis, to allow the synthesis of molecules that this strain would not normally produce. Additionally, a path- way for salt-dependent glycerol production is discovered and a likely candidate enzyme is presented. Finally, the use of multicompartment systems (cocultures) allows the combina- tion of otherwise incompatible processes in the same culture. Chapter2 reviews recent progress made in genetically manipulated cyanobacteria for the conversion of CO2 into fuels and chemical feedstocks. Methods, concepts, results and some perspectives are discussed. In Chapter3 , mutant strains of Synechocystis sp. PCC6803 are presented that have been en- gineered for the production of meso-2,3-butanediol from light, water and carbon dioxide. This chapter investigates how the nature and abundance of a cosubstrate (NAD(P)H) that is required for the last step of the pathway influences the yield of 2,3-butanediol. In Chapter4 , the [butanediol]/[acetoin] ratios measured in Chapter3 are used to estimate intracellular [NAD(P)H]/[NAD(P)] ratios. In Chapter5 , a mutant strain of Synechocystis sp. PCC6803 is presented which harbours the phosphoglycerol phosphatase 2 enzyme of Saccharomyces cerevisiae and produces glycerol from light, water and carbon dioxide. The chapter investigates the effects of mild salt stress on the glycerol production. This increase is shown to be independent of the heterologous enzyme, i.e. the wild type strain already produced glycerol under these stress conditions. In Chapter6 , the pathway by which wild-type Synechocystis produces glycerol under mild salt stress is investigated with a set of inacitvation mutants. A genome-scale model of Syne- chocystis is extended to incorporate different candidate reactions for the hydrolysis of glu- cosylglycerol and to predict the benefit such a reaction would confer onto the cyanobacte- rium. In Chapter7 , a means of production for enantiopure S,S-2,3-butanediol is presented. This approach relies on the usage of the NADPH/NADP pool as the driving force for the reduc- tion of acetoin, while the NADH/NAD pool of a heterotrophic bacterium is used to oxidise the 2,3-butanediol enantiomer with the undesired stereo-configuration. Chapter8 covers the dehydration of polyols to yield alcohols with increased hydrophobic- ity and heating values. Since the enzymatic pathway that leads to these molecules contains oxygen-sensitive steps, these reactions are investigated in chemotrophic bacteria such as recombinant E. coli and Lactobacillus reuteri in addition to recombinant Synechocystis. Chapter9 puts the results obtained in this thesis into a broader perspective.

2 ENGINEERINGCYANOBACTERIAFOR DIRECTBIOFUELPRODUCTION

FROM CO2

Philipp SAVAKIS, Klaas J. HELLINGWERF

For a sustainable future of our society it is essential to close the global carbon cycle. Oxidised forms of carbon, in particular CO2, can be used to synthesise energy-rich organic molecules. Engineered cyanobacteria have attracted attention as catalysts for the direct conversion of CO2 into reduced fuel compounds. Proof of principle for this approach has been provided for a vast range of commodity chemi- cals, mostly energy carriers, such as short chain and medium chain alcohols. More recently, research has focused on the photosynthetic production of compounds with higher added value, most notably terpenoids. Below we review the recent developments that have improved the state-of-the-art of this approach and speculate on future developments.

This chapter has been published in Current Opinion in Biotechnology 33:8, 8-14 (2015) [102].

17 18 2.E NGINEERINGCYANOBACTERIAFORDIRECTBIOFUELPRODUCTIONFROM CO2

INTRODUCTION:‘LIGHT-DRIVENCONVERSION’

YANOBACTERIA are photosynthetic prokaryotes that can use photon energy to ultimately C transfer electrons from water to carbon dioxide, generating more reduced molecules in the process. The introduction of heterologous, mostly catabolic, pathways into the metab- 2 olism of cyanobacteria allows production of a wide range of fuel and commodity products from CO2, light and water [21, 103]. In the recent past this approach has matured so that by now for a large range of compounds proof of principle has been provided (for review see: [104]; Angermayr, Thesis, 2014). Quan- titative evaluation of these production systems leads to the conclusion that in many cases the majority (i.e. >50%) of the CO2 fixed by the engineered cyanobacterium is directly con- verted into product [105]. Angermayr et al. introduced the term ‘biosolar cell factories’ for these organisms/systems (see also Figure 2.1). At the laboratory scale biosolar cell facto- ries have now proven to be functional for sucrose, ethanol, L-lactic acid and 2,3-butanediol [105–108].

hν

NADP

CO 2 H2O

C NADPH a r O b 2 ATP ADP o n u C pt a ak lv e in - B

e

n

growth & s

o

n

maintenance -

B

a

t s

c s

ss h

u a a e l b m m c

y io d c ro p o t

n

i

n

o

b

r a c (excreted) product

Figure 2.1: Schematic representation of a cyanobacterial cell factory. In the thylakoids, photon en- ergy drives water splitting, reduction of NADP and phosphorylation of ADP. Carbon dioxide gets as- similated in the Calvin-Benson-Bassham cycle at the cost of NADPH and ATP.Fixed carbon can then either be channelled into biomass or into a production pathway. 19

For a limited range of compounds pilot-scale facilities have been set up and are currently being tested for economic competitiveness. To a significant extent the rapid expansion of the range of products that can be made by cyanobacteria, engineered to carry out ‘photofer- mentative’ metabolism, was facilitated by a spill-over of knowledge from conventional fer- mentation approaches, in particular from Escherichia coli and Saccharomyces cerevisiae, which have been engineered to produce a wide range of products. 2 In the cyanobacterial field, most metabolic engineering studies have been carried out us- ing the two-model organisms Synechocystis sp. PCC 6803 (Synechocystis 6803) and Syne- chococcus elongatus sp. PCC 7492. Synechococcus sp. PCC 7002 (Synechococcus 7002) and Anabaena sp. PCC 7120 (Anabaena 7120), however, are receiving increased attention. A similar approach of ‘direct conversion’ is also possible for eukaryotic oxygenic phototrophs, like green algae or brown algae. However, the generally much more complex genetic engi- neering required for these organisms has hampered rapid progress so far [109].

BIOFUELSANDOTHERPRODUCTSOFENGINEEREDCYANOBACTE- RIA

With fuel applications in mind, there are several interesting classes of molecules available. Figure 2.2 gives an overview of heterologous pathways that have been introduced into cy- anobacteria and the corresponding product titres that were achieved. Hydrogen, although versatile in its applications, will not be discussed in this review. Short chain alcohols can be used as drop-in automotive fuels. The highest titres for a pho- tosynthetically produced biofuel have been reported by Gao et al. for ethanol (5.5 g/L af- ter 26 days) [107]. Cyanobacterial production of acetone [110], isopropanol [111] and 1,2- propanediol [112] was reported recently. Lan and Liao increased butanol titres [101, 113]. 2,3-Butanediol synthesis was achieved in Synechococcus 7942 and Synechocystis 6803 [108, 114, 115]. Shen and Liao reported production of 2-methyl-1-butanol in Synechococcus 7942 [116]. Because of their high energy content, fatty acids and their alcohol derivatives and alkane derivatives are attractive fuels. Following the work on Synechocystis 6803 [117], photosyn- thetic production and excretion of free fatty acids (FFAs), through overexpression of a thio- esterase, were recently also achieved in Synechococcus 7942 [118] and in Synechococcus 7002 [119]. Using an alternative approach, that is, overexpression of the endogenous acyl-ACP reductase, Kaiser et al. demonstrated excretion of FFAs in Synechococcus 7942 [120]. In the same study, production of triacylglycerols and wax esters was reported. Overproduction of alkane biosynthesis genes [121] from various cyanobacteria in Synechocystis 6803 led to an increase of heptadecane and heptadecene content [122]. Mutants harbouring NADPH- dependent fatty acyl-CoA reductase showed increased levels of C15–C17 fatty alcohols [123]. Terpenoids are an extremely diverse class of molecules, highly interesting for a wide variety of fuel-chemical, bulk-chemical, and fine-chemical applications. Production of isoprene in cyanobacteria was first reported by Lindberg et al. [124]. Recently, also production of β- phellandrene was demonstrated [125, 126]. Limonene production has been reported in An- abaena 7120 [127], Synechocystis 6803 [128] and Synechococcus 7002 [129]. Sesquiterpenes are attractive jet fuels or precursors thereof. Photosynthetic production of the sesquiter- pene a-bisabolene was demonstrated in Synechococcus 7002; inactivation of glycogen syn- 20 2.E NGINEERINGCYANOBACTERIAFORDIRECTBIOFUELPRODUCTIONFROM CO2

thesis in this strain did not positively influence terpenoid production [129]. Englund et al. showed that deletion of squalene-hopene cyclase in Synechocystis 6803 leads to a mutant that accumulates squalene [130]. The terpenoid precursors dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP) can be synthesised via the methylerythritol phosphate (MEP) or via the mevalonate pathway. Cyanobacteria utilise the former. Over- 2 production of MEP pathway enzymes led to increased carotenoid levels [131] and increased limonene yield [127, 128]. Cells expressing the heterologous mevalonate pathway in combination with an isoprene synthase showed increased isoprene titres [132]. Thermal recycling of fossil-derived plastics contributes to CO2 emission. Conversion of the CO2 produced as the direct result of thermal recycling of polymers into new monomers would contribute to closing the carbon cycle. For the monomers ethylene [133] and L- lactate [106, 134, 135] increased yields were reported. Proof of principle was provided for the production of D-lactate [136] and 3-hydroxybutyrate [122].

GENERALCHALLENGESFORTHEDESIGNOFPRODUCTIONSYS- TEMS

A compound of interest can be produced through the introduction of a reaction that con- verts an endogenous metabolite. If the compound of interest is an endogenous metabolite, then its intracellular concentrations can be increased by removal of a consuming reaction. Many compounds are excreted from and/or leak out of the cytoplasm into the culture me- dium. For cyanobacteria, currently little is known about the underlying processes. It was shown that exporter proteins can lead to extracellular accumulation of products that other- wise would not be able to diffuse through the membrane [137]. In selected examples, overall product formation was stimulated through such proteins [105]. Fermentative pathways are attractive for biofuel synthesis as many lead to the formation of reduced compounds with high heat of combustion. In the native host, these pathways are typically operating under conditions with limited oxygen supply and can therefore include oxygen sensitive conversions, which may conflict with oxygenic photosynthesis. Mutually incompatible processes can be either changed or separated. Thus, replacing oxy- gen-sensitive CoA-acylating butyraldehyde dehydrogenase with oxygen-tolerant CoA-acyl- ating propionaldehyde dehydrogenase increased butanol titres [113]. Separation can either be spatial [138] or temporal. Spatial separation can be achieved in single cells through pro- tein complexes or specialised micro-compartments, in multicellular strains through spe- cialised cells, and in cocultures [139, 140] through different organisms. In filamentous dia- zotrophic cyanobacteria, vegetative cells carry out oxygenic photosynthesis, while oxygen- sensitive nitrogen fixation takes place in dedicated heterocysts. Harnessing the power of the latter, Ihara et al. targeted oxygen-sensitive formate dehydrogenase to heterocysts of Anabaena 7120, which resulted in the direct reduction of CO2 to formate [141]. Temporal separation of oxygenic photosynthesis and nitrogen fixation is realised in unicellular dia- zotrophs [142]. These strains provide potentially interesting targets for genetic engineering. Storage compounds allow temporal separation of photon energy harvesting and utilisation. During nitrogen starvation, cyanobacteria can accumulate up to 41% of dry cell weight as glycogen [143]. Synechocystis 6803 can accumulate up to 9.5% and 11% of its dry cell weight 21

Isoprene DMAPP Limonene, Phellandrene A Bisabolene CO DXP MEP HMBPP GPP FPP 2 Lactate IPP

Acetolactate Isobutyraldehyde Isobutanol

CO2 Mannitol Ru1,5BP 3PG GAP 2PG PEP Pyruvate Acetoin 2,3-Butanediol

G1P CO Acetaldehyde Ethanol F1,6BP 2 2 DHAP 1-Propanol Ru5P Methylglyoxal Sucrose F6P E4P 2-Ketobutyrate 2-Methyl-1-butanol R5P Xu5P 1,2-Propanediol Acetyl CoA Acetoacetyl CoA HMG CoA Mevalonate Mevalonate PP S7P S1,7BP Glycogen Acetoacetate Acetone Isopropanol CO2 multiple reaction steps Hydroxybutyryl CoA Crotonyl CoA Butyryl CoA Butanol

storage compound Malate OA Citrate Hydroxybutyrate molecule derived from Malonyl CoA Polyhydroxybutyrate G1P Free fatty acids Fumarate Isocitrate F6P Malonyl ACP

GAP Ac(et)yl ACP Acetoacyl ACP Fatty aldehydes Succinate SSA OG Pyruvate CO CO2 2 Acetyl CoA GABA Glutamate ethylene Enoyl ACP Hydroxyacyl ACP Fatty alcohols Alkanes 1000 B

ethanol 100

2,3-butanediol lactate isobutyraldehyde lactate lactate 10 2,3-butanediol isobutanol mannitol 3-hydroxybutyrate n-butanol lactate sucrose isobutanol 2,3-butanediol 1,2-propanediol 2-methyl-1-butanol 1 n-butanol FFA sucrose FFA concentration / mmol/L concentration 0.1

limonene acetone isoprene 0.01 n-butanol limonene α-bisabolene limonene 0.001 0 5 10 15 20 25 30 35 time / day

Figure 2.2: (a) Intermediary metabolism of a typical cyanobacterium under photoautotrophic growth conditions, including heterologous, biofuel-forming pathways. Fuels are coloured accord- ing to the metabolite they originate from. Major carbon sinks are underlined. Abbreviations: 2 PG: 2-phosphoglycerate, 3 PG: 3-phosphoglycerate, ACP: acyl carrier protein, CoA: coenzyme A, DHAP: dihydroxyacetone phosphate, DMAPP: dimethylallylpyrophosphate, DXP: 1-deoxyxylulose- 5-phosphate, E4P: erythrose-4-phosphate, F1,6BP: fructose-1,6-bisphosphate, F6P: fructose-6- phosphate, G1P: glucose-1-phosphate, G6P: glucose-6-phosphate, GABA: γ-hydroxybutyrate, GAP: glyceraldehyde-3-phosphate, (continued overleaf) 22 2.E NGINEERINGCYANOBACTERIAFORDIRECTBIOFUELPRODUCTIONFROM CO2

as polyhydroxybutyrate (PHB) under nitrogen starvation and phosphorus starvation, re- spectively [144]. Inactivation of storage pathways should therefore allow a greater fraction of the fixed car- bon to be directed into product. Through this approach, partitioning to product can only be increased in conditions under which storage pathways would be active in the wild-type 2 organism. Inactivation of the glycogen synthesis pathway has shown success under nitro- gen deplete conditions [145, 146]. Inactivation of PHB biosynthesis increased yields of 3- hydroxybutyrate [122] and led to a 70% increase in heptadecane and heptadecene content, compared to the strain that still produced PHB [122]. For lactate production, no positive effect of this mutation could be observed [146]. While for an ethanol producing Synechocys- tis strain, inactivation of PHB synthesis did not lead to a significant increase in productiv- ity, a PHB deficient mutant harbouring an additional copy of the ethanol production cas- sette showed a twofold increase in enzyme activity and, intriguingly, a threefold increase in ethanol titre [107]. This illustrates that in optimising productivity, actual limitations should be identified. To this end, a sensitivity analysis [147] was recently carried out for lactate producing Synechocystis strains [134]. At very high expression levels it could be shown that control was shifted away from the lactate dehydrogenase into other parts of the product- forming pathway, as expression of pyruvate kinase only stimulated product formation in this latter strain [106]. An alternative to the removal of competing pathways, is the manipulation of driving forces. In cyanobacteria, decarboxylation reactions [108, 115, 116, 148–150] and cleavage of phos- phoester bonds [151] have been used for this. Coupling of an uphill reaction to ATP-hydrolys- is has been successful [101]. NADPH can be directly recycled by the thylakoids and is pre- sumably more abundant than NADH. NADH-dependent reactions can therefore be driven through expression of a transhydrogenase [112, 115, 135, 137] or by replacement with en- zymes that utilise NADPH [107, 112, 115]. In the pursuit of ever-higher titres, product toxicity can limit yield. A number of studies have investigated transcriptional responses induced by added or produced solvent [152– 155]. Anfelt et al. identified, among other proteins, HspA to increase tolerance of Syne- chocystis towards butanol. Production of free (particularly unsaturated) fatty acids in Syne- chococcus 7492 seems to be limited by negative physiological effects [118], most probably product intercalation in cellular and thylakoid membranes [156]. For volatile products con- tinuous extraction can be used for sustained production[128, 129, 148]. This alleviates not only product toxicity, but also potential product (feedback) inhibition.

Figure 2.3 (continued from previous page): GPP: geranyl pyrophosphate, HMBPP: 4-hydroxy-3- methylbut-2-enyl diphosphate, HMG-CoA: hydroxymethylglutaryl CoA, IPP: isopentenyl pyrophos- phate, MEP: 2-methylerythritol-4-phosphate, OA: oxaloacetate, OG: oxoglutarate, PEP: phospho- enolpyruvate, Ru1,5BP: ribulose-1,5-bisphosphate, Ru-5-P: ribulose-5-phosphate, R-5-P: ribose-5- phosphate, S1,7BP: sedoheptulose-1,7-bisphosphate, S7P: sedoheptulose-7-phosphate, SSA: succinic semialdehyde, Xu-5-P: xylulose-5-phosphate. (b) Production overview. Maximum titres (in the re- spective study) of excreted products are shown at the time points indicated. The time axis does not distinguish between constitutive systems and inducible systems, in which concentrations are typi- cally measured x days after induction. Colours used reflect the cyanobacterial native metabolite that the product originates from. 23

Although eventual large-scale production will most probably occur under a day-night regime, most laboratory studies have investigated productivity under continuous light. For a lactate producing strain, productivity under light-dark conditions did not significantly differ from production under constant light [135]. The industrial application of processes developed on the laboratory scale requires upscal- ing. Details of designing photobiorectors for these purposes are beyond the scope of this 2 review. For this the reader is referred to [157]. Upscaling involves growing cyanobacteria in large quantities. The more cell doublings are required to achieve the desired amount of biomass, the greater the probability that a spontaneous mutation that increases growth rate will be selected for. For production systems this means that any mutant that diverts fixed carbon away from a production pathway will be enriched (See [158] for an overview on genetic instability in cyanobacteria). Therefore it may be of importance to separate the growth phase and the production phase, so that mutations leading to decreased produc- tivity will not be positively selected for. To this end, production pathways can be placed under tight control. Huang and Lindblad developed a tightly regulated promoter system [159] but the fact that it is induced by anhydrotetracyclin might forestall large scale appli- cations. Ideally, inexpensive inducers, such as light, would be used. Thus, Miyake et al. de- veloped a greenlight inducible lysis system, based on the cpcG2 promoter of Synechocystis 6803 [160]. Advances in the genetic manipulation of cyanobacteria were reviewed recently [92]. Increasing complexity of heterologous pathways, and the need to manipulate host me- tabolism have led to the development and improvement of a number of counterselection systems based on B. subtilis sacB [117, 150, 161–163], E. coli mazF [164], and organic acid sensitivity conferred by AcsA [165]. Metabolic engineering of heterotrophs has benefitted considerably from metabolic mod- els. For cyanobacteria, various genome scale models have been constructed [36, 166, 167]. Although highly desirable, the construction of dynamic models is thus far limited by the availability of kinetic data [168]. Instead, flux balance analysis (FBA) is used. FBA predicts a flux distribution in which an objective function is optimised (e.g. maximisation of biomass formation) [169]. In a system, where growth does not depend on product formation (such as in a photosynthetic cell factory), biomass formation as the objective function would lead to a flux distribution that does not allow for product formation, while optimisation for product formation would not predict growth. Therefore, current models of production strains use product formation as objective function, while growth is fixed to a certain value [170, 171]. Such a priori constraints limit the predictive value of these models, reducing their applica- bility considerably.

THEIDEALCYANOBACTERIALHOST

The ideal production host is hard to specify. Rather, different desired properties can be for- mulated, depending on the nature of the process. Generally, genetic manipulation should be straightforward and engineered strains should show a high degree of stability. Growth should be fast and robust, as well as photosynthetic efficiency and carbon fixation rate could be high. To use off-gas, growth at elevated CO2 concentrations should be possible. Growth in seawater would reduce competition with the freshwater use for food purposes. Product excretion and flocculation can facilitate harvest- ing. Cultures should show resistance to product consuming contaminants and grazers. 24 2.E NGINEERINGCYANOBACTERIAFORDIRECTBIOFUELPRODUCTIONFROM CO2

SCIENTIFIC AND ECONOMIC PROSPECTS Commercial fuels usually are mixtures of compounds. Similarly, production of biofuel mix- tures by photosynthetic microbes could be advantageous; even more so as many enzymes show inherent promiscuity which may lead to product diversification. Mixtures with differ- 2 ent chain lengths were reported for alkanes [122], fatty acids [117–120, 156, 172] and fatty alcohols [123] producing strains. Furthermore, Synechococcus 7942 engineered to produce 2-methyl-1-butanol accumulated 1-propanol and isobutanol as by-products[116]. This ap- proach, however, shows distinct disadvantages for the production of commodity chemicals. At present, the cost of the most developed biofuel, plant-derived biodiesel, is still several times that of fossil fuels. As cyanobacteria do not need to devote fixed carbon to the syn- thesis of trunks, roots etc., production yields for a direct conversion approach per m2 could be significantly higher. With additional costs for downstream processing and for operation of photobioreactors and, most significantly for maintaining axenic conditions, the price for solar biofuels is still considerably higher than for the fossil competitor product. This is true in particular when no cost is placed on carbon emission. Therefore, for a development towards a more mature technology based on ‘direct conversion’ it may be wise to initially concentrate on the production of higher-value-added compounds. Future developments in strain selection and engineering, bioreactor design and processing technology then may pave the way for the production of fuels, using the direct conversion approach that can economically compete with their fossil counterparts.

ACKNOWLEDGEMENTS The authors thank Filipe Branco dos Santos and Pascal van Alphen for helpful discussions and S. Andreas Angermayr for critical review of the manuscript. This review was written within the research programme of BioSolar Cells, co-financed by the Dutch Ministry of Eco- nomic Affairs. 3 SYNTHESISOF meso-2,3-BUTANEDIOLBY Synechocystis SP. PCC6803

Philipp SAVAKIS, S. Andreas ANGERMAYR, Klaas J. HELLINGWERF

The direct and efficient conversion of CO2 into liquid energy carriers and/or bulk chemicals is crucial for a sustainable future of modern society. Here we describe the production of 2,3- butanediol in Synechocystis sp. PCC6803 expressing a heterologous catabolic pathway de- rived from enteric- and lactic acid bacteria. This pathway is composed of an acetolactate synthase, an acetolactate decarboxylase and an acetoin reductase. Levels of up to 0.72 g/l (corresponding to 8 mmol/L) of C(4) products, including a level of 0.43 g/l (corresponding to 4.7 mmol/L) 2,3-butanediol production are observed with the genes encoding these three en- zymes integrated into the cyanobacterial genome, as well as when they are plasmid encoded. Further optimization studies revealed that Synechocystis expresses significant levels of aceto- lactate synthase endogenously, particularly under conditions of restricted CO2 supply to the cells. Co-expression of a soluble transhydrogenase or of an NADPH-dependent acetoin reduc- tase allows one to drive the last step of the engineered pathway to near completion, resulting in pure meso-2,3-butanediol being produced.

This chapter has been published in Metabolic Engineering 20, 121-130 (2013) [115].

25 26 3. meso-2,3-BUTANEDIOL SYNTHESIS BY Synechocystis

INTRODUCTION

TMOSPHERIC CO2 levels are increasing since the beginning of the industrial revolution. A This increase adds to the greenhouse effect and thus to climate change. The main source of CO2 emission is the use and combustion of fossil fuel [173]. As fuel demand is increasing, a renewable, non-fossil source of a liquid energy fuel is urgently needed as an alternative. Fuel compounds derived from CO2, water and sunlight and synthesised by pho- tosynthetic (micro)organisms are renewable by definition and their production is sustain- able when they are produced in a process with a negative carbon footprint. Its high heating 3 value [174] and ability to increase the octane number [97] of a fuel make 2,3-butanediol a promising drop-in fuel. Dehydration of 2,3-butanediol yields methyl ethyl ketone (bu- tanone), which can be used both as a solvent, a fuel additive, and a precursor for the pro- duction of 2-butanol. The biological conversion of 2,3-butanediol to 2-butanol is realised in a number of lactobacilli [175]. Microbial production of 2,3-butanediol in fermentation- based processes has had a long history and the role of this compound as an intermediate in the synthesis of butadiene, a precursor of synthetic rubber, led to fermentation processes reaching pilot plant scale during World War II. In the second half of the 20th century this process research was abandoned due to cheaper petrochemical routes to butadiene [176]. In recent years, interest in the bio-based production of 2,3-butanediol is increasing with expected compound annual growth rates of 3.2% for 2013- 2018 [177]. Current research ef- forts are focusing in particular on unlocking inexpensive carbon sources for this process, such as lignocellulosic feedstock or food industry wastes [176]. Using genetically modified Synechocystis sp.PCC6803 (hereafter Synechocystis ) strains, we describe a route in which the need for a fixed carbon source is eliminated entirely. These recombinant strains harbour a fermentative pathway derived from lactic acid- and enterobacteria, which are well-known for their versatility with respect to fermentative metabolism [178–180]. In these classes of organisms, the enzymatic pathway leading to 2,3-butanediol involves two successive de- carboxylation steps, which constitute a high thermodynamic driving force for the pathway (Fig. 3.1). We disregarded alternative pathways occurring in fungi [181], since these utilise acetaldehyde and acetyl-CoA, respectively. We reasoned that acetaldehyde as an interme- diate in the pathway might not allow a significant flux to acetoin due to its volatility and toxicity (compare Fig. S 3.1). Previous efforts in utilising acetyl-CoA for the production of useful C4 compounds in cyanobacteria have led to low product titers, namely below 1 mil- limolar [101, 182]. In contrast to these examples, the synthesis of isobutyraldehyde, origi- nating from the valine biosynthesis pathway, i.e. from pyruvate via acetolactate, did result in significant amounts of product formation, exceeding 10 millimolar [148]. However, in situ product recovery had to be applied to reach these titers and to deal with the toxicity of isobutyraldehyde. Significantly, both 2,3-butanediol and its immediate precursor, acetoin, showed low toxic- ity to cyanobacterial cells, as reflected in an IC50 of growth in batch culture of 375 mM and 95 mM, respectively (Fig. S 3.1). In the time this manuscript was written, a study appeared in the Proceedings of the National Academy of Sciences [108], dealing with D-2,3-butanediol synthesis in E. coli and the cyanobacterium Synechococcus elongatus PCC7942. 27

3

Figure 3.1: The biosynthetic pathway, derived from lactic acid bacterial metabolism, to produce 2,3- butanediol from pyruvate. Two subsequent irreversible reactions are catalyzed by an ALS, acetolactate synthase and an ALDC, acetolactate decarboxylase both releasing one CO2 molecule. The NAD(P)H dependent reaction is catalyzed by an AR, acetoin reductase (BDDH, butanediol dehydrogenase). Acetolactate synthase is also part of the native metabolism of Synechocystis sp. PCC6803. Depicted in gray are several prominent high-value products derived from 2,3-butanediol.

MATERIALS AND METHODS

Reagents. All chemicals were purchased from Sigma-Aldrich unless stated otherwise. Pwo DNA Polymerase was from Roche. All other DNA modifying enzymes were from Fermentas. Oligonucleotides were from Biolegio (The Netherlands) and are listed in Tab. S 3.1. Strains and growth conditions. All cloning procedures were carried out in Escherichia coli XL1 (blue) (Stratagene), grown in liquid LB medium at 37◦C in a shaking incubator at 200 rpm, or on solidified LB plates, containing 1.5% (w/v) agar. When appropriate, media were supplemented with antibiotics needed for propagation of (a) specific plasmid(s) or se- lection for (a) marker(s). Concentration of antibiotics used, alone or in combination, were 50 µg/mL kanamycin, and100 µg/mL ampicillin. The same growth and selective condi- tions were used for the E. coli J53 (pRP4) helper strain (obtained from A. Wilde, Giessen University, Germany) used in bacterial conjugation (via tri-parental mating) procedures unless otherwise noted. Synechocystis sp. strain PCC6803 (a glucose-tolerant derivative, obtained from D. Bhaya, University of Stanford, US) was routinely cultivated in BG-11 me- dium [71] (Cyanobacteria BG-11 freshwater solution, Sigma) at 30◦C in a shaking incubator at 120 rpm (Innova 43, New Brunswick Scientific) under constant moderate white light illu- mination (30–40 µE/m2/s; measured with a LI-250 light meter, LI-COR) provided by 15 W cool fluorescent white lights (F15T8-PL/AQ, General Electric). All kanamycin resistant mu- tant Synechocystis strains were grown in a medium containing 20 µg/mL kanamycin. For plates, BG-11 medium was supplemented with 1.5% (w/v) agar, plus where indicated:5 mM glucose, 0.3% (w/v) sodium thiosulfate and respective concentrations of kanamycin for re- sistant strains. Concentrated stocks of cells were routinely stored at –80◦C in BG-11 medium supplemented with 5% dimethyl sulfoxide. Cells were re-streaked on plates and grown to 28 3. meso-2,3-BUTANEDIOL SYNTHESIS BY Synechocystis

stationary phase in a pre-culture before they were re-diluted to an OD730 (Spectrophotome- ter Lightwave II, Biochrom) of 0.1 at the start of each experiment. Growth was monitored by recording OD730. For high carbon conditional growth cultures were supplemented with 50 mM NaHCO3 pH 8.0 (Sigma). For cultures buffered at the respective pH the following buffers were used: For pH 6.5: 25 mM TES-KOH, for pH 7.5: 25 mM HEPES-NaOH, for pH 8.0: 25 mM TES-KOH, for pH 9.0: 25 mM CAPSO, and for pH 11.0: 25 mM CAPS. All buffers were purchased from Sigma. Plasmid construction. All plasmids were constructed according to standard cloning tech- 3 niques [183]. Briefly, genes were either amplified by PCR or synthesized in codon-optimi- zed form at Genscript, digested and ligated into appropriately cut vectors. Selection was carried out on LB solidified with1.5% agar and containing appropriate selection markers. An acetolactate synthase (als) gene was amplified from genomic DNA of Enterococcus fae- calis V583 [184]. An acetolactate decarboxylase (aldc)[185] and an acetoin reductase (butA) [186] gene were amplified from Lactococcus lactis sp. cremoris. Genes were cloned into the pQE30 vector (Qiagen) with BamHI and KpnI and HindIII, respectively, placing them under the control of the T5 promoter present in pQE30. The aldc and the butA genes, in- cluding the promoter sequence, were subsequently placed downstream of als, using KpnI and SmaI and SmaI and SalI, respectively, creating three adjacent gene cassettes. Each gene carries the PT5 and RBS from the pQE30 vector. The complete unit, including the transcriptional terminator present in pQE30, was excised from pQE30 with ZraI and NheI and cloned into the integration plasmid pHKH001 [135] cut with XbaI and EcoRV. Next, an acetolactate synthase (als) gene was chosen from Leuconostoc lactis NCW1 [187]. An ace- tolactate decarboxylase (aldc) gene was chosen from Brevibacillus brevis[188, 189] and an acetoin reductase (ar) gene was chosen from Enterobacter sp. The Enterobacter ar was cho- sen based on the KM value published for the AR of Enterobacter aerogenes [190]of which the sequence was not available at the beginning of this study (but became available during the submission period of manuscript) but shows high sequence similarity to the ar used [191]. The nucleotide sequences were modified according to standard codon optimization procedures [192]. A codon usage table was created based on the 50 most highly expressed protein-coding genes in Synechocystis published by Mitschke et al. [193] using the Count codon program [194] of the Codon Usage Database. The resulting codon usage table was then used to optimize the sequence with the OPTIMIZER application [192] leading to an elevated codon adaptability index (data not shown). Recognition sites for restriction en- donucleases to be used for modifications of the final operon were eliminated with OPTI- MIZER. In the case of the Brevibacillus brevis ALDC the sequence of the mature enzyme is preceded by a signal peptide that may lead to secretion of the enzyme from the cell. Partial sequencing of the polypeptide produced in Brevibacillus brevis and Bacillus subtilis yielded slightly different N-termini [195]. The codons for the first 28 amino acids were therefore re- placed by ATG, prior to all other optimization processes. A core Shine–Dalagarno sequence (50-GGAGG-30) and a randomized sequence of 6 nucleotides as a spacer to the start codon were placed in front of every gene [196]. The promoter Ptrc [197] was placed upstream of the als gene. The whole operon was synthesized at GenScript (NJ, US). The operon was cloned into the mobilizable pVZ321 [78] (obtained from A.Wilde, Giessen University, Ger- many) in the EcoRI and XbaI sites, using EcoRI and NheI, thus creating pVZ001. For the construction of pVZ002, pVZ001 was digested with SpeI and BamHI, cutting out the als 29 gene, blunted with Klenow fragment (Fermentas), after which the plasmid was religated. pSB1AC3_TrcSTHtt was used as source for the sth of Pseudomonas aeruginosa [135]. The cassette was cut with EcoRI and SpeI and cloned into the EcoRI and XbaI sites of the mo- bilizable conjugation plasmid pVZ001, creating pVZ003. To create pVZ006, the Leuconos- toc lactis NCW1 acetoin reductase gene[198] was synthesized at GenScript (NJ, US), ampli- fied with primers Nde1-NCW1_ar_f and PvuI-NCW1_ar_r, digested with NdeI and PvuI and ligated into pVZ002 cut with NdeI and PacI. The aldc gene on pVZ001 was amplified with primers BamHI_Bb_aldc_f and SacI_Bb_aldc_r. The ar gene on pVZ001 was amplified with primers BamHI_Ent_ar_f and SacI_Ent_ar_r. The ar gene on pUC57LlNCW1AR was am- plified with primers BamHI_LlNCW1_ar_f and SacI_ LlNCW1_ar_r. Products were digested 3 with BamHI and SacI and cloned into the appropriately cut pQE30 vector. For a list of plas- mids used and constructed in this study see Tab. S 3.2. Mutant construction. Natural transformation of Synechocystis was performed essentially as described in [135] and [199]. After full segregation was achieved, mutants were verified regarding the correct incorporation of the genes of interest with colony PCR, employing gene-specific primers and the insertion flanking primers, respectively (Tab. S 3.1) and via sequencing of the PCR product. Conjugation to Synechocystis was performed by tri-parental mating with E. coli J53 (pRP4) as helper strain and and E. coli XL1 (blue), carrying pVZ001, pVZ002, pVZ003 and pVZ006, respectively as donor strain. The conjugation procedure was performed essentially as described in [78]. Briefly, relevant E. coli cultures were inoculated from overnight cultures and grown for 2.5 h at 37◦C without antibiotic pressure. Cells were harvested by centrifugation for 8 min at 2500 rpm and resuspended in fresh LB medium. Subsequently, 900 µl of both cell suspensions were mixed and concentrated by centrifu- gation, resuspended in 100 µl fresh LB medium and incubated for 1 h at 30◦C. 800 µl of a growing Synechocystis culture were then added, cells were mixed and concentrated by cen- trifugation and resuspended in 30 µl fresh BG-11 medium. The suspension was spread onto sterile filter membranes (Gelman Science, Supor-200 0.2 µM filter membrane) and placed on 1.5% (w/v) agar BG-11 plates, supplemented with 5% LB. The plates were covered with paper to shield from intense light and incubated overnight in a light incubator at 30◦C. The next day the filter membranes were washed with 400 µl BG-11 medium, and the washed-off cells streaked on 1.5% (w/v) agar BG-11 plates, with 20 µg/mL kanamycin. Plates were incu- bated in a lit incubator at 30◦C until single colonies did appear. Plasmid carrying mutants were verified using colony PCR, employing gene-specific primers (Tab. S 3.1). For the construction of the recombinant ethanol-producing strain, Syn SAA012, the pyru- vate decarboxylase (pdc) and the alcohol dehydrogenase (adhII) of Zymomonas mobilis were cloned into the integration plasmid pPSBTC with SalI and EcoRI from a synthesized construct (GeneArt, Germany) which resembles the open reading frame sequences and in- tergenic region found in [149]. This strategy places the genes of interest directly down- stream of the psbA2 structural gene (slr1311), creating a transcriptional fusion. The same Shine–Dalgarno sequence as mentioned above was used. To construct the integration plas- mid pPSBTC the two homologous regions stretching from chromosomal position 7510 to 8311 and from 8312 to 9113 [75], were amplified from genomic DNA of Synechocystis , and cloned into the XhoI and SalI and BamHI and NotI sites of pBluescript II SK(+) (Stratagene), respectively. The kanamycin resistance cassette (aphX), was amplified from pHKH001 [135] and cloned into the SmaI site of pBluescript II SK(+). For the construction of the Synechocystis strains carrying the NADH-dependent acetoin re- 30 3. meso-2,3-BUTANEDIOL SYNTHESIS BY Synechocystis

ductase from Enterobacter sp., named Syn AR and the NADPH dependent acetoin reductase from Leuconostoc lactis NCW1, named Syn ARP, genes of interest were amplified from the respective genomic DNA. For the latter the Leuconostoc citreum KM20 (GenBank accession no. DQ489736) was used to design a pair of oligonucleotide primers (Tab. S 3.1). Genomic DNA of Leuconostoc lactis NCW1 strains was isolated using a DNeasy Blood & Tissue Kit (Qi- agen) following the manufacturer’s instruction. Genes were fused with the promoter Ptrc and on pSEQ_Trc into PstI and SpeI sites and further cloned into the multiple cloning site of the integration plasmid pHKH001 [135]. Sequencing of DNA fragments was performed at Macrogen Europe (The Netherlands) with dedicated primers (Tab. S 3.1). For a list of strains 3 used and constructed in this study see Tab. S 3.2. Protein overproduction and purification. Proteins were overproduced in Escherichia coli M15. Catabolic acetolactate synthase from Enterococcus faecalis was overproduced from pQE30 Ef als. Brevibacillus brevis acetolactate decarboxylase was produced from pQE30 Bb aldc. Acetoin reductases from Enterobacter sp. and Leuconostoc lactis NCW1 were over- produced from pQE30 Es ar and pQE30 Ll ar, respectively. Production and purification were carried out as follows: Briefly, Escherichia coli harboring the overproduction plas- mid and pREP4 were grown in LB medium containing 100 µg/mL ampicillin and 25 µg/mL kanamycin at 37◦C. At an OD600 of 0.4–0.6 expression was induced by addition of IPTG to a final concentration of 0.1 mmol/L. Production was allowed to continue for 3 h at 37◦C, after which the cells were harvested by centrifugation (7000 rpm, 10 min, 4◦C). Cells were resus- pended in lysis buffer (50 mmol/L sodium phosphate pH 7.5, 300 mM NaCl, 20 mM imida- zole), treated with lysozyme (final concentration 1 mg/mL, 30 min incubation at 25◦C) and lysed by ultrasound (Branson Sonifier 250, Duty cycle 40%, Output control 6, 4×20 pulses). Suspensions were cleared by centrifugation (18,000 rpm, 30 min, 4◦C), supernatants were loaded onto a His-Trap FF column (GE Healthcare Life Sciences) and eluted in an imida- zole gradient ranging from 20 mmol/L to 500 mmol/L. Proteins were dialyzed twice against imidazole-free lysis buffer and flash-frozen or used immediately. For a list of plasmids and strains used and constructed in this study see Tab. S 3.2. Preparation of cell extracts and enzymatic activity assays of heterologously expressed produced enzymes. Crude cell-free (cfe) extract of Synechocystis cells was prepared as de- scribed earlier [135]. Briefly, the cells were harvested in the mid-exponential growth phase, opened by bead-beating, and cell debris was removed by centrifugation. The protein con- centration of the cfe was determined with the colorimetric BCA protein assay kit (Pierce). Acetolactate synthase activity was assayed in the presence of 10 mM MgCl2, 0.075 mM FAD, 0.1 mM thiamine pyrophosphate and 100 mM sodium phosphate, pH 7.0 at 30◦C for 2–18 h. The reactions were started by addition of sodium pyruvate to a final concentration of 20 mM. After incubation at 30◦C, 0.1 volumes of 50% H2SO4 were added to decarboxylate acetolactate. Decarboxylation was allowed to continue for 30 min at 37◦C, after which the acetoin concentration was determined using the Voges Proskauer test [200] as modified by [201]. Quantification was achieved with a calibration curve with pure acetoin. Acetolactate decarboxylase (ALDC) activity was measured in cell-free extracts coupled to the acetoin reductase assay. NADH consumption was monitored at 340 nm. Briefly, the reaction was performed in 96-well plates in a 100 mM sodium phosphate buffer pH 7.0, 5 mM MgCl2, 0.1 mM thiamine pyrophosphate, 2.5 µg/mL purified acetolactate synthase of Enterococcus faecalis, 20 mM sodium pyruvate and 0.1 µg/mL crude cell free extract. The reaction mix was incubated for 2 h at 30◦C to allow build-up of acetolactate from pyruvate 31 by the ALS, which can further be converted by the ALDC to acetoin (compare also reaction scheme presented in Fig. 3.1). After 2 h, NADH, to a final concentration of 300 µM, was added together with 1.0 µg/mL purified acetoin reductase of Brevibacillus brevis to facili- tate acetoin reduction. The coupled consumption of NADH was monitored in an endpoint assay and compared to the absorbance change of a calibration curve of known concentra- tions of acetoin. A minor complication of the assay was that the acetoin (Sigma-Aldrich) used for the calibration is a racemate, however in the cell extracts we expect only produc- tion of R-acetoin (see the Results section for further details) thus values for the calibration were doubled. The conclusion that ALDC activity is present in the samples is derived from the observation of the build-up of acetoin within the 2 h incubation time. For calculations 3 of the ALDC activity we have assumed saturating substrate concentrations (acetolactate), supplied by the ALS from pyruvate. Background activity and activity in the wild type cell extracts were observed due to non-enzymatic spontaneous decarboxylation of acetolactate to acetoin. Acetoin reductase (AR) activity assays were performed similar to the ALDC assays in trans- parent 96-well plates. Briefly, the reaction mixture contained 100 mM sodium phosphate, pH 7.0, 300 µM NADH, 5.0 mM MgCl2, and 0.1 µg/mL crude cell free extract; the reac- tion was started with the addition of 35 mM acetoin; oxidation of NADH was monitored at 340 nm. The enzymatic activity is determined by monitoring the consumption of NADH per second and is expressed in nmol/mg total protein/min. The transhydrogenase (STH) activity assay was performed as described earlier [135]. SDS/PAGE analysis. Crude cell-free extracts from cells grown in BG11 medium buffered at pH 9.0 with 25 mmol/L CAPSO were heated for 10 min at 80◦C in SDS/DTT loading dye and applied to NuPAGE®Novex®4-12% Tris-Bis Gels (Life Technologies), used with a MES/SDS buffer. After fixation, the gels were stained with Page Blue®a Coomassie G-250 stain (Fer- mentas) for 1 h. A total amount of 50 µg protein was analyzed per lane. Transcript analysis by qRT-PCR. Synechocystis cells were harvested in the mid-exponential growth phase opened by bead-beating and cell debris was removed by centrifugation. Prior to use the glass beads were washed in 4 N HCl and in ethanol and autoclaved twice. RNA was isolated using the RNeasy mini kit (Qiagen) and further used to prepare cDNA with the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). RNA quantity was deter- mined on a Nanodrop 1000 spectrophotometer (Thermo Scientific) and quality assessed on a 1% agarose gel to assure lack of RNA degradation. For reverse transcription to yield cDNA the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) employing the M- MuLV Reverse Transcriptase and random hexamer primers was used. qRT-PCR was per- formed on the cDNA in an Applied Biosystems 7300 Real Time PCR system using the Power SYBR®Green PCR Master Mix (Life Technologies) employing gene specific primers, de- signed with Primer3 software (Life Technologies) (Tab. S 3.1). The relative abundance was calculated and normalized with the ∆∆CT method to rnpB which is used as a housekeeping control [202] in samples from different conditions or different mutant strains. C4 product and ethanol detection and quantification by high-performance liquid chro- matography (HPLC). Batch culture samples were harvested at selected time points. To determine the concentration of acetoin, meso-2,3-butanediol and of the D-/L-isoforms of butanediol, a 500 µl aliquot was taken. Cells were removed by centrifugation for 2 min at 14,000 rpm at room temperature and supernatant samples were stored at 4◦C. Of the resulting supernatant 250 µl were used in HPLC analysis. HPLC sample preparation was 32 3. meso-2,3-BUTANEDIOL SYNTHESIS BY Synechocystis

performed as follows: The 250 µl supernatant was treated with 25 µl of 35% Perchloric acid (VWR) and incubated for 10 min on ice. Then, 14 µl 7 M KOH (VWR) was added, samples were mixed and the precipitate was removed by centrifugation for 2 min at 12,000 rpm at room temperature. The supernatant was filtered (Sartorius Stedin Biotech, minisart SRP 4, 0.45 µm). Separation of the products in a 20 µl aliquot was achieved with a Rezex ROA- Organic Acid H+ (8%) column (Phenomenex), using a refractive index detector (Jasco, RI- 1530); operating with 7.2 mM H2SO4 as eluent with a flow of 0.5 ml/min and a column tem- perature of 85◦C. The order of elution was diacetyl, acetoin, meso-2,3-butanediol and D-/L- 2,3-butanediol. For ethanol detection the column temperature was set to 45◦C. AZUR 4.5 3 (Datalys) software was used for data analysis. Peaks were identified and quantified us- ing internal standards. Acetoin (Fluka), meso-2,3-butanediol (Sigma), R,R-2,3-butanediol (Sigma), S,S-2,3-butanediol (Sigma), diacetyl (Alfa Aesar) ethanol (Scharlau) were purchased at the highest purity available. In our setup, the two optically active isoforms of 2,3-butane- diol, D-/L-2,3-butanediol could not be distinguished from each other, and by consequence they appear as one cumulative peak. Identification of optically active 2,3-butanediol isomers. Synechocystis cells were har- vested by centrifugation (14,000 rpm, 2 min, 4◦C). Supernatants were saturated with so- dium chloride and extracted with one volume of ethyl acetate. 1 µL was injected into an Agilent 6890N Network GC system equipped with a Chirasil dex CB column (Agilent Tech- nologies, inner diameter: 0.25 mm, length: 25 m, film: 25 µm), a flame ionization detector and a GC Pal autosampler (CTC analytics). Conditions were the same as elsewhere [203]: Front inlet temperature was set to 250◦C, oven temperature was set to 80◦C for 10 min, then increased to 120◦C at 10◦C/min and then to 200◦C at 40◦C/min. Helium was used as car- rier gas. The order of elution was D-acetoin, L-acetoin, D-2,3-butanediol, L-2,3-butanediol, meso-2,3-butanediol. Growth curve fitting. To facilitate growth rate- and production rate determination and car- bon partitioning calculations, we decided to smooth the growth curves. We were prompted to do so due to standard deviations from the average optical density, especially at later time points in our growth experiments and the complex limitations to which the cyanobacte- ria are subjected in batch culture. Thus, a growth curve was fitted over the obtained data points. We evaluated several mathematical functions [204] and decided for a second order polynomial, which showed the best fit, particularly at later time points. We used the Excel Solver (Microsoft Office Professional Package 2010) to estimate the parameters, using least absolute values as criteria for the quality of the fit. No significant trend in the residuals was visible. Rate and carbon partitioning calculations. From the data of the fitted growth curve and the actual product values at respective time points the growth rate and production rate were deduced. Dry cell weight was calculated from the OD730, assuming an average cell

composition of C4H7O2N. Carbon fixation rate, QCO2 , and the resulting yield, Ybiomass were calculated as published previously [135] (and references therein). Partitioning rates were calculated based on a C6 compound since six molecules of CO2 need to be fixed to produce one C4 product molecule. Partitioning rates are expressed as percent of total carbon fixation

rate (which equals the sum of Qbiomass and QC6pr oduct . 33

3

Figure 3.2: Construction and time course of growth and extracellular product (acetoin and meso- butanediol) formation. (a) Schematic presentation of the genetic modifications in Syn SAA018 and (b) in Syn pVZ001. (c) Growth and respective product formation over time in strain SAA018 and (d) Syn pVZ001. The values are obtained from the average of biological replicates; the error bars indicate the standard deviation (n=3); if error bars are not visible, they are smaller than the respective data point symbol.

RESULTS AND DISCUSSION Integration of the butanediol-biosynthesis pathway. To achieve the goal of construction of a cyanobacterial cell factory, initially, we introduced the genes encoding: (i) acetolac- tate synthase (ALS) from Enterococcus faecalis, (ii) acetolactate decarboxylase (ALDC) from Lactococcus lactis sp. cremoris and (iii) acetoin reductase (AR) from Lactococcus lactis sp. cremoris, into the chromosome of Synechocystis PCC6803 (Fig. 3.2a). Jointly, these three genes constitute a butanediol-biosynthesis pathway. The ALS enzymes selected have a catabolic function in their host [187, 205]. We selected a catabolic ALS rather than an an- abolic AHAS (acetohydroxy acid synthase) because the latter ones exhibit feed-back reg- ulation by branched chain amino acids [206]. The chromosomal insertion was achieved by targeting the slr0168 locus of the Synechocystis chromosome by means of double ho- mologous recombination. In the resulting mutant, Syn SAA018, expression of each of the three heterologous genes is controlled individually by PT5, which allows for low, unreg- ulated expression [207]. The t0 transcriptional terminator of lambda phage was placed downstream of each of the three genes. This terminator has not been characterized in cy- anobacteria and should hence be considered a putative terminator. Detectable amounts of acetoin and butanediol started to accumulate in the supernatant of Syn SAA018 cultures two days after inoculation; 12 days after inoculation, product titers reached 0.025 mmol/L and 0.021 mmol/L, for acetoin and butanediol, respectively (Fig. 3.2c and d). To improve product levels we designed an optimized alternative of this 2,3-butanediol pathway. For this we selected enzymes which show enhanced performance (Tab. S 3.3), namely the ALS of Leuconostoc lactis NCW1, the ALDC of Bacillus brevis and the AR of Enterobacter sp. 638. The corresponding genes were codon-optimized for Synechocystis and synthesized as a single operon under control of Ptrc (Fig. 3.2b). This latter promoter is one of the strongest currently available in Synechocystis and drives constitutive expression. The Bio- Brick BBa_B0015 transcriptional terminator was placed downstream of each of the genes 34 3. meso-2,3-BUTANEDIOL SYNTHESIS BY Synechocystis

[acetoin] biomass A [meso-BD] B diverted C carbon 6 [D/L-BD] C products Syn pVZ002 4 polynomial fit 10 0.05 10 0.04 8 100

730 6 80 0.03 1

OD 60 3 4 0.02

c / mmol/L 40 2 20 0.01

0.1 products]/dt / mmol/L/h 4 0 carbon partitioning / % 0 0.00

0 5 10 15 20 25 30 0 5 10 15 20 25 d[C t / d t / d [acetoin] [acetoin] C [meso-BD] D [meso-BD] [D/L-BD] [D/L-BD] Syn WT Syn WT Syn pVZ03 Syn pVZ06 2.5 2.5 10 10 2.0 2.0

730 1 1.5 730 1 1.5

OD 1.0 OD 1.0 c / mmol/L 0.1 0.1 c / mmol/L 0.5 0.5 0.01 0.0 0.01 0.0 0 2 4 6 8 10 12 0 2 4 6 8 10 12 t / d t / d E B. brevis Enterobacter sp. Syn pVZ002

P. aeruginosa L. lactis B.NCW1 brevisEnterobacter sp. Syn pVZ03

B. brevisL. lactis NCW1 Syn pVZ06

Figure 3.3: Schematic construction and time course of growth and improved extracellular product (acetoin and meso-butanediol) formation. (a) Growth plot and stacked bar representation of daily total production and detailing the single fractions of acetoin, meso-butanediol and D-/L-butanediol in Syn pVZ002. (b) Fixed carbon partitioning of product over biomass and corresponding volumetric production rate in pVZ002. (c) Growth and total production of acetoin, meso-butanediol (continued overleaf) 35 of interest [208]. This terminator has been used but was not characterized in Synechocystis [196]. This artificial operon was introduced into Synechocystis on a self-replicating plas- mid, derived from pVZ321 [78]. The resulting strain, Syn pVZ001 accumulated detectable amounts of acetoin and butanediol in the supernatant two days after inoculation. After 12 days, acetoin, meso-2,3-butanediol and D-/L-2,3-butanediol accumulated in the super- natants to concentrations of 0.21 mmol/L, 0.22 mmol/L and 0.1 mmol/L, respectively (Fig. 3.2b). Employing the endogenous acetolactate synthase activity under changing Ci regimes. Characterization of the ALS from Leuconostoc lactis NCW1, supposedly more suitable for 2,3-butanediol production than the enzyme from Enterococcus faecalis, revealed very low 3 activity in subsequent in vitro measurements (Tab. S 3.4). This, in combination with the higher production rates in pVZ001, suggested that the activity of acetolactate synthase, necessary for 2,3-butanediol production in Synechocystis , was in fact provided by (an) en- zyme(s) endogenous to Synechocystis . Indeed, acetolactate is a biosynthetic precursor of the amino acids valine and leucine. Therefore, an enzyme capable of catalyzing the con- version of pyruvate into acetolactate must be present in Synechocystis . ALS activity was determined in cell-free extracts of all Synechocystis strains used in this study, including wild type strain (Tab. S 3.4 and Supplementary Fig. 3.2). To test whether or not a heterologous acetolactate synthase is a prerequisite for 2,3-butane- diol production in Syn pVZ001, we constructed a mutant harboring a truncated version of the butanediol operon, in which the als gene was deleted (Fig. 3.3a, e) and introduced it into Synechocystis via the self-replicating plasmid, which yielded strain Syn pVZ002. We then compared 2,3-butanediol production in the two strains, i.e. with the complete and with the truncated pathway, under a range of conditions (Fig. S 3.3). To our surprise, the strain with the truncated pathway showed higher product titers under all conditions tested. In pVZ002, the aldc and the ar gene are closer to the promoter than in pVZ001. This can lead to higher transcription efficiency and consequently to a higher concentration of the latter two enzymes in the 2,3-butanediol pathway. To corroborate this, we carried out a transcript analysis of als, aldc and ar in pVZ001 and pVZ002. As expected, no heterologous als transcript was detected in pVZ002. The heterologous aldc and ar are up-regulated >2.5- fold, and >1.5-fold, in pVZ002 compared to pVZ001 (Tab. S 3.5). Furthermore, acetolactate decarboxylase activity in crude cell free extracts of pVZ002 consistently shows a higher ac- tivity than in pVZ001 and pVZ003 (Tab. S 3.6). Acetoin reductase activity in crude cell free extracts is increased in pVZ002 compared to pVZ001 (and pVZ003, which shares an identical genetic make-up with pVZ001 with respect to als, aldc and ar) (Tab. S 3.7). Taken together, the elevated production rates in the mutant harbouring the truncated pathway correlate well with the increased availability of enzymes for the two last steps of the pathway. Since

Figure 3.4 (continued from previous page): and D-/L-butanediol in Syn pVZ003, co-expressing a soluble transhydrogenase. (d) Growth and total production of acetoin, meso-butanediol and D-/L- butanediol in Syn pVZ006, expressing a NADPH dependent acetoin reductase. (e) Schematic repre- sentation of the mutant strains. Cells were grown in BG11 medium buffered with 25 mM CAPSO at an initial pH of 9.0. CO2 was the only carbon source in these experiments. The values obtained are from the average of biological triplicates; the error bars indicate the standard deviation (n=3); if error bars are not visible they are smaller than the respective data point symbol or bar. 36 3. meso-2,3-BUTANEDIOL SYNTHESIS BY Synechocystis

Syn pVZ006

Syn pVZ001

meso-butanediol

L-2,3-butanediol

3 D-2,3-butanediol

acetoin

5 10 15 t / min

Figure 3.5: Synechocystis harboring acetolactate decarboxylase from Brevibacillus brevis and acetoin reductase from Enterobacter sp. produces meso-2,3-butanediol and L-2,3-butanediol. Supernatants of cultures of Syn pVZ001 and pVZ006 were compared to pure butanediol isomers (Sigma).

ALS activity of Synechocystis wild type was high enough to support butanediol production and since acetolactate can undergo spontaneous decarboxylation to acetoin the question arose if a heterologous acetolactate decarboxylase is a requirement for butanediol produc- tion. To test this, we constructed mutants PCC6803 AR and PCC6803 ARP, harboring the acetoin reductases of Enterobacter sp. and Leuconostoc lactis NCW1, respectively. None of these mutants produced any 2,3-butanediol, indicating that spontaneous decomposition of acetolactate does not contribute to butanediol formation in a significant way. Product concentrations were higher when the cells were cultured in the absence, rather than in the presence, of additional dissolved inorganic carbon (Ci, i.e. 50 mmol/L sodium bicarbonate). We then investigated ethanol production in an engineered Synechocystis strain expressing a heterologous pyruvate decarboxylase and an alcohol dehydrogenase both orig- inating from Zymomonas mobilis (Fig. S 3.4a) under similar conditions (Fig. S 3.3b and c). Production of ethanol from pyruvate was unchanged when additional sodium bicarbonate was added to the BG-11 medium (Fig. S 3.4b). This indicated that these heterologous path- ways, even though originating from the same metabolite, are subject to different control mechanisms under changing Ci regimes. Evaluating production over a wide range of differ- ent pH- and medium conditions, including the addition of bicarbonate, shows a dramatic decrease in production when cultures were supplemented with bicarbonate (Fig. S 3.3). In- terestingly, the growth rate during the exponential growth phase is highly increased under this condition (Fig. S 3.3). Unlike the cultures grown at pH 9, bicarbonate supplemented cultures showed stagnation of growth after 15–20 d and mild bleaching towards the end of long term cultivation experiments (i.e. 30 days) (Fig. S 3.3 and data not shown). Ex- cess carbon at the beginning of the experiment and long term culturing might result in an imbalance of the C–N ratio of the cell [209]. We set out to test if the increase in butane- diol production at low Ci can be rationalized with the up-regulation of sll1981, encoding an acetolactate synthase endogenous to Synechocystis [210], as indicated by transcriptome [209, 211, 212] and proteome [213] data obtained under comparable conditions. Both puta- 37 tive als genes of the Synechocystis genome, sll1981 and slr2088 [75] show a down-regulation (0.31 and 0.42 fold respectively) upon bicarbonate addition, as opposed to pH 9 conditions for the wild type. Interestingly sll1981 shows no large expression change in the mutants pVZ001 and pVZ002. slr2088 is even up-regulated >2.0 fold under bicarbonate conditions in the two mutants investigated (Tab. S 3.8). Both latter results remain elusive, suggesting a different regulation of sll1981 and slr2088 in the mutant strains, and do not fit the hypothe- sis. It is evident, however, that the abundance (activity) of the (putative, native) acetolactate synthase does not influence the production rates of butanediol in a significant way in the mutants tested. Long term culturing and accumulation of acetoin and butanediol. The highest 2,3-butane- 3 diol concentration (after 29 days of batch-culturing; see Fig. 3.3a) was achieved in BG11 me- dium supplemented with 25 mM CAPSO buffer at an initial pH of 9. At this time after inoc- ulation, acetoin and butanediol had accumulated in the culture medium to concentrations of 1.4 mmol/L and 6.5 mmol/L, respectively (note that 2,3-butanediol was produced as a mixture of the meso-isomer, which accumulated to 4.7 mmol/L and of S,S-2,3-butanediol, which showed a final titer of 1.8 mmol/L ( Figs. 3a and 4)). Notably, Syn pVZ002 showed consistently higher product titers than strain pVZ001, under comparable growth conditions (Fig. S 3.3). To increase 2,3-butanediol production potentially a larger fraction of the central carbon me- tabolism could be diverted into the heterologous pathway. This would increase the overall amount of products formed and would leave the respective product ratios untouched. Dif- ferent stages of the growth phase showed dynamic changes in the volumetric production rate of the C4 products and consequently different carbon partitioning ratios. Thus, so far up to 30% of the fixed carbon was diverted into the heterologous pathway (Fig. 3.3b). Changing the cofactor dependence increases butanediol titers. The ratio between the two products, acetoin and 2,3-butanediol could be shifted in favor of the latter one, increasing both product-yield and -purity. In the Synechocystis strains harboring pVZ001 and pVZ002, the conversion of acetoin into 2,3-butanediol is catalyzed by an NADH dependent acetoin reductase. Assuming chemical equilibrium (in both cases the capacity of the acetoin reduc- tase reaction surpasses the actual production rate by two orders of magnitude), it can be derived from the mass action law that the ratio of [2,3-butanediol] to [acetoin] is propor- tional to the ratio of [NADH] to [NAD+] (Eq. 3.1). [2,3 butanediol] [NAD(P)H] − Keq[H+] (3.1) [acetoin] = [NAD(P)] Reduction equivalents provided by photosystem II are produced as NADPH. Hence, the NADPH pool is more reduced under photoautotrophic conditions than the NADH pool [214]. To get a more reduced intracellular NADH/NAD+ pool, we therefore sought to couple it to the NADPH/NADP+ pool. This was achieved through the introduction of the sth gene of Pseudomonas aeruginosa, which encodes a soluble transhydrogenase. Soluble transhy- drogenases reversibly catalyze the NADPH-dependent reduction of NAD+. Cyanobacterial NADH-dependent lactate production has been increased by the introduction of a transhy- drogenase [135, 137] to shift the availably of co-factor to a favourable ratio. Indeed, Syn pVZ003, harbouring sth along with the operon for 2,3-butanediol biosynthesis, produced 2,3-butanediol and acetoin in a ratio much higher than 1 (Fig. 3.3c, e). Activity of STH was exclusively detected in mutant strain Syn pVZ003, but not in the wild type nor in other mu- tant strains (Tab. S 3.9). The STH protein is clearly visible on a Coomassie stained SDS/PAA 38 3. meso-2,3-BUTANEDIOL SYNTHESIS BY Synechocystis

gel (in fact as the only one of all proteins heterologously expressed in this study), which provides further proof for its presence in the soluble fraction of the crude cell free extract (Fig. S 3.5). As an alternative approach, we constructed a similar biosynthetic route for 2,3-butanedi- ol production, employing an NADPH-dependent acetoin reductase instead of an NADH- dependent one. This allows the direct use NADPH, which is generated in the light reactions of photosynthesis, for product formation. For this we selected the acetoin reductase from Leuconostoc lactis NCW1 [198] and introduced it into Synechocystis , generating Syn pVZ006. This strain produced meso-2,3-butanediol. In the culture supernatant, acetoin was barely 3 detectable, i.e. amounting to concentrations of less than 0.020 mmol/L, which is the detec- tion limit of the HPLC method used. This showed that a nearly complete shift in the product ratio had been achieved and also indicated a high stereospecificity of the Leuconostoc ace- toin reductase since no optically active butanediol was detected ( Figs. 3d and 4). Enzymatic decarboxylation of acetolactate yields the R-isomer of acetoin. The acetoin re- ductases employed in this study stem from meso-butanediol forming organisms and hence install an S-configured stereocenter at the ketone moiety of R-acetoin (Fig. 3.6). In cultures with the NADH dependent acetoin reductase of Enterobacter sp., acetoin is present in the supernatant at all times. In these cultures, high pH can lead to racemization of acetoin via an enolate intermediate. The R-isomer is reduced to meso-butanediol, while the S-isomer of acetoin is reduced to S,S-butanediol. With the NADPH dependent enzyme from Leu- conostoc lactis NCW1, acetoin concentrations in the supernatant are so low that S-acetoin cannot build up to high concentrations, therefore these cultures produce meso-butanediol exclusively. 39

CONCLUSION

In summary, highest product titers of the C4 products achieved in this study are 8 mM, which equals 0.72 g/l. CO2-based, photosynthesis-driven, production of fuels and chemical feedstock is an emerg- ing field and encouraging results were presented recently [148]. The development of pro- cesses, in which cells are used as catalysts for the synthesis of such products has received great interest. In almost all engineered photosynthetic microbes to date, the majority of carbon taken up by the cells is still used to create biomass. We define a photosynthetic cell factory as an organism that channels the predominant fraction of fixed CO2 (i.e.>50%) into 3 product. Using a non-optimized host strain, we have shown here channeling of an encour- aging maximum of 30% of total carbon fixed to measured biomass into product, indicating that we are on the right track (with Syn SAA012 we achieve channeling of 70% of total car- bon fixed to measured biomass into ethanol (see Fig. S 3.3d)). It is relevant to note that if a very large fraction of fixed CO2 is channeled into a product (carbon sink), the total carbon fixation rate can increase ([105] and Fig. S 3.3e). Further advances in the understanding of existing limitations (like in the rate of CO2 fixation [148]), removal of competing path- ways (van der Woude, A., unpublished) and new developments in metabolic engineering will help reaching the goal of a photosynthetic 2,3-butanediol cell factory, i.e. a cyanobac- terium that partitions more than 50% of its fixed carbon into butanediol. Further optimization strategies could include the heterologous expression of an additional ALS. The expression level of ALDC and AR could be further increased by placing the operon under control of multiple strong promoters, a strategy recently employed in Escherichia coli for the overexpression of green fluorescent protein and subsequently applied in poly- hydroxybutyrate production in the same host [215]. Alternatively, multiple copies of the butanediol operon could be introduced into the host, as shown recently for ethylene [133] and ethanol producing Synechocystis [107]. In these latter two examples, metabolic flux to the fuel compound seems to be controlled largely by the capacity of the heterologous en- zymes. A recent study on lactic acid production employing a lactate dehydrogenase from Lactococcus lactis in Synechocystis shows that the enzyme concentration can control the production rate by a large extend, emphasizing the need for a systematic search of the bot- tleneck of an introduced pathway [134]. A similar comparison between Syn pVZ001 and the truncated pathway in Syn pVZ002 can be made. The yield of the C4 products, i.e. the sum of acetoin and butanediol (Fig. 3.2a), is higher in pVZ002, which can be rationalized by a higher level of the ALDC enzyme. ALS activity appears similar in all conjugative strains tested (including wild type) (Tab. S 3.4). C4 product yield is controlled by ALS and ALDC because AR activity is much higher than the activity of these two enzymes. Given that ALDC activities in Syn pVZ002 are higher than in Syn pVZ001, while ALS activities are not signifi- cantly different, it can be concluded that ALDC controls C4 product yield more than ALS in this case. High in vitro activity of ALS could only be observed in Syn SAA018 which carries an ALS with high activity (compare Tab. S 3.1 and S 3.4). This mutant, however, does not accumulate high levels of butanediol or acetoin (Fig. 3.1). All other mutants exhibit only a fraction of this high activity and yet show higher productivity in vivo. While in vitro ALS activity is certainly underestimated in Tab. S 3.4, control of this step on the yield of C4 prod- ucts can be considered minor. While the acetoin reductase activity measured in cell-free extracts of Syn pVZ006 is lower than in the other mutants tested, it is still quite higher than the in vivo production rates (Tab. S 3.6). In Syn pVZ006, buildup of acetoin would be an 40 3. meso-2,3-BUTANEDIOL SYNTHESIS BY Synechocystis

indicator of insufficient acetoin reductase activity. Since acetoin is only formed in traces, it follows that the capacity of this reaction must be high enough to convert all of the acetoin to butanediol, leading to the hypothesis again, that a large part of the control of this pathway lies in the acetoacetate decarboxylase step. Ultimately, optimal enzyme concentrations will have to be found, high enough to allow a high flux through the pathway but so low that en- zyme production does not pose a metabolic burden for the cells. Under such conditions, cyanobacterial autotrophic ‘cell factories’ for short-chain alcohols may surpass the some- what pessimistic view on their performance foreseen in [216]. In summary, in this study, we were able to successively increase production of butanediol 3 from carbon dioxide using genetically modified Synechocystis from an initial 0.025 mmol/L after 12 d to a final 1.8 mmol/L after 12 d (interpolated), corresponding to a more than 70- fold increase. Highest butanediol titers were 6.5 mmol/L after 29 days, which, although currently lower than yields in fermentation based processes, presents an encouraging first step towards a sustainable process that is independent from fixed carbon. During the initial submission period of this manuscript a study on (R,R)-2,3-butanediol production with Synechococcus elongatus PCC7942 appeared [108] employing a similar, but distinct pathway. Both, the similarities (i.e. the general strategy) and differences (i.e. the choice of specific enzymes, resulting in enantio-pure and/or racemic butanediol being pro- duced and the respective optimization strategies) report significant improvement in the area of metabolic engineering in cyanobacteria for the purpose of employing the latter as cell factories. As mentioned above, an advantage identified in this study is the successful optimization step of employing the NADPH dependent highly stereospecific acetoin reduc- tase of Leuconostoc resulting in meso-2,3-butanediol, but none of the optical active butane- diol isomers. Both, an interesting similarity and difference in the engineered production of butanediol by Synechoccus PCC7942 and Synechocystis sp. PCC6803 can be observed when comparing the toxicity of the product and the intermediate substrate acetoin. As mentioned in the intro- duction we have found IC50 values of 375 mM (34˜ g/L) butanediol and 95 mM (8˜ g/L) acetoin for Synechocystis , whereas [108] find the same value for butanediol, but much lower values for acetoin in Synechococcus. The differences could be due to the experimental setup. We apply the compounds at the start of the batch culture, hence at a very low OD, and observe reduction of initial growth rate, whereas Oliver et al. add the compounds at OD 0.4 and ob- serve a stop of growth after 24 h for concentrations of 0.2 and 1.0 g/L acetoin. Synechocystis and Synechococcus show different tolerance to different chemicals, for example Synechocys- tis is more tolerant to ethanol [217].

CONFLICT OF INTEREST All authors declare a competing financial interest because of jointly having filed a patent (patent pending) related to the presented line of work. K.J.H. is affiliated with Photanol B.V., The Netherlands.

ACKNOWLEDGMENTS We thank Ivo van Stokkum and Jeroen B. van der Steen for help with developing a proce- dure for a mathematical fit of the growth curves. We thank Orawan Borirak, Sacha Stelder and Mark Daniels for help with cloning and strain construction. We thank Peter Schoen- 41 makers, Andjoe Sampat and Ngoc A Dang for the use of their GC setup. We thank David van Brussel with help for setting up initial experiments for activity measurements. We thank the anonymous reviewers for their helpful suggestions that markedly increased the quality of this manuscript. This project was carried out within the research program of BioSolar Cells, co-financed by the Dutch Ministry of Economic Affairs, Agriculture and Innovation.

SUPPLEMENTARY INFORMATION 3 42 3. meso-2,3-BUTANEDIOL SYNTHESIS BY Synechocystis

Table S 3.1: Primers used in this study

Name Sequence NdeI-NCW1 AR f ACACATATGCGGCAAGGAGGTTCATAATGACCAAA PvuI-NCW1 AR r CCGACGATCGTTAATGGAATACCATACCACC Als_Ef_f GGTGGATCCATGAGTAAAAAAGGATCAGATATCATAG Als_Ef_r GGAGGTACCTCAATAAAGTTGATCTGGTAACAATGTTTTA Aldc_Ll_f GGTGGATCCATGTCAGAAATCACACAACTTTTTCAAT Aldc_Ll_r GGAGGTACCTCATTCAGCTACATCAATATCTTTTTTCAAA 3 butA_Ll_f GGTGGATCCATGTCTAAAGTTGCAGCAGTTACTG butA_Ll_r GGAAAGCTTTCAATGAAATTGCATTCCACCATCAAC pdc_zm_BB_f AATGAATTCGCGGCCGCTTCTAGAGGAGGACTAGCATGAGTTATACTGTC GGTACCTATTTAGC pdc_zm_BB_r AATCTGCAGCGGCCGCTACTAGTACTAGAGGAGCTTGTTAACAGGCTTAC adh_zm_BB_f AATGAATTCGCGGCCGCTTCTAGAGGAGGACTAGCATGGCTTCTTCAAC TTTTTATATTCC adh_zm_BB_r AATCTGCAGCGGCCGCTACTAGTATTAGAAAGCGCTCAGGAAGAGT Hom1Xho_f TTTACTCGAGTGTTGTACCTTCTTCCAACGCTATCGG Hom1Hind_r TTTAAAGCTTTTAACCGTTGACAGCAGGAGCGG Hom2Bam_f_II TAAAGGATTCTTCCTTGGTGTAATGCCAACTGAATAATCTGC Hom2Not_r_II TAAAGCGGCCGCTTACAAATGATTAGGGGCTTG Kan903_f AAATCCCGGGAAAGCCACGTTGTGTCTCAAAATC Kan903_r AAATCCCGGGCGCTGAGGTCTGCCTCGTGAA Psb_seq_f CAGTTGTGTCGCCCCTCTACA Psb_seq_r CCATAATTCCTCTATCGAAGATGGA H1_seg_f TGTCGCCGCTAAGTTAGA H2_seg_r CTGTGGGTAGTAAACTGGC BamHI_Bbrev_aldc f ATTAGGATCCATGACTGTACCCGCCCCCCCT SacI_Bbrev_aldc r TGTGGAGCTCTTATTTCCGTTCGGATTCAGCTTGGTGTACTT BamHI_Ent_ar f ATTAGGATCCCAAAAAGTTGCTCTCGTAACC SacI_Ent_ar r TGTAGAGCTCTTAATTGAATACCATCCCACC BamHI_llNCW1_ar f ATTAGGATCCACAAAAGTAGCATTAGTAACAGGAG SacI_llNCW1_ar r TATAGAGCTCTTAATGAAATACCATACCACCATC AREnt_na_Spe_f ACTAGTAGGAGGACTAGCATGCAAAAAGTTGCTCTCGTAACC AREnt_na_Pst_r CTGCAGTTAATTGAATACCATCCCACCG ARPLeu_Spe_f ACTAGTAGGAGGACTAGCATGACAAAAGTAGCATTAGTAACAGG ARPLeu_Pst_r CTGCAGTTAATGAAATACCATACCACCATCAAC Qsll1981_f CCATTTCCAAGGCCAAAAAC Qsll1981_r GCTTCGGCTCGGATGGT Qslr2088_f TGCCCAGGTGCAGGAATT Qslr2088_r TTCCCATCAGGGTGGTTGTT QrnpB_f GCGCACCAGCAGTATCGA QrnpB_r CCTCCGACCTTGCTTCCAA QalsNCWI_f AGTCCGTGGCCCTGTTTG QalsNCWI_r AATGTTATTGGGATCTTGAATTTCG QaldcBB_f CGACAGCACCGGTAAATTATCC QaldcBB_r GTAACGGCGAAGGGAGTTTTC QarEnt_f CACAGGCGGGCCATACAG QarEnt_r CCCCCTAACCGCAAATTTG Table S 3.2: Strains and plasmids used in this study

Strain and plasmid Description Origin E. coli XL-1 (blue) Cloning host strain Stratagene E. coli J53 (pRP4) Conjugation helper strain Wilde A. E. coli M15 pREP4 Protein production strain Qiagen E. coli M15 pREP4 pQE30 Ef als Used to produce recombinant Enterococcus faecalis V583 ALS This study E. coli M15 pREP4 pQE30 Bb aldc Used to produce recombinant Brevibacillus brevis ALDC This study E. coli M15 pREP4 pQE30 Es ar Used to produce recombinant Enterobacter sp. AR This study E. coli M15 pREP4 pQE30 Ll ar Used to produce recombinant Leuconostoc lactis NCW1 AR This study Synechocystis sp. PCC6803 Wild type, glucose tolerant, naturally transformable Bhaya D. pHKH001 Integration vector disrupting slr0168 in the Synechocystis genome with kanamycin cassette [135] pPSBTC Integration vector for transcriptional coupled insertion downstream to psbA2 (slr1311) This study in the Synechocystis genome with the kanamycin cassette pSB1AC3_TrcSTHtt Source for the P.aeruginosa sth cassette Ptrc::sth::tt [135] pSEQ_Trc trc promoter on pSEQ_Trc [135] pVZ321 Mobilizing and self-replicating plasmid for Synechocystis [78] pUC57 Ll NCW1 AR Codon optimized ar of Leuconostoc lactis NCW1 This study pHKH018 Integration vector with E. faecalis als, L. lactis aldc, L. lactis butA cassettes This study pPSBEtOH Integration vector at psbA2 locus with Z. mobilis pdc and adhII This study pHKHAR Integration vector with Enterobacter sp. ar cassette This study pHKHARP Integration vector with Leuconostoc lactis NCW1 ar cassette This study Syn SAA018 Synechocystis sp. PCC6803 PT5::als::PT5::aldc::PT5::butA::Kmr ALS of E. faecalis, ALDc and This study ButA of L. lactis Syn SAA012 Synechocystis sp. PCC6803 PpsbA2::psbA2s::pdc::adhII::Kmr PDC and ADHII of Z. mobilis This study Syn pVZ001 self-replicating plasmid with Ptrc::als::aldc::ar Kmr ALS of L. lactis NCWI, ALDc of This study B. brevis and AR of Enterobacter sp. Syn pVZ002 self-replicating plasmid with Ptrc::aldc::ar Km ALDc of B. brevis and AR of Enterobacter sp. This study Syn pVZ003 pVZ001 and P.aeruginosa sth cassette This study Syn pVZ006 self-replicating plasmid with Ptrc::aldc::ar Kmr ALDc of B. brevis and AR of Leuconostoc This study lactis NCW1 Syn AR Synechocystis sp. PCC6803 Ptrc::arEnt::Kmr AR of Enterobacter This study Syn ARP Synechocystis sp. PCC6803 Ptrc::arLeu::Kmr AR of Leuconostoc lactis NCW1 This study 44 3. meso-2,3-BUTANEDIOL SYNTHESIS BY Synechocystis

Table S 3.3: Enzymes employed for the conversion of pyruvate to butanediol via acetolactate and acetoin. This biosynthetic pathway is found in lactic acid bacteria, enterobacteria and bacilli. Ace- tolactate synthase (ALS), acetolactate decarboxylase (ALDC) and acetoin reductase/butanediol dehy- drogenase (AR/BDDH). n.a. not available.

Enzyme Gene Donor Substrate KM for Product name Organism substrate ALS [218] als Enterococcus Pyruvate 40 mMi Acetolactate faecalis 3 ALS [187] als Leuconostoc Pyruvate 10 mMi Acetolactate lactis NCW1 ALDC [185, 219] aldc Lactococcus Acetolactate 10 mM Acetoin lactis sp. cremoris ALDC [189] aldc Brevibacllus Acetolactate 0.06 mM Acetoin brevis AR/BDH [186, 220] butA Lactococcus Acetoin n.a. Butanediol lactis sp. cremoris AR/BDH [190] ar Enterobacter Acetoin 0.4 mM Butanediol sp. AR/BDH [198] ar Leuconostoc Acetoin n.a. Butanediol lactis NCW1

i Enzymes were chosen according to their kinetic properties found after a search in the BRENDA database [221]. Detailed references are given in the Table S. Two of a few characterized catabolic ALS (cALS) enzymes, L. lactis and E. faecalis showed a high KM of 50 mM and 40 mM for pyruvate as determined earlier [184, 218]. Based on that information initially this catabolic ALS of E. faecalis was chosen for the construction of Syn SAA018, to improve on that, the ALS of Leuconostoc lactis NCW1 was chosen for Syn pVZ001 and derivative mutant strains, due to its reported (lower) KM of around 10 mM for pyruvate [187]. During the submission period of this manuscript, how- ever the cALS of E. faecalis V583 was investigated in greater detail revealing FAD-independent activity with a KM of 1.37 mM [205]. Consequently, we do not detect any (extra) ALS activity in the mutant carrying theLeuconostoc ALS. 45

Table S 3.4: Acetolactate synthase (ALS) activities from cell-free extracts of wild type and Synechocystis mutant strains grown in BG11 medium under the conditions indicated. Errors represent standard deviation of at least three biological replicates.

Strain Condition ALS activity [nmol/mg potein/min] Synechocystis WT 25 mM CAPSO pH 9.0 0.042 0.0063 ± Synechocystis WT 50 mM NaHCO 0.042 0.011 3 ± Synechocystis pVZ001 25 mM CAPSO pH 9.0 0.049 0.030 3 ± Synechocystis pVZ001 50 mM NaHCO 0.031 0.033 3 ± Synechocystis pVZ002 25 mM CAPSO pH 9.0 0.037 0.032 ± Synechocystis pVZ002 50 mM NaHCO 0.073 0.13 3 ± Synechocystis pVZ003 25 mM CAPSO pH 9.0 0.026 0.017 ± Synechocystis pVZ003 50 mM NaHCO 0.047 0.007 3 ± Synechocystis pVZ006 25 mM CAPSO pH 9.0 0.013 0.017 ± Synechocystis pVZ006 50 mM NaHCO 0.029 0.010 3 ± Synechocystis SAA018 25 mM CAPSO pH 9.0 1.03 0.038 ± Synechocystis SAA018 50 mM NaHCO 28.63 0.71 3 ±

Table S 3.5: Gene expression data of plasmid borne over-expressed heterologous genes in the strains ∆∆ Syn pVZ001 and Syn pVZ002 obtained with the Q-PCR. According to the 2− CT method the fold- changes, relating Syn pVZ002 with pVZ001, of the investigated transcript are shown. N.d. for not detected the mean and standard deviation are derived from biological duplicates and technical trip- licates

gene strain Fold-change als Synechocystis pVZ002 n.d. aldc Synechocystis pVZ002 2.79 0.68 ± ar Synechocystis pVZ002 1.66 0.36 ± 46 3. meso-2,3-BUTANEDIOL SYNTHESIS BY Synechocystis

Table S 3.6: Acetolactate decarboxylase (ALDC) activities from cell-free extracts of wild type and Syne- chocystis mutant strains. The activity expressed in mmol/gDW/h serves as comparison with the bu- tanediol production rates. The wild type activity detected can be evaluated as non-enzymatic catalysis in the assay, possibly due to the instability of the acetolactate that has been build-up by the acetolac- tate synthase employed in the assay (section3 for details on the ALDC activity assay). As observed by [108] the ALDC activity in the assay might not strictly be under saturating conditions, but is in- ferred from the assay setup that employs ALS. Thus the activity mainly serves the comparison of the in-parallel assessed cell extracts. Errors represent standard deviation of at least three biological repli- cates. 3 Activity (nmol/mg protein/min) Capacity (mmol/gDW/h) Synechocystis WT 22.92 4.92 0.69 0.15 ± ± Synechocystis pVZ001 53.92 5.00 1.62 0.15 ± ± Synechocystis pVZ002 69.05 5.44 2.07 0.16 ± ± Synechocystis pVZ003 41.39 3.33 1.24 0.10 ± ± Synechocystis pVZ006 19.51 4.85 0.59 0.15 ± ± Synechocystis SAA018 39.38 3.96 1.18 0.12 ± ± Synechocystis AR 6.42 3.91 0.19 0.12 ± ± Synechocystis ARP 7.61 3.00 0.23 0.09 ± ±

Table S 3.7: Acetoin reductase (AR) activities from cell-free extracts of wild type and Synechocystis mutant strains. The activity expressed in mmol/gDW/h serves as comparison with the butanediol production rates. The wild type activity detected reflects the NADH (and NADPH respectively) ox- idation, catalysed by unknown components of the crude cell free extract, and can be considered a background activity. (see section3 for details on the AR activity assay). Errors represent standard deviation of at least three biological replicates

Activity (nmol/mg protein/min) Capacity (mmol/gDW/h) Synechocystis WT 1.60 0.38 0.05 0.01 ± ± Synechocystis SAA018 12.92 9.69 0.39 0.29 ± ± Synechocystis pVZ001 204.99 25.53 6.15 0.77 ± ± Synechocystis pVZ002 282.66 58.07 8.48 1.74 ± ± Synechocystis pVZ003 153.22 52.69 4.60 1.58 ± ± Synechocystis AR 14.89 16.25 0.47 0.48 ± ± Synechocystis WT i 1.46 1.00 0.04 0.03 ± ± Synechocystis pVZ006 i 3.48 0.67 0.10 0.02 ± ± Synechocystis ARP i 19.97 4.83 0.60 0.14 ± ±

i The NADPH dependent acetoin reductases were assayed with NADPH as co-factor in the reaction. Wild type data is presented for both, the NADH (upper part of the Table S) and NADPH (lower part of the Table S) oxidation, respectively. 47

Table S 3.8: Gene expression data of two putative native als genes (sll1981 and slr2088) of Synechocystis ∆∆CT investigated at pH 9 and with the addition of bicarbonate. According to the 2− method the fold-changes, bicarbonate over pH 9 condition, of the investigated transcripts are shown. Mean and standard deviation are derived from biological duplicates and technical triplicates. gene strain Condition Fold-change sll1981 Wild type bicarbonate 0.31 0.01 ± slr2088 Wild type bicarbonate 0.42 0.02 ± sll1981 Synechocystis pVZ001 bicarbonate 0.94 0.03 ± slr2088 Synechocystis pVZ001 bicarbonate 2.41 0.08 3 ± sll1981 Synechocystis pVZ002 bicarbonate 1.14 0.01 ± slr2088 Synechocystis pVZ002 bicarbonate 2.30 0.01 ±

Table S 3.9: Transhydrogenase (STH) activities in cell-free extracts of wild type and Synechocystis mu- tant strains. Errors represent standard deviation of at least two biological replicates.

Activity (nmol/mg protein/min) Synechocystis WT 0.10 0.06 ± Synechocystis SAA018 0.08 0.04 ± Synechocystis pVZ001 0.12 0.07 ± Synechocystis pVZ002 0.10 0.05 ± Synechocystis pVZ003 103.6 34.2 ± Synechocystis pVZ006 0.04 0.01 ±

a b Syn WT sigmoidal fit Syn WT sigmoidal fit

100 100

80 80

0 0 60 0 60 0 1 1

* *

x x a a m m µ µ 40 / 40 / µ µ

20 20

0 0

1 10 100 1000 0.1 1 10 100 1000 Log([meso-butanediol]) / mmol/L Log([acetoin]) / mmol/L

Figure S 3.1: Synechocystis exhibits high tolerance to meso-butanediol and acetoin. Cells were grown in BG11 medium buffered with 10 mM TES to a pH of 8.0 with racemic acetoin or meso-butanediol added to different concentrations. Growth rates were normalized to maximum growth rates and plot- ted against the concentration of the additive. 48 3. meso-2,3-BUTANEDIOL SYNTHESIS BY Synechocystis

Figure S 3.2: Cell-free extracts of Synechocystis wild type and mutants catalyze the conversion of pyru- vate to acetolactate. Cuvettes show the Voges-Proskauer assay [200] modified by Yang [201], which gives a pink color in the presence of acetoin. “+” indicates presence of pyruvate, “-“ indicates its 3 absence. WT means wild type Synechocystis , 01, 02, 03, 06 correspond to Synechocystis mutants har- boring the respective pVZ plasmid. T5 indicates the Syn SAA018 mutant, which carries the butane- diol pathway downstream of the T5 promoter. 9 and BC indicate growth at pH 9.0 and in presence of50 mmol/L sodium bicarbonate, respectively. 49

Condition Synechocystis pVZ001

10 12 10 Unsupple- 11 D/L-BD 10 8 8 meso-BD mented 10 6 acetoin BG11 730 9 6 1 4 8 pH OD 4 c/mmol/L 7 c/mmol/L 2 2 0.1 6 0 5 0 0 5 10 15 20 25 30 0 5 10 15 20 25 30 3 t/d t/d 10 12 10 BG11 D/L-BD 11 10 8 8 meso-BD medium 10 6 acetoin + 50 mM 730 9 6 NaHCO 1 4 8 pH 3 OD 4 c/mmol/L 7 c/mmol/L 2 2 0.1 6 0 5 0 0 5 10 15 20 25 30 0 5 10 15 20 25 30 t/d t/d 10 12 10 BG11 11 D/L-BD 10 8 8 meso-BD medium 10 6 acetoin + 25 mM 730 9 6 1 4 8 pH TES/KOH OD 4 c/mmol/L 7 c/mmol/L pH 6.5 2 2 0.1 6 0 5 0 0 5 10 15 20 25 30 0 5 10 15 20 25 30 t/d t/d 10 12 10 BG11 D/L-BD 11 10 8 8 meso-BD medium 10 6 acetoin + 25 mM 730 9 6 HEPES/ 1 4 8 pH OD 4 NaOH c/mmol/L 7 c/mmol/L 2 2 pH 7.5 0.1 6 0 5 0 0 5 10 15 20 25 30 0 5 10 15 20 25 30 t/d t/d 10 12 BG11 10 D/L-BD 11 10 8 8 meso-BD medium 10 6 acetoin + 25 mM 730 9 6 TES/KOH 1 4 8 pH OD 4 pH 8.0 c/mmol/L 7 c/mmol/L 2 2 0.1 6 0 5 0 0 5 10 15 20 25 30 0 5 10 15 20 25 30 t/d t/d 50 3. meso-2,3-BUTANEDIOL SYNTHESIS BY Synechocystis

Condition Synechocystis pVZ001

10 12 10 BG11 11 D/L-BD 10 8 8 meso-BD medium 10 6 acetoin + 25 mM 730 9 6 CAPSO 1 4 8 pH OD 4 pH 9.0 c/mmol/L 7 c/mmol/L 2 2 0.1 6 0 5 0 3 0 5 10 15 20 25 30 0 5 10 15 20 25 30 t/d t/d 10 12 10 BG11 11 D/L-BD 10 8 8 meso-BD medium 10 6 acetoin + 25 mM 730 9 6 CAPS 1 4 8 pH OD 4 pH 11.0 c/mmol/L 7 c/mmol/L 2 2 0.1 6 0 5 0 0 5 10 15 20 25 30 0 5 10 15 20 25 30 t/d t/d

Condition Synechocystis pVZ002 10 12 Unsupple- 10 D/L-BD 11 10 8 8 meso-BD mented 10 6 acetoin BG11 730 9 6 1 4 8 pH OD 4 c/mmol/L 7 c/mmol/L 2 2 0.1 6 0 5 0 0 5 10 15 20 25 30 0 5 10 15 20 25 30 t/d t/d 10 12 BG11 10 D/L-BD 11 10 8 8 meso-BD medium 10 6 acetoin + 50 mM 730 9 6 NaHCO 1 4 8 pH 3 OD 4 c/mmol/L 7 c/mmol/L 2 2 0.1 6 0 5 0 0 5 10 15 20 25 30 0 5 10 15 20 25 30 t/d t/d 10 12 10 BG11 11 D/L-BD 10 8 8 meso-BD medium 10 6 acetoin + 25 mM 730 9 6 TES/KOH 1 4 8 pH OD 4 pH 6.5 c/mmol/L 7 c/mmol/L 2 2 0.1 6 0 5 0 0 5 10 15 20 25 30 0 5 10 15 20 25 30 t/d t/d 51

Condition Synechocystis pVZ002 10 12 BG11 10 D/L-BD 11 10 8 8 meso-BD medium 10 6 acetoin + 25 mM 730 9 6 HEPES/ 1 4 8 pH OD 4 NaOH c/mmol/L 7 c/mmol/L 2 2 pH 7.5 0.1 6 0 5 0 0 5 10 15 20 25 30 0 5 10 15 20 25 30 3 t/d t/d 10 12 BG11 10 D/L-BD 11 10 8 8 meso-BD medium 10 6 acetoin + 25 mM 730 9 6 TES/KOH 1 4 8 pH OD 4 pH 8.0 c/mmol/L 7 c/mmol/L 2 2 0.1 6 0 5 0 0 5 10 15 20 25 30 0 5 10 15 20 25 30 t/d t/d 10 12 BG11 10 D/L-BD 11 10 8 8 meso-BD medium 10 6 acetoin + 25 mM 730 9 6 CAPSO 1 4 8 pH OD 4 pH 9.0 c/mmol/L 7 c/mmol/L 2 2 0.1 6 0 5 0 0 5 10 15 20 25 30 0 5 10 15 20 25 30 t/d t/d 10 BG11 12 10 D/L-BD 11 10 8 8 meso-BD medium 10 6 acetoin + 25 mM 730 9 6 CAPS 1 4 8 pH OD 4 pH 11.0 c/mmol/L 7 c/mmol/L 2 2 0.1 6 0 5 0 0 5 10 15 20 25 30 0 5 10 15 20 25 30 t/d t/d

Figure S 3.3: Time course of growth, total extracellular C4 compounds, pH and stacked bar representa- tion of total production detailing the fraction of acetoin, meso- and D/L-2,3-butanediol in Syn pVZ001 and Syn pVZ002, respectively. Culture conditions are indicated in the leftmost column. In both blocks, the left graphs show optical density (black circles, left y-axis), extracellular pH (open triangles, black right y-axis) and total extracellular C4 compounds (green squares, green right y-axis). The right graphs show the composition of the extracellular C4 compounds in a stacked bar representation. 52 3. meso-2,3-BUTANEDIOL SYNTHESIS BY Synechocystis

a Syn SAA012 Z. mobilis HOM1 pdc adhII KmR HOM2

slr1311 slr1312 Genomic integration site

Syn SAA012 pH 9 b Syn WT NaHCO3 Syn SAA012 NaHCO3 c Syn WT pH 9 Syn SAA012 NaHCO [EtOH] Syn SAA012 pH 9 [EtOH] 3 25 25 10 10 20 20

3 ) )

730 1 15 730 1 15

log(OD 10 log(OD 10 [EtOH] / mmol/L [EtOH] / mmol/L 0.1 0.1 5 5

0.01 0 0.01 0 0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20 t / d t / d

d Diverted C3 Carbon e Biomass EtOH [mmol/L/h] Syn WT Syn SAA012 0.100

/h 1.5 dw

100 0.075 /h

80 1.0 0.050 60

40 0.5 Carbon partitioning / %

0.025 d[EtOH]/dt / mmol/L 20 carbon fixation rate / mmol/g 0 0.000 0.0 0 5 10 15 20 0 5 10 15 20 t / d t / d

Figure S 3.4: Ethanol production over time with a modified Synechocystis strain. (A) Strain Syn SAA012 harbours the pdc and adhII genes from Z. mobilis, transcriptionally coupled to the psbA2 (slr1311) gene, in the chromosome. (B) Growth of Syn SAA012 is shown in BG11 medium supplemented with 50 mmol/L NaHCO3 and (C) in BG11 medium supplemented with 25 mM CAPSO, pH 9, respectively. Also shown are ethanol concentrations at respective time points for the two conditions for strain Syn SAA012. (D) Fixed carbon partitioning of product over biomass and corresponding volumetric production rate in Syn SAA012 in the presence of added bicarbonate. (E) Total carbon fixation rate for (B), comparing carbon fixed into biomass for wild type and biomass plus C3 diverted carbon (taking into account that PDC eliminates one CO2 molecule) for Syn SAA012. The wild type did not produce detecTable S amounts of ethanol under the conditions tested. The values are obtained from the aver- age of biological replicates; the error bars indicate the standard deviation (n=3); if error bars are not visible they are smaller than the respective data point symbol. 53

3

Figure S 3.5: SDS/PAGE picture of a 4-12% Bis-Tris gel of the soluble fraction of crude cell free extracts of Synechocystis wild type and mutant strains, highlighting the overexpressed soluble transhydroge- nase (STH) from P.aeruginosa expressed in Syn pVZ003 (molecular weight 51.1 kDa; see white arrow). Other overexpressed proteins are not visible, potentially present at lesser amounts and/or covered by native proteins visible at the same position in the gel (molecular weight of the ALS of Enterococcus and L. lactis NCW1 are 61 kDa; ALDC of L. lactis and (truncated) B. brevis are 27 and 29 kDa; AR of L. lactis and L. lactis NCW1 are 27 kDa).

Enterobacter sp. AR Leuconostoc lactis NCW1 sp. AR

acetoin acetoin S,S-2,3-butanediol meso-butanediol R,R-2,3-butanediol 21.5 22.0 22.5 23.0 23.5 24.0 24.5 25.0 25.5 21.5 22.0 22.5 23.0 23.5 24.0 24.5 25.0 25.5

t / min t / min

Figure S 3.6: The acetoin reductases of Enterobacter sp. and Leuconostoc lactis NCW1 are specific for S-configured stereocenters. The three isomers of 2,3-butanediol were incubated with the respective recombinant acetoin reductase and with NAD(P) at 37◦C for 16 h. Then, the sample composition was analysed by HPLC. Acetoin formation was only observed in isomers with S-stereocenters (i.e. S,S-2,3- butanediol and meso-butanediol). R,R-butanediol was not oxidized.

4 REDOXCOSUBSTRATELEVELSIN MUTANT Synechocystis STRAINS

Philipp SAVAKIS, Klaas J. HELLINGWERF

Directly measuring intracellular concentrations of redox cosubstrates is complicated by their reactivity towards oxygen and extremes of pH. Here, we analyse data from the previous chap- ter with an indirect method, which estimates intracellular [NAD(P)H]/[NAD(P)] levels from the extracellular ratios of [acetoin] and [2,3-butanediol]. It is found that in Synechocystis the NADH pool becomes more reduced towards stationary phase and is generally more reduced in a strain harbouring energy independent transhydrogenase. The NADPH pool is more reduced than the NADH pool.

55 56 4.R EDOX COSUBSTRATE LEVELS IN MUTANT Synechocystis

INTRODUCTION

ANY enzymatic pathways contain redox reactions, where a redox cosubstrate1 serves M as an electron donor or acceptor. Often, these cosubstrates are nicotinamide adenine dinucleotide derivatives. The section below will focus on NADP(H) and NAD(H)2. Reduc- tion reactions (e.g. ketone to secondary alcohol) are coupled to the stoichiometric oxidation of the reduced cosubstrate (NAD(P)H), while oxidation reactions (e.g. secondary alcohol to ketone) are coupled to the stoichiometric reduction of the oxidised cosubstrate (NAD(P)). The enzymatic pathway for the synthesis of 2,3-butanediol has already been introduced in chapter3 3. In the final step of this biosynthetic pathway, acetoin reductase catalyses the NAD(P)H dependent reduction of acetoin to 2,3-butanediol. In this chapter the acetoin reductase reaction will be used as an indicator reaction to determine intracellular NADH/- NAD ratios. 4 At chemical equilibrium and constant pH, the ratio of the redox cosubstrates is proportional to the ratio of the oxidised and the reduced substrate, as demonstrated for the reduction of acetoin to butanediol: [acetoin] [H+] [NAD(P)] Keq · , (4.1) [2,3 butanediol] = [NAD(P)H] − + 4 where [H ] and Keq are the proportionality constants . It follows, that the ratio of the redox cosubstrates can be estimated from the ratio of the substrates, if the redox reaction is in equilibrium. For convenience, [square brackets], com- monly denoting molar concentrations, will be omitted in the following. Rearrangement of Eq. 4.1 yields

1 2,3 butanediol NAD(P)H H+ − · , (4.5) Keq · acetoin = NAD(P)

1The word cosubstrate is more accurate than cofactor or coenzyme. The latter are typically used for non-covalently bound molecules that assume a catalytic role, i.e. the same cofactor is used in multiple turnovers. The term cosubstrate indicates that the molecule is used stoichiometrically. This means that only one molecule of the starting material can react per cosubstrate molecule. 2For more comprehensive information, see [222]. 3Briefly, the condensation reaction of two molecules of pyruvate (catalysed by acetolactate synthase) yields ace- tolactate, whose decarboxylation (catalysed by acetolactate decarboxylase) yields acetoin, which is reduced to 2,3-butanediol in a reaction that is mediated by acetoin reductase. 4 At chemical equilibrium, the velocity of a chemical reaction in one direction is equal to the velocity into the op- posite direction. Therefore, the net composition of the mixture (indicated by the concentration of the individual molecules) does not change over time:

d d [acetoin] [2,3 butanediol] 0 , (4.2) dt = dt − =

where [a] is the concentration of the respective molecule. Substitution with appropriate terms for the rates gives

k1 [acetoin] [NAD(P)H] [H+] k 1 [2,3 butanediol] [NAD(P)] , (4.3) · · · = − · − ·

where ki are the kinetic constants for the respective direction. Rearrangment of Eq. 4.3 yields

k1 [2,3 butanediol] [NAD(P)] : Keq − · , (4.4) k 1 = = [acetoin] [NAD(P)H] [H+] − · · the mass action law for the reaction. Keq is named the equilibrium constant of the reaction. Further rearrange- ment results in Eq. 4.1. 57 which is equivalent to

pH 1 2,3 butanediol NAD(P)H 10− − · , (4.6) Keq · acetoin = NAD(P)

For biological systems, an apparent equilibrium constant, K’eq, is defined at physiological -pH’ conditions (e.g. pH’) instead of Keq [17, 223]. Division of Eq. 4.6 by 10 gives

1 2,3 butanediol NAD(P)H − , (4.7) pH pH’ K’eq · acetoin = NAD(P) 10 · − While calculating the redox cosubstrate ratios according to Eq. 4.1 requires accurate de- pH pH’ termination of the intracellular pH, the ratio NAD(P)/NAD(P)H 10 − can be inferred · pH pH’ without this knowledge [223]. Canelas et al. determined NAD/NADH 10 − levels in · 4 Saccharomyces cerevisiae using a heterologous mannitol phosphate dehydrogenase as the indicator enzyme [223]. In this publication, a number of criteria were identified that need to be fulfilled to estimate the intracellular cosubstrate ratio: The enzyme shows absolute specificity for the cosubstrate and is fast enough to ensure near-equilibrium. Keq is known. The observed reaction is confined to one compartment in which the reactants are equally distributed. The reactant concentrations can be deter- mined precisely. In the original publication, this latter criterion refers to the intracellular concentrations of the reactants. For the following application, however, this is not crucial. Given that acetoin as well as butanediol can exchange between the cytosol and the extra- cellular medium5, the extracellular ratio of acetoin/butanediol should reflect their intracel- lular ratio6, and by extension, the NAD(P)/NAD(P)H ratio (Eq. 4.7). In the acetoin/butanediol system, the addition of acetion to strains that express acetoin reductase faces the problem that commercially available acetoin is racemic and that the respective enantiomers will be converted at different rates. Ferone therefore investigated the exogenous addition of meso-butanediol to acetoin-reductase expressing mutants [224]. There, it was shown that the NADPH/NADP ratio was low when cells were incubated in the dark, and increased rapidly when the culture was irradiated. A simple way to add optically pure acetoin is to synthesise it in the cell that harbours the ace- toin reductase. In chapter3, acetoin and butanediol titres were determined in two different mutant strains under a wide range of extracellular pH values (Fig. S 3.3). In the following, these data will be reexamined from the aspect of intracellular redox cosubstrate ratios.

5Butanediol is formed, when acetoin is added to Synechocystis harbouring acetoin reductase and vice versa. 6Under the assumption that diffusion is fast enough. 58 4.R EDOX COSUBSTRATE LEVELS IN MUTANT Synechocystis

MATERIALS AND METHODS 7 Calculation of K’eq. K’eq was calculated with the eQuilibrator tool [225] . The intracellular pH of Synechocystis was reported to lie between 7.5 and 7.8 [226]8. In the same publication, low ionic strength was mimicked with 10 mL of a 0.05 M HEPES solution to which 2 mL of 9 D2O were added [226] . Since the pKa of HEPES is 7.5, at pH 7.5, the ratio of dissociated and non-dissociated HEPES is 1. Undissociated HEPES is zwitterionic and it was therefore assumed that it does not contribute to the ionic strength, I [227]. I is defined as:

n 1 X I ci zi , (4.8) = 2 i 1 =

ci = concentration of ion i, zi = charge of ion i. With a concentration of 0.021 M of undisso- 4 ciated HEPES, I amounts to 0.021 M. With a pH of 7.5 and an ionic strength of 0.021, K’eq is calculated to be 756 M. Under the same conditions, a K’eq value of 1700 is calculated for the NADPH dependent reduction reaction. See Fig. S4.1 for the dependency of K’eq on pH and ionic strength for both reactions. pH 7.5 Calculation of NAD(P)H/(NAD(P) 10 − ). Raw data were obtained from chapter3, pub- · lished in [115]. For the calculation of ratios, the sum of the concentrations of meso-butanediol and optically active butanediol was divided by the concentration of acetoin and by K’eq, 756 M (NADH dependent reaction) and 1700 M (NADPH dependent reaction), respectively. Since there were only two time points at which an acetoin concentration could be deter- mined for the NADPH dependent reaction, for the remaining points, it was assumed that the concentrations were at the detection limit (0.02 mM [115]). The data points in this graph (Fig. 4.2) therefore represent the lowest ratio that is supported by the data.

RESULTS AND DISCUSSION In chapter3, the production of 2,3-butanediol was surveyed under a wide range of growth conditions and it was shown that strains Syn pVZ001 and Syn pVZ002 differ greatly in bu- tanediol productivity (Figs. 3.3,S3.3). A closer examination of the data with Eq. 4.7, however, reveals that the corresponding cal- culated NADH/NAD ratios are remarkably similar (Fig. 4.1A-G). This indicates that NADH/- NAD homeostasis in strain Syn pVZ002 is unimpaired, even though twice as much carbon is devoted to the heterologous pathway as compared to Syn pVZ001. Consequently, pro- ductivity cannot be increased by further increasing the amount of acetoin reductase as the acetoin/butanediol ratio is determined by NADH/NAD homeostasis. Only by manipulation of NADH/NAD levels can the yield further be increased. This is indeed experimentally demonstrated in chapter3. There, the introduction of energy independent transhydrogenase from Pseudomonas aeruginosa led to a shift in the ratio of pH 7.5 butanediol/acetoin. A calculation of the corresponding NADH/(NAD 10 − ) reveals that · 7 Note that calculation of Keq values with data from different sources yields very different results [224]. This should be kept in mind when the NADH/NAD values presented here are compared to values reported elsewhere. 8Note that this was for strain 6308. To the best of my knowledge, no published data are available for 6803. For convenience, an intracellular pH of 7.5 will be assumed for strain 6803. 9This assumption does not seem to be based on experimental data. To the best of my knowledge, there is no publication that reliably measures ionic strength in Synechocystis. Therefore, the value from Lawrence et al. will be used to calculate the ionic strength in the following. 59 the NADH pool is more reduced in these strains (Fig. 4.2A). For Syn pVZ001 and Syn pVZ002, in the cultures that were buffered to pH values between 6.5 and 9.0, the NADH/NAD ratio increases towards the later phase of the experiment (days 14 to 28, Fig. 4.1). It is unclear whether a similar trend would have been observed for the Syn pVZ003 mutant, as this growth experiment was stopped at day 14. The reason why this increase is observed is not known at the moment. One possible ex- planation would be that the cells become carbon deficient in the later stage of the growth experiment. This would mean that electrons from water splitting are partially stored in the NADH pool. The notion that carbon limitation is responsible for the increase in the pH 7.5 NADH/(NAD 10 − ) ratio is supported by the data from the cultures that were supple- · pH 7.5 mented with bicarbonate, where NADH/(NAD 10 − ) continuously decreases. · A greater degree of reduction in the NAD(P)H pool in response to carbon limitation was e.g. observed in [228]. In this study, however, fluorescence based methods were used to measure NAD(P)H/NAD(P) ratios. Distinguishing NADH and NADPH by fluorescence is not possi- 4 ble. By measuring extracellular ratios of product to substrate, NADH/NAD and NADPH/NADP can be distinguished, if the enzyme displays sufficiently high specificity. In chapter3, a mutant strain harbouring NADPH dependent acetoin reductase from Leunonostoc lactis NCW1 was constructed and analysed. This strain is suited to estimate the intracellular pH 7.5 NADPH/(NADP 10 − ) ratio. The determination, however, is complicated by the sim- · ple fact that acetoin was only detected in the last two time points of the growth experi- ment presented in Fig. S 3.3 and even there remained barely detectable. To give at least a lower estimate, undetected acetoin was set to 0.02 mM, the detection limit of the HPLC. pH 7.5 NADPH/(NADP 10 − ) ratios calculated with these concentrations are indeed higher than · pH 7.5 the NADH/(NAD 10 − ) measured for any other strain (FIg. 4.2B). · pH 7.5 Some parameters that are necessary for the calculation of NAD(P)H/(NAD(P) 10 − ), and · from this, NAD(P)H/NAD(P), cannot be reliably determined. To estimate the error this will incur, these parameters were varied around the values that were used for the actual calcu- lations. pH 7.5 When NAD(P)H/(NAD(P) 10 − ) is plotted against butanediol/acetoin and K’ (Eq. 4.7), · eq the resulting surface is described by f(x) = x/y (Fig. 4.3A). Qualitatively, the same depen- pH 7.5 dency is observed, when NAD(P)H/(NAD(P) 10 − ) is plotted against butanediol/ace- · toin at constant K’eq (not shown). As far as the experimental determination of extracellu- lar metabolite ratios10 is concerned, the uncertainty for acetoin is greater than for butane- diol. The reason for this is that the acetoin concentrations are generally lower than the butanediol concentrations. Under the assumption that noise is constant throughout the chromatogram, this leaves greater uncertainties to be expected for acetoin. This becomes a problem particularly in the early phases of the growth experiments, where acetoin is barely detectable. Therefore, e.g. the initial seemingly erratic behaviour of the NADH/NAD ratio in Syn pVZ001 at pH 11 (Fig. 4.1G) is very likely a result of poor quantification at the respective time points.

10by HPLC, see chapter3. 60 4.R EDOX COSUBSTRATE LEVELS IN MUTANT Synechocystis

0.012 BG11 plain BG11 pVZ001 ) A B pVZ002 0.010 50 mM NaHCO3 pH-7.5 0.008

0.006

0.004

0.002 NADH/(NADx10 0.000 0 7 14 21 28 0 7 14 21 28 0.012 ) C BG11 pVZ001 D BG11 pVZ001 0.010 25 mM TES/KOH pVZ002 25 mM HEPES/NaOH pVZ002 4 pH-7.5 pH 6.5 pH 7.5 0.008

0.006

0.004

0.002 NADH/(NADx10 0.000 0 7 14 21 28 0 7 14 21 28 0.012

) E BG11 pVZ001 F BG11 pVZ001 0.010 25 mM TES/KOH pVZ002 25 mM CAPSO pVZ002

pH-7.5 pH 8.0 pH 9.0 0.008

0.006

0.004

0.002 NADH/(NADx10 0.000 0 7 14 21 28 0 7 14 21 28 0.012 t / d ) G BG11 pVZ001 0.010 25 mM CAPS pVZ002 pH-7.5 pH 11.0 0.008

0.006

0.004

0.002 NADH/(NADx10 0.000 0 7 14 21 28 t / d

Figure 4.1: The NADH/(NAD 10pH 7.5) ratio increases in buffered cultures towards stationary phase. · − The ratios are shown for plain BG11 medium (A), BG11 medium supplemented with bicarbonate (B), BG11 medium buffered to initial pH values of 6.5 (C), 7.5 (D), 8 (E), 9 (F) and 11 (G). Values for Syn pVZ001 are shown as black squares and values for Syn pVZ002 are shown as grey diamonds. Values were calculated from previously published data (See Fig. S 3.3. Error bars denote standard deviations of at least two biological replicates. 61

A ) B 80 80 pH-7.5 pVZ003 pVZ006 pVZ006 lower estimate 60 60

40 40

20 20

xNAD(P)H/(NAD(P)x10 0 0 -3 0 7 14 21 28 0 7 14 21 28 10 t / d t / d 4

Figure 4.2: In Synechocystis, the NADPH pool is more reduced than the NADH pool. Introduction of energy independent transhydrogenase into Synechocystis leads to a more reduced NADH pool (A). Black pentagons denote NADH/(NAD 10pH 7.5) ratios, shown against time. Hexagons stand for · − NADPH/(NADP 10pH 7.5) ratios (B). Black hexagons denote ratios calculated from measured data. · − Grey hexagons stand for ratios that were calculated with the acetoin concentration set to the detec- tion limit of the HPLC system (0.02 mM). These values are to be read as the lowest possible ratio that is supported by the data. The data were obtained from chapter3, published in [115]. The strains were grown in BG11 medium, supplemented with 25 mM CAPSO, pH 9.0. Error bars denote standard devia- tions of at least two biological replicates. Error bars that are not visible are smaller than the respective data point symbol.

A B pH-7.5

1000 0.20 NADH

800 NAD

NADH 0.15 NADx10 600 0.10 106 400 105 8.5 104 200 103 0.05 8.0 102 101 eq 7.5 0 K’ 0 10 0.00 pH 10-1 20 7.0 10 9 8 -2 15 7 6 5 10 10 4 3 2 1 5 0 6.5 butanediol 10-3x NADH acetoin NADx10pH-7.5

Figure 4.3: Estimation of errors in the determination of the NADH/NAD ratio. (A) Dependence of NADH/(NAD 10pH 7.5) on butanediol/acetoin and K’ . (B) Dependence of NADH/NAD on · − eq NADH/(NAD 10pH 7.5) and pH. Simulations are shown for NADH/NAD. The respective simulations · − for NADPH/NADP,however, give the exact same result and have therefore been omitted. 62 4.R EDOX COSUBSTRATE LEVELS IN MUTANT Synechocystis

pH 7.5 At a given NAD(P)H/(NAD(P) 10 − ) value, the NAD(P)H/NAD(P) ratio can be calculated · by multiplication with 10pH-pH’. This means that errors in pH estimation will potentially have a large influence on the calculated NAD(P)H/NAD(P) ratio11. Under the assump- tion that the intracellular pH within one experiment does not change, trends in NAD(P)H/ pH 7.5 (NAD(P) 10 − ) still reflect trends in NAD(P)H/NAD(P). · Absolute NAD(P)H/NAD(P) ratios have been reported e.g. by Takahashi et al [214]. There, NADPH/NADP ratios of 1.4 and NADH/NAD of 0.25 were reported for exponentially grow- ing Synechocystis [214]. While the general trend, that the NADPH pool is more reduced than the NADH pool is observed in this chapter as well, the absolute values determined here are consistently lower than those reported in [214]. In order to calculate the rations, assump- tions had to be made that were backed by little to no experimental data. This should be kept in mind when comparing absolute values determined here to published ones. In conclusion, examining the ratios of the substrate and the product of a redox reaction is a 4 convenient method to survey if this reaction is a step that limits productivity. In Syn pVZ001 and Syn pVZ002, acetoin reductase activity is not limiting. This was already hypothesised in the previous chapter. Optimisation efforts therefore should focus on acetolactate synthase and acetolactate decarboxylase. In the mutant Synechocystis strains analysed here, the NADH pool tends to get more reduced towards stationary phase. Furthermore, the introduction of a transhydrogenase leads to a more reduced NADH/NAD pool (Fig. 4.2A). The NADPH pool generally is more reduced than the NADH pool, as shown by strain pVZ006 (Fig. 4.2B).

SUPPLEMENTARY INFORMATION

A 10 B 10

8 8 eq

eq 6 6 x K’ x

x K’ x 4 4

4 4 10

10 2 2 0 0.5 0 0.5 5 5 6 0.4 0.4 7 0.3 7 0.3 8 0.2 8 0.2 pH 9 0.1 I pH 9 0.1 I 10 0.0 10 0.0

+ Figure S 4.1: Dependency of K’eq of the reactions acetoin + H + NADH butanediol + NAD (A) + and acetoin + H + NADPH butanediol + NADP (B) on pH and ionic strength. K’eq values are displayed as white circles. The projections on the xz and the yz plane are shown as black squares. The eQuilibrator tool was used to calculate K’eq with pH and ionic strength as input.

11 Note that errors in pH measurement also influence K’eq in a non-linear fashion (Fig. S 4.1). 63

) 20 BG11 plain pVZ001 BG11 pVZ001 A pVZ002 B pVZ002 pH-7.5 50 mM NaHCO3 15

10

5 NADH/(NADx10 -3

10 0 6 7 8 9 10 11 6 7 8 9 10 11

) 20 BG11 pVZ001 BG11 pVZ001 C pVZ002 D pVZ002 pH-7.5 25 mM TES/KOH 25 mM HEPES/NaOH 15 pH 6.5 pH 7.5 4

10

5 NADH/(NADx10 -3

10 0 6 7 8 9 10 11 6 7 8 9 10 11 ) 20 E BG11 pVZ001 F BG11 pVZ001

pH-7.5 25 mM TES/KOH pVZ002 25 mM CAPSO pVZ002 15 pH 8.0 pH 9.0

10

5 NADH/(NADx10 -3

10 0 6 7 8 9 10 11 6 7 8 9 10 11 ) 20 G BG11 pH

pH-7.5 25 mM CAPS 15 pH 11.0

10

5 NADH/(NADx10

-3 pVZ001 pVZ002 10 0 6 7 8 9 10 11 pH

Figure S 4.2: Intracellular NADH/(NAD 10pH 7.5) ratios do not correlate with extracellular pH. The · − ratios are shown for plain BG11 medium (A), BG11 medium supplemented with bicarbonate (B), BG11 medium buffered to initial pH values of 6.5 (C), 7.5 (D), 8 (E), 9 (F) and 11 (G). Values for Syn pVZ001 are shown as black squares and values for Syn pVZ002 are shown as grey diamonds. Values were calculated from previously published data (See Fig. S 3.3. Error bars denote standard deviations of at least two biological replicates.

5 PHOTOSYNTHETICPRODUCTIONOF GLYCEROLBYARECOMBINANT CYANOBACTERIUM

Philipp SAVAKIS, Xiaoming TAN, Wei DU, Filipe BRANCODOS SANTOS, Xuefeng LU, Klaas J. HELLINGWERF

Cyanobacteria are prokaryotic organisms capable of oxygenic photosynthesis. Glycerol is an important commodity chemical. Introduction of phosphoglycerol phosphatase 2 from Sac- charomyces cerevisiae into the model cyanobacterium Synechocystis sp. PCC6803 resulted in a mutant strain that produced a considerable amount of glycerol from light, water and CO2. Mild salt stress (200 mM NaCl) on the cells led to an increase of the extracellular glycerol con- centration of more than 20%. Under these conditions the mutant accumulated glycerol to an extracellular concentration of 14.3 mM after 17 days of culturing.

This chapter has been published in Journal of Biotechnology 195:10, 46-51(2015) [229].

65 66 5.P HOTOSYNTHETICGLYCEROLPRODUCTION

INTRODUCTION

YANOBACTERIA are prokaryotic organisms capable of carrying out oxygenic photosyn- C thesis. They are widely distributed in nature [230] and have very simple growth require- ments [71]. Glycerol is a commodity chemical that is routinely used as a solvent, lubricant, humectant or sweetener, amongst other applications. It is a versatile building block in chemical syn- thesis [231] and can be used as a carbon source by many chemoheterotrophic organisms [232]. Furthermore, numerous processes have recently been developed to convert glycerol into higher value-added compounds [233]. Traditionally, fermentative processes would be used for glycerol production from sugars, based on the metabolic capacities of yeasts such as Saccharomyces cerevisiae [234]. In recent years, glycerol has been produced instead as a by-product of biodiesel formation. However, the production of plant-derived biodiesel competes with the use of arable land for food production, and therefore ultimately with food supply. The development of fully sustainable production processes is highly desirable, preferably those that utilize readily available carbon and energy sources, such as CO2 and sunlight, 5 and that do not require arable land. Within this context, it would be highly pertinent to engineer glycerol producing cyanobacteria that would convert CO2 and water into glycerol fueled by (sun)light.

A intracellular extracellular Dihydroxyacetone phosphate (DHAP) CO2

glpD P gpp i

Glucose-1-phosphate (G1P) Glycerol-3-phosphate Glycerol Glycerol ATP glpK ADP ATP ADP Pi ADP-glucose Glucosylglycerol-phosphate ggpS ggpP

Pi Glucosylglycerol B C 1 2 3 4 slr0168 5 kb

1.5 kb

R 0.5 kb Ptrc RBS gpp Km

Figure 5.1: (A) Simplified view of conversion of CO2 into glycerol and glucosylglycerol in Synechocys- tis. (B) Schematic representation of the genetics of the construction of the glycerol-producing mutant. (C) Colony PCR of the glycerol producing strain. 1: PSA002; 2: WT Synechocystis; 3: pPSgpp2 (positive control); 4: no template (negative control).The primers used were H1 seg F and H2 seg R. 67

Many yeasts (e.g. Saccharomyces cerevisiae) synthesise glycerol in response to adverse en- vironmental conditions, most notably osmotic stress, exploiting its natural properties as an osmoprotectant [235]. Additionally, it can also be accumulated extracellularly when di- hydroxyacetone phosphate (DHAP) functions as a physiological electron acceptor under anaerobic conditions [236]. In either case, glycerol production occurs from the conversion of DHAP in a two-step path- way. Firstly, DHAP is reduced to glycerol-3-phosphate (G3P) in an NADH-dependent re- action, catalysed by one of the two isozymes of glycerol-3-phosphate dehydrogenase (Gpd 1 and Gpd 2). The subsequent dephosphorylation of G3P, yielding glycerol, can be cat- alyzed by two alternative phosphoglycerol phosphatase isoforms (gpp1 and gpp2, encod- ing Gpp1p and Gpp2p, respectively) that are present in S. cerevisiae. Expression of gpp1 is increased under anaerobic conditions [237], while the levels of phosphoglycerol phos- phatase 2 (Gpp2p) are increased in response to osmotic stress [238]. When facing hyperosmotic conditions, the cyanobacterium Synechocystis sp. PCC6803 (Syne- chocystis) accumulates the compatible solute glucosylglycerol in the cytoplasm [88]. This osmoprotectant is derived from glycerol-3-phosphate and ADP-glucose. Glucosylglycerol phosphate synthase (GgpS) catalyzes the glycosylation of glycerol-3-phosphate to yield glu- cosylglycerol phosphate. The subsequent dephosphorylation of the latter metabolite to glu- 5 cosylglycerol is catalyzed by glucosylglycerol phosphate phosphatase (GgpP) [239] (Fig. 5.1A). Here, we apply a combination of different metabolic engineering approaches to the green biotechnology workhorse Synechocystis, aiming at the direct conversion of CO2 into glyc- erol. First, we use genetic engineering by expressing a heterologous enzyme catalyzing the dephoshorylation of glycerol-3-phosphate, which leads to an extracellular accumulation of glycerol up to 11.6 mM after 17 days. We then resort to the manipulation of environmental conditions to further increase glyc- erol production. We impose salt stress with the goal of increasing intracellular glycerol- 3-phosphate concentrations. Serendipitously, this was found to already lead to the extra- cellular accumulation of glycerol in the wild type strain of Synechocystis. We subsequently confirm that these two effects are additive and can be combined, increasing production of glycerol during a similar period by more than 20%. As Synechocystis does not require a com- plex medium for growth, the resulting glycerol is produced in a chemically simple matrix, which will facilitate subsequent downstream processing (Fig.S 5.1)[240].

RESULTS

Mutant construction. Wild type Synechocystis was transformed with plasmids pPSgpp1 and pPSgpp2, harboring the respective phosphoglycerol phosphatase under control of the strong, constitutive, Ptrc promoter and a kanamycin resistance cassette. This vector allows for chromosomal integration at the neutral slr0168 locus [241](Fig. 5.1B). Genetic analysis with PCR revealed that transformants harbouring gpp1 grew slowly, and were difficult to segregate (data not shown). In contrast, transformants harboring gpp2 segregated more readily (Fig. 5.1C). Photosynthetic glycerol production. The resulting mutant, termed Synechocystis PSA002 (see Tab. 5.1), excreted glycerol to high concentrations into the culture medium, leading to as much as 11.6 mM in a period of 17 days after inoculation (Fig. 5.2), when the biomass in the culture increases gradually to about 2 gram dry weight per liter. In addition, after 68 5.P HOTOSYNTHETICGLYCEROLPRODUCTION

A OD glycerol Syn WT + 200 mM NaCl Syn WT Syn PSA002 + 200 mM NaCl Syn PSA002 20 18 10 16 14 12

730 1 10 5 OD 8 c / mmol/L 6 0.1 4 2 0.01 0 0 2 4 6 8 10 12 14 16 t / d B 100 Syn PSA002 + 200 mM NaCl Syn WT Syn PSA002 50

% carbon into product 0

1-7 7-9 9-12 12-13 13-15 15-17 time period / d

Figure 5.2: (A) Introduction of a phosphoglycerol phosphatase from Saccharomyces cerevisiae into Synechocystis leads to high level production of glycerol from CO2. Extracellular glycerol is shown for both wild type Synechocystis and Synechocystis PSA002 in plain BG11 medium (squares, circles), and in BG11 medium supplemented with 200 mM NaCl (diamonds, triangles). Closed symbols: growth. Open symbols: extracellular glycerol concentration. (B) Percentage of the total amount of fixed car- bon that is diverted into the glycerol synthesis pathway. Error bars represent standard deviations of three biological replicates. Error bars not visible are smaller than the respective data point symbol. 69

PSA002 WT 10

1

0.1

730 0.01 730 OD 0.001

0.0001 0.4 0.2

0.0 0.00001 c(glycerol)/OD 0 5 10 15 20 25 t / d 5

Figure 5.3: Serial transfers of Synechocystis PSA002 (blue) and wild type Synechocystis sp. PCC6803 (red). (A) Growth (measured as OD730) as a function of time. Eight days after each inoculation, the cultures were diluted 10,000-fold with fresh medium. Bars show extracellular glycerol concentration divided by cell density. Error bars represent standard deviations of three biological replicates. Error bars that are not visible are smaller than the respective data point symbol.

2.5

2.0

1.5 730 1.0 OD

0.5

0.0 0 2 4 8 16 31 63 125 250 300 400 500 c(glycerol) / mM

Figure 5.4: Glycerol tolerance of wild type Synechocystis sp. PCC6803. The bars show cell densities 2 days after inoculation to an OD730 of 0.028. The cells were grown in a 96 well plate in BG11 medium containing 50 mM NaHCO3 with the amounts of glycerol indicated. Error bars represent standard deviations of seven biological replicates. 70 5.P HOTOSYNTHETICGLYCEROLPRODUCTION

prolonged serial cultivation for nearly 20 generations, the glycerol-production phenotype of Synechocystis PSA002 remained unaltered, as indicated by the constant ratio of glycerol producedover biomass formed (Fig. 5.3). Glycerol was reported to be toxic to Synechocystis already at very low concentrations [242]. Our initial result (see above) could not confirm this. Therefore, we investigated glycerol tolerance of our wild type strain (Fig. 5.4). It can clearly be seen that we could only observe an inhibitory effect at glycerol concentrations higher than 125 mM. In the study by Ortega-Ramos et al., a glucose-sensitive wild-type strain was used. Toxicity of both glucose and glycerol was alleviated through overexpres- sion of the endogenous hydrogenase of Synechocystis. Glucose- and glycerol toxicity were therefore proposed to be related. The strain used in our study is glucose-tolerant. If glucose and glycerol indeed present related stresses, then it is possible that a glucose-tolerant strain also shows increased tolerance to glycerol. Effect of salt stress. When facing osmotic stress, Synechocystis produces glucosylglycerol (Fig. 1A). We speculated that salt stress would lead to an increased intracellular glycerol-3- phosphate concentration and therefore to more substrate for the heterologously introduced phosphatase. Therefore, we cultivated the wild type organism in BG11 medium containing increasing concentrations of NaCl (0-1 M,Fig. S 5.2). We observed that 200 mM NaCl had a 5 minor, if any effect on growth and that 600 mM NaCl led to a pronounced increase in the length of the lag phase, prior to exponential growth (Fig. S 5.2). At even higher NaCl con- centrations, no growth could be observed within the time window tested (11 d). We then cultivated the wild-type strain in plain BG11 medium and in medium containing 200 and 400 mM NaCl and analyzed the composition of the extracellular medium. To our surprise, the wild type Synechocystis strain excreted glycerol in low concentrations into the growth medium (Fig. 5.5). At 200 mM NaCl, we found a positive impact on glycerol pro- duction, while the difference in growth (rate) was negligible, as compared to the unstressed condition. At 400 mM NaCl, growth rate and final yield were affected to a significant extent. We therefore tested if this observation could be used to further increase glycerol production in Synechocystis PSA002, by comparing glycerol production in cells growing in the presence and absence of 200 mM NaCl (Fig. 5.2A) We observed that glycerol production due to salt stress and glycerol production due to expression of a heterologous phosphoglycerol phos- phatase was cumulative. Ultimately, this results in the conclusion that the fraction of fixed carbon that was diverted into the glycerol-production pathway exceeded 50% in the Synechocystis PSA002 mutant

Table 5.1: Strains and primers used in this study

Strain Genotype Comment Synechocystis sp. PCC6803 Obtained from D. Bhaya, Stanford University Synechocystis Syn PSA001 ∆slr0168::Ptrc::gpp1::KmR Synechocystis Syn PSA002 ∆slr0168::Ptrc::gpp2::KmR

Oligonucleotide name Sequence 50-30 H1 seg F TGTCGCCGCTAAGTTAGA H2 seg R TGTCGCCGCTAAGTTAGA 71 under mild salt stress (Fig. 2B).

Syn WTc(glycerol)c(NaCl) 0 mM 200 mM 400 mM 10 2

1

730 1 OD 0.1 5 c / mmol/L

0.01 0 0 2 4 6 8 10 t / d

Figure 5.5: Wild type Synechocystis sp. PCC6803 produces glycerol under salt stress. Cells were culti- vated in BG11 medium, supplemented with an amount of NaCl as indicated. Closed symbols: growth; open symbols: extracellular glycerol concentration. Error bars represent standard deviations of three biological replicates. If the error bars are not visible, then they are smaller than the respective data point symbol.

DISCUSSION

The proof-of-principle of CO2-, water-, and sunlight-based production of glycerol presented in this study is the first step toward truly sustainable glycerol production directly from CO2. This glycerol production was achieved through heterologous expression in Synechocystis of a single enzyme, namely Gpp2p from S. cerevisiae. Glycerol produced via this route is largely free of impurities, which facilitates downstream processing. It is yet unclear how the glycerol produced leaves the cyanobacterial cell. In S. cerevisiae, FpsI, a member of the MIP (major intrinsic protein) family is required to facilitate glycerol transport out of the cytosol [243]. Synechocystis harbours aquaporins and MIP family pro- teins but as of now, none have been characterised with respect to their ability to transport glycerol. In S. cerevisiae, overexpression of fpsI was shown to increase glycerol production. In Synechocystis, potential yield limitations due to possible feed-back inhibition could be alleviated through overproduction of FpsI or a related transport protein. Gpp1p and Gpp2p from S. cerevisiae exhibit more than 90% sequence identity at the pro- tein level. In vitro, they display a very similar maximal activity ((Vmax), although Gpp1p has 72 5.P HOTOSYNTHETICGLYCEROLPRODUCTION

a slightly higher substrate affinity under the conditions assayed (KM of 3.1 and 3.9 mM, re- spectively) [238]. Expression of the heterologous Gpp1p did not result in a fully segregated mutant, while expression of Gpp2p did. Investigating the details of why gpp1 expression was unsuccessful is beyond the scope of this proof of concept study. More importantly, the phenotypic stability of the latter genetic construct developed in this study has been confirmed. Although often disregarded [158], this aspect is crucial to ensure the success of any eventual application at an industrial scale and ought to be considered already at this proof of concept stage [168]. To this end, separation of the growth and production phase through the use of regulatory genetic elements would be a potential step toward more stable strains. Further increases in productivity could be achieved through the use of different phosphatases in combination with manipulations of host metabolism, i.e. deletions of glpK and/or ggpS. In this study, going beyond classical genetic engineering has proven crucial to make Syne- chocystis PSA002 behave as a cell factory, i.e. having 50% or more of the fixed carbon re- routed to product [115]. In this case, a simple manipulation of growth conditions helped to further divert carbon flux toward product formation. Supplementing the culture medium with salt, in addition to increasing glycerol production by more than 20%, has the advantage 5 of inhibiting the growth of many contaminants of cyanobacterial cultures that otherwise could consume the product of interest [105]. Again, this knowledge may prove beneficial in the light of a scale-up process. The source of glycerol produced under salt stress is at present unknown. Further studies will be necessary to elucidate the biochemical route to glycerol under salt stress in the wild type organism. The measured carbon partitioning coefficient, which is comparable with those achieved for several other compounds that are produced at high levels such as sucrose or ethanol [105, 107] and appreciably higher than the partitioning coefficient achieved for many other compounds, shows promise for the next phase of optimization of glycerol synthesis from light, water and carbon dioxide.

MATERIALS AND METHODS

Reagents. All chemicals were purchased from Sigma-Aldrich at the highest purity available. Growth conditions. Synechocystis sp. PCC6803 (obtained from D. Bhaya, Stanford Univer- sity) was grown using a shaking incubator at 30◦C and at 120 rpm (Innova 43, New Brunswick Scientific) under continuous irradiation (15 W cool fluorescent white light, F15T8-PL/AQ, General electric) at 30-40 µE/m2/s. Precultures were inoculated from plate and grown to stationary phase. These cultures were then diluted to a starting (OD730 of 0.1. All production experiments were carried out using BG11 medium supplemented with 10 mM TES buffered at a pH of 8.0. Kanamycin-resistant mutant Synechocystis strains were grown in BG11 me- dium supplemented with 10 mM TES, pH 8.0 and 50–100 µg/mL kanamycin. In liquid cul- tures, cell growth was monitored by following the optical density at 730 nm. For glycerol tolerance measurements, cells were grown in transparent 96-well plates at 700 rpm in un- buffered BG11 medium containing 50 mM NaHCO3 and the indicated amounts of glycerol. Plates contained BG11, 0.3% Na2S2O3 (w/v), 1.5% agar (w/v), and were buffered with 10 mM TES at pH 8.0. Kanamycin was used at 50 µg/mL where appropriate. Plasmids. Plasmids pPSgpp1 and pPSgpp2 were ordered at Genscript. The respective glycerol- 73

1-phosphatase gene was codon optimized for use in Synechocystis and placed under the control of the Ptrc promoter. Downstream of the phosphatase gene there was a aphII cas- sette, allowing for selection on kanamycin. The entire construct was flanked by regions homologous to the slr0168 locus of theSynechocystis chromosome. Mutant construction. Synechocystis cultures were grown to an (OD730 of 0.2–0.4 and con- centrated 20–50 fold. Then, 3 µg plasmid DNA were added, and the cells were incubated in Eppendorf tubes for 5 h under white light. Cells were then transferred onto a membrane, which was placed on BG11 plates without selective pressure. Following a 20 h incubation in a lit incubator, the membrane was transferred onto BG11 plates containing 50 µg/mL kanamycin. Single colonies appeared after 6–12 d. Segregation status of the mutants was monitored using primers H1 seg F and H2 seg R (Tab. 5.1). Glycerol detection and quantification by high performance liquid chromatography (HPLC). 1 mL aliquots culture were harvested by centrifugation (14,500 rpm, 5 min, 21◦C). The su- pernatant was filtered (Sartorius Stedin Biotech, minisart SRP 4, 0.45 µm) and analyzed by HPLC (Rezex ROA-Organic Acid H+ (8%) column (Phenomenex), column temperature: 85◦C) equipped with a refractive index detector (Jasco, RI-1530); operating with 7.2 mM H2SO4 as eluent with a flow of 0.5 mL/min. Peaks were identified and quantified using ex- ternal standards. 5 Carbon partitioning calculations. Calculations were essentially carried out as in [115, 135], assuming a cellular composition of of C4H7O2N. Carbon partitioning was calculated for ev- ery biological replicate individually and then averages and standard deviations were calcu- lated.

ACKNOWLEDGEMENTS This article was written within the research program of BioSolarCells, co-financed by the Dutch Ministry of Economic Affairs. WD is supported by the China Scholarship Council. FBdS is supported by the Netherlands Organization for Scientific Research (NWO) through VENI grant 863.11.019. 74 5.P HOTOSYNTHETICGLYCEROLPRODUCTION

SUPPLEMENTARY INFORMATION

c(NaCl)/mM 10 0 200 400 600

730 1 800 1000 OD

5 0.1

0 2 4 6 8 10 12 t / d

Figure S 5.1: Growth of Synechocystis in BG11 with different salt concentrations. Data points repre- sent single observations.

TES

glycerol

0 5 10 15 20 25 30

t / min

Figure S 5.2: Glycerol excreted by Synechocystis PSA002. 6 IDENTIFICATION OF A NOVEL PROTEINFROM Synechocystis SP. PCC 6803 INVOLVED IN RE-ASSIMILATIONOFTHEOSMOLYTE GLUCOSYLGLYCEROL

Philipp SAVAKIS, Xiaoming TAN, Kuo SONG, Xuefeng LU, Klaas J. HELLINGWERF, Filipe BRANCODOS SANTOS

When subjected to mild salt stress, the cyanobacterium Synechocystis sp. PCC 6803 produces small amounts of glycerol through an as yet unidentified pathway. Here, we show that this glycerol is a degradation product of the main osmolyte of this organism, glucosylglycerol (GG). Inactivation of ggpS, encoding the enzyme that catalyses the first step of GG-synthesis abolished de novo synthesis of glycerol, while the ability to hydrolyze exogenously supplied glucoslylglycerol was maintained. Inactivation of glpK, encoding glycerol kinase, had no ef- fect on glycerol synthesis. Inactivation of slr1670, encoding a GHL5-type putative glycoside hydrolase, abolished de novo synthesis of glycerol, as well as hydrolysis of GG, and led to in- creased intracellular concentrations of this osmolyte. A protein homologous to the one en- coded by slr1670 occurs in a wide range of cyanobacteria, proteobacteria and archaea. In cyanobacteria, it often occurs with GG-synthesis.

This chapter has been published in Frontiers in Microbiology 7:1350, (2016) in modified form under the title Slr1670 from Synechocystis sp. PCC 6803 Is Required for the Re-assimilation of the Osmolyte Glucosylglycerol.

75 76 6.S LR1670 IS INVOLVED IN GLUCOSYLGLYCEROL RE-ASSIMILATION

INTRODUCTION PON increases in extracellular osmolarity, many bacteria synthesise small organic mol- U ecules that raise the intracellular osmotic pressure. In cyanobacteria, the nature of the osmolyte correlates with the hosts’ osmotolerance: strains with a low salt tolerance produce sucrose, moderately halotolerant strains utilise glucosylglycerol, and highly tolerant strains use glycine betaine [79]. The moderately halotolerant cyanobacterium Synechocystis sp. PCC 6803 (hereafter: Syne- chocystis) uses glucosylglycerol as its primary osmolyte [88], but is also capable of salt- induced sucrose synthesis. Glucosylglycerol is synthetised via a two-step pathway from central metabolites (Fig. 6.1): In the first step, glucosylglycerol phosphate is synthesised from ADP-glucose and glycerol-3-phosphate, in a condensation reaction catalysed by glu- cosylglycerol phosphate synthase (GgpS). Cleavage of the phosphate moiety is subsequent- ly accomplished by glucosylglycerol phosphate phosphatase (GgpP). Synechocystis harbours a transporter that is used for reuptake of GG that is lost due to leak- age [244]. A third osmolyte used by bacteria such as E. coli is the disaccharide trehalose [245]. Al- though incapable of synthesis, Synechocystis can take up exogenously supplied trehalose and use it as an osmoprotectant [89]. In cells to which trehalose has been added, the to- tal concentration of glucosylglycerol decreases over time, which suggests that this molecule can be catabolised [89]. 6 Some marine cyanobacteria have been demonstrated to be able to ferment a part of their osmolytes. Thus, Microcoleus chthonoplastes ferments some of its glucosylglycerol in the dark, re-assimilating the glucose part, while the glycerol part is excreted [246]. Yet, a metab- olic pathway for glucosylglycerol re-assimilation has so far remained elusive [90], although recently, a dedicated GG phosphorylase was discovered in Bacillus selenitireducens [247]. We reported elsewhere that under mild salt stress, wild-type Synechocystis cells synthesize small quantities of glycerol [229]. Here, we provide evidence supporting the notion that glucosylglycerol is the precursor of this glycerol under salt stress. Further, we demonstrate that a previously unassigned protein, Slr1670, is directly involved (and required for) GG re- assimilation. 77

A CO2 Dihydroxyacetone phosphate B sll1085 sll1566 ssl3076 slr1670 slr1672 NAD(P)H GlpD (sll1085) glpD ggpS ggpR glpK NAD(P)

Synechocystis sp. PCC 6803 ADP-Glucose Glycerol-3-phosphate

Synechocystis sp. PCC 6803 ∆slr1670 KmR GgpS (sll1566) Synechocystis sp. PCC 6803 ∆glpK CmR Glucosylglycerol phosphate ADP Synechocystis sp. PCC 6803 ∆ggpS KmR

GgpP (slr0746) GlpK (slr1672) Synechocystis sp. PCC 6803 ∆slr1670∆glpK KmR CmR

Glucosylglycerol ATP

ORF

disrupted ORF KmR glycerol

Figure 6.1: A) Proposed overview of Glycerol and glucosylglycerol metabolism. B) Genes involved in GG synthesis and mutant strains used in this study. Arrows represent the direction of transcription of the respective open reading frame.

MATERIALS AND METHODS

Chemicals were purchased from Sigma-Aldrich, unless stated otherwise. Glucosylglycerol (51% aqueous solution) was purchased from Bitop (Germany). Culturing conditions. Escherichia coli was grown in LB medium at 37◦C with shaking at 200 6 rpm. Selection of transformants was carried out at 37◦C on LB medium solidified with 1.5% (w/v) agar. Where appropriate, ampicillin, kanamycin and chloramphenicol were added at 100 µg/mL, 50 µg/mL, and 35 µg/mL, respectively. For batch experiments, Synechocystis cells were grown in a shaking incubator (Innova 43, New Brunswick Scientific, 120 rpm, 30◦C) under fluorescent white light (15 W cool fluores- cent white light, F15T8-PL/AQ, General electric, incident light intensity 30-40 µE/m²/s) in BG11 medium, supplemented with 10 mM TES/KOH and adjusted to an initial pH of 8.0. Cells were inoculated to an OD730 of 0.1 from a pre-culture. Where indicated, NaCl was added to a concentration of 200 mM to non-adapted cells. For salt tolerance experiments, cells were grown in transparent 96-well plates (Greiner bio- one cellstar, F-bottom with breathe easy seals (Diversified Biotech)) under white light (GE PL/AQ F15T8) at 30◦C and shaking at 700 rpm in BG11 medium buffered to an initial pH = 8.0 with 10 mM TES/KOH and supplemented with NaHCO3 to a concentration of 50 mM. Synechocystis mutants were selected under white light (10 µE/m²/s) at 30◦C on BG11 me- dium solidified with 1.5% (w/v) agar and supplemented with 10 mM TES/KOH pH = 8.0, plus 0.3% (w/v) Na2S2O3 and selection markers where appropriate. Extraction of genomic DNA. Cells were grown until an OD730 of around 1 and 1 mL was harvested by centrifugation (12,000 rpm, 1 min, RT). The supernatant was discarded and the cells were resuspended in 200 µL TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). Next, 200 mg glass beads (0.1 mm diameter) were added and the sample was vortexed for 5 min. The sample was cleared by centrifugation (5 min, 12,000 rpm, RT), and the super- natant was transferred to a fresh tube. Then, phenol/chloroform/isoamylalcohol (25/24/1, v/v/v) (200 µL) was added, the sample was mixed and centrifuged (5 min, 12,000 rpm, RT). The aqueous phase was transferred to a fresh tube and washed with 200 µL of chlorofor- m/isoamylalcohol (24/1, v/v). After centrifugation (5 min, 12,000 rpm, RT), 40 µL 5 M NaCl 78 6.S LR1670 IS INVOLVED IN GLUCOSYLGLYCEROL RE-ASSIMILATION

and 400 µL ethanol were added. After thorough mixing, the sample was placed at -20◦C for 30 min. Then, the DNA was pelleted (10 min, 12,000 rpm, RT) and washed with 500 µL 70% pre-cooled ethanol. After centrifugation (5 min, 12,000 rpm, RT), the supernatant was re- moved and the pellet was dried on a bench-top incubator at 30◦C for 15 min. The pellet was dissolved in 30 µL nuclease-free water and the solution used immediately, or kept at -20◦C. Plasmid construction. For the construction of pMD18-T1670, slr1670 was amplified from genomic DNA of Synechocystis with primers Slr1670-BamHI and Slr1670-XhoI and intro- duced into pMD18-T. For the construction of pMD18-T1670Km, pMD18T1670 was digested with NcoI, blunted with T4 DNA polymerase and ligated with the Km resistance cassette (obtained from pRL446 [248] by digestion with PvuII). The glpK gene was amplified from the genomic DNA of Synechocystis by PCR with primers glpK-Fwd and glpK-Rev, and cloned into the pMD 18-T vector (Takara, Japan). The resultant plasmid was digested by NheI, blunted by T4 DNA Polymerase (Fermentas), and ligated with the blunted chloramphenicol resistance gene cassette, resulting in the plasmid pXT323. Construction of mutant Synechocystis strains. Mutant strains of Synechocystis were con- structed essentially as described previously ([135, 199]). Briefly, 10 mL cells were grown to an OD730 of 0.2 – 0.6, centrifuged and concentrated twenty- to fiftyfold. Then, plasmid DNA (1-3 µg) was added and the cells were incubated in tubes at 30◦C for 5 h. Cells were grown for 16 h on a membrane filter, on plates without selective pressure. Next, cells were transferred onto plates containing the respective selection marker. Colonies appeared after 1-2 weeks. Identity of transformants was confirmed by colony PCR, using appropriate primers. Segre- 6 gation of mutant strains was achieved in liquid culture. For construction of the slr1670 in- sertion strain, WT Synechcocystis was transformed with pMD18-T1670Km. For construction of the double insertion mutant ∆slr1670∆slr1672, Synechocystis ∆slr1672 was transformed with pMD18-T1670Km segregation was followed by PCR (NheI-slr1670 fw,BamHI-slr1670 rv). Analysis of intra- and extracellular metabolites using HPLC. For analysis of extracellular metabolites, samples were cleared by centrifugation (14,500 rpm, 10 min, 21◦C) and re- maining particles were removed with a syringe, fitted with a filter (Sartorius Stedin Biotech, minisart SRP 4, 0.45 µm pore size). Samples were then analyzed by HPLC (column: Rezex + ROA-Organic Acid H (8%) (Phenomenex); column temperature: 85◦C); detector (Jasco, RI- 1530); eluent: 7.2mM H2SO4; flow: 0.5 mL/min). Identification and quantification was done using external standards (detection limit 0.05 mmol/L). ∼ For extraction of GG from cyanobacteria, 1 mL of a culture was harvested by centrifugation (15,000 rpm, 10 min, 4◦C). Pellets were resuspended in 1 mL 80% (v/v) ethanol and incu- bated at 65◦C for 4.5 h. After re-centrifugation (15,000 rpm, 10 min, 21◦C), the supernatant was transferred to a fresh tube and the liquid was evaporated using a stream of nitrogen. The residue was dissolved in 1 mL of water, filtered and analyzed by HPLC as described above. Preparation of cell-free extract. Pre-cultures were grown without additional NaCl. 200 mL of Synechocystis WT and 200mL of Synechocystis ∆slr1670 were grown to OD730 of 1.95 and 1.97, respectively in BG11 medium supplemented with 200 mM NaCl. Cells were harvested by centrifugation at 3,900 rpm for 15 min. Cell pellets were resuspended in 50 mL lysis buffer (25 mM Tris pH 7.5, 100 mM NaCl, 0.5 mM EDTA, 5 mM MgCl2) and centrifuged at 3,900 rpm and 4◦C for 15 min. This was repeated once. Then, pellets were resuspended in 5 mL of lysis buffer, 1 mL aliquots were prepared, flash-frozen in liquid nitrogen or used 79

Table 6.1: Reactions added to the genome-scale model of Synechocystis

Gene Reaction name Reaction IDi Equationi association GG transport via diffusion R_glcglyctpp M_glcglyc_c - (cytosol to periplasm) ↓ M_glcglyc_p Glucosylglycerol R_GLCGLYCHyd M_h2o_c + M_glcglyc_c slr1670 Hydrolase Glucosylglycerol R_GLCGLYCPhosphorylase M_pi_c + M_glcglyc_c slr1670 Phosphorylase ↓ M_g1p_c + M_glyc_c Glucosylglycerol storage R_GLCGLYCstorage M_glcglyc_c - (cytosol to sink) i Abbreviations: M_glcglyc, glycosylglycerol; M_h2o_c, water; M_glc_DASH_D, D-glucose; M_glyc, glycerol; M_pi, inorganic phosphate; M_g1p, glucose-1-phosphate.

immediately. An aliquot was lysed (500 mg acid-washed glass beads (0.1 mm ) in a 2 mL ® screwcap tube, 3 20 s at 6,000 rpm, samples were cooled on ice for 2 min between lysis × steps). Then, samples were cleared by centrifugation (30 min, 15,000 rpm, 4◦C) and kept on 6 ice. Enzymatic assays. Glucosylglycerol hydrolase activity was assayed in lysis buffer with 5 mM glucosylglycerol as substrate. The mixture was heated up to 30◦C, after which the reaction was started by addition of cell-free extract (50 µL in a total volume of 500 µL). 200µL Sam- ples were taken ca. 30 s and 48 h after the start of the reaction. Samples were quenched immediately by incubation at 95◦C for 10 min. Samples were centrifuged at 15,000 rpm for 10 min, filtered (Sartorius Stedin Biotech, minisart SRP 4, 0.45 µm pore size) and analysed by HPLC. Genome scale model. We studied the newly identified GG degradation pathway in the con- text of a genome-scale metabolic network model previously reported for Synechocystis [36]. Although glucosylglycerol was already present in this earlier reconstruction along with the reactions needed for its synthesis and exchange, key missing steps made it impossible for the latter to carry any flux in silico. In order to fix this, and additionally include the re- utilization of glycosylglycerol, reactions for transport, metabolism and storage were added to the model (Tab. 6.1). Flux balance analysis of this new version of the Synechocystis stoichiometric model were carried out in the on-line modeling platform FAME [250] using newly developed visualization tools specific for this organism [171]. Constraining the rele- vant reactions simulated the different derivative strains constructed in this study. The stoi- chiometric impact on the genome-scale metabolic network of the different glycosylglycerol breakdown pathways here postulated was assessed by including the two alternative routes in the model. Comparisons between model versions mimicking different strains and con- ditions were established using biomass maximization as the objective function, while con- straining the exchange flux of glycosylglycerol according to experimental measurements, as detailed elsewhere [251]. Increased fitness was calculated as the maximum of the ob- jective function (BOFmax ) for the unconstrained utilisation reaction (i.e. GG phosphorol- 80 6.S LR1670 IS INVOLVED IN GLUCOSYLGLYCEROL RE-ASSIMILATION

Table 6.2: Strains used in this study

Strain Genotype Source Synechocystis sp. PCC 6803 X. Xu, Institute of Hydrobiology, Chinese Academy of Sciences Synechocystis ∆ggpS ∆ggpS::KmR [249] Synechocystis ∆glpK ∆glpK::CmR This study Synechocystis ∆slr1670 ∆slr1670::KmR This study Synechocystis ∆glpK∆slr1670 ∆glpK::CmR∆slr1670::KmR This study

Table 6.3: Plasmids used in this study

6 Plasmid Description Source pRL446 [248] pMD18T TA cloning vector TaKaRa clonetech pMD18Tslr1670 This study pMD18Tslr1670KmR construction ∆slr1670 This study pXT323 construction ∆glpK This study

Table 6.4: Primers used in this study

Primer Sequence Slr1670-BamHI AGGATCCATGAAAACATTGAATCGTATCCATCTG Slr1670-XhoI TCTCGAGGCATTCCTTCTTCGAGCGA Slr1670-NheI fw GATAACGCTAGCATGAAAACATTGAATC Slr1670-BamHI rv ATGGATCCCTAGCATTCCTTCTTCGAGC glpK-Fwd CGCCATATGACAGCAAAACATAATCAG glpK-Rev TCTCGAGGATGGAAGCAATGTCAC 81 ysis, hydrolysis and/or glycerol phosphorylation constrained between 0 and ) divided by ∞ BOFmax for the respective flux constrained to zero. Phylogenetic analyses. PSI-BLAST (24.09.2015) of the Slr1670 sequence against non-redun- dant protein sequences was carried out using the following parameters: expect threshold: 10, word size: 3, matrix: BLOSUM62, Gap existence cost: 11, Gap extension cost: 1, Con- ditional compositional score matrix adjustment. For the second iteration, sequences with a coverage >90% (91/96 hits) were selected. The resulting hits (71) had sequence cover- age <40% and were left out of the subsequent alignments. Alignments were constructed using MEGA6 (ClustalW algorithm; pairwise alignment: Gap opening penalty: 6, gap exten- sion penalty: 0.1; Multiple alignment: Gap opening penalty: 6, gap extension penalty: 0.2; Protein weight matrix: BLOSUM; Residue-specific penalties: on; Hydrophilic penalties: on; Gap separation distance: 4; end gap separation: off; use negative matrix: off; delay divergent cutoff: 30%). The best model for the phylogenetic analysis was found using the following parameters: Tree to use: neighbour-joining tree; statistical method: Maximum likelihood; Gaps/missing data treatment: partial deletion; site coverage cut-off: 95%; branch swap fil- ter: very strong. The best model for the cyanobacterial subset (Fig. 6.5) was LG+G [252]. The best model for the entire set of proteins was LG+G+F [252]. The outgroup (hexokinase 1 of Saccharomyces cerevisiae) was used to root the trees and was not included in the figures.

RESULTS

When grown in presence of 200 mM NaCl, wild type Synechocystis accumulates small amounts 6 of glycerol in the extracellular medium ([229] and Fig. 6.2A) The transcript of glpK, encoding glycerol kinase was reported to be upregulated under salt stress conditions [253]. We expected that in the absence of other assimilation reactions, a strain deficient in glpK would produce an increased amount of glycerol when facing salt stress. Interestingly, glycerol production remained unaltered in a strain, in which glpK was disrupted with a chloramphenicol resistance cassette (Fig. 6.2A,C and Fig. 6.3A,C). This finding suggests that alternative enzymes for glycerol assimilation exist in this cyanobacte- rium. Alternatively, GlpK might not be involved in the assimilation of glycerol under these conditions. To test whether the appearance of glycerol in the extracellular medium under is dependent on the presence of intracellular glucosylglycerol, we analysed a mutant strain, in which ggpS is disrupted via insertion of a kanamycin resistance cassette [249]. This strain was sensitive to moderately high salt concentrations (400 mM NaCl, Fig. S6.1), as observed previously [254]. Growth at lower concentrations (i.e. 200 mM), however, was unaffected (Fig. S6.1F). In the supernatant of this strain, no glycerol could be detected upon the addition of salt (Fig. 6.2B). This indicated that GG is a precursor to glycerol under these conditions. Within the genomic context of ggpS and glpK, there was also a gene, whose transcript was shown to be upregulated under salt stress conditions [253, 255, 256]. The open reading frame upstream of glpK, slr1670, has a translated length of 885 amino acids. The Slr1670 protein belongs to the GHL5 family of hypothetical glucoside hydrolases [257]. In order to investigate the role of Slr1670, we constructed a mutant strain, in which its ORF was disrupted by a kanamycin resistance cassette (Fig. 6.1B). Growth of the Slr1670 disruption strain was unaffected by salt (Fig. S6.1A,D,G,J). No glycerol was detectable in the extracellu- lar medium when this strain was grown in BG11 medium supplemented with 200 mM NaCl (Fig. 6.2D). 82 6.S LR1670 IS INVOLVED IN GLUCOSYLGLYCEROL RE-ASSIMILATION

A 10.0 2.5 B 10.0 2.5 WT ΔggpS glycerol 2.0 glycerol 2.0 1.0 1.0

730 1.5

730 1.5 OD 1.0 OD 1.0 0.1 0.1 c / mmol/L c / mmol/L 0.5 0.5

0.01 0.0 0.01 0.0

C 10.0 2.5 D 10.0 2.5 ΔglpK Δslr1670 glycerol 2.0 glycerol 2.0 1.0 1.0

730 1.5

730 1.5 OD 1.0 OD 1.0 0.1 0.1 c / mmol/L c / mmol/L 0.5 0.5

0.01 0.0 0.01 0.0 0 2 4 6 8 10 0 2 4 6 8 10 6 t / d t / d

Figure 6.2: Inactivation of slr1670 or sll1566 (ggpS) abolishes glycerol production under mild salt stress. Squares represent wild type (A), diamonds the ggpS inactivation mutant (B), circles the glpK inactivation mutant (C) and triangles the slr1670 inactivation mutant (D). Filled symbols represent optical density values and correspond to the left axes; empty symbols represent extracellular glycerol concentrations and correspond to the right axes. Cells were grown in BG11 medium buffered to an initial pH of 8.0 with 10 mM TES/KOH and supplemented with 200 mM NaCl. Error bars represent standard deviation of three biological replicates. Error bars that are not visible are smaller than the respective data point symbol. 83

OD730 no GG + GG OD730 no GG + GG glycerol no GG + GG glycerol no GG + GG A 10.0 5.0 B 10.0 5.0 WT ΔggpS

1.0 1.0 730 730 2.5 2.5 OD OD 0.1 0.1 c / mmol/L c / mmol/L

0.01 0.0 0.01 0.0

OD730 no GG + GG OD730 no GG + GG glycerol no GG + GG glycerol no GG + GG C 10.0 5.0 D 10.0 5.0 ΔglpK Δslr1670

1.0 1.0 730 730 2.5 2.5 OD OD 0.1 0.1 c / mmol/L c / mmol/L

0.01 0.0 0.01 0.0 0 2 4 6 8 10 0 2 4 6 8 10 6 t / d t / d

Figure 6.3: Glucosylglycerol degradation is abolished in the slr1670 inactivation mutant. The ggpS dis- ruption strain cannot synthesise glucosylglycerol, but does produce glycerol in the presence of extra- cellular GG. Cells were grown in BG11 medium buffered to an initial pH of 8.0 with 10 mM TES/KOH, and supplemented with 200 mM NaCl in the presence and absence of 10 mM glucosylglycerol.

no GG + GG OD no GG + GG OD730 730 glycerol no GG + GG glycerol no GG + GG A 10.0 5.0 B 10.0 5.0 WT ΔglpKΔslr1670

1.0 1.0 730 730 2.5 2.5 OD OD 0.1 0.1 c / mmol/L c / mmol/L

0.01 0.0 0.01 0.0 0 2 4 6 8 10 0 2 4 6 8 10 t / d t / d

Figure 6.4: Glycerol production with and without GG addition in the Synechocystis ∆slr1670, ∆slr1672 mutant. Squares denote wild-type, hexagons the double insertion mutant. Filled, black symbols de- note gowth in BG11 medium supplemented with 10 mM GG and 200 mM NaCl, filled, grey symbols denote growth in BG11 medium supplementd with 200 mM NaCl. Open symbols denote GG concen- trations, half-filled symbols glycerol concentrations under the respective condition. Error bars denote standard deviations of at least two biological replicaes. 84 6.S LR1670 IS INVOLVED IN GLUCOSYLGLYCEROL RE-ASSIMILATION

GgpS GgpP GlpK Aphanocapsa montana 97 Nodosilinea nodulosa Microcoleus chthonoplastes 97 Geitlerinema sp. PCC7105 * Cyanothece sp. ATCC51142 91 Lyngbya confervoides 100 Nodosilinea nodulosa Geitlerinema sp. PCC7407 Rubidibacter lacunae 97 Dactylococcopsis salina 99 Halothece sp. PCC7418 Microcoleus chthonoplastes filamentous cyanobacterium ESFC-1 97 Spirulina subsalsa Geitlerinema sp. PCC7105 * Arthrospira platensis 100 Arthrospira sp. PCC8005 Cyanobacterium aponinum 100 Cyanobacterium stanieri PCC7202 Stanieria cyanosphaera 98 Myxosarcina sp. GI1 99 6 Pleurocapsa sp. PCC7319 Pseudanabaena sp. PCC7367 * Pseudanabaena sp. PCC7367 * Synechocystis sp. PCC6714 93 Synechocystis sp. PCC6803 Synechococcus sp. PCC7002 * Leptolyngbya sp. PCC7376 97 Synechococcus sp. PCC7002 * Acaryochloris marina 99 Acaryochloris sp. CCMEE 5410

% identity distance bootstrap value * : species with multiple homologues 0.2 no hit10% 20% 30% 40% 50%60%70% 80% 90%100% % coverage

Figure 6.5: Slr1670 homologues are present in a variety of cyanobacterial-, bacterial- and archaeal species. A phylogenetic tree is shown for cyanobacterial species for which a complete genome se- quence was available (See supplement for the full tree). Yellow-green boxes visualize presence of proteins homologous to GgpS, GgpP and GlpK in the respective genome. Grey boxes indicate that no significant hit was obtained. Yellow triangles mean low coverage/similarity while green triangles indicate high coverage/similarity. 85

150 *

* 100

50 c(GG) / mmol/L

0

WT slr1670 Δ

Figure 6.6: Inactivation of slr1670 leads to increased accumulation of intracellular glucosylglycerol. Cells were grown in BG11, supplemented with 200 mM NaCl and 10 mM TES buffer, at an initial pH of 8.0 to OD730 values of 6-7. For each strain, three biological replicates were analyzed. For the ex- 6 traction, three technical replicates were taken from every biological replicate. Intracellular concen- trations were calculated assuming a culture with an OD730 of 1 contains 0.2 gram dry weight per liter and assuming that 1 mg dry weight corresponds to 1 µL intracellular volume. Error bars show stan- dard deviations calculated over all data points for a given strain (9). The asterisk denotes statistical significance (P<0.001). 86 6.S LR1670 IS INVOLVED IN GLUCOSYLGLYCEROL RE-ASSIMILATION

10 9 8 0 48 h 0 48 h 7 6 5 4

c(GG) / mmol/L 3 2 1 0

WT slr1670 Δ

Figure 6.7: slr1670 is involved in hydrolysis of glucosylglycerol. The concentration of glucosylglyc- erol drops when incubated with cell-free extracts of wild-type Synechocystis, while levels remained 6 comparable after incubation with cell-free extracts of the slr1670 inactivation mutants.

R_EX_GLCGLYC_LPAREN_e_RPAREN Sink GGe R_GLCGLYCtex

GG R_GLCGLYCtpp p R_GLCGLYCABCpp_syn

ATP + H O ADP-Glucosec c 2 c R_GGPS ADP + HPO 2- GG-Phosphate GG R_GL c 4 c c R_GGPP c C GLYCst ylase R_GL orage Glycerol-3-phosphatec CGL Sink HPO 2- H O Y hosphor 4 c 2 c CH CP yd Y GL C Glucosec + Glycerolc

R_GL

Cytosol Glucose-1-Phosphatec + Glycerolc Periplasm Extracallular

unidirectional reaction bidirectional reaction Metabolitecompartment R_eaction name

Figure 6.8: Schematic representation of the modifications of the genome-scale model of Synechocystis sp. PCC 6803. The reactions R_EX_GLCGLYC_LPAREN_e_RPAREN, R_GLCGLYCtex, R_GLCGLYCtpp, R_GLCGLYCABCpp_syn, R_GLCGLYCstorage, R_GLCGLYCHyd and R_GLCGLYCphosophorylase were added (see listing B.1 for details.) 87

We then added glucosylglycerol and analysed the extracellular glycerol concentration (Fig. 6.3). In the wild type strain, the concentration of extracellular glycerol was increased as compared to the control condition (no additional GG, Fig. 6.3A). The ggpS disruption strain accumulated extracellular glycerol only when GG was added (Fig. 6.3B). The amount of glyc- erol was lower than in the wild type strain. When GG was supplied to the ∆glpK strain, the extracellular accumulation of glycerol was increased and comparable to wild type (Fig. 6.3C). In all strains tested, the addition of extracellular glucosylglycerol led to an increase in growth rate in the exponential phase (Fig. 6.3A-D). When glucosylglycerol was added exogenously to the slr1670 disruption mutant, no glycerol formation could be observed (Fig. 6.3D). These findings are indicative of Slr1670 playing a role in glycerol production from glucosylglycerol. To make sure that the absence of glycerol formation could not be attributed to polar effects (e.g. (over-) expression of glpK from the promoter of the kanamycin resistance cassette), a double insertion mutant deficient in both slr1670 and glpK was constructed. Under salt stress, this strain failed to accumulate glycerol in the extracellular medium (Fig. 6.4B). Inter- estingly, at the last sampling point, despite very similar optical densities, this strain was con- siderably more yellow (i.e. showed considerably more chlorosis) than the wild-type strain, used as a control. The slr1670 deletion strain showed increased intracellular concentrations of Glucosylglyc- erol (Fig. 6.6), corroborating the hypothesis that Slr1670 is involved in GG degradation. Taken together, these findings suggest that Slr1670 is involved in the degradation of gluco- sylglycerol to glycerol, presumably through the hydrolysis of the hemiacetal bond. We used genome-scale modeling, to analyse whether a possible growth advantage could be 6 conferred by the ability to degrade GG. We started with the model published by Nogales et al. [36] and extended it with previously published and the new information acquired here. Since inactivation of the ABC transporter involved in GG uptake leads to extracellular ac- cumulation of GG [244, 258], a leakage reaction from the cytoplasm to the periplasm was introduced. In this state, the model would not synthesise GG when maximizing growth. Therefore, a sink reaction for cytoplasmic GG was introduced. Since it is at this point un- known, whether cleavage of GG occurs via hydrolysis or phosphorolysis (as described by [247] for the B. selenitireducens enzyme), both reactions were included. Phosphorolysis of GG similar to the enzyme of B. selenitireducens yields phosphorylated glucose [247]. We therefore expected a clear preference for this reaction over the hydrolysis reaction. Accordingly, we simulated growth for phosphorolytic cleavage for a range of GG uptake fluxes qGG and divided the obtained rates by those obtained for hydrolytic cleavage (Fig. S6.2A). At low uptake rates, no difference in growth rate could be obtained for either phosphorolysis or hydrolysis. Only at very high uptake rates was the phosphorolysis reac- tion beneficial, but even there, the increase was minor (<2%). Interestingly, the genome- scale model makes similar predictions for the benefit of glycerol utilisation: for low values of qGG , no benefit for the assimilation of glycerol is predicted (Fig. S6.2B). Even at very high values, the increase in ratio is minor (<2%). To estimate a physiological range for qGG , we used the glycerol production data from the strains supplemented with extracellular GG (Fig. 6.3). The ∆ggpS mutant is unable to syn- thesise GG, and extracellular glycerol consequently must stem from the degradation of ex- ogenous GG, exclusively (Fig. 6.3B). We therefore used the glycerol production values from this strain to constrain qGG in the genome-scale model. Next, we simulated photo(hetero)- trophic growth (µ) at 30 µE/(m2 s) with the resulting q values for wild type, ∆ggpS, ∆glpK · GG 88 6.S LR1670 IS INVOLVED IN GLUCOSYLGLYCEROL RE-ASSIMILATION

and ∆slr1670. To estimate, if the degradation of GG results in increased fitness for these qGG uptake values, we divided the growth rate of the respective strain by the growth rate of the ∆slr1670 mutant and compared these values to the experimentally determined data (Tab. 6.5). In the exponential phase, (day 0-4) there is excellent agreement between simula- tion and experiment. Between day 4 and day 6 and between day 8 and day 10, the predicted increase is lower than in the experiment. As the culture increases in density, the absolute value of photons that a single cell perceives, decreases. This results in slower growth (light limitation). Under the assumption that the contribution of GG utilisation to growth remains constant, the difference in growth (expressed as the ratio) between a strain that utilises GG and one that cannot, will therefore be amplified under low light conditions. Since we used a constant photon uptake rate for the simulations, it is expected that at later timepoints, the effect of GG utilisation is underestimated. In the strains that have functional slr1670, on day 8, there is a drop in optical density (Fig. 6.3), which results in negative values for the experimentally determined growth rates. Since the model can only predict growth, a com- parison between the experimental and simulated ratios is not meaningful for this and the adjacent intervals. The cause of this transient drop in OD is at present unknown. A possible explanation is that the degradation of GG and utilisation of the glucose part lead to a drop in intracellular osmotic pressure. Transient efflux of water could then reduce the cell volume and hence, the optical density. Until now, Slr1670 is annotated as a hypothetical protein, so we decided to study its phy- logenetic distribution. A PSI BLAST of the translated sequence of Slr1670 yielded around 6 90 homologues, which are distributed among the cyanobacteria, α-proteobacteria and Ar- chaea (See Fig. 6.5 for a condensed tree, including only the cyanobacterial species and Fig. 6.3 for the full phylogenetic tree). In many cyanobacterial strains, also homologues to ggpS, ggpP and glpK could be identified (Fig. 6.5). Notably, a homologue was also found in Microcoleus chthonoplastes, a strain that has been shown to ferment GG under dark con- ditions, thereby utilizing the glucose moiety, but excreting glycerol [259].

DISCUSSION

Production of one GG molecule requires fixation of 9 CO2 molecules. From a cellular/physi- ological point of view, GG is an energetically costly compound with often transient use only. It is therefore conceivable that systems have evolved to reduce energy losses related to the synthesis of this osmolyte. The GgtABCD system takes up GG that is lost from the cells due to leakage from the cellular cytoplasm; inactivation of this system leads to pronounced ex- tracellular accumulation of GG [244, 258]. Upon transition from a high-salt to a low-salt environment, osmoprotective compounds are no longer needed to maintain turgor pres- sure, so cells able to salvage the nine carbon atoms of GG are at an advantage. In line with this, the adjusted genome scale model of Synechocystis (see section6) predicted an increase in growth rate when extracellular GG was made available (Tab. 6.5). In the first part of the growth experiments, there is excellent agreement of the simulated and the experimental data. Inactivation of glycerol kinase did not lead to increased glycerol concentrations under salt stress or when GG was supplied in the culture medium. The annotation of the respective ORF was based on homology, in the absence of biochemical evidence. Therefore, the gene may have been misannotated. 89

Marine cyanobacteria may ferment a portion of their osmoprotectants [246]. Interestingly, M. chthonoplastes ferments GG, using only the glucose and excreting the glycerol [259]. In M. chthonoplastes, GG was used as a substrate for fermentation during the night. The genome of M. cthonoplastes encodes a homologue of Slr1670 (Fig 6.5). At this moment, it is not known if the degradation of GG observed under continuous light in Synechocystis, a freshwater organism, serves a regulatory purpose or is the result of a con- stitutive reaction, that becomes more meaningful in the dark. During culture growth under constant continuous light, the average amount of light per cell decreases. The individual cells, however, perceive something different. At a given moment, the cells located close to the light source receive light at a high intensity, while a cell in the centre of the culture re- ceives fewer photons, or none at all. Increases in culture density therefore lead to increased periods of darkness from the perspective of an individual cell. This might explain why fer- mentative pathways become active during the day. Disregard of glycerol as a fermentation substrate in both Synechocystis and M. chthonoplas- tes is in agreement with the prediction from the genome scale model, where glycerol util- isation does not lead to increased growth at physiological levels for the GG assimilation reaction (Fig. S6.2B). The mutant strains deficient in degradation of GG are valuable tools to study the kinetics of GG synthesis. Under industrial production settings, Synechocystis may be exposed to variations in osmotic stress. It is important to be able to simulate the behavior of this organism under such con- dition in order to facilitate its usage in the direct conversion of CO2 to products of interest in a sustainable fashion [168]. The addition in the model of the ability to synthesize, trans- 6 port and degrade glycosylglycerol is a necessary step towards the accurate simulation of the growth of Synechocystis while in such conditions. Additionally, disruption of slr1670 allows to construct strains with increased levels of GG, an interesting commodity chemical [260]. In conclusion, we have demonstrated a function for a previously non-annotated protein that has turned out to be involved in the re-assimilation of glucosylglycerol. The protein shows homology to glucoside hydrolases and is found in a wide range of cyanobacteria, where its presence correlates with the presence of GG synthesis. Furthermore, homologues of this protein are found in the α-proteobacteria and in the domain of the Archaea. Often, homologues indentified had previously been annotated as alpha amylases. These annota- tions, however, are purely based on homology and are not supported by any experimental evidence. It is also of interest to note that some cyanobacterial species exhibited multiple homologues of Slr1670. Genome-scale modeling revealed that the increase in growth rate caused by the utilisation of the glucose part of GG outweighs the use of the glycerol part by far. This is in line with observations in M. chthonoplastes, in which only the glucose part of GG is utilised by the cells. Studies with the purified enzyme will help to elucidate its kinetic properties and shed light on the mechanism of cleavage for GG. 90 6.S LR1670 IS INVOLVED IN GLUCOSYLGLYCEROL RE-ASSIMILATION

Table 6.5: Growth rates of Synechocystis wild type, ∆ggpS and ∆glpK divided by the growth rate of ∆slr1670. qGG values were calculated using glycerol production data of the ∆ggpS mutant (Fig. 6.3).

µ/µ /%

interval qGG / ∆ggpS ∆glpK / d mmol WT gDW h ∆slr1670 ∆slr1670 ∆slr1670 · sim.i exp.ii sim.i exp.ii sim.i exp.ii 0-2 0.008 103.9 100.9 2.4 103.9 105.7 2.3 103.9 102.8 3.5 ± ± ± 2-4 0.015 107.4 109.4 11.0 107.4 106.1 12.2 107.4 104.6 10.3 ± ± ± 4-6 0.014 106.9 122.8 5.6 106.9 123.3 5.4 106.9 128.7 11.2 ± ± ± 6-8 0.009 104.6 -73.7 36.8 104.6 -42.8 24.6 104.6 -106.9 21.7 ± ± ± 8-10 0.003 101.5 135.7 41.8 101.5 93.0 31.6 101.5 120.9 59.8 ± ± ±

i sim.: simulated; at physiological uptake fluxes of GG, the model does not predict a difference for the mode of cleavage (phosphorolytic vs. hydrolytic, Fig. S6.2A). Similarly, utilisation of the glycerol part does not lead to in- creased growth (Fig. S6.2B). ii exp.: experimental; error margins represent standard deviations of three replicates.

ACKNOWLEDGEMENTS 6 The authors thank E. Kilias for help with the construction of phylogenetic trees. 91

SUPPLEMENTARY INFORMATION

WT WT WT ∆slr1670 ∆glpK ∆ggpS 1.0 0.8 A B C

730 0.6

OD 0.4 0.2

1.0 0.8 D E F 6

730 0.6

OD 0.4 200 mM NaCl 0.2

1.0 } 0.8 G H I

730 0.6

OD 0.4

0.2 400 mM NaCl

1.0 } 0.8 J K L

730 0.6

OD 0.4 0.2 600 mM NaCl 0 1 2 3 0 1 2 3 0 1 2 3 } t / d t / d t / d

Figure S 6.1: Growth of Synechocystis mutants is compared in BG11 medium supplemented with 10 mM TES/KOH to an initial pH of 8.0 and with varying amounts of NaCl. The experiments were carried out in 96-well plates. OD values are shown as the difference of the OD and the OD of wells with the same condition but without cells. In some cases, this can lead to ODs 0. Error bars repre- < sent standard deviations of 8 biological replicates. 92 6.S LR1670 IS INVOLVED IN GLUCOSYLGLYCEROL RE-ASSIMILATION

Phosphorolysis, glycerol utilisation • 100 Phosphorolysis, glycerol utilisation • 100 Hydrolysis, glycerol utilisation Phosphorolysis, no glycerol utilisation Phosphorolysis, no glycerol utilisation • 100 Hydrolysis, glycerol utilisation • 100 Hydrolysis, no glycerol utilisation Hydrolysis, no glycerol utilisation A 101.6 B 101.6

101.2 101.2

100.8 100.8 µ / µ / 100.4 100.4

100.0 100.0 0.001 0.01 0.1 1.0 0.001 0.01 0.1 1.0

qGG / (mmol/gDW • h) qGG / (mmol/gDW • h)

Figure S 6.2: Ratios of simulated growth rates in response to varied GG uptake fluxes. (A): Compar- ison between phosphorolytic and hydrolytic cleavage of GG. At physiological GG uptake rates, there is no difference between phosphorolysis and hydrolysis. At very high, unphysiological, uptake rates, phosphorolysis of GG leads to higher growth rates than hydrolysis. This effect is slightly larger, when glycerol is not used. (B): Influence of glycerol utilisation. At physiological uptake rates, utilisation of 6 glycerol does not increase growth rate. At very high uptake rates, a minor effect is predicted. 93

A - Full tree Cyanobacteria (54 entries)

Purple bacteria (17 entries)

Archaea (17 entries)

Spirochaeta africana DSM 8902

Spirochaeta africana

0.3 Salinispira pacifica B - Cyanobacteria subtree

6 Aphanocapsa montana BDHKU210001 Oscillatoriophycideae MULTISPECIES Leptolyngbya sp. PCC 6406 Leptolyngbya sp. KIOST-1 Nodosilinea nodulosa Leptolyngbya sp. Heron Island J Leptolyngbya sp. PCC 7375 Synechococcus sp. PCC 7335 Acaryochloris marina Acaryochloris sp. CCMEE 5410 Synechococcus sp. PCC 7002 Synechococcus sp. PCC 7002 Synechococcus sp. NKBG042902 Synechococcus sp. NKBG15041c Leptolyngbya sp. PCC 7376 Synechococcus sp. NKBG042902 Synechococcus sp. PCC 7002 Leptolyngbya sp. PCC 7376 Synechocystis sp. PCC 6714 Synechocystis sp. PCC 6803 Arthrospira platensis Arthrospira sp. PCC 8005 TJSD091 Arthrospira sp. Arthrospira platensis filamentous cyanobacterium ESFC-1 Spirulina subsalsa Coleofasciculus chthonoplastes Myxosarcina sp. GI1 Pleurocapsa sp. PCC 7319 Stanieria cyanosphaera Geitlerinema sp. PCC 7105 Dactylococcopsis salina Halothece sp. PCC 7418 Rubidibacter lacunae Geitlerinema sp. PCC 7407 Cyanobacterium aponinum Cyanobacterium stanieri PCC 7202 Pseudanabaena sp. PCC 7367 Pseudanabaena sp. PCC 7367 Pseudanabaena sp. PCC 7367 Leptolyngbya sp. KIOST-1 confervoides Lyngbya Nodosilinea nodulosa Cyanothece sp. CCY0110 51142 Cyanothece sp. ATCC Coleofasciculus chthonoplastes Coleofasciculus chthonoplastes PCC 7420 Geitlerinema sp. PCC 7105 Synechococcus sp. WH 5701 Synechococcus sp. WH 5701 Cyanobium sp. PCC 7001 Cyanobium sp. PCC 7001 Synechococcus sp. PCC 7336 Leptolyngbya sp. Heron Island J 100 100 100 96 100 100 98 84 100 100 100 100 100 100 100 100 100 100 92 84 100 100 92 100 100 100 100 100 100 100 94 94 0.2 94 6.S LR1670 IS INVOLVED IN GLUCOSYLGLYCEROL RE-ASSIMILATION

C - Purple bacteria subtree 100 Rhodovulum sulfidophilum NHU 02465 Rhodovulum sulfidophilum 100 Roseibacterium elongatum 0.07 100 Jannaschia aquimarina Jannaschia aquimarina 100 100 Roseivivax atlanticus 100 Roseivivax halodurans 100 Roseivivax isoporae Puniceibacterium sp. IMCC21224 96 Maribius sp. MOLA 401 96 Oceaniovalibus guishaninsula 100 Citreicella sp. SE45 100 Pelagibaca bermudensis 96 100 Citreicella sp. 357 Salipiger mucosus Salinarimonas rosea Thalassobaculum salexigens

D - Archaic subtree Haloferax sp. Arc-Hr Haloferax sulfurifontis 100 0.2 Haloferax denitrificans 6 100 Haloferax alexandrinus 100 Haloferax prahovense 100 Haloferax mediterranei Haloferax elongans 100 100 Haloferax larsenii Halogeometricum borinquense Halogranum salarium Halorubrum lipolyticum 82 Halorubrum kocurii 100 Halorubrum aidingense 98 Halorubrum sp. 5 Halapricum salinum 100 Haloarcula sp. SL3 Haloarcula vallismortis

Figure S 6.3: Phylogenetic distribution of Slr1670 homologues. (A) Full tree; for the sake of clarity, large clades are shown collapsed. The corresponding subtrees are shown below. (B) Cyanobacteria subtree. The overwhelming majority of entries belongs to the class Oscillatoriophycideae. Stanieria, Myxosarcina and Pleurocapsa are the only representatives of another class, Pleurocapsales. (C) Purple bacteria subtree. All species in this subtree belong to the class α-proteobacteria. With the exception of Salinarimonas rosea (Rhizobiales) and Thalassobaculum salexigens (Rhodospirillales), all entries belong to the order Rhodobacterales. (D) Archaea subtree. All species in this subtree belong to the class Halobacteria. All species belong to the order Haloferacales, with the exception of Halapricum and Haloarcula, which belong to the Halobacteriales. Bootstrap values (500 repetitions) are shown on the nodes. Values 80 are not shown. ≤ 7 A COCULTURINGAPPROACHFOR GENERATIONOFOPTICALLYACTIVE 2,3-BUTANEDIOLISOMERS

Philipp SAVAKIS, Jeroen B. VANDER STEEN, Filipe BRANCODOS SANTOS, Klaas J. HELLINGWERF

The stereospecific production of alcohols is of great importance for the chemical industry. Often, materials are only available as racemic mixtures, necessitating dedicated resolution techniques. Here, we describe a method for light-driven dynamic resolution at mild temper- atures (30 ◦C). We use a consortium of a mutant strain of the cyanobacterium Synechocystis sp. PCC6803 that harbours an S-specific, NADPH dependent acetoin reductase and a mu- tant strain of the enterobacterium Escherichia coli, harbouring an R-specific, NADH depen- dent acetoin reductase. Together, these two strains convert R-configured stereocentres into S-configured stereocentres. We chose acetoin/butanediol as a model system and show the con- version of racemic acetoin into (S,S)-2,3-butanediol. Furthermore, we show that the pathway can be expanded to allow for synthesis of (S,S)-2,3-butanediol from CO2, light and water.

95 96 7.( S,S)-BUTANEDIOL SYNTHESIS BY A COCULTURE

INTRODUCTION HIRAL entities differ from their mirror image, like the left hand differs from the right C (for a brief overview, see chapter1). In synthetic chemistry, in particular for pharma- ceutical applications, it is of paramount importance to control the stereochemical outcome of a given reaction [261]. This can be achieved by conversion of prochiral molecules into chiral products with a chi- ral catalyst. Alternatively, stereocentres that are already present in the starting material (e.g. sugars, amino acids), can be used to control the stereochemistry of the product [262]. Resolution techniques. With appropriate techniques, racemic mixtures can also serve as the starting point for stereoselective synthesis (see [263] for a review). In synthetic chemistry, resolution refers to the complete conversion of a racemate into a diastereomeric mixture. Kinetic resolution (see [264] for a review) is a separation technique for enantiomers, in which a catalyst facilitates conversion of one isomer much faster than of the other1,2 . Thus, when the reaction is stopped at less than 50% conversion, the product and recovered start- ing materials have increased enantiomeric excess (ee). A logical consequence of kinetic resolution is that the yield is limited to 50% of the starting material, since conversion be- yond this value would decrease the ee of the product. If there is a way to constantly racemise the enantiomeric mixture, yield can increase to 100% in a process referred to as dynamic kinetic resolution [266]3. Dynamic resolution is possible if the following conditions are met:

h the preference of the catalyst for one of the enantiomers is high enough. 7 h there is a method to constantly racemise the starting material. h there is a way to continuously remove product or there is a driving force that ensures that the backreaction from product to starting material is disfavoured.

This last point is of crucial importance for any chemical process. Biological reduction reactions. NADPH is the key reductant in anabolic reactions, such as amino acid and fatty acid biosynthesis [17]. NAD, on the other hand, often is the electron acceptor in catabolism [17]. The cyanobacterial redox pool is dominated by NADP(H) [214] while in chemotrophic organisms NAD(H) is the more abundant redox cofactor [267].

1Interestingly, while kinetic resolution techniques based on enzymes or metal catalysts are employed more and more often by the chemical industry, one of the earliest examples of kinetic resolution was shown with living organisms (i.e. Pasteur’s work on the growth of Penicillium glaucum on racemic ammonium tartrate, referenced in [264]) 2Typically, the separation of the product from the starting material is much simpler than separation of the two enantiomers. In biocatalysis, a well-studied example involves lipase-catalysed esterification reactions (reviewed in [265]). While separation of two enantiomers involves comparatively complicated techniques such as selec- tive crystallisation or even chiral chromatography (very costly on a large scale), esters and alcohols can often be separated on the basis of polarity, by extraction with an apolar solvent. 3As an illustration, consider a racemic mixture of a chiral alcohol. The R-isomer of the alcohol reacts signifi- cantly faster with a lipase to form an ester. If there is a catalyst (or a combination of catalysts) in the reac- tion that oxidises the alcohol to the ketone and subsequently reduces it without stereogenic control, the lipase continuously removes the R-isomer from the redox system, while the S-isomer remains. After each successive oxidation-reduction cycle, the amount of the S-isomer is reduced by 50%, until all starting material is converted into the enantiomerically pure ester. Thus, this technique allows one to make enantiomerically pure products from racemic starting material with yields of 100% 97

One of many examples of reactions where stereocontrol is desirable is the reduction of a ketone to the corresponding alcohol (see [268] for an overview on enzymatically produced alcohols) Enzymes that can catalyse this reaction are called alcohol dehydrogenases or alde- hyde reductases (E.C. number: [1.1.1.1]). The portfolio of alcohol dehydrogenases with favourable properties is steadily increasing through the discovery of new enzymes and the alteration of existing ones [269]. In alcohol dehydrogenases, the reduction of the carbonyl group is coupled to the stoichio- metric oxidation of NADPH or NADH. These compounds are very costly, and regeneration systems are therefore a requirement for large-scale use (see [270] for a brief review). An in- expensive way to regenerate redox cofactors is the use of a cosubstrate that is oxidised in a coupled reaction by either an isolated enzyme (e.g. glucose getting oxidised by glucose dehydrogenase) or in the context of a whole-cell biocatalyst. While continuous acquisition of a cosubstrate on an industrial scale involves a direct mon- etary cost, the utilisation of e.g. carbohydrates may lead to the accumulation of byprod- ucts, which may complicate product recovery, thereby indirectly increasing costs. H2 as an electron source is cheap and does not give rise to complex byproducts. Hydrogen storage, however may be cost and energy intensive. In photosynthetic organisms, photon energy is harnessed to reduce NADP,with H2O as the ultimate electron donor. Water is still inexpensive and a requirement for the vast majority of bioprocesses. Sunlight does not cost anything. Based on these considerations, cyanobac- teria have been employed for the stereocontrolled reduction of ketones to the correspond- ing alcohols. Literature examples include 4-chloroacetophenone, ethylbenzoylacetate and pentafluoroacetophenone with impressive yields and high enantiomeric excess [271, 272].

2,3-butanediol. 2,3-Butanediol has three different isomers, the (S,S)(L)-, (R,R)(D)-, and the meso ((R,S)) isomer (see Fig. 7.1 for biosynthetic pathways and side reactions). Most 7 organisms decarboxylate S-acetolactate to R-acetoin, with inversion [273]. R-acetoin is re- duced to either the D or the meso-isomer of 2,3-butanediol, depending on the specificity of the acetoin reductase. Furthermore, S-acetolactate can undergo non-enzymatic oxidative decarboxylation to di- acetyl [186], which can be reduced to S-acetoin and further to (S,S)-butanediol. To gener- ate (S,S)-butanediol from meso-butanediol, the R-stereocentre must be isomerized to the S-form. This can be achieved by subsequent oxidation to the ketone and stereoselective reduction to the alcohol. A two-step system involving Klebsiella pneumoniae4 and Bacillus subtilis5 has been shown to convert glucose to (S,S)-butanediol [274]. K. pneumoniae con- verts glucose to meso-2,3-butanediol. After purification, B. subtilis oxidises the R-stereo- centre, yielding S-acetoin. After a second purification step, K. pneumoniae reduces S-acetoin to (S,S)-butanediol. While this approach gives (S,S)-butanediol in satisfactory yield from glucose, the intermediary purification steps increase the cost of the process. We reasoned that cyanobacteria are ideally suited to perform the reduction reaction. Syne- chocystis harbouring NADPH dependent acetoin reductase from Leuconostoc lactis NCW1 can reduce racemic acetoin to a mixture of L- and meso-butanediol [115]. If meso-butane- diol can be oxidised to S-acetoin, then subsequent reduction by the Leuconostoc acetoin reductase would yield S,S-butanediol.

4S-specific acetoin reductase 5R-specific acetoin reductase 98 7.( S,S)-BUTANEDIOL SYNTHESIS BY A COCULTURE

Condensation Decarboxylation RedOx OH (NAD(P)(H))

pyruvate S-acetolactate CO2 R-acetoin O O O O YdjL OH - ALS O (R,R)-butanediol 6 CO2 2 - O ALDC NCW1 AR O OH OH enzymatic non-

CO2 rac. OH

CO2 YdjL OH O diacetyl O (R,S)-butanediol reductase NCW1 AR O OH OH diacetyl S-acetoin

OH (S,S)-butanediol

Figure 7.1: Schematic overview of the synthesis of different stereoisomers from CO2. The high- lighted pathway shows the synthetic route to (S,S)-2,3-butanediol designed in this chapter. Abbre- viations: ALS: acetolactate synthase; ALDC: acetolactate decarboxylase; YdjL: R-specific acetoin re- ductase of Bacillus subtilis; RedOx: reduction/oxidation, NCW1 AR: acetoin reductase of Leuconostoc lactis NCW1; rac.: racemisation.

MATERIALS AND METHODS 7 Unless stated otherwise, all chemicals were purchased from Sigma-Aldrich. Culturing. Escherichia coli XL1 was grown in LB medium at 37◦C and with shaking at 200 rpm with appropriate selection markers. For selection of transformants, E. coli was grown at 37◦C on LB medium with 1.5% (w/v) agar and appropriate selection markers. For coculturing experiments, E. coli was grown overnight in LB medium supplemented with 50 µg/mL kanamycin at 37◦C and 200 rpm. The culture was harvested by centrifugation (2,500 rpm, 5 min) and the cells were washed twice with 1 mL BG11 medium supplemented with 10 mM TES/KOH, pH 8.0. Then, the cells were added to the culture to the indicated final OD600. In the coculturing experiment for the reduction of acetoin to (S,S)-butanediol, the growth medium was supplemented with 1.76 mM NH4Cl. Synechocystis cells were grown in a shaking incubator (Innova 43, New Brunswick Scien- tific, 120 rpm, 30◦C) under 15 W cool fluorescent white light (F15T8-PL/AQ, General elec- tric, light intensity 30-40 µE/m2/s) in BG11 medium, supplemented with 10 mM TES/KOH pH 8.0. Cells were inoculated to an OD730 of 0.1 from a preculture. Plasmid construction. pPS1313 was ordered at Genscript, NJ. This plasmid is a pHKH [135]6 derivative, which contains ydjL7, and the NADH kinase gene from Pichia membran- ifaciens, each under the control of Ptrc. Digestion of pPS1313 with XbaI and AvrII excised the NADH kinase gene, and ligation of the backbone resulted in pPS1315, which harbours

6This plasmid does not replicate in Synechocystis and allows for chromosomal integration at the neutral site slr0168 and selection with kanamycin. 7encodes R-specific [275] acetoin reductase [276] from B. subtilis) 99 ydjL under the control of Ptrc. pMLK83 [277] was digested with EcoRI and self-ligated to create pJBS1303. This removed the gusA gene from the plasmid, and resulted in the genotype: ’amyE(up)-neo-amyE(down)’ bla. In other words: it can be used to insert the neo (KmR) gene into the amyE gene via dou- ble cross-over. Mutant construction. Briefly, Synechocystis was grown to an OD730 of 0.2-0.4 in BG11 me- dium buffered at an initial pH of 8.0 with 10 mM TES/KOH. 10 mL were harvested by cen- trifugation (4,000 rpm, 15 min, RT) and resuspended in 500 µL of BG11 medium. 1-3 µg of the respective plasmid were added and the cell suspension was incubated in an Eppendorf 2 tube for 5 h at 30◦C and 30-50 µE/m /s. Cells were harvested by centrifugation (4,000 rpm, 15 min, RT), resuspended in 200 µL and streaked on a filter membrane placed on solidified BG11 medium (supplemented with 1.5% (w/v) agar and containing 0.3% (w/v) Na2S2O3 and buffered at pH 8.0 with 10 mM TES/KOH). Following overnight incubation, the membrane was transferred to BG11 plates containing 0.3% (w/v) Na2S2O3 and 50 µg/mL kanamycin. Transformants appeared after 6-12 days. JBS13005 was created by transforming PB2 with pJBS1303. Correct disruption of the amyE locus was confirmed with a starch test. Detection of extracellular molecules using high performance liquid chromatography (HPLC). Ca. 1 mL of a given culture was harvested, the cells were removed by centrifugation (14,500 rpm, 5 min, 21◦C), the supernatant was filtered (Sartorius Stedin Biotech, minisart SRP 4, 0.45 µm) and analyzed by HPLC (Rezex ROA-Organic Acid H+ (8%) column (Phenomenex), column temperature: 85◦C) with a refractive index detector (Jasco, RI-1530); eluent: 7.2 mM H2SO4, flow of 0.5 mL/min. Peaks were identified and quantified using external standards. Colorimetric determination of acetoin. Where indicated, acetoin was measured using the Voges-Proskauer test [200], following the modified protocol described in [201]. A curve with acetoin standards was used for quantification. 7

RESULTS Generation of enantiomerically pure (S,S)-butanediol from racemic acetoin. When racemic acetoin was added to a culture of Synechocystis harbouring ydjL, encoding an R- specific [275], NADH dependent acetoin reductase [276] from Bacillus subtillis, it was re- duced to a mixture of meso- and optically active butanediol (Fig. 7.2). Since in Synechocystis, the NADPH pool is more reduced than the NADH pool [214], we expected that the use of an NADPH dependent acetoin reductase would allow to shift the equilibrium towards butanediol (Eq. 3.1)8, as demonstrated previously [115]. In a culture of Synechocystis ARP harbouring the acetoin reductase from Leuconostoc lactis NCW1, initially, 50% of the acetoin were converted into meso-2,3-butanediol. More slowly, the remaining 50% were converted into optically active 2,3-butanediol (Fig. 7.3). Leuconos- toc lactis NCW1 acetoin reductase has been reported to be S-specific (Fig. S3.6). This means that the optically active butanediol in this experiment is (S,S)-butanediol. The apparent delay in the production of optically active butanediol indicates that this enzyme prefers R- over S-acetoin.

8Note that these considerations only apply to a situation in which chemical equilibrium was established. In Fig. 7.2, there is no indication that equilibrium has been reached, since the butanediol/acetoin ratio increases until the last sampling point. 100 7.( S,S)-BUTANEDIOL SYNTHESIS BY A COCULTURE

Table 7.1: Strains used in this study

Strain Genotype Source B. subtilis PB2 wild-type [278] B. subtilis WN1075 ∆ydjL [276] B. subtilis JBS13005 thrC2 amyE::neo This study E. coli XL1 Blue endA1, gyrA96(nalR) thi–1 recA1 relA1 lac Stratagene q glnV44 F’[::Tn10 proAB+ lacI ∆(lacZ)M15] hsdR17 (rK− mK+) E. coli XL1 pPS1315 endA1, gyrA96(nalR) thi–1 recA1 relA1 lac This study q glnV44 F’[::Tn10 proAB+ lacI ∆(lacZ)M15] hsdR17 (rK− mK+) pHKH[135] Ptrc::ydjL B. subtilis Synechocystis D. Bhaya, sp. PCC6803 Stanford Syn pVZ006 harbours pVZ321 with Ptrc::aldc::ar::KmR [115] ALDC of Brevibacillus brevis and AR of Leuconostoc lactis NCW1 Syn ARP ∆slr0168::ar::KmR [115] AR of Leucnonostoc lactis NCW1 Syn ydjL ∆slr0168::ydjL::KmR This study

7

Table 7.2: Plasmids used in this study

Plasmid Description Source pMLK83 amyE(up)-gusA-neo-amyE(down)-bla [277] pJBS1303 amyE(up)-neo-amyE(down)-bla This study pPS1313 pHKH [135] with Pichia membranifaciens NADH kinase This study and Bacillus subtilis ydjL (R-specific, NADH dependent acetoin reductase) pPS1315 pHKH [135] with Bacillus subtilis ydjL This study 101

acetoin meso-butanediol R,R-butanediol 10 12 Syn ydjL(NADH) 10 1 8

730 0.1 6 OD 4 0.01 c / mmol/L 2 0.001 0 0 2 4 6 8 t / d

Figure 7.2: Reduction of acetoin by Synechocystis harbouring ydjL from B. subtilis. Cells were cultured in BG11 medium supplemented with 5 mM racemic acetoin. Error bars denote standard deviations of three replicates. Error bars that are not visible are smaller than the respective data point symbol.

acetoin 7 meso-butanediol S,S-butanediol 10 12 Syn AR(NADPH) 10

1 8

730 6

OD 0.1 4 c / mmol/L 2 0.01 0 0 2 4 6 8 t / d

Figure 7.3: Reduction of acetoin by Synechocystis harbouring acetoin reductase from L. lactis NCW1. Cells were cultured in BG11 medium supplemented with 5 mM racemic acetoin. Error bars denote standard deviations of two replicates. Error bars that are not visible are smaller than the respective data point symbol. 102 7.( S,S)-BUTANEDIOL SYNTHESIS BY A COCULTURE

acetoin 2.0

1.5

1.0

c / mmol/L 0.5

0.0 0 100 200 300 t / min

Figure 7.4: Oxidation of meso-2,3-butanediol by E. coli harbouring ydjL. Error bars denote standard deviations of three replicates. Error bars that are not visible are smaller than the respective data point symbol. Acetoin concentrations were determined using the Voges-Proskauer test.

acetoin acetoin meso-2,3-butanediol meso-2,3-butanediol S,S-2,3-butanediol S,S-2,3-butanediol 2.0 2.0 7 Syn AR Syn AR + E. coli ydjL 1.0 1.0 1.5 1.5 730 730 1.0 1.0 OD 0.1 OD 0.1 c / mmol/L c / mmol/L 0.5 0.5

0.01 0.0 0.01 0.0 0 2 4 0 2 4 t / d t / d

Figure 7.5: Solar driven stereocontrolled conversions inspired by dynamic kinetic racemisation. Racemic acetoin is converted to (S,S)-2,3-butanediol. Synechocystis ARP harbours an S-specific, NADPH-dependent acetoin reductase (from Leuconostoc lactis NCW1). E. coli harbours an R-specific, NADH dependent acetoin reductase (ydjL from B. subtilis). Synechocystis reduces R-acetoin to meso- 2,3-butanediol and S-acetoin to (S,S)-butanediol. E. coli oxidises meso-butanediol to S-acetoin, which is then reduced to (S,S)-butanediol by Synechocystis ARP.Arrows indicate addition of racemic acetoin to a concentration of 1 mmol/L. Error bars represent standard deviations of two biological replicates. Data on the reduction of acetoin by recombinant Synechocystis only are single, representative obser- vations. 103

Therefore, Synechocystis ARP can be used for the light-driven kinetic resolution of racemic acetoin; if the reaction were stopped at the right moment (between day 2 and 3 in Fig. 7.3), meso-butanediol would be the only product, while the starting acetoin would be enriched with the S-isomer. At the last sampling points of the experiment, all of the acetoin was converted into butane- diol (Fig. 7.3). We therefore reasoned that if meso-butanediol were to be converted back into S-acetoin, (S,S)-butanediol could be synthesised from racemic acetoin. If oxidation and reduction were to happen in the same compartment, effectively an acetoin- powered transhydrogenase9 reaction would be generated, as NAD would indirectly be re- duced by NADPH:

NADPH + R acetoin NADP + meso butanediol + NAD + meso butanediol NADH + S acetoin + NADPH + S acetoin NADP + (S,S) butanediol = 2 NADPH + NAD + R acetoin 2 NADP + NADH + (S,S) butanediol

Recombinant Synechocystis with high expression levels of soluble transhydrogenase from Pseudomonas aeruginosa was shown to be genetically unstable [135]. To avoid instability, and also to create favourable conditions for the reduction and the oxi- dation reaction, we sought to divide them into different compartments. Thus, Synechocystis ARP would catalyse the NADPH dependent, S-specific reduction of acetoin to butanediol, while the R-configured stereocentre would be oxidised by a second bacterium, harbouring an NADH dependent10, R-specific acetoin reductase. We selected YdjL as the R-specific acetoin reductase. Bacillus subtilis can utilise acetoin as 7 a carbon source. We therefore selected Escherichia coli as the chemotroph. YdjL-mediated oxidation of acetoin could be driven to completion by continuous removal of NADH (Eq. 3.1). We supposed that in metabolically active Escherichia coli, NADH formed from the oxidation of meso-butanediol would continuously donate electrons to the electron transport chain, consequently being converted back to NAD. Indeed, when E. coli harbouring ydjL were transferred to BG11 medium containing meso-butanediol, acetoin was formed over time (Fig. 7.4). When both strains were combined in one culture, racemic acetoin was completely con- verted into (S,S)-butanediol (Fig. 7.5). Generation of enantiomerically pure (S,S)-butanediol from light, H2O and CO2. Previ- ously, we showed that meso-2,3-butanediol can be produced from light, water and CO2 by engineered Synechocystis strians [115]. We expected that the combination of a mutant, which makes meso-butanediol from CO2 with a strain, that selectively oxidises R-configured stereocentres would result in the synthesis of (S,S)-2,3-butanediol from CO2. As a proof on concept, we cultured Syn pVZ06 [115] in BG11 medium, with CO2 as the only carbon source. This strain produces meso-2,3-butanediol from CO2,H2O and light. Next, we removed the cells and added Bacillus subtilis JBS13005 (B. subtilis). This bacterium har- bours the R-specific acetoin reductase ydjL [275, 276]. After the meso-butanediol was ox-

9Energy-independent soluble transhydrogenase catalyses the reaction NADPH + NAD NADP + NADH. 10This is not a requirement. NADPH would work just as well if the NADPH/NADP ratio were to be shifted such that it would allow the oxidation of butanediol. 104 7.( S,S)-BUTANEDIOL SYNTHESIS BY A COCULTURE

HO

O HO HO OH

OH OH OH Supernatant after incubation with

Syn pVZ06 ty / A U i B. subtilis en s t n I Syn ARP

21.0 22.5 24.0 25.5 t / min

Figure 7.6: Sequential incubation of BG11 medium with Synechocystis pVZ06, Bacillus subtilis and Synechocystis harbouring acetoin reductase from Leuchonostoc lactis NCW1 leads to the generation of (S,S)-2,3-butanediol from CO2, sunlight and water.

idised to S-acetoin, we removed the cells and added Syn ARP, which led to reduction to (S,S)-butanediol (Fig. 7.6). We then sought to culture both Synechocystis pVZ06 and E. coli ydjL in BG11 medium and analysed the extracellular medium composition (Fig. 7.7). Addition of E. coli ydjL indeed led to synthesis of (S,S)-butanediol from light, water and CO2 (Fig. 7.7D), although substantial 7 amounts of meso-2,3-butanediol remained in the mixture.

DISCUSSION Here, we show the conversion of racemic acetoin into (S,S)-butanediol using a consor- tium of genetically modified Synechocystis and E. coli. The conversion of racemic start- ing material into enantiopure product is similar to dynamic kinetic resolution and most closely resembles the type III DYKAT approach shown in [279]. The use of two different re- dox couples (NADPH/NADP vs NADH/NAD), in two separate cellular compartments, how- ever, also places the thermodynamic sink of the pathway at the desired end product; S- specific reduction is driven by the NADPH/NADP ratio in Synechocystis, which remains >1 due to electrons derived from water. R-specific oxidation is driven by the NAD/NADH ra- tio, which stays <1, ultimately, due to transfer to oxygen, generating water. Thus, (S,S)- butanediol would be produced regardless of the starting material (R-acetoin, S-acetoin, (R,R)-butanediol, (meso)-butanediol all lead to the same product). Furthermore, we show that the pathway to (S,S)-butanediol can be extended such, that it is initiated from CO2. This was achieved with a genetically modified Synechocystis strain, which produces meso-butanediol from CO2, light and water [115] and with both a native producer of (R,R)-butanediol (B. subtilis) and a genetically modified E. coli strain. In this co- culturing approach, no fixed carbon has been added other than the citrate that is present in the original BG11 medium. Cyanobacteria have been demonstrated to shed carbohydrates, which heterotrophic organisms can grow on [280]. E. coli and B. subtilis could consequently 105

A B

growthmeso-BD Syn pVZ06 CO2 Syn pVZ06 + E. coli ydjL 10.0 2

E. coli ydjL OH 1.0 Syn pVZ06 730 OH 0.1 1 OD

O OH 0.01 c / mmol/L

0.001 0 OH OH 0 5 10 t / d C D growthacetoin growthL-BD Syn pVZ06 Syn pVZ06 Syn pVZ06 + E. coli ydjL Syn pVZ06 + E. coli ydjL 10.0 2 10.0 2

1.0 1.0 730 730 0.1 1 0.1 1 OD OD c / mmol/L 0.01 0.01 c / mmol/L 7 0.001 0 0.001 0 0 5 10 0 5 10 t / d t / d

Figure 7.7: Synthesis of S,S-2,3-butanediol from CO2 by a microbial consortium. (A): Schematic rep- resentation of the consortium. (B): meso-2,3-butanediol concentrations. (C): acetoin concentrations. (D): (S,S)-butanediol concentrations. To ease understanding, the growth curves are shown in panels B, C and D. The cells were grown in BG11 medium at an initial pH of 7.0 (10 mM TES/KOH). Data points denote single, representative observations. Squares describe a culture with Synechocystis pVZ06, ex- clusively. Hexagons indicate a culture with Synechocystis pVZ06 and E. coli ydjL. Filled symbols stand for OD values, half-filled symbols mark the concentration of the respective metabolite. 106 7.( S,S)-BUTANEDIOL SYNTHESIS BY A COCULTURE

A B

O Syn ARP Syn ARP E. coli ydjL OH O2 OH NADPH

OH NADP OH NADP S NAD NADPH H O NAD red 2 OH O H O 2 O2 H2O O NADH OH O2 NADH OH S OH ox OH

Figure 7.8: Schematic representation of Synechocystis/E. coli coculture systems. (A) System for the conversion of racemic acetoin to (S,S)-butanediol. (B) Expansion of the idea for reduction of oxi- dised substrate molecules (Sox ) to reduced substrate molecules (Sred ). Electron flow is depicted as a translucent arrow.

be fed by Synechocystis, eliminating the need to supplement a source of fixed carbon. Ad- ditionally, the catabolism of these molecules in the spent medium by the chemotroph can increase the purity of the product. Acetoin shows considerable toxicity to Synechocystis, but the ultimate product, butanediol, is tolerated at much higher concentrations [115]. High yields of optically active butanediol could therefore be reached if acetoin were added at a slow rate. This would keep transient 7 acetoin concentrations low and circumvent toxic effects of acetoin. Although resembing dynamic kinetic resolution, the pathway to (S,S)-butanediol presented here does not rely on a racemisation step. Acetoin can spontaneously racemise, in partic- ular when the pH of the culture is high. Theoretically, the racemisation of acetoin would introduce an NAD and NADPH consuming loop into the pathway highlighted in Fig. 7.1. Thus, the efficiency would be reduced. Since the thermodynamic sink of the system is (S,S)- butanediol, however, the final yield would be unaltered. The data presented here suggests that racemisation of acetoin is not a big issue, since its intermediary concentration is very low (Fig. 7.7C). Added traits that reduce the growth rate but do not confer a benefit to an organism may reduce its genetic stability11. In this system, however, this will likely not pose a problem. While continuous allocation of NADPH to acetoin reduction can be a drain on the cyano- bacterial metabolism, this does not necessarily lead to unstable cultures; even when acetoin reductase is continuously produced by the cyanobacterium, it is only active in the presence of substrate. Thus, the culture can be grown until a certain density is reached, after which acetoin can be added. Only then does the presence of acetoin reductase become a true burden to the cell. During growth, there is no strong selective pressure against acetoin re- ductase. In a culture with high cell density, an acetoin reductase deficient mutant then has difficulty taking over the culture. The cost of acetoin reduction (and thus, increased NADPH utilisation) for Synechocystis could be quantified by following growth and acetoin reductase

11By which is meant that when mutations arise, they can be selected for and consequently propagate themselves through the culture. 107 activity in the presence of different amounts of acetoin. These considerations on strain sta- bility are only valid for the culture, in which acetoin is used as the starting material. When the entire butanediol biosynthesis pathway is expressed in Synechocystis, the strain is still likely less stable. Conceptually, in this coculturing system, electrons are transported from water via the cya- nobacterial metabolism and the metabolism of a heterotrophic organism to a ketone and to O2, recycling water (Fig. 7.8A). Thus, the acetoin/butanediol system can be seen as an inter-organismal soluble electron carrier system. In the electron transport chain of the het- erotroph, NADH oxidation is used to translocate protons, which can drive ATP synthase. An alternative application could occur through introduction of another reductase (Fig. 7.8B). This system would allow one to carry out solar-powered reduction reactions that are diffi- cult to accomplish in cyanobacteria. Furthermore, introduction of RubisCO and phospho- ribulokinase would generate a CO2-fixing "heterotroph" that derives electrons from photo- synthesis. In this study, the culture density was determined through measuring the optical density at 730 nm. This means that there was no distinction between the photoautotroph and the chemotroph. Future studies should therefore consider the use of instruments that can quantify both organisms, such as a flow cytometer [281] or a Coulter counter [282] In conclusion, we present here a method for the one-pot conversion of a racemic mixture of an alcohol into an enantiomerically pure alcohol at the theoretical yield, and the extension for this pathway to start from CO2 using acetoin/butanediol as a model system. The use of an NADPH-specific acetoin reductase introduces a thermodynamic driving force to the final product. This approach is not limited to the acetoin/butanediol system. It is applicable to any racemic mixture of alcohols, for which the following conditions are met: 7

h none of the starting materials nor products are catabolised by either strain.

h all starting and intermediary molecules are soluble in water.

h the product molecules are not toxic at low concentrations, or can be removed effi- ciently.

h all products are mobile over all membranes involved (via diffusion or dedicated trans- port systems).

h enzymes of appropriate stereospecificity are available for the NADPH catalysed re- duction.

ACKNOWLEDGEMENTS We thank Pascal van Alphen for helpful insight and discussions. We thank Wayne L. Nichol- son from the Dept. of Microbiology & Cell Science, University of Florida, Space Life Sciences Lab for providing Bacillus subtilis WN1075.

8 ON POLYOL DEHYDRATION

Philipp SAVAKIS, S. Andreas ANGERMAYR, Alejandra DE ALMEIDA, Mariateresa FERONE, Klaas J. HELLINGWERF

Dehydratase reactions can convert diols into carbonyl compounds. Subsequent reuduction yields an alcohol. Here, we demonstrate the dehydration of meso-butanediol to butanone and in a separate reaction, the reduction of butanone to 2-butanol in recombinant Syne- chocystis strains. Additionally, the conversion of CO2 into 1,3-propanediol is demonstrated with a consortium of a recombinant Synechocystis strain and Lactobacillus reuteri.

109 110 8.T OWARDSSYNTHESISOFBUTANONEAND 2-BUTANOL BY Synechocystis

INTRODUCTION

HE elimination of H2O from a diol yields a carbonyl compound. Geminal diols (hy- T drates)1 readily lose water and will not be discussed beyond this point. If one of the hydroxy groups in a vicinal diol2 belongs to a primary alcohol, an aldehyde can be formed through dehydration. Removal of water from vicinal diols that only have secondary hydroxy groups yields ketones, exclusively (Fig. 8.1). In dehydration reactions, the formal oxidation number of the carbon atom that will trans- form into the carbonyl atom is increased by 1. The oxidation number of the carbon atom that will lose the oxygen atom is reduced by 1. The net oxidation state of the molecule thus remains unchanged. Hence, dehydration reactions are redox neutral processes. A number of economically attractive molecules can be derived from polyols through dehy- dration. First and foremost, 1,3-propanediol is used in the production of PTT (Polytrimethy- lene terephthalate). Native producers of 1,3-propanediol include Klebsiella pneumoniae, Citrobacter freundii and Clostridium acetobutylicum. However, recombinant bacteria have been employed in commercial processes to produce 1,3-propanediol, as well (see [283] and [178] for a review article). Dehydration of 2,3-butanediol yields butanone, which can be reduced to 2-butanol3 (Fig. 8.1). Butanone and 2-butanol are promising fuels, based on their high heating value (2- butanol: 35920 J/mol) and low hygroscopicity. Industrially, 2-butanol is produced through the hydration of n-butene [284]. 2-Butanol is found in fermented beverages and distillates thereof ([285] and references there- in). Hieke and Vollbrecht speculate that lactobacilli are responsible for the bulk of the 2- butanol production [285]. Chemically, the simplest way to remove H2O from a larger molecule involves Lewis acid/ base catalysis. In nature, this indeed is the preferred mechanism for diols that are activated by a carbonyl moiety. Carbonyl groups can stabilise negative charges in the α-position4. Thus, in α,β-hydroxycarbonyl compounds, the proton at Cα is abstracted more easily than in the corresponding unsubstituted diol. 8 For the conversion of unsubstituted diols, nature has evolved a class of enzymes that have been named dehydratases5.

1Geminal diol: R1 R2

HO OH

OH R3

2Vicinal diol: R1 R4

R2 OH

32-butanol is the simplest chiral alcohol. Interestingly, in industry, the majority of 2-butanol is converted to bu- tanone [284] O

Ci Cβ 4 Atom numbering in carbonyl compounds: Cα Cα

5In the older literature, the name dehydrase is also used 111

vicinal diol carbonyl compound final product OH H + NAD(P)H

1,2-propanediol n-propanol

H O - H O - NAD(P) 2 O OH

OH H OH OH + NAD(P)H

glycerol OH 1,3-propanediol

H O - H2O O - NAD(P) OH

OH + NAD(P)H

2,3-butanediol s-butanol

H O - H2O O - NAD(P) OH

Figure 8.1: Removal of a water molecule from a diol (first column) leads to formation of a carbonyl compound (second column); 1,2-propanediol gives propionaldehyde, glycerol gives 3- hydroxypropionaldehyde (reuterin). Elimination of H2O from meso-2,3-butanediol gives butanone. Reduction leads to the corresponding alcohol (third column).

6 These enzymes are dimers of heterotrimers ((αβγ)2)[286] and rely on radical-based mech- anisms for dehydration. Labelling studies [290] have shown that during dehydration, an oxygen atom migrates. (see Fig. 8.2 for a minimal mechanism and [291] for a theoretical study). Among the dehydratases there are two related classes of enzymes that mediate the vitamin 7 B12-dependent dehydration of diols :

h Diol dehydratase (DDH, E.C. number: [4.2.1.28])

h Glycerol dehydratase (GDH, E.C. number: [4.2.1.30])

The best studied dehydratases are those from the organism that was deposited as Aerobac- 8 ter aerogenes ATCC 8724. This strain has hence undergone renaming to Klebsiella oxytoca ATCC 8724. In the older literature, however, Klebsiella pneumoniae and Aerobacter aero- genes ATCC 8724 are used synonymously. K. oxytoca diol dehydratase shows considerable activity on 1,2-propanediol and ethylene glycol [293]8. Glycerol is a substrate for both diol dehydratase and glycerol dehydratase, but was also shown to inactivate both enzymes. [294, 295]9. Permeabilised Klebsiella pneumoniae cells inactivated by glycerol were shown to regain dehydratase activity when ATP and Mg2+ or Mn2+ were added [296]. This observation led to the discovery of diol dehydratase-reactivating factors in K. pneumoniae and K. oxytoca (encoded by ddrAB)[297]. Both reactivases are het- erodimers ((αβ)2)[298, 299] and are thought to act through subunit displacement: When

6Diol dehydratase was initially reported to have two [287] or four [288] subunits. Functional enzyme was recombi- nantly produced from an operon encoding three subunits [289]. Therefore, it was concluded that diol dehydratase has three subunits. X-ray structures have confirmed the (αβγ)2 quarternary structure [286] 7 There are also enzymes that can mediate the reaction without vitamin B12 [292] 8In this study, it was reported that the enzyme showed no activity on 2,3-butanediol. It was not specified, however, which isomer was tested. 9Interestingly, cultivation of Klebsiella in the presence of glycerol induces diol dehydratase 112 8.T OWARDSSYNTHESISOFBUTANONEAND 2-BUTANOL BY Synechocystis

the Co-C bond in dehydratase is split, the cobalamin cofactor remains bound to the en- zyme. Likely, displacement of the β-subunit of reactivase by the β-subunit of diol dehy- dratase weakens the interaction of the dehydratase and the cobalamin [300]. As a result, the damaged cofactor can be replaced with new adenosylcobalamin. Interestingly, while GDH reactivation factors are specific to GDH, DDH reactivation factors were shown to also restore GDH activity [300]. Organisms that have evolved dehydratases, typically separate them from the cytosol with specialised bacterial microcompartments, not unlike the cyanobacterial carboxysomes (see [301] and [302] for current review articles on bacterial microcompartments). In those or- ganelles, the substrate concentration is increased relative to the cytoplasmic concentration, while the oxygen concentration is decreased. Carbonyl compounds, and aldehydes in par- ticular, can exhibit considerable toxicity towards living cells. Generation (and subsequent reduction to the corresponding alcohols) of these molecules in a distinct compartment can therefore help to avoid adverse effects. Synechocystis does not produce the dehydratase cofactor vitamin B12. Instead, it produces the chemically related compound pseudovitamin B12 [303]. In Synechococcus elongatus PCC 7942, functional GDH could be assembled without additional vitamin B12 supplementation [304]. It seems that pseudovitamin B12 can substitute vitamin B12 in this enzyme. For DDH, substrate promiscuity was first demonstrated in the 1970’s; a number of terminal vicinal diols are substrates for this enzyme. Crucially, Toraya et al. found that propanediol dehydratase from Klebsiella also converts 2,3-butanediol, albeit at considerably lower rates than the physiological substrates [294]. 2,3-butanediol represents the only non-terminal vicinal diol that acts as substrate for DDH. Of the three isomers of 2,3-butanediol, only meso-butanediol is accepted as substrate and inactivator. D- and L-2,3-butanediol act as competitive inhibitors, exclusively [294, 305]. Recently, Yoneda et. al reported that GDH of K. pneumoniae accepts meso-butanediol as substrate. In this study, E. coli was also engineered to synthesise butanone from glucose [306]. Previously, we have shown the synthesis of meso-butanediol by recombinant Synechocys- 8 tis from light, water and carbon dioxide [115]. We reasoned that the substrate promiscuity of DDH10 would allow the extension of this pathway to the formation of 2-butanol via bu- tanone (Fig. 8.2B). Since dehydratases rely on a cobalamin cofactor, these enzymes are susceptible to inacti- vation by light and the diradical O2, making functional expression in an oxygen-producing phototroph a challenge. We therefore used approaches where the dehydratase step was implemented in Synechocystis as well as in a chemotrophic bacterium.

MATERIALS AND METHODS Chemicals were purchased from Sigma Aldrich, unless stated otherwise. Culturing. E. coli was cultured routinely in LB medium in a shaking incubator at 37◦C and 200 rpm. Synechocystis was grown routinely in BG11 medium, buffered to an initial pH of 8.0 with 10 mM TES/KOH, at 30◦C and 120 rpm under white light (15 W cool fluorescent , F15T8-PL/AQ, General Electric; light intensity: 20-50 µE/m2/s) For growth on plates, media

10At the time this project was started, this was the only dehydratase for which substrate promiscuity had been demonstrated. 113 were solidified with 1.5% (w/v) Bacto agar. For Synechocystis, plates were additionally sup- plemented with 0.3% (w/v) Na2S2O3. Antibiotics were added where appropriate. Mutant construction. E. coli XL1 PDD and E. coli XL1 PDD DDR were constructed through heat-shock transformation of E. coli XL1 with pPS1310 and pPS1320, respectively. Syne- chocystis PDD and Synechocystis PDD DDR were constructed respectively through chro- mosomal integration of PA1l acO 1::pddABC (from pPS1310) and Ptrc::ddrAB ::PA1l acO 1:: − − pddABC (from pPS1320) at slr0168. Plasmid construction. For the construction of pPS1320, pPS1310 was digested with XbaI and PstI and the fragment containing pddABC was ligated into pPS1309, digested with SpeI and PstI. Western blotting. Proteins were separated according to size by SDS-PAGE, and transferred onto a nitrocellulose membrane. Then, the membrane was washed with 800 mL of PBS buffer, blocked for 1 h with PBS/milk powder (5% w/v), and incubated for 2 h with rabbit α-PDD antiserum in PBS/milk powder (5% w/v). The membrane was washed again with 1 L PBS and incubated for 1 h with GARPO (Goat anti rabbit peroxidase) in PBS/milk powder (5% w/v) and washed again with 500 mL PBS. Bands were visualised using the ECL detec- tion kit (GE healthcare). The image was created as an overlay of the luminescence channel (detects luminescence from the peroxidase reaction) and the 700 nm channel (detects the protein marker). Detection and quantification of external metabolites using HPLC. Supernatants were fil- tered (Sartorius Stedin Biotech, minisart SRP 4, 0.45 µm) and the flow through analysed by HPLC (stationary phase: Rezex ROA-Organic Acid H+ (8%) column (Phenomenex), column temperature: 85◦C), refractive index detector: Jasco, RI-1530; mobile phase: 7.2 mM H2SO4, 0.5 mL/min. Peaks were identified and quantified using external standards. Dehydratase reaction in E. coli under microaerobic conditions. For this test, conditions were essentially as described in Yoneda et al. [306]. Briefly, E. coli was grown overnight in modified M9 medium (modified M9: M9 + 10 g/L glucose, 5 g/L yeast extract, 10 mg/L thi- amine, 50 µg/mL kanamycin). In a 15 mL tube, 5 mL modified M9 was inoculated with 50 µL of the overnight culture and grown for 3 h at 37◦C. Then, cyanocobalamin was added to a concentration of 1 µM, IPTG was added to a concentration of 0.1 mM and one of the 8 following compounds was added to a concentration of 5 g/L: meso-2,3-butanediol, glycerol or 1,2-propanediol. The tube was closed and incubated for 24 h at 30◦C in a culturing wheel. After incubation, 1 mL was harvested by centrifugation for 3 min at 15,000 rpm at 4◦C. The samples were filtered and subsequently analysed by HPLC. Dehydratase reaction in concentrated suspensions of E. coli. E. coli XL1 harbouring pHKH pddABC, pHKH pddABCddrAB or pHKH were grown in LB medium supplemented with 0.1 µg/mL cyanocobalamine at 37◦C and 200 rpm for 24 h. Cells were harvested by centrifu- gation (4,000 rpm, 15 min) and washed once in BG11 medium supplemented with 25 mM TES, pH 8.0. Then, the cells were resuspended to a calculated OD600 of 100 in BG11 medium supplemented with 25 mM TES, pH 8.0, Km20 and either of the following: 25 mM meso- 2,3-butanediol or 25 mM 1,2-propanediol. Cells were incubated at 37◦C for 24 h in 1.5 mL Eppendorf tubes. Then, the cells were pelleted by centrifugation (15,000 rpm, 15 min) and the supernatant was analysed by HPLC with respect to (exo)metabolite formation. Dehydratase reaction in Synechocystis. Cells were incubated in closed Eppendorf tubes wrapped in aluminium foil. Wild-type Synechocystis and Synechocystis PDD DDR were in- oculated to an OD730 of 2.8 in BG11 medium buffered at pH 8.0 with 10 mM TES/KOH and 114 8.T OWARDSSYNTHESISOFBUTANONEAND 2-BUTANOL BY Synechocystis

supplemented with 0.01 mg/mL cyanocobalamin, 20 mM meso-butanediol, 5 mM glucose and incubated for 17 d at 30◦C. Then, the samples were cleared by centrifugation (14,500 rpm, 5 min, RT) and the supernatant was analysed by HPLC. Conversion of CO2, light and water into 1,3-propanediol. Wild type Synechocystis and Syn PSA002 were grown in BG11 medium buffered to an initial pH of 8.0 with 10 mM TES/KOH. The cultures were inoculated with an OD730 of 0.1. L. reuteri was grown overnight in MRS medium at 30◦C, harvested by centrifugation (4,000 rpm, 15 min, RT) and washed with BG11 medium, buffered at pH 8.0 with 10 mM TES/KOH. Then, the L. reuteri cells were added to the Synechocystis cultures to an OD730 value of 0.1.

RESULTS AND DISCUSSION

Earlier, we demonstrated synthesis of meso-2,3-butanediol from CO2, light and water [115]. To extend the heterologous pathway used in this study to 2-butanol (Fig. 8.2B), two reac- tions need to be added: the dehydration of meso-butanediol to butanone and the reduction of butanone to 2-butanol. Interestingly, we could show that Syn ARP could reduce butanone to 2-butanol (Fig. 8.3). Conversion of butanone, however, was slow, and a large fraction evaporated before it could be converted to 2-butanol. Likely, acetoin is the preferred substrate for this enzyme. In a final production strain, however, the volatility of butanone should not be a problem. Ac- etaldehyde, the precursor of ethanol shows greater volatility; yet, ethanol-producers so far are the best genetically modified cyanobacteria in terms of titre [102]. We analysed recombinant E. coli for their ability to convert meso-butanediol into butanone. Recently, Yoneda et al. reported the reaction of meso-butanediol to butanone using recom- binant E. coli harbouring glycerol dehydratase plus the cognate reactivase from K. pneu- moniae. In this publication, a mutant strain was reported, in which the pathway from glu- cose to butanone was successfully implemented [306]. Under the conditions reported in [306], we could not detect formation of butanone in recombinant E. coli harbouring diol 8 dehydratase or the combination of diol dehydratase and diol dehydratase reactivase from K. oxytoca. Even extended incubation times (up to 2 d) did not lead to detectable amounts of butanone. We therefore repeated the dehydratase reactions at increased cell densities (OD600 values of 10 and 100, respectively, Fig. 8.4). In recombinant E. coli harbouring propanediol dehy- dratase of K. oxytoca, conversion of the native substrate, 1,2-propanediol could be demon- strated at both cell densities. The dehydratase activity was increased in strains harbour- ing the cognate reactivase (Fig. 8.4A,B). The dehydratase reaction yields propionaldehyde, which likely is reduced by endogenous alcohol dehydrogenases to 1-propanol. For the al- ternative substrate, meso-butanediol, conversion could be demonstrated, but was signifi- cantly less efficient (Fig. 8.4C,D). Under ’normal’ growth conditions (cultures grown in the light in Erlenmeyer flasks with cot- ton stoppers) no conversion of meso-butanediol to butanone could be demonstrated with Synechocystis harbouring either PddABC or PddABC and DdrAB from K. oxytoca, despite the fact that soluble diol dehydratase was produced (Fig. 8.5B). Diol dehydratase is inactivated by oxygen. Furthermore, the volatility of butanone under these culturing conditions (Fig. 8.3) makes its detection difficult. We therefore reasoned that conversion possibly might be improved and better monitored under microanaerobic 115

A B OH OH pyruvate acetolactate acetoin + Ado-CH2 HO HO O O O O - Ado-CH 3 2

H H H O- O- O - CO2 - CO2 H OH OH HO + Ado-CH HO 3 + NADPH - Ado-CH HO H 2 HO H - NADP

OH + NADPH O HO NH O Ado = 2 - NADP N N

- H O H O N N 2 HO propionaldehyde HOH OH butanol butanone butanediol

Figure 8.2: Minimal mechanism for the dehydratase reaction (adapted from Eda et al. [291]) (A) and synthetic pathway for the synthesis of 2-butanol from pyruvate (B) .

butanone 2-butanol 5 8

2.5 c / mmol/L

0 0 1 2 3 4

Figure 8.3: Recombinant Synechocystis harbouring acetoin reductase from Leuconostoc lactis NCW1 can reduce butanone to 2-butanol. A substantial fraction is lost due to evaporation. Error bars denote standard deviations of three biological replicates. Error bars that are not visible are smaller than the respective data point symbol. 116 8.T OWARDSSYNTHESISOFBUTANONEAND 2-BUTANOL BY Synechocystis

A n-propanol B 1 mM

E. coli PDD DDR E. coli PDD Intensity AU / E. coli pHKH 30 31 32 33 34 35 36 30 31 32 33 34 35 36 t / min t / min

C butanone D 1.0 mM 0.5 mM 0.1 mM

E. coli PDD DDR E. coli PDD Intensity AU / E. coli pHKH 31 32 33 34 35 36 37 31 32 33 34 35 36 37 t / min t / min

Figure 8.4: Dehydratase reactions catalysed by recombinant E. coli, harbouring K. oxytoca propane- diol dehydratase (pddABC) or a combination of dehydratase and reactivase (ddrAB). A strain harbour- ing just pHKH is shown as a control. Conversion of 1,2-propanediol to n-propanol in a highly concen- trated (A) and very highly concentrated suspension (B). Conversion of meso-butanediol to butanone in a highly concentrated (C) and very highly concentrated (D) cell suspension.

8 Table 8.1: Strains used in this study

Strain Genotype Source E. coli XL1 Blue endA1, gyrA96(nalR) thi–1 recA1 relA1 lac Stratagene q glnV44 F’[::Tn10 proAB+ lacI ∆(lacZ)M15] hsdR17 (rK− mK+) E. coli XL1 PDD XL1 pHKH pPS1310 This study E. coli XL1 PDD DDR XL1 pHKH pPS1320 This study Synechocystis sp. wild-type D. Bhaya PCC 6803 Synechocystis ARP ∆slr0168 Ptrc::arLeu::KmR [115] (AR of Leuconostoc lactis NCW1) Synechocystis PDD ∆slr0168::PA1l acO 1::pddABC::KmR This study − Synechocystis PDD DDR ∆slr0168::Ptrc::ddrAB::PA1l acO 1:: This study − pddABC::KmR Synechocystis PSA002 ∆slr0168::Ptrc::gpp2::KmR [229] Lactobacillus reuteri wild-type [307] 117

A B

Butanone 0.5 mM Butanone 0.1 mM WT Syn PDD Syn PDD DDR

Syn PDD DDR

Intensity / AU Syn WT

30 31 32 33 34 35 36 t / min

Figure 8.5: Recombinant Synechocystis can convert meso-2,3-butanediol into butanone. (A) Conver- sion of meso-butanediol to butanone by Synechocystis PDD DDR. Chromatograms of the pure stan- dard is shown on top. Chromatograms of culture supernatants are shown in the bottom half. Dotted lines are included to aid the eye. (B) soluble diol dehydratase is produced in recombinant Synechocys- tis. Western blotting after denaturing SDS-PAGE of cell-free extracts of the mutant strains carrying dehydratase alone or in combination with the reactivating enzyme, clearly showed a band between 65 and 80 kDa (for comparison: PddA: 60 kDa, PddB: 24 kDa, PddC: 19 kDa).

8

Table 8.2: Plasmids used in this study

Plasmid name Description Source pPS1309 pHKH Ptrc::ddrAB Genscript pPS1310 pHKH PA1l acO 1::pddABC Genscript − pPS1320 pHKH Ptrc::ddrAB::PA1l acO 1::pddABC This study − 118 8.T OWARDSSYNTHESISOFBUTANONEAND 2-BUTANOL BY Synechocystis

A Syn WT B Syn PSA002 10 5 10 5 /L /L 5 4 4 5 1 1 730 3 730 3 OD 2 OD 2 0.1 0.1 1 1 cell counts / 10 0.01 0 0.01 0 cell counts / 10 0 2 4 6 8 10 0 2 4 6 8 10 t / d t / d

7d 7d 2d 2d 1d 1d

Intensity / AU 20 21 22 23 24 25 Intensity / AU 20 21 22 23 24 25 t / min t / min C Syn WT D Syn PSA002 L. reuteri L. reuteri Syn WT + L. reuteri Syn PSA002 + L. reuteri 10 5 10 5 /L /L 5 4 5 4 1 1 730 3 730 3 OD 2 OD 2 0.1 0.1 8 1 1 cell counts / 10 cell counts / 10 0.01 0 0.01 0 0 2 4 6 8 10 0 2 4 6 8 10 t / d t / d 1,3-propanediol

7d 7d 2d 2d 1d 1d

Intensity / AU 20 21 22 23 24 25 Intensity / AU 20 21 22 23 24 25 t / min t / min

Figure 8.6: Synthesis of 1,3-propanediol from CO2 by a consortium of a recombinant Synechocystis strain and L. reuteri. OD730, cell counts and chromatograms for WT (A), Syn PSA002 (B), WT and L. reuteri (C), Syn PSA002 and L. reuteri (D). Only the consortium of Syn PSA002 and L. reuteri showed production of a small amount of 1,3-propanediol. Arrows show timepoints at which L. reuteri was added. 119 or anaerobic conditions. When Synechocystis harbouring PddABC and DdrAB from K. oxy- toca was incubated for extended time periods (17 d) under microaerobic conditions in the dark, a small fraction of exogenously supplied meso-butanediol indeed was converted into butanone (Fig. 8.5A). Recently, Hirokawa et al. showed the conversion of glycerol to 3-hydroxypropionaldehyde (reuterin) and the subsequent reduction to 1,3-propanediol in Synechococcus elongatus PCC 7942 [304]. We, however, were unable to demonstrate conversion of glycerol to reuter-in11 or 1,3-propanedeiol in Synechocystis. One explanation for this might be an inability of Syne- chocystis to take up glycerol12. 2-butanone is mobile across the cyanobacterial membranes. Dehydratase activity could be demonstrated in a mixed culture of recombinant Synechocys- tis and Lactobacillus reuteri (L. reuteri) (Fig. 8.6). In this experiment, Syn PSA002 produces glycerol from CO2, light and water [229] and L. reuteri performs the conversion of glycerol to reuterin. At this moment, it is unclear whether the reduction to 1,3-propanediol is carried out by L. reuteri or by Syn PSA002. In L. reuteri, reduction of reuterin to 1,3-propanediol is dependent on the presence of substrates like glucose, which were not added exogenously in this experiment. Cyanobacteria, however, have been reported to shed carbohydrate com- pounds into the culture medium [280], which might be taken up by the lactic acid bac- terium. The genome of Synechocystis encodes at least two alcohol dehydrogenases (Slr0942 and Slr1192). It is conceivable that one of these enzymes shows activity towards reuterin. In conclusion, we could demonstrate the dehydration of meso-butanediol to butanone in both recombinant E. coli and Synechocystis. However, the activity of the heterologously ex- pressed dehydratase with the non-native substrate 2,3-butanediol, needs to be significantly increased if this reaction is to be run on a meaningful scale in cyanobacteria. Additionally, we could show the conversion of CO2 to 1,3-propanediol using a consortium of recombi- nant Synechocystis and L. reuteri.

ACKNOWLEDGMENTS We thank T. Toraya and K. Mori (Okayama University) for kindly providing antibodies di- rected against Diol dehydratase. 8

11On the HPLC system, detection of reuterin was difficult, as the peaks were very broad and overlapping with others. 12In chapter6, upon incubation of cell-free extracts of Synechocystsi with glucosylglycerol, the small amount of contaminant glycerol was consumed, even when the cell-free extracts were previously purified from small mol- ecules, via dilution and subsequent concentration with a protein concentration filter (data not shown). When wild-type Synechocystis was grown in the presence of small amounts of glycerol in the dark, (5 mM), no con- sumption was evident, as judged by HPLC (data not shown)

9 GENERALDISCUSSION

The Stone Age came to an end not for a lack of stones And the Oil Age will end, but not for a lack of oil. Ahmed Zaki Yamani

121 122 9.G ENERALDISCUSSION

HE aim of this chapter is to put the research that was carried out in the course of this the- T sis into a broader perspective. In the introduction to this thesis, it has already been laid out how cyanobacterial biotechnology can aid to close the carbon cycle, while not affecting other resource cycles. In the following section, methods for the optimisation of metabolic pathways as well as the cyanobacterial host will be discussed. Subsequent sections address challenges regarding strain stability and contamination. The chapter closes with consider- ations on the economic feasibility of this approach.

CHASSIS OPTIMISATION Conceptually, to introduce a heterologous enzymatic pathway into an organism is to add a very small set of reactions to a very large set of reactions. The majority of publications in the genetic engineering of cyanobacteria has so far focused on the implementation and op- timisation of these few additional reactions. In Chapter2, progress in this field is reviewed. Further information can be found in other more recent reviews [308–310]. One way to increase the flux through a metabolic pathway is to increase the ac- Metabolic control analysis. Essentially, J, tivities of the enzymes that constitute this the flux through an enzymatic reaction, is pathway1. At some point, however, fur- determined by the activity of the respective ther optimisation of this added pathway enzyme. In order to analyse the contribu- does not result in increased product lev- tion of an individual enzymatic reaction i els. Thus, Angermayr et al. used metabolic to the total flux through the pathway, Jtotal , control analysis (see box for a short expla- lnJ can be plotted against ln[Ei ]. The slope nation and [147] for a review) to determine of this curve is given as the derivative: at which fluxes the productivity of lactate- ∂lnJtotal synthesising Synechocystis is insensitive to ∂[Ei ] further optimisation [134]. In this study, This derivative is the control coefficient. it was shown that even in the mutant with When the control coefficient is 1, the re- the highest LDH activity, control still fully spective enzymatic reaction is said to have resided on the heterologous pathway. In a full control. follow-up study, Angermayr et al. demon- strated that even higher LDH activities re- sulted in a partial loss of control [106]. In 9 the resulting mutant strains, lactate titres could be increased, when pyruvate availability was increased2. The largest3 bottlenecks therefore have to be identified prior to any attempts at chassis op- timisation. For a recent review on the subject, see [311]. Matching the flux through an added pathway with the fluxes from central metabolism is one step towards a highly productive cyanobacterial cell factory. Another approach is to increase the total amount of carbon that is fixed by a cell. This seems to happen naturally in mutants, in which the heterologous pathway consitutes a significant carbon sink4, but can also be engineered. Atsumi et al. demonstrated that overproduction of RuBisCO, in combi-

1Among other things, by increasing the concentrations, substituting with more active enzymes, enzyme engineer- ing, allosteric activators. 2Via the knockdown of phosphoenolpyruvate carboxylase. 3Or, rather, the smallest. 4see e.g. Chapter3,[105]. 123 nation with a heterologous pathway, increased isobutyraldehyde titres [148]. Alternatively, the inactivation of competing metabolic reactions can increase the amount of carbon that can be converted into product. As an example, van der Woude et al. showed that inactiva- tion of glycogen biosynthesis led to increased lactate production in Synechocystis [146]. Other approaches may include increasing the amount of energy that is available to the cy- anobacterial cell. Cyanobacterial photosystems can only utilise a fraction of incident solar radiation. In an effort to harness a broader wavelength range for the growth of Synechocys- tis, Chen et al. recently reported the functional expression of proteorhodopsin [312]. Light- induced proton pumping could contribute to the proton gradient accross the thylakoid and cytoplasmic membranes and thus allows higher rates of ATP synthesis. Thus, the energy that seemingly is wasted in processes such as cyclic electron flow around PS I5 could be utilised elsewhere. Rather than adding new photoactive proteins, the existing complexes could be altered such that they utilise a greater part of the solar spectrum. In dense cultures, a truncation of the light harvesting antennae has been suggested to in- crease the amount of photons available for each cell. It was shown that under high light conditions, a phycocyanin deficient strain of Synechocystis accumulated more biomass than the wild type strain [313]. Research in the fundamentals of the cyanobacterial transcriptome, proteome and metabolome as well as physiology and regulatory systems will further the understanding of these organisms. With increased understanding, heterologous pathways will be better integrated with general metabolism, thereby allowing higher fluxes and increased stability. If cyanobacteria-based organic molecules are to compete economically with those derived from other sources, all aspects of the cell need to be optimised and improved. Therefore, holistic approaches have to be taken for optimisation [314]. Future approaches will have to focus on the isolation of new strains.

COSUBSTRATE SPECIFICITY AND ALTERATIONS In wild-type Synechocystis, the NADPH pool is larger and more reduced than the NADH pool [214]. In Chapter4, the same was concluded for mutant strains of Synechocystis that bear heterologous enzymatic pathway for the synthesis of 2,3-butanediol. Chemotrophs rely on substrate catabolism for energy generation. In glycolysis, electrons from the degradation of glucose are transferred to NAD, yielding NADH. Under aerobic con- 9 ditions the electrons can be relayed to oxygen, forming water. Under anaerobic conditions, the cells use fermentative pathways to rid themselves of excess NADH. Many fermentation products are potentially interesting fuels and a wealth of fermentation pathways has already been introduced into cyanobacteria (chapter2). When a given enzymatic pathway is transferred to a heterologous organism, the cosubstrate preference of both pathway and host can be matched or mismatched. If there is a match, there is no problem. If cosubstrate preferences are mismatched, two different approaches can be taken

6 h Increasing the availability of the required cosubstrate .

5One way to adjust ATP/NADPH ratios. 6Either through genetic engineering or through changes in culturing conditions. The latter is more applicable to chemotrophs and will therefore not be discussed here. Examples are referenced in [222]. 124 9.G ENERALDISCUSSION

h Changing the specificity of the reaction step.

In Chapter3, both strategies are demonstrated. Overexpression of a soluble transhydrogenase along with an NADH dependent acetoin re- ductase increased butanediol yields in recombinant Synechocystis. Similar results have been obtained for lactate [135, 137]. The substitution of NADH-dependent acetoin reductase with an NADPH-dependent isozyme increased butanediol titres even further. Comparable results were published for 3-hydroxybutrate [122] in Synechocystis as well as for isobutanol [148], n-butanol [101] and 1,2-propanediol [112] in Synechococcus elongatus. If there is no enzyme available that has the required preference, two strategies are conceiv- able:

h Changes in substrate preference of an NADPH-dependent enzyme

h Alteration of cosubstrate specificity of an NADH-dependent enzyme

Li and Liao demonstrated the first approach [315]; in this study, the NADPH-dependent glu- tamate dehydrogenase from E. coli was engineered to accept 2-oxo-phenylbutyric acid as a substrate. In specificity alterations through protein modification, several outcomes are possible7: The affinity for the non-physiologcal cosubstrate can be increased, the affinity for the native co- substrate can be decreased, or both. Naturally, the last option is the preferred one. When both cosubstrates are accepted with comparable affinity, a system that resembles an energy- independent transhydrogenase can result. This may lead to a redox imbalance [135]. NADH and NADPH are very similar in terms of structure, the only difference being a phos- phorylated 20-hydroxy group in the adenosine moiety of NADPH. There is no crucial differ- ence in the folds of enzymes utilizing either cosubstrate; yet, a given enzyme often shows near-exclusive specificity for one of the two. Specificity must therefore be conferred through interaction of the apoprotein with the 20-hydroxy group of NADH and the 20-phosphate group of NADPH, respectively. There are a number of NAD(P) binding motifs, the most prominent of which is the Ross- mann fold8 [318], consisting of a six-stranded β sheet whose strands are connected by α helices in the fashion (βαβαβ)2. These domains employ an GXGXXG motif located in a small loop between the β1 strand and the α1 helix to bind the pyrophosphate moiety of 9 dinucleotides [319]. Since traditional biotechnology has largely relied on the catabolism of chemotrophic organ- isms, more research efforts have been devoted to change the preferred co-substrate from NADPH to NADH9. Chen et al. changed the cosubstrate specificity of isopropyl-malate de- hydrogenase from NADH to NADPH [322]. Similarly, Ehsani et al. changed the cosubstrate specificity of Bdh110 [323]. Conceptually, using the organism’s preferred cosubstrate has two effects: Assuming that

k k 7cosubstrate specificity alteration can be defined as cat,NADPH / cat,NADH [316]. This ratio alone, however, KM,NADPH KM,NADH should not be used to decide whether a mutant should be used, as decreased preference of the native cosub- strate and increased preference of the non-native cosubstrate can give similar values. 8 Other examples of dinucleotide binding domains include the (β/α)8 barrel found in NADP- dependent aldose and aldehyde reductases and in the 3-α-hydroxysteroid/dihydrodiol dehydrogenase [317]. 9For examples, see references in [222, 320, 321]. 10Encodes butanediol dehydrogenase. 125 the enzymatic reaction is fast enough, a more favourable balance of the oxidised and the re- duced cosubstrate provides a greater driving force. An effect related to this is that recycling of the preferred cosubstrate is faster and may allow for higher fluxes through the pathway. From these two considerations, it follows that the effect of a cosubstrate substitution de- pends on the position of the reduction step within a pathway. Thus, changing the speci- ficity of a reaction upstream of a slow or an irreversible reaction will have no effect; chang- ing the availability of substrate for a downstream reaction will only increase fluxes when the downstream reaction is not saturated. The effect of a cosubstrate substitution must then be greatest in the last step of a pathway (as is the case in Chapter3). cosubstrate substitutions in the acetyl-CoA derived n-butanol pathway, heterologously ex- pressed in Synechococcus elongatus, follow these predictions [101]: There are four reduc- tion reactions in this pathway and NADPH-dependent enzymes were available for three of these steps. Substitution of the two reduction reactions at the end of the pathway (butyryl- CoA to butyraldehyde and subsequently, n-butanol) had a profound effect. In comparison, the resulting changes in titre were negligible when the reduction reaction further upstream (acetoacetyl-CoA to hydroxybutyryl-CoA) was altered. Ultimately, the flux through the entire pathway will have to be streamlined, and changing the cosubstrate dependence of a metabolic pathway is only one of many conceivable opti- misation strategies (for a recent review, see [310]).

GENETIC STABILITY No genome is stable. Mutations can arise in a number of ways, including errors in DNA replication and recombination, invasion of the host cell with foreign DNA, exposure to mu- tagens11, and incorrect repair after damage to DNA [17]. Mutations that arise in a single cell will typically not be visible on the population level, un- less they are selected for under the culturing conditions. Mutations that cause severe im- balances within a cells are typically selected against. In Chapter5, we attempted to create a mutant strain of Synechocystis that harboured gpp112 of S. cerevisiae for the production of glycerol from CO2, light and water. Segregation of this strain proved difficult and was eventually accompanied by loss of phenotype. In the same chapter, we could demonstrate the stable production of glycerol by a mutant strain har- bouring gpp2 for nearly 20 generations13. Genetic instability in cyanobacteria seems to be frequently observed, but not often reported 9 [158]. Hence, only a handful of examples have ended up in the literature, among them ethylene- [324], lactate [135] and butanediol-producing Synechococcus elongatus [325] and mannitol-producing Synechocystis [151]. Conceptually, when a pathway is simply added to the metabolism of a cell14, the rate at which this transgenic cell can divide is often decreased due to a combination of several ef-

11Molecules or ions with extended cyclic π-systems (e.g. ethidium cations) can intercalate between DNA bases, causing errors in transcription and, crucially, DNA replication. Reactive molecules can cause breaks in the sugar phosphate backbone or modify the chemical structure of DNA bases. Oxygenic photosynthesis produces a va- riety of highly reactive oxygen species (ROS). While cyanobacteria have repair mechanisms to both neutralise these species and to undo the damage caused by them, the repair mechanisms can fail. 12gpp1 encodes phosphoglycerol phosphatase 1. 13At which point, the experiment was stopped. There was no reason to assume that glycerol production was re- duced at this point. 14This also includes approaches, where enzymes that are part of a native pathway are overproduced. 126 9.G ENERALDISCUSSION

fects: The genome size and the total amount of mRNA15 increases. Enzymes are produced that do not benefit the cell and therefore are a burden on the metabolism. Metabolites are continuously diverted from central metabolism into the heterologous pathway. While the effects of the added pathway on the genome, transcriptome and proteome may be minor, the effect on the metabolome may be more significant. Crucially, central metab- olism and the heterologous pathway are competing for resources. Especially when a large fraction of the metabolites is channeled into the pathway, mutations that shift the balance towards central metabolism16 will make more material available for growth. Cells bearing such mutations consequently are at an advantage and, given time, will ac- cumulate in the population, reducing its overall productivity. The more cell doublings are required (e.g. to bring a bioreactor to a certain optical density), the more probable it is for such mutations to arise. The earlier they do, the more likely it is that they take over the cul- ture. A decrease in the frequency in which mutations arise17 delays this problem. Accordingly, the runtime of a batch process could be increased, but once an undesired mutation arises, it will get fixed. Fundamentally, two strategies can be envisioned to counter this.

h Coupling production to survival

h Decoupling growth and production

Coupling production to survival. When a cell relies on a metabolic pathway for survival, loss-of-function mutations in this pathway lead to growth defects and consequently cannot propagate themselves. Coupling a heterologous pathway to the survival of the cell would thus ensure functionality of this pathway in a growing culture. One function of a living cell is to harness the energy within a substrate and to maintain a balance of energy carriers under a wide range of substrate uptake fluxes. Chemotrophic organisms catabolise substrates such as glucose to generate energy, which drives biosynthesis. Energy homeostasis (i.e. ATP/ADP,NAD(P)H/NAD(P) ratios) in the face of varying substrate input is achieved through alternative and redundant metabolic routes and a suite of regulation mechanisms. For photoautotrophs, the substrate that is harnessed for energy generation are photons, so the ATP/ADP and NAD(P)H/NAD(P)-ratio need to be resilient to changing photon influx. 9 This is achieved through alternative electron flow and other mechanisms, such as exciton annihilation or non-photochemical quenching. Conceptually, one way to increase the genetic stability18 of a strain which bears a heterol- ogous pathway is to have this pathway neutralise a metabolic imbalance. If a reduction reaction in a heterologous pathway is the only means by which a cell can rid itself of NADH, the survival of the cell is coupled to the presence of that functioning pathway. Although strain stability was not the prime objective of either of the two studies that will be discussed in the following, their comparison nonetheless is an illustrative demonstration of the challenges that metabolic engineering in photoautotrophs is facing, relative to those in

15Or the fraction of mRNA that does not have a function in cell growth. 16e.g. mutations that lead to impaired function or that reduce the concentrations of one or more enzymes in the pathway. 17e.g. deletion of recombinases or increase in polymerase fidelity. 18By which is meant the absence of selection for loss-of-function mutations. 127 chemotrophs. Shen et al. demonstrated increased butanol production in an E. coli strain deficient in fdrBC, ldhA, adhE and pta19 which carried a heterologous pathway from Clostridia [326]. Deletion of phospho-transacetylase increases the amount of acetyl-CoA that can be made available to the butanol pathway, which originates from this metabolite. Inactivation of fumarate reductase, as well as lactate and alcohol dehydrogenase, lowers the organism’s capacity to oxidise NADH under anaerobic conditions. This means that a metabolic imbal- ance is achieved through the prior reduction of metabolic robustness. Similarly, stability of an added mutation can be increased in cyanobacteria if a metabolic imbalance is rescued by a heterologous metabolic pathway. A prime example is given by Angermayr et al. [135]. There, a presumed imbalance in the NADH/NAD pool, introduced through the production of soluble transhydrogenase (energy-independent STH20, encoded by sth), can be rescued through the concomitant introduction of an NADH-depen-dent lac- tate dehydrogenase (LDH). There is, however, a crucial difference between these two approaches: In the example given by Shen et al., pathways that mitigate metabolic imbalances are taken away and replaced with the heterologous pathway. In the study by Angermayr et al., an added metabolic im- balance is rescued by an added heterologous pathway. While it is exceedingly unlikely that NADH-consuming systems evolve from scratch, the approach of Angermayr et al. is vulner- able to mutations in sth21. There is a set of deletion mutations that are predicted to reduce the photoautotroph’s ca- pacity for redox cofactor homeostasis [327]. These mutations will come at the cost of pho- tosynthetic robustness. Since for low-value molecules such as fuels, cultivation will most likely only be commer- cially feasible when sunlight is used, light influx is expected to vary periodically (day/night rhythm) as well as erratically (weather, culture mixing). With this in mind, sacrificing alter- native electron flow pathways will likely lead to poor growth characteristics in large-scale applications, where photons originate from the sun. Potentially, solar cells could be used to convert sunlight into electricity, which could in turn be converted to photons that are tailored to the photosystems using LEDs. This could elimi- nate fluctuations due to day/light-rhythms and weather. Sudden changes of light regime as a consequence of culture mixing, however would not be circumvented with this approach. A compelling idea is followed by Du et al. (personal communication): In some steps of biomass precursor biosynthesis, acetate ions are eliminated from precursor molecules. These 9 acetate ions are then reassimilated by the cells. In Synechocystis, Du et al. removed genes that are involved in acetate uptake, generating mutant strains in which the production of acetate is coupled to amino acid biosynthesis and by extension, survival. These mutant strains have been show to display a stable acetate-producing phenotype. While this ap- proach is novel in its coupling of production and survival, it is limited to molecules that are byproducts of essential biosynthetic reactions.

19fdrBC: encodes subunits of fumarate reductase, ldhA: encodes lactate dehydrogenase A, adhE: encodes alcohol dehydrogenase E and pta: encodes phosphotransacetylase. STH 20STH catalyses the reduction of NAD by NADPH and vice versa: NADPH + NAD NADP + NADH. 21Functioning STH increases the NADH/NADPH and the NADH/NAD ratio. Impaired or lost function of STH restores the wild-type ratios. Since lower amounts of NADH result in lower amounts of lactate, sth mutants have more fixed carbon at their disposal, will show a higher growth rate and ultimately outcompete cells with functioning STH. 128 9.G ENERALDISCUSSION

Decoupling growth and production. Since in cyanobacteria, coupling production and sur- vival will come at the cost of photosynthetic robustness, a more feasible approach would be to completely decouple growth and production. Accordingly, a culture would be allowed to grow to a certain density, at which transcription of the genes encoding a heterologous path- way is induced. Mutations affecting this pathway can and will arise during the growth phase but will not be selected for, since there is no flux through the pathway and consequently no competition with central metabolism. Heterotrophs can metabolise carbon sub- Regulation of protein levels. The intra- strates when there is no growth [328]. Thus, cellular concentration of an enzyme over physiological uncoupling can be reached time is dependent on the frequency with by constraining growth conditions [329]. which the polypeptide is produced and This has not been experimentally demon- on its lifetime within a cell. The rate at strated in cyanobacteria. which a protein is synthesised is in turn Decoupling can be achieved in a number dependent on the amount of mRNA, the of ways, all of which involve the regula- frequency with which ribosomes bind to tion of protein levels22. The frequency with it, and the rate of translation. The amount which an RNA polymerase binds to a pro- of a given mRNA again is a function of the moter can be modulated through different rate at which it is produced and degraded, promoters. When separation of growth and respectively. The rate of mRNA synthesis production is to be achieved, constitutive depends on the frequency with which RNA promoters have to be ruled out (see [92] for polymerase binds to the promoter region a list of inducible promoters in cyanobac- and on the speed with which it transcribes teria). the gene. All of the steps mentioned here Addition of chemicals for induction of gene are potential targets for the fine-tuning of expression is a handy process in the labo- absolute and relative enzyme levels in a ratory, but will be cumbersome on a large (heterologous) metabolic pathway. scale. Moreover, when low-cost molecules are to be produced, the cost of the inducer may outweigh that of the molecule that is synthesised. Alternatives include promoters that are inactive in growing cells and active in non-growing cells. An interesting approach would be to repurpose quorum sensing23. Best-characterised in Vibrio fisheri, evidence for quorum sensing machinery was demonstrated in cyanobacte- 9 ria [331–334]. The transfer of such systems to producer strain would lead to induction at a certain culture density. The induction density could be altered by varying the levels of the enzymes that produce the signaling molecule24. More feasible systems are those that are repressed by a nutrient that is exhausted as the culture reaches its target density. Examples include the nirA promoter from Synechococcus – + elongatus PCC7942 (induced by NO3 , repressed by NH4 ) or the isiA promoter (repressed by

22An approach in which an inactive enzyme is produced during the growth phase and activated in the production phase will ultimately not be viable, as the enzyme, even in the inactive form, presents a burden to the cell. 23Essentially, cells synthesise small amounts of a molecule that is mobile accross bacterial membranes and can therefore diffuse from one bacterium into another. The bacteria harbour promoter system(s) that respond to the molecule, but only when it is present in concentrations that surpass a certain threshold. Since the amount of the signalling molecule depends on the amount of bacteria, a response will only occur in dense cultures. See [330] for a review. 24Note that this approach is vulnerable to mutations in the signal molecule pathway. 129

Fe3+) (both referenced in [92]). Gene expression would then be switched on automatically, making intervention unnecessary. Furthermore, promoters that are inducible with light of a wavelength range different from that required for growth could be utilised. There, expres- sion could be induced manually (e.g. by changing a colour filter on top of the reactor) when the target density is reached. Examples of light-inducible systems include those involved in chromatic adaptation. Miyake et al. demonstrated a mutant Synechocystis strain that har- boured lytic genes of T4 bacteriophage under control of the green light-inducible cpcG225 promoter. Lysis could be demonstrated upon exposure to green light, suggesting that regu- lation of this promoter is tight [160]. In addition to the frequency of DNA-binding of RNA polymerase, its progress along the gene can be impaired through DNA-binding elements. In Synechocystis, Yao et al. [335] demon- strated multiple gene repression using CRISPRi [336]. Recently, a correlation between the enzymatic activity of and the flux through a heterologous pathway was demonstrated in Synechocystis [134]. With this in mind, the use of strong inducible promoters becomes ob- vious. Tight repression, however is even more crucial, to avoid activity of the heterologous pathway during the growth phase. Higher mRNA levels could then be achieved through amplification of the initial signal using an orthogonal polymerase system (e.g. the T7 polymerase system, functionality of which was demonstrated in Anabaena over two decades ago [337]). While there are many well- characterised promoter systems for biotechnologically important organisms such as E. coli and S. cerevisiae, there are only a handful of studies in cyanobacteria, where inducible pro- moter systems are characterised. Further research will be necessary to gain insight into the mechanisms by which gene expression in cyanobacteria is regulated, to quantify time- resolved responses and to ultimately increase robustness in induction systems. In a highly interesting conceptual study in E. coli, Soma et al. designed a metabolic tog- gle switch that, upon addition of IPTG, results in expression of a heterologous isopropanol biosynthesis pathway and, crucially, in downregulation of citrate synthase, effectively stop- ping growth [338]. With appropriate molecular tools, similar approaches could be realised in cyanobacteria. Ultimately, stability is a question of timescale. If cultures are to be grown in large-scale re- actors, their phenotype only has to be stable for the time that a particular reactor is in use, in order for yield to be maximised. 9 CHALLENGESINTHEUSEOF BACTERIAL MONOCULTURES Most published research concerning genetically manipulated cyanobacteria describes lab- oratory studies of pure cultures (Chapter2,[104, 310, 339, 340]). In agriculture, it has long been known that monocultures are characterised by high per plant yields, but are prone to infection by pathogens, since the genetic markup of all members is similar or even identical. Analogous thoughts apply to liquid cultures in biotechnology. This means that chemicals could theoretically be used to keep contaminants at bay. If a production system was completely shut off from its surroundings, contamination could not occur. Although perhaps evident, it should still be mentioned that contamination only poses a problem, when the culture is affected. For large scale applications of cyanobacteria that produce low-value chemicals, maintaining axenicity would be theoretically possible,

25encodes the phycobilisome rod-core linker peptide CpcG2. 130 9.G ENERALDISCUSSION

but likely not economically justifiable.Indeed, contamination is a severe problem in cyano- bacterial culturing [341]. Fundamentally, contaminants can threaten productivity in a microbial monoculture in sev- eral ways:

h The producers can be consumed.

h The contaminant and the producer can compete for resources.

h The product can be consumed.

Producer consumption. Cyanobacteria can be consumed by phages. Bacteriophages are viruses that affect bacteria. For Synechocystis, no phages have been identified so far. In higher organisms, viral infections can be combatted with immune systems. The bacterial analogue of immune systems is the CRISPR26/Cas system. Since CRISPR/Cas functions at the DNA/RNA level, recombinant DNA technology potentially allows to realise "prokaryotic vaccination"; vaccine development should be trivial if the nature of the phage is known. Such a strategy can be successful if bioreactors are plagued by known phages. Since phages can rapidly destroy entire cultures, it is doubtful whether this technique would be fast enough to save an infected batch. The next batch, however, would be resistant to infection by this particular phage. Given that phages can mutate, a likely result would be an arms race. An alternative mechanism, by which producers are threatened, is consumption through bacteria or eukaryotic grazers. Grazers are organisms that can consume others. Grazing can be specific and unspecific. For microalgal bioreactors, different species of zooplankton have been identified27. Specific grazer-prey interaction could be fought through modifica- tion of the cyanobacterial cell surface as demonstrated in [343]. While response times for such approaches would be too slow to have any meaningful impact on specific grazing, un- specific grazing would not be affected at all. One way to address parasitism in agriculture is to utilise biological pest control [344]. Thus, zooplanktivores could be used to keep zooplankton at bay ([345] and references therein). Competition for resources and product consumption. Cells are not only made up of car- bon, so, along with the product, other nutrients will likely be taken up by a contaminant. If it is known, which species usually affect cyanobacterial cultures, other carefully selected species can be deliberately introduced so that this particular ecological niche is occupied 9 [345]28. Current approaches include the use of extreme conditions (high pH and salinity) to ward off contamination [341]. From there, it is a logical step to consider more extreme conditions, i.e. to utilise extremophiles. Note that this merely reduces the risk of contamination but does not abolish it altogether; invasion by strains that thrive under extreme conditions is less likely, but not impossible. Contaminated reactors aside, the development and application of extremophilic cyanobac- teria as cell factories is interesting in that some have the capacity to survive conditions in extraterrestrial habitats [346]. Strains of Chroococcidiopsis have been isolated from Antarc- tic deserts as well as from the Atacama and the Mojave desert, places that are considered terrestrial analogues of Martian and Lunar surfaces [346, 347]. Consequently, these strains

26Clustered regularly interspaced short palindromic repeats. See [342] for a review. 27Referenced in [341]. 28This approach would be vulnerable to contaminants that have superior nutrient uptake kinetics. 131 have been suggested as ’photosynthetic pioneers’ [346] to pave the way for the colonisation of Mars [347]. Aside from the colonisation of entire planetary bodies, cyanobacteria have been envisioned as part of self-sustaining communities [348]. Thus, in the experimental MELiSSA29 program [349, 350], Arthrospira is planned to provide food as well as oxygen and fix the CO2 generated by both the crew and by anaerobic waste degradation reactors, using only photon energy. In addition to CO2 recycling and oxygen production, engineered cyanobacteria can synthesise essentially any desired molecule on location [351], making transport unnecessary.

PRODUCTIVECO-CULTURING Contaminants are truly problematic only if they negatively affect production. If selected carefully, however, adding organisms in a culture of cyanobacteria can even be beneficial. co-culturing potentially offers four advantages:

h Nutrients can be made more accessible

h The likeliness of malignant contamination is reduced

h New chemical pathways and reactions can be realised

h Resource re-utilisation is possible

Nutrient accessibility. Under nutrient-deficient conditions, algal growth was shown to be improved by chemotrophs (See [352], where the excretion of siderophores by Halomonas allowed the cultivation of Dunaliella at concentrations of Fe3+ that normally cannot sustain growth). Reduced likeliness of contamination. This point was already addressed in the previous section. New reactions and pathways. From the perspective of chemical pathway design, having an additional compartment can be extremely rewarding30; reaction sequences that otherwise would not be possible could be realised with ease. In Chapters7 and8, such systems are demonstrated. Specifically, in Chapter7, compart- mentalisation provides thermodynamic feasibility for a sequence of redox reactions that otherwise would partly lack driving forces. Additionally, a system is outlined, in which water-derived electrons are transported from Synechocystis to a chemotroph, namely E. coli. 9 The utilisation of these electrons opens the door for chemical conversions that would not be attainable in cyanobacteria. In Chapter8, an oxygen-sensitive reaction step was out- sourced to a chemotrophic organism. This approach is conceptually identical with the one recently published by Wang et al. [353]. Resource recycling. A co-culture is essentially a synthetic ecosystem. On earth, all elements go through cycles (See Chapter1). There is no reason to assume that smaller scale resource re-utilisation cycles, confined to a bioreactor, are technically infeasible. Thus, waste prod- ucts of one organism could be selectively converted into more desirable compounds. The result would be a decrease in contaminant molecules and as a consequence, potentially cheaper downstream processing. Challenges.

29Micro Ecological Life Support System Alternative. 30See [138] for an outstanding review on spatial separation of metabolic processes. 132 9.G ENERALDISCUSSION

The design of co-cultures faces many challenges, the largest Tragedy of the com- of which is stability. In principle, however, stable interac- mons. From the per- tions are possible and can be selected for. The trivial proof spective of an individual, of this statement is that symbiosis can be found in nature, al- sharing a resource with beit growth of natural systems may be too slow for industrial other individuals, it is application. beneficial to maximize In order to illustrate stability problems of engineered sym- gains for oneself. Profit, bioses in reactor contexts, in the following, a hypothetical ex- then, is privatized, while ample will be discussed. losses (i.e. depletion of Consider a co-culture of strains A and B, in which A excretes the resource) are shared 31 resources that B requires for growth and vice versa . If A and among the community. B are grown in a well-mixed medium that does not contain these resources and that is continuously removed32, the cul- ture faces the tragedy of the commons (See box): Let mutations arising in A yield A*33, which competes with A for niche ª. If A* can utilize resources from, but not provide resources to, the community (i.e. it uses the resources more efficiently), it has an advantage over A and will eventually seize ª. As A* takes over ª, levels of B, whose growth depends on A, increase more slowly and even- tually decrease. Drops in B lead to drops in A and A* and eventually, collapse. The problem is absent in structured co-cultures and in strains that are hardwired to share resources. This refers to organisms in which the production phenotype is coupled to the survival of the strain. Considerations on how this may be achieved can be found in the second section of this chapter. When the co-culture is structured, i.e. when both strains are fixed or approximately fixed, and if nutrient diffusion is sufficiently slow on a cell-division scale, A* has difficulty taking over the population, since cells of this mutant cannot sustain growth of B in their immedi- ate vicinity and will thus be at a disadvantage compared to A. In nature, micro-structuring is achieved in a number of environments, including micro- bial mats and biofilms, by filamentous cyanobacteria that generate heterocysts and, as the prime example, endosymbiotic relationships. Also, emulsions can serve as a model of micro-structured systems. In a fascinating paper, Bachmann et al. demonstrated that when Lactococcus lactis is grown under nutrient limi- tation in emulsion, after serial transfers, mutants showing a high yield based on substrate 9 had been selected for [354]. When two or more strains share a droplet –given substrate limitation– the occurrence of cheaters is selected against34. It is important to highlight at this point, that when selective pressure is changing, no pheno- type is stable. Applied to this strategy, it means that a symbiotic relationship evolved in an emulsion-based system would revert when placed in a different environment, e.g. a biore- actor. Ultimately, the ecology of reactor systems has to be carefully studied, and mutual inter- actions between producers and benign contaminants can be exploited to increase overall rates and yields. Thus, a transition from synthetic biology to synthetic ecology [355] will

31e.g. A provides fixed carbon, B provides fixed nitrogen. 32e.g. turbidostat or chemostat regime. 33Sometimes termed a "cheater". A* reaps benefits without making a contribution. 34Note that this is not a rational approach in which genetic information within either strain is purposefully modi- fied. Rather, this method applies selective pressure towards a stable symbiotic relationship. 133 increase resilience of algal and cyanobacterial production systems.

ECONOMIC CONSIDERATIONS AND CONCLUDING REMARKS At this point in time, molecules generated by genetically engineered cyanobacteria cannot economically compete with those derived from fossil sources or fermentation processes, since prices of the latter two are lower. In this context, a price can be defined as "[the] amount of money (or a material equivalent) expected, required, or given in payment for a commodity [...]" [356]. In the simplest case, the price of a commodity is determined by supply and demand. In order for a producer to make a profit, the price at which product is sold must be higher than the production costs. The fossil fuels that society is based on are cheap, because the extraction of raw materials is simple. Fossil fuels are the result of the deposition and decay of organic matter over eons, but their utilisation is a relatively new phenomenon. Since the rate of fossil fuel consump- tion is orders of magnitude higher than the rate of their deposition, supply will eventually dwindle, and prices will increase, making other sources economically competitive. The greatest part of the organic matter that fossil pools were derived from was created with photon energy as the primary input. The same is true for the sugar that is consumed in fermentation processes. When overall efficiencies are calculated, this needs to be kept in mind. Fuel prices do not capture the ecological consequences of fossil fuel combustion, some of which have been discussed in the introduction to this thesis. These consequences are al- ready manifest. Therefore, it is of great importance that alternatives be developed, sooner, rather than later. Processes that rely on cyanobacteria may involve an inititally higher capital expenditure than those that rely on already established technologies, like fossil fuels. The reasons for the high production cost of fuels derived from photosynthetic microbes are manifold (among others: genetic instability, slow growth, low productivity, low product titres, contamination). None of these problems seem fundamentally unsolvable and ap- proaches to overcome some have been addressed earlier in this chapter. Specifically, devising mutants, in which loss of phenotype is selected against or not selected for, will increase the duration per production cycle possible and consequently the number 9 of production cycles per year. This results in higher annual productivity (ton/year) and in lower operating costs, because of reduced down times35. Tailoring metabolic pathways to the preferred resource use of the organism36 (or vice versa) improves the process yield (tonoutput/toninput). Future fundamental research will allow for faster and more efficient growth37 and chassis optimisation will increase production yields and rates, thus increasing annual process pro- ductivity (ton/year), which allows for lower prices (€/ton). When stable synthetic ecosystems are developed, in which water, photons and carbon diox- ide are the only input and fuel is the only output, built-in recycling of resources will reduce

35Charge and discharge of production vessel, cleaning, sterilisation and process restart. 36See the section on cofactor specificity in this chapter. 37Taken to its extremes, synthetic biology allows to make artificial "minimal cells", which convert energy more effientily than natural organisms. 134 9.G ENERALDISCUSSION

overhead costs. Decreasing the likeliness of contamination would allow a given process to run longer, and in consequence increase annual productivity. If externalities, such as the detrimental effect the use of fuels has on ecosystems, are taken into account, cyanobacteria-derived fuels may already be cheaper than established fuels. Although the production of cyanobacteria-derived fuels and chemical feedstocks is a rela- tively recent research field, impressive results have already been achieved. If this technology is to be applied, it will have to compete economically with many alternative approaches. Further studies should now also address economic feasibility, because a bewildering array of alternative (bio)chemical conversions can be proposed based on the availability of the same amount of solar energy. With future development, this technology has the potential to change the energy basis of society. In this process, changes will have to be made on every level of politics and economics, possibly even starting with the curricula.

ACKNOWLEDGEMENTS I am indebted to Sabrina Zaini, for insightful suggestions on economic considerations.

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APPENDIX

155

A ABBREVIATIONS

Abbreviation Explanation Ado 5-deoxy-adenosyl ADP Adenosine diphosphate AEF alternative electron flow ALDC acetolactate decarboxylase ALS acetolactate synthase Amp Ampicillin APS Ammonium peroxydisulfate, (NH4)2S2O8 AR acetoin reductase ATCC American Type Culture Collection ATP Adenosine triphosphate B. brevis Brevibacillus brevis B. subtilis Bacillus subtilis BCC Berkeley Culture Collection BOFmax maximum of the bioobjective function BSA Bovine serum albumin CaCl2 Calcium chloride CAPS N-cyclohexyl-3-aminopropanesulfonic acid CAPSO 3-(Cyclohexylamino)-2-hydroxy-1-propanesulfonic acid CBB Calvin Benson Bassham Cm Chloramphenicol CO2 carbon dioxide CoA Coenzyme A cyt b6f cytochrome b6f Da Dalton DDH Diol Dehydratase DHAP Dihydroxyacetone phosphate DMSO dimethyl sulfoxide DNA Deoxyribonucleic acid d day dNTP Deoxynucleotide triphosphate E4P Erythrose-4-phosphate E. aerogenes Enterobacter aerogenes E. coli Escherichia coli (continued overleaf)

157 158A BBREVIATIONS

Abbreviation Explanation (continued from previous page) EDTA Ethylenediaminetetraacetic acid ee enantiomeric excess exp experimental F6P Fructose-6-phosphate FBA flux balance analysis FBP Fructose-1,6-bisphosphate Fig. Figure FNR Ferredoxin and Ferredoxin:NADP oxidoreductase G3P L-Glycerol-3-phosphate Ga Gigayears = 109 years GAP Glyceraldehyde-3-phosphate GARPO Goat anti rabbit peroxidase GC Gas chromatography GDH Glycerol Dehydratase GG 2-(β-D-glucosyl)-sn-glycerol-3-phosphate gpp glycerol phosphate phosphatase h hour H2O Water H3BO3 Boric acid HEPES 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid His Histidine HPLC High performance liquid chromatography I Ionic strength IPTG Isopropyl β-D-1-thiogalactopyranoside IR infrared K. pneumoniae Klebsiella pneumoniae K. oxytoca Klebsiella oxytoca kan kanamycin KCl Potassium chloride Km kanamycin L. lactis NCW1 Leuconostoc lactis NCW1 L. reuteri Lactobacillus reuteri LB Lysogeny Broth LDH lactate dehydrogenase LED light emitting diode M mol per litre M mol per litre M. chthonoplastes Microcoleus chthonoplastes Mb Megabases MgCl 6H O Magnesium chloride hexahydrate 2· 2 min minute mM millimoles per litre MMP molecular microbial physiology Mn Manganese mRNA messenger RNA n. noun Na2S2O3 Sodium thiosulfate NaCl Sodium chloride NAD Nicotinamide adenine dinucleotide (oxidised) NADH Nicotinamide adenine dinucleotide (reduced) NADP Nicotinamide adenine dinucleotide phosphate (oxidised) NADPH Nicotinamide adenine dinucleotide phosphate (reduced) NCBI National Center for Biotechnology Information nm nanometer NTP nucleotide triphosphate (continued overleaf) ABBREVIATIONS 159

Abbreviation Explanation (continued from previous page) OD optical density ORF open reading frame PAGE polyacrylamide gel electrophoresis PHB polyhydroxybutyrate PS Photosystem R5P Ribose-5-phosphate rac. racemisation RNA Ribonucleic acid rpm rotations per minute Ru5P Ribulose-5-phosphate RuBP Ribulose-1,5-bisphosphate RubisCO Ribulose-1,5-bisphosphate carboxylase/oxygenase S. cerevisiae Saccharomyces cerevisiae S7P Sedoheptulose-7-phosphate SBP Sedoheptulose-1,7-bisphosphate SDS sodium dodecyl sulfate P.aeruginosa Pseudomonas aeruginosa PBS Phosphate-buffered saline PCC Pasteur Culture Collection PDD Propanediol dehydratase ppmv parts per million by volume PTT Polytrimethylene terephthalate RT room temperature s second s secondary S Svedberg sim. simulated sn stereospecifically numbered sp. species STH soluble transhydrogenase Tab. Table TE Tris/EDTA TEA Triethanolamine TES N-[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid Tris Tris-(hydroxymethyl)-aminomethane UCYN uncultured cyanobacterium v volume vs versus w weight WT wild type X5P Xylose-5-phosphate Xu5P Xylulose-5-phosphate z charge

B ALTERATIONS OF THE GENOME-SCALEMODELOF Synechocystis

Listing B.1: Reactions added to the genome scale model of Synechocystis SUBSYSTEM: Transport Abbrevatiation: R_glcglyctpp NAME: GG transport via diffusion (cytosol to periplasm) GENE ASSOCIATION:

161 162 B.A LTERATIONS OF THE GENOME-SCALEMODELOF Synechocystis

SUBSYSTEM: Confidence Level: 2 GENE ASSOCIATION: slr1670 EC Number: AUTHORS:

163

SUBSYSTEM: Confidence Level: 2 GENE ASSOCIATION: slr1670 EC Number: AUTHORS:

SUBSYSTEM Confidence Level: 2 GENE ASSOCIATION: EC Number: AUTHORS:

ACKNOWLEDGEMENTS

A PhD thesis always is a joint effort. So, even though my name is on the cover page, I could never have done this alone. I would like to thank everyone that contributed in any way to this thesis. Most notably, Klaas Hellingwerf, for giving me the chance to work on this interesting topic, for fruitful discussions and for the trust and freedom to explore ideas of my own. Filipe Branco dos Santos, who agreed to become my copromotor, for lots of hour-long five- minute discussions, inspiration, support, and for great collaboration. The entire Qingdao Metabolic engineering group, most notably, Xuefeng Lu, for hosting me in his research group. This was an amazing opportunity I will never forget. Xiaoming Tan, for going out of his way to help me. Kuo Song, for his friend- ship. Song Kuo, I hope I have not seen you for the last time. Li Cheng, for help with trans- lations, chromatography and endless patience. Lei Zhang, for interesting insights and dis- cussions. Peizhen Ma, for sharing her bench with me. The MMP group at the University of Amsterdam, most notably, Jos Arents and Dennis Rijnsburger, without whom the lab would have sunk into chaos a long time ago. Andreas Angermayr and Pascal van Alphen for amazing discussions, crazy ideas and a great time inside and outside of the lab. Jeroen van der Steen, for countless Wordfeuds, sound advice and for collaboration on a super-interesting chapter. Pascal van Alphen, for translating the summary of this thesis into Dutch. Aniek van der Woude, for transforming the lab into a space where research is possible. Federico Muffatto and Joeri Jongbloets, for helping in the last and in the very last minute, respectively. Wei Du for an inspiring work-ethic and for always being calm. Joeri Jong- bloets, for multicultivator experiments. The members of the 12:30 lunch group: Aleksandra Bury, Alice Sorgo, Andreas Anger- mayr, Clemens Heilmann, Milou Schuurmans, Nadine Händel, Orawan Borirak, Pascal van Alphen, Soraya Omardien, Que Chen, Wei Du. Angie Vreugdenhil, for making the lab more hospitable to squirrels. Milou Schuurmans, for sharing the burden of writing in spirit. All coauthors of the chapters. Marijke Duyvendak, for finding even the most antiquated and obscure book chapters, re- search articles and conference proceedings. Hans Matthijs, for always being helpful, enthusiastic and friendly. Estelle Kilias, for her help on phylogenetic analyses. Merijn Schuurmans, for introducing me to the FACS and the CASY counter. Ngoc A Dang, Andjoe Sampat, Peter Schoenmakers for allowing me to use their GC setup. The people that were involved in proofreading: Klaas Hellingwerf, Jeroen van der Steen, Federico Muffatto, Sabrina Zaini. The members of the committee, Michel Haring, Jeroen Hugenholtz, Ron Wever, Annegret Wilde, Paul Hudson, for agreeing to read my thesis and for being there on the day of the

165 166 defence. Paul and Annegret, for traveling far to be there. Andreas Angermayr and Jeroen van der Steen, for agreeing to become paranimfs. TBSC, for funding, inspiring meetings and for giving me practical insight into how a patent is written. Julia, Kersti, Noreen and Philipp, for friendship and support, particularly towards the end of the writing period. Zuletzt möchte ich auch meiner Familie danken. Ohne euch wäre ich nicht der Mensch, der ich heute bin. LISTOF PUBLICATIONS

5. P.Savakis, X. Tan, C. Qiao, K. Song, X. Lu, K. J. Hellingwerf, F.Branco dos Santos Slr1670 from Synechocystis sp. PCC 6803 Is Required for the Re-assimilation of the Osmolyte Glucosylglycerol, Frontiers in Microbiology 7:1350 (2016).

4. P.Savakis, K. J. Hellingwerf, Engineering cyanobacteria for direct biofuel production from CO2, Current Opinion in Biotechnology 33, 8-14 (2015).

3. P.Savakis, X. Tan, W. Du, F.Branco dos Santos, X. Lu, K. J. Hellingwerf, Photosynthetic produc- tion of glycerol by a recombinant cyanobacterium, Journal of Biotechnology 195, 46-51 (2015).

2. P.Savakism, S. A. Angermayrm, K. J. Hellingwerf, Synthesis of 2,3-butanediol by Synechocystis sp. PCC6803 via heterologous expression of a catabolic pathway from lactic acid- and enterobac- teria, Metabolic Engineering 20,121-130 (2013).

1. P. Savakism, S. de Causmaeckerm, V. Angerer, U. Ruppert, K. Anders, L.-O. Essen, A. Wilde, Light-induced alteration of c-di-GMP level controls motility of Synechocystis sp. PCC6803, Molec- ular Microbiology 85:2, 239-251 (2012).

m: equal contribution

167