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Engineering oleaginous yeast Yarrowia lipolytica for production of fatty acid-derived compounds
Marella, Eko Roy
Publication date: 2020
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Engineering oleaginous yeast Yarrowia lipolytica for production of fatty acid-derived compounds
Eko Roy Marella PhD Thesis, March 2020
The Novo Nordisk Foundation Center for Biosustainability Technical University of Denmark
Engineering oleaginous yeast Yarrowia lipolytica for production of fatty acid-derived compounds
PhD Thesis by Eko Roy Marella
Main supervisor: Senior Researcher Dr. Irina Borodina Co-supervisors: Dr. Guokun Wang, Dr. Carina Holkenbrink, Dr. Sudeep Agarwala
The Novo Nordisk Center for Biosustainability Technical University of Denmark
Preface
This PhD thesis serves as a partial fulfilment of the requirements for obtaining a PhD degree at the Technical University of Denmark. The works included in the thesis were carried out at the Novo Nordisk Foundation Center for Biosustainability in the period of 15th of February 2017 to 31st of March 2020 under the supervision of Senior
Researcher Dr. Irina Borodina, Dr. Guokun Wang, and Dr. Carina Holkenbrink from the Novo Nordisk Foundation Center for Biosustainability. I also worked at Ginkgo
Bioworks, Inc. for the period of 1st of April 2019 to 30th of September 2019 under the supervision of Dr. Sudeep Agarwala. The PhD project has been funded by the
European Union's Horizon 2020 research and innovation programme under the
Marie Sk odo ska-Curie grant agreement No 722287 (PAcMEN).
Eko Roy Marella
Kongens Lyngby, March 2020
i Abstract
With a human population estimated to reach 9.7 billion in 2050, activities to fulfil the growing needs for food, energy, and materials will cause increasing environmental damages. Biobased production offers potential solutions by enabling microorganisms to produce a plethora of compounds. Covering products from alkanes to polyunsaturated fatty acids (PUFAs), production of fatty-acid derived compounds (FADCs) in microbes could contribute to realizing more sustainable productions.
During optimization of FADC production in microbes, metabolic engineering efforts need to address various pathway bottlenecks and cellular limitations. Examples in resolving these challenges are described in the first chapter of this thesis. Yarrowia lipolytica offers the advantage of naturally producing large pool of acetyl-CoA, the main precursor for FADC, compared to the model yeast Saccharomyces cerevisiae. Owing to this, Y. lipolytica is arguably the host-of-choice for production of FADCs. Such potential has motivated the development of well-characterized genetic tools for this yeast, which further encourages the use of Y. lipolytica as bioproduction host.
Taking advantage of the increasingly available genetic tools, in this thesis Y. lipolytica was engineered to produce lactones, which are used for giving fruity and milky notes in foods and beverages. Conventional production methods of lactones require hydroxylated fatty acids as starting material. Mainly sourced from plants, series of extraction processes add costs to the production aside from time and land requirements. Through metabolic engineering, production of flavor lactones from more available, non-hydroxylated fatty acids oleic- and linoleic acid was realized and optimized.
PUFAs are arguably the most valuable FADC to date. Dietary requirement of these essential fatty acids is mainly supplied from seed oils and marine fish, both entails environmental concerns on land and in the ocean. PUFAs production in Y. lipolytica had been reported before. To complement the existing engineering strategies, the last work in this thesis borrowed from acyl-editing mechanism in plants. The work demonstrated improvement of linoleic acid content in the cell upon introducing an acyl-editing enzyme. Since linoleic acid is the precursor of all PUFAs, this new strategy could be employed for production of other PUFAs.
Overall, this thesis provides the research community insights to overcome strain engineering challenges in pathways connected to fatty-acid metabolism. The presented research offers a new application of biobased production and a novel approach for metabolic engineering which could inspire future efforts in optimizing FADC production in Y. lipolytica and other microbes.
ii Dansk Resumé
Verdens samlede befolkning estimeres at nå 9,7 milliarder i 2050, og de aktiviteter, som skal sikre nok mad, energi, og materialer, vil komme til at forårsage øget skade på miljøet. Biobaseret produktion tilbyder potentielle løsninger på dette ved at gøre mikroorganismer i stand til at producere et utal af forskellige kemiske forbindelser. Mikroorganismer, der kan syntetisere fedtsyreafledte forbindelser (FADCer), vil eksempelvis kunne bidrage til bæredygtig produktion af forskellige stoffer lige fra alkaner til flerumættede fedtsyrer (PUFAer).
Når mikrobiel FADC-produktion skal optimeres, er det nødvendigt at anvende metabolic engineering til at adressere forskellige biosyntetiske flaskehalse og cellulære begrænsninger. Denne afhandlings første kapitel beskriver eksempler på løsning af disse udfordringer. Sammenholdt med modelgæren Saccharomyces cerevisiae har Yarrowia lipolytica fordelen af en naturlig stor pulje af acetyl-CoA, som er den vigtigste byggesten for FADCer. Netop derfor er Y. lipolytica sandsynligvis den foretrukne vært for produktion af FADCer. Dette potentiale har motiveret udvikling af velkarakteriserede genetiske værktøjer for denne gær, hvilket yderligere har tilskyndet anvendelsen af Y. lipolytica som vært for biobaseret produktion.
I denne afhandling blev Y. lipolitica modificeret til at producere lactoner, som anvendes til at forstærke frugt- eller mælkeagtige smage og dufte i føde- og drikkevarer, ved at gøre brug af disse nye, tilgængelige genetiske værktøjer. Konventionelle produktionsmetoder af lactoner kræver hydroxylerede fedtsyrer som startmateriale. Disse udvindes fra plantemateriale gennem en række af oprensningsprocesser, hvilket øger produktionsomkostningerne og kræver både tid og landbrugsjord til dyrkning. Ved hjælp af metabolic engineering opnåede og optimerede vi produktion af smagslactoner fra de mere tilgængelige uhydroxylerede fedtsyrer.
PUFAer er uden tvivl de mest værdifulde FADCer til dato. Det daglige indtag af disse essentielle fedtsyrer sker hovedsageligt gennem frøolier og havfisk, hvilket medfører miljøudfordringer både på landjorden og i havet. PUFA-produktion i Y. lipolytica er tidligere rapporteret. For at supplere de eksisterende metabolic engineering strategier lånte afhandlingens sidste studie fra planters acyl- redigeringsmekanisme. Cellens linolensyreindhold blev forbedret efter introduktion af et acylredigerende enzym. Da linolsyre er forstadiet til alle PUFAer, kan denne nye strategi potentielt anvendes til produktion af andre PUFAer.
Samlet set bidrager denne afhandling med ny viden omkring, hvordan udfordringer relateret til konstruktion af stammer med biosynteseveje, der er forbundet til fedtsyremetabolismen, kan afhjælpes. Den beskrevne forskning demonstrerer en ny anvendelse af biobaseret produktion og en ny tilgang til metabolic engineering, som kan inspirere fremtidige tiltag inden for optimering af FADC- produktion i Y. lipolytica og andre mikroorganismer.
iii Acknowledgements The last 3 years have been a noteworthy period for my scientific journey as well as personal development. I and my PhD could not be as what it has been without the people, as things did not happen by themselves. Irina, thank you for being a tough supervisor, and for always being honest about where I should improve. Thank you for your trust, so I could train my independence and follow my curiosity. Thank you for your making possible all the training, courses, and secondment, which are paramount for my development as a scientist. I would like to thank all the people involved in the works in this thesis. Carina and Guokun, as my co-supervisors, thank you for your insights and guidance in the lab, during meeting, and when writing the manuscripts. Thank you for being patient with me. Marie, thank you very much for introducing the art of fatty acid analysis, and your delicious cakes. Thank you for being there in the lab during my first days and for helping me taking off with my project! Kanchana, thank you for being a great resource on Yarrowia engineering. Thank you for teaching me the USER cloning and the EASYCLONEYALI toolbox. Verena and Farshad, thank you for the opportunity for collaborating with you. Hanne, I am glad I have learned many things about GC/MS analysis from you. Thank you for sharing your knowledge and for being very accommodating. Lars, thank you for teaching me on the freeze drier and the SpeedVac. They have been invaluable in my project. Jolanda and Suresh, it was a really fun bioreactor experiment. Thank you for accommodating my fat strain and the oily cultivations in the PPP lab. Lea, the Dansk Resumé is there because of you! Thanks for coming to me and offered the help! I also would like to thank my fellow PhD students who have been important part of the science and the life outside the lab. Jonathan, I am really glad that I have had a comrade like o . Thanks for always challenging my views and providing insights about anything. I e learned a lot from o . David, as someone who shares so much similar feelings about living in another country, thank you for being a constant friend. Anne-Sofie, thank you for lifting up the spirit during tough times, and for enlightening me about living in Denmark. And thank you for the Dansk Resumé with all the comments on my abstract. Vasil, thank you for your friendship. Thank you for helping me to see the positive sides of the story, and for dragging me out of the lab once in a while! Wasti, thank you for the company during long lab hours, and for being someone I could always talk to. Ksenia, thank you for reminding me to be spontaneous and more flexible. Steven, thank you for the Indonesian dish! And for being an example in project management. Jonathan, thank you for introducing the miraculous RedTaq for Yarro ia colon PCR! That has made e er one s life easier. As an international student who spent so much time in the lab, the lab members have been influential parts in my PhD. I am glad that we have become
iv friends, and not only colleagues. Javier, my project did not freeze when I left them for 6 months thanks to you. Thank you for your constructive criticism about my techniques and for being an exemplary lab citizen. Matej, I really enjoyed supervising an ever-curious and enthusiastic student like you. Thank you for the tough questions and for making me learn more. Karolis, it was great to have someone in the lab as eager to learn as I was. Thanks for infecting your great spirit. Larissa, your positivity and hardworking attitude have been inspiring. Thank you sharing DNA aliquots and for always into discussing Yarrowia! Laura, thank you for being an example in doing good science, for your suggestions on being more social, and for the good atmosphere you created in the group. Paulina, thank you for being a friend inside and outside the lab. Mahsa, the lab lightened up when you were around. Thanks for the energy and the insightful talks. Konrad, thank you for getting me out of the lab and showing the beautiful nature in Sjælland! Nick, Iben, Behrooz, Jane, Adam, and Alexanda thank you for your wisdom and insights. For past and present member of yeast labs, Chloé, Diogo, Dorota, Emilja, Eric, Indre, Katarzyna, Luisa, Manu, Marc, Marcos, Mathias, Tom, Veronica, thank you for all the helps and the great times inside and outside the lab! This PhD and living in Denmark would not be as enjoyable as it has been without all of you. I thank all CfB people without whom my PhD would not go as smooth as it has been. Susanne, Birte, Rebeca, and Darko, thank you for arranging my contract, secondment, PAcMEN training, and PhD administration. Anne and Anders, thank you for making the communication articles about my lactone study. Ann, Lise-Lotte, and all of the media team, thank you for providing constant supply of media and clean labwares. For the IT, HR, and facility team,s thank you for keeping CfB up and running. I spent 6 months Ginkgo Bioworks where I learned a great deal on science, orking in a compan , and li ing in the US. I o ld like to thank I e orked and met there. Elena, thank you for convincing Ginkgo to accept me as an intern. Sudeep, I on t enjo orking and li ing in the US ithout your constant support and encouragement. Thank you for giving me the time to adjust and learn, to let me test my ideas, and for being an example as a leader. Ruchita, thank you for helping me get onboarded at Ginkgo, for being a friend, and for your trust, understanding, and support as a colleague. Wesley, I am so glad you were there in Boston! Thank you so much for your help and hospitality while I was in Boston, from my arrival to my departure. Thank you for not letting me be alone during weekends and holidays. Rose, thank you for being a friend I spent so much time with while in Boston. Thanks for helping me with getting social in Boston. Tante Amanda, thank you for cooking the Indonesian food, and for your help and encouragement! Punita, thank you for your insights on the fermentation, and thanks for being a friend. Silvia, Jeff, Nate, Ming, Adam, Jenifer, and Natalia, thanks for teaching and helping me on the protein works. Now I love the western blot. Swami, Ishaan, Naveed, Cam, Bob, Gabe, and Jim, thank you for the insights and support on OE, NGS, automation, and python stuff. Andrea,
v Dan, Fiona, Huey-Ming, Krystina, Naveed, Rose, thank you for the great times outside the lab. Will, Jason, Swami, Bob, Ted, Krishna, and Dave, thank you for convincing Ginkgo to have me back as an OE! Adek, terima kasih untuk semangat dan dukungannya. Terima kasih untuk terus percaya kalau abang bias sukses. Bapak dan mama, terima kasih untuk doa kalian. Terima kasih sudah mendidik aku menjadi seorang yang bisa mandiri dalam hidup dan dalam berpikir. Terima kasih karena sudah membiarkan aku pergi pertama ke Belanda, lalu ke Denmark, dan lanjut ke Amerika, sehingga aku bisa mencapai apa yang ingin aku capai. Walaupun itu sulit buat mama dan bapak karena kita harus berjauh-jauhan, tetapi terima kasih pak, ma, pada akhirnya aku bisa mengerjakan apa yang aku cita-citakan.
vi List of publications
The following scientific articles (published and in preparation) are included in this thesis:
1. Marella ER, Holkenbrink C, Siewers V, Borodina I: Engineering microbial fatty acid metabolism for biofuels and biochemicals. Curr Opin Biotechnol 2017, 50:39 46.
2. Darvishi F, Ariana M, Marella ER, Borodina I: Advances in synthetic biology of oleaginous yeast Yarrowia lipolytica for producing non-native chemicals. Appl Microbiol Biotechnol 2018, 102:5925 5938
3. Marella ER, Dahlin J, Dam MI, ter Horst J, Christensen HB, Sudarsan S, Wang G, Holkenbrink C, Borodina I: A single-host fermentation process for the production of flavor lactones from non-hydroxylated fatty acids. Metab Eng 2019, doi:10.1016/j.ymben.2019.08.009
4. Marella ER, Dahlin J, Sáez-Sáez J, Christensen HB, Wang G, Borodina I: Engineering of acyl-editing pathway for an enhanced production of linoleic acid in the oleaginous yeast Yarrowia lipolytica. Manuscript in preparation
Additionally, minor contributions have been provided in the following article:
1. Dahlin J, Holkenbrink C, Marella ER, Wang G, Liebal U, Lieven C, Weber D, McCloskey D, Ebert BE, Herrgård MJ, Blank LM, Borodina I: Multi-Omics Analysis of Fatty Alcohol Production in Engineered Yeasts Saccharomyces cerevisiae and Yarrowia lipolytica. Front Genet 2019, 10
vii Table of Contents
Preface...... i
Abstract ...... ii
Dansk Resume ...... iii
Acknowledgements ...... iv
List of publications...... vii
Table of contents ...... viii
Chapter 1 - Introduction ...... 1
Chapter 2 - Engineering microbial fatty acid metabolism for biofuels and biochemicals ...... 11
Chapter 3 - Advances in synthetic biology of oleaginous yeast Yarrowia lipolytica for producing non-native chemicals ...... 29
Chapter 4 - A single-host fermentation process for the production of flavor lactones from non-hydroxylated fatty acids ...... 60
Chapter 5 - Engineering of acyl-editing pathway for an enhanced production of linoleic acid in the oleaginous yeast Yarrowia lipolytica ...... 109
Chapter 6 Perspectives ...... 152
viii
1
Introduction
Chapter 1
1.1. A briefing in fermentation-based production
The United Nations predicted that there will be 9.7 billion people in 2050 from about 7.7 billion in 2019. The population growth, along with the consumption pattern, has pushed the planet to its limit for providing human needs [1]. With the current means of fulfilling demands, more cases of environmental degradation have been happening and more intense effect climate changes are being felt throughout the world
[2,3]. Therefore, alternative ways of production are needed [4].
Fermentation-based production, by enabling production using microorganisms, has provided alternatives in producing many compounds with less negative impacts [5]. As an example, the production of polylactic acid biopolymer has
30-60% lower greenhouse gas (GHG) emissions compared to that of conventional plastics [6]. Several compounds relevant to the global demands have successfully produced at commercial scale using fermentation, such as amino acids [7], bioplastics
[8], drugs [9,10], and omega-3 fatty acids [11]. According to an analysis, with a total market size of over 127 billion USD, the fermentation-based chemical industry is expected to grow at more than 4% annually [12].
Early applications of fermentation technology dealt with the production of relatively few compounds (compared to today) with the aid of microorganisms that are naturally capable of producing them. Bioethanol production by Saccharomyces cerevisiae [13] and glutamic acid production by Corynebacterium glutamicum [14] are well-known examples. Today, many different hosts have been engineered to produced numerous native and non-native chemicals [15]. This pursuit in the engineering of microbes for optimized production of chemicals is the domain of metabolic engineering [16,17].
2 Chapter 1
1.2. Metabolic engineering for fatty acid-derived compounds
Among the compounds that can be produced in microbes, this thesis took particular interest in fatty-acid derived compounds (FADC). FADC includes compounds such as fatty alcohols, free fatty acids, fatty acid esters, flavor lactones, and polyunsaturated fatty acids. Examples of their applications are lubricants, cosmetics, flavor and fragrance, biodiesel, and polymers (Figure 3.2).
Chapter 2 of this thesis captures metabolic engineering works for optimizing microbial FADC production. The diversity of produced compounds demonstrates the convenience of using microbes for the production of compounds. The universal nature of biochemical reactions allows the transplantations of pathways from different kingdoms which eventually provide scientists extensive options for optimization [18].
As an example from this thesis (Chapter 4), enzymes from bacteria and bat made a suitable combination for the production of lactone naturally found in fruits [19].
After introducing the enzymes for the main biosynthesis, production levels are usually low and need to be increased to achieve a cost-effective project [17]. In metabolic engineering, strategies to boost production usually involve overexpression of biosynthetic enzymes, optimizing metabolic pathways for precursor supply, omics analyses, minimizing degradation of intermediates and final products, and enhancing metabolites trafficking and transport [20,21]. It is evident in Table 2.1 and Table 3.3 that to achieve a substantial increase in production, multiple strategies had to be implemented.
1.3. The oleaginous yeast Yarrowia lipolytica
A dimorphic fungus with a big capacity to produce, utilize, and store lipids,
Yarrowia lipolytica are naturally able to sustain the production of cytosolic acetyl-
3 Chapter 1
CoA, the precursor of fatty acid biosynthesis [22,23]. Under nitrogen limitation, wild- type Y. lipolytica could store lipid at more than 30% of its dry weight [24], giving it
e eag ea e.
This propensity implies that fewer engineering works would be required to produce FADC when Y. lipolytica is used, compared to when the model yeast
Saccharomyces cerevisiae. As an example, when both yeasts were tested for the production of fatty alcohol in minimal glucose medium, Y. lipolytica produced three times more fatty alcohols despite 7.5 times lower glucose uptake rate. The rate of acetyl-CoA formation was ten times higher in Y. lipolytica, and S. cerevisiae channel a large portion of the carbon into ethanol [23]. This example illustrates the potential of Y. lipolytica for acetyl-CoA-demanding products. In agreement with this, most of the compounds produced in Y. lipolytica (see Table 3.3. for a summary), are indeed compounds like FADC, terpenoids, and sterols which take cytosolic acetyl-CoA as their biosynthetic precursor [25].
Besides the capability to synthesize lipid (lipogenesis) and store it in a large amount, Y. lipolytica is also endowed with the ability to efficiently degrade lipids
(lipolysis). Lipolysis is the main challenge for the FADC production in Y. lipolytica.
Fatty acid degradation occurs mainly in peroxisome and is initiated by acyl-CoA oxidase [26,27]. Consequently, minimizing lipolysis was addressed by either deleting the peroxisome biogenesis factor (PEX10) [28,29] or deleting all [30,31] or some [32
34] of the peroxisomal acyl-CoA oxidases (POX1-6). On the other hand, efficient lipolysis is also desired in a few cases such as lactone production from hydroxylated fatty acids. As an example, the industrial conversion of castor oil (containing ricinoleic acid, a hydroxy fatty acid) to -decalactone has been done with Y. lipolytica [35].
4 Chapter 1
1.4. Not all fats are equal
Many of the microbial FADC are produced to substitute similar products obtained from traditional petrochemical or farming [36]. As an example, fatty alcohols and fatty acid methyl esters have been produced from vegetable oils or via petrochemical routes [37]. Consequently, many of biobased processes are bound with such product-price caps, which are often difficult to attain as strain engineering activities to achieve it are costly [18].
One way to get around this issue is to produce high-value chemicals in microbes [38]. Such compounds are usually found in very little amount in natural sources like plants, which make extraction expensive, or very difficult to produce in petrochemical processes, e.g. due to the stereochemical requirements. In FADC, products with requiring modification of the hydrophobic tail at a specific carbon of fatty acids is a great example of such compounds.
With this motivation, the two research works in this thesis were conceived.
Production of flavor lactones requires hydroxy fatty acids with the hydroxy group at the fourth or fifth carbon, counting from the carboxyl end, which further esterify with the carboxy group to form the lactone ring (Figure 4.1). Unlike terminal hydroxylation, as in the case of fatty alcohols or hydroxy fatty acids, introducing a hydroxy group in the middle of the hydrophobic tail requires the works of enzymes. In Chapter 4, it is described that the 4- and 5-hydroxy fatty acids could be obtained by first introducing a hydroxy group at carbon 10 and 13 of 18-carbon fatty acids. Lactone production was obtained by further chopping the fatty acids, via modified -oxidation, into twelve and ten carbon, respectively (Figure 4.1). With these strains, flavor lactones could be made from non-hydroxylated, more abundant oleic- and linoleic acid in single hosts [19].
5 Chapter 1
A similar consideration applies for PUFAs production, which require desaturation at a specific carbon with correct stereospecificity. The second research work, described in Chapter 5, dealt with the biosynthesis of linoleic acid, a PUFA which also serves as the precursor for important PUFAs such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) [39,40]. EPA is arguably the most valuable microbial
FADC products reported in the literature [19]. Although some fungi such as
Mortierella alpina are excellent in making certain PUFAs, difficulties in genetic engineering and cultivation of these fungi made them less amenable hosts. Y. lipolytica, with the expanding genetic toolbox (Table 3.3) and the ease of its cultivation, makes an excellent alternative. On the enzymatic side, variants of desaturases have been used and tested for PUFA production in microbes [28,31,41
43]. Indeed, overexpressing the desaturases have been the colloquial strategy for microbial PUFA production. However, many of these desaturases acts on the endoplasmic reticulum- (ER-) membrane phospholipids [44], while accumulation as triacylglycerol (TAG) is desired to obtain high accumulation and thus, high titer
(Figure 5.1). Chapter 5 described that manipulating the fatty acid traffic between acyl-
CoA and ER-membrane phospholipids could improve the accumulation of linoleic acid. As many desaturases act on ER-membrane as well, the strategy could be applied for the production of other PUFAs.
1.5. The structure of this thesis
As might have been hinted from the above introduction, this thesis first introduces the development of metabolic engineering in microbial FADC production
(Chapter 2). In Chapter 3 the utility of Y. lipolytica as a production host is elaborated, from the availability of synthetic biology toolboxes as well as the spectrum of compounds that have been produced. Chapter 4 presents the work in enabling Y.
6 Chapter 1 lipolytica for the production of flavor lactones from non-hydroxylated fatty acids.
Chapter 5 describes the strategy to improve linoleic acid production via rerouting the fatty acid pathway towards TAG. Eventually, I put my thoughts on what can be done to accelerate the metabolic engineering of Y. lipolytica for FADC production as well as how to make the strain engineering efforts more efficient. This last chapter is a condensation of what I have learned while during my PhD projects, as well as conferences, courses, seminars, and conversations with fellow scientists.
I hope that this thesis could be valuable for the readers at any scientific level.
For the metabolic engineering community in general, I hope the thesis provides knowledge and insights that could be used as a steppingstone for future research.
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8 Chapter 1
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29. Palmer CM, Miller KK, Nguyen A, Alper HS: Engineering 4-coumaroyl-CoA derived polyketide production in Yarrowia lipolytica through a β-oxidation mediated strategy. Metab Eng 2020, 57:174 181.
30. Matthäus F, Ketelhot M, Gatter M, Barth G: Production of Lycopene in the Non- Carotenoid-Producing Yeast Yarrowia lipolytica. Appl Environ Microbiol 2014, 80:1660 1669.
31. Imatoukene N, Verbeke J, Beopoulos A, Taghki AI, Thomasset B, Sarde C-O, Nonus M, Nicaud J- M: A metabolic engineering strategy for producing conjugated linoleic acids using the oleaginous yeast Yarrowia lipolytica. Appl Microbiol Biotechnol 2017, doi:10.1007/s00253-017-8240-6.
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2
Engineering microbial fatty acid metabolism for biofuels and biochemicals
Eko Roy Marella, Carina Holkenbrink, Verena Siewers, Irina Borodina
This chapter is from a published article in Current Opinion of Biotechnology journal
Marella ER, Holkenbrink C, Siewers V, Borodina I. Engineering microbial fatty acid metabolism for biofuels and biochemicals. Curr Opin Biotech. 2018; 50:39-46 (doi: 10.1016/j.copbio.2017.10.002) Chapter 2
Abstract
Traditional oleochemical industry chemically processes animal fats and plant oils to produce detergents, lubricants, biodiesel, plastics, coatings, and other products.
Biotechnology offers an alternative process, where the same oleochemicals can be produced from abundant biomass feedstocks using microbial catalysis. This review summarizes the recent advances in the engineering of microbial metabolism for production of fatty acid-derived products. We highlight the efforts in engineering the central carbon metabolism, redox metabolism, controlling the chain length of the products, and obtaining metabolites with different functionalities. The prospects of commercializing microbial oleochemicals are also discussed.
2.1. Introduction
Oleochemicals are a large group of fatty-acid derived compounds with an unprecedented application range: biodiesel, detergents, soaps, personal care products, industrial lubricants, plastic enhancers, bioplastics, emulsifiers, coatings, food and feed additives, and others [1]. Oleochemicals have traditionally been derived from vegetable oils and animal fats via chemical or enzymatic processes [2,3]. However, the limited availability, sustainability, and high cost of feedstocks limit the growth of this sector [4]. The long-term solution for this problem is sought in the expansion of feedstock range to more abundant lignocellulosic biomass [5] and its conversion via chemical and microbial.
Using a microbial chassis in comparison to the traditional conversion of plant oils and animal fats presents a number of advantages. Firstly, feedstock availability is expanded from edible plant oils and animal fats to abundant first-generation and second-generation biomass feedstocks. Secondly, the feedstock-product dependence
12 Chapter 2 is eliminated as the desired oleochemicals can be obtained directly using an engineered cell factory from any feedstock, that is, a feedstock can be chosen based on the market price and availability. Finally, complex oleochemicals that cannot be obtained from natural sources because of low abundance can be produced by introducing novel synthetic biochemical pathways into platform chassis.
Microbial chassis must be extensively engineered in order to produce oleochemicals at high titer, rate, and yield for commercial exploitation [6]. The supply of metabolic precursors, acyl-CoAs, and redox co-factor NADPH need to be boosted
(Figure 2.1). The chain length of the products needs to be controlled to obtain the required properties. One must also implement heterologous enzymes that will functionalize fatty acyl-CoAs into final products: hydroxylated and desaturated fatty acids, fatty alcohols, hydrocarbons, waxes, lactones, and others.
This review highlights the recent advances in the engineering of microbial metabolism towards the optimized production of natural and synthetic fatty-acid derived products. The microbial hosts covered in this review include common industrial workhorses: the bacterium Escherichia coli, the yeast Saccharomyces cerevisiae, and the oleaginous yeast Yarrowia lipolytica.
2.2. Engineering the central carbon metabolism
A common starting point for engineering microbial hosts is increasing the supply of fatty acid metabolic precursors: acetyl-CoA, malonyl-CoA, and fatty acyl-
CoAs. In E. coli, it is effective to limit the fermentative pathways towards lactate, acetate, succinate, and ethanol, which consume acetyl-CoA. To circumvent the negative effects of these deletions on the cellular metabolism, these pathways can be first downregulated in the production phase, for example, Wu et al. applied CRISPR-
13 Chapter 2 based interference for repression of fermentative pathways and achieved 36% increase of the medium-chain fatty acid (MCFA) titer [7] (Table 2.1).
The yeast S. cerevisiae does not naturally produce cytosolic acetyl-CoA at high levels and, to overcome this limitation, strategies circumventing the native pyruvate dehydrogenase reaction have been developed previously [8,9]. Recent studies reported the implementation of cytosolic acetyl-CoA generation via heterologous
ATP:citrate lyase (ACL). Zhou et al. [10 ] optimized a synthetic chimeric citrate lyase pathway by combining the expression of ATP:citrate lyase from Mus musculus, malic enzyme from Rhodosporidium toruloides, and overexpression of native mitochondrial citrate transporter and malate dehydrogenase, thus obtaining a 20% increase in the free fatty acid titer in S. cerevisiae. A strain that was additionally engineered for decreased degradation and activation of fatty acids and overexpression of acetyl-CoA carboxylase and fatty acid synthase, produced 1 g/L and 10.4 g/L of free fatty acids in correspondingly shake flask and fed-batch cultivations, the highest titers reported to date. A similar strategy, though using the ATP:citrate lyase from Y. lipolytica, was applied by Ghosh et al. [11] and resulted in a small improvement of the free fatty acid titer by 5%. The authors further carried out 13C metabolic flux analysis and identified that a large flux of acetyl-CoA was channelled into malate via malate synthase (Mls1p) and that additional carbon was lost to glycerol via glycerol-3-phosphate dehydrogenase (Gpd1p). Downregulation of MLS1 and deletion of GPD1 in addition to
ACL over- expression, resulted in a 70% improvement over the reference strain, achieving $0.8 g/L of free fatty acids in shake flasks.
Another important control point to increase the supply of malonyl-CoA is acetyl-CoA carboxylase (ACC), which is allosterically inhibited by C16-C20 saturated acyl-CoAs. This inhibition can be alleviated by deletion of fatty acid-CoA ligases (e.g.,
14 Chapter 2
FAA1 and FAA4 in S. cerevisiae), which activate free fatty acids to acyl-CoAs [13]. Such a strategy is however not viable for production of acyl-CoA-derived metabolites and here an alternative approach was used by Qiao et al. [14 ]. They overexpressed stearoyl-CoA desaturase in lipid-producing Y. lipolytica to decrease the concentration of stearoyl-CoA, the main inhibitor of ACC. This resulted in a 3-fold increase of the lipid yield, reaching 84.7% of the theoretical maximum without growth impairment
[14 ].
Furthermore, through comparative genomic and transcriptomic analysis of lipid-overproducing strains, Liu et al. [15] identified a mutant Mga2p regulator, which leads to increased unsaturated fatty acid biosynthesis and lipid accumulation. Yet another approach is to apply dynamic regulatory modules to balance the precursor and product biosynthesis and avoid inhibition. A 2.1-fold improvement of the free fatty acid titer was obtained in E. coli with a regulatory circuit responding to malonyl-CoA concentration by upregulating either synthesis or consumption [16]. Shin et al. bypassed the ACC pathway by expressing methylmalonyl-CoA carboxyltransferase and phosphoenolpyruvate carboxylase, thus increasing the FFA titer about two- fold [17].
2.3. Engineering the redox metabolism
Fatty acid biosynthesis demands NADPH as a reducing co-factor, for example,
14 NADPH molecules are needed to produce one molecule of palmitoyl-CoA from acetyl- CoA. In Y. lipolytica, 13C metabolic flux analysis identified the oxidative pentose phosphate pathway as the primary source of NADPH for lipid biosynthesis [18].
However, generation of 2 NADPH through the pentose phosphate pathway costs one carbon, affecting the maximum theoretical yield.
15 Chapter 2
Table 2.1. Metabolic engineering strategies for fatty acid-derived metabolites Goals and genetic modifications (Note 1) Achievements Host Ref. Improving acetyl-CoA supply Inducible adhE, pta, poxB, ldhA, frdA) 1.36x C6-C10 FFA titer Ec [7] Acetate recycling ( acs) 3.71x intracellular AcCoA Ec [7] PCC MCC 2xlevel FFA Ec [17] ADH2, ALD6, acsFR 1.6-1.8x FAEE titer Sc [9] YlACL, MLS1, d1 1.67x FFA titer Sc [11] ACL route ( MmusACL RtME MDH3 CTP1) 1.18x FFA titer Sc [10 ] PDH-bypass ( ScPDC1 EcAldH) 1.57x lipid titer Yl [12 ] Pyruvate- formate lyase route ( EcPflA EcPflB) 1.47x lipid titer Yl [12 ] Carnitine shuttle ( ScCAT2) 1.75x lipid titer Yl [12 ] Non-oxidative PPP ( AnPK BsPta) 1.62x lipid titer Yl [12 ] Increasing acetyl-CoA carboxylase flux Minimize acyl-CoA pool ( faa1 faa4 MmusTes) 4x ACC transcript level Sc [13] ACB1 ACC1FR 1.2-1.3x FAEE titers Sc [9] OLE1 ACC1 DGA1 3x lipid yield, 84.7% TM Yl [14 ] Chain-length control RBO ( RtBktB fadB EgTER ydiI) fadA e B 1.1 g/L C6-C10 FFA (SF) Ec [29] RBO ( RtBktB fadB EgTER ydiI) acs CRISPRi 3.8 g/L C6-C10 FFA (SF) Ec [7] RBO ( cytFOX3 YlKR YlHTD cytoETR1 EcEutE) 12 mg/L C6-C10 FFA (SF) Sc [30] CaBdhB FASI replacement ( EcFAS fas2 RcFatB) ~54 mg/L C14 (SF) Sc [27] MPT swapping with Tes 380 mg/L C14 (SF) Yl [12 ] FAS mutagenesis 118 mg/L C6-C8 FFA (SF) Sc [23] Chimeric RtFAS-AcTes ( cRtFAS-AbTes) 1.4 mg/L C6-C8 FFA, 0.3 Sc [2 •] Product diversification (Note 2) mg/L C10-C12 FFA (SF) Fatty alcohols (MmarCAR BsSfp EcAHR EcFadD) 2.15 g/L fatty alcohols (FB) Yl [12 ] Akanes ( SeFAR SeADO SeFd/FNR) 1.31 g/L alkanes (FB) Ec [46] Alkenes ( JeOleT HEM3 ctt3 cta ccp1) 3.7 mg/L (FB) Ec [47] FAEEs (ER-localized AbAtfA) 136 mg/L (SF) Yl [1 ••] Linoleic acid (Fvd12) 1.3 g/L (SF) Rt [51] GLA ( Mald6) 71.6 mg/L (SF) Yl [35] EPA ( heterologous-C16/C18E, d12, d9, d8, d5, d17) >25% DCW Yl [36 ,37] VLC-WE ( elo3 ELO2 MaqFAR SciWS ACC1FR) 15 mg/L VLC-WE (SF) Sc [48] CBL lipid (cocoa GPAT, LPAT, and DGAT) Titer not mentioned Sc [38] HFA ( fadD ACC Te BmCYP102AI) 58.7 mg/L HFA (SF) Ec [40] Methyl ketones ( RtFAS-ShMks2 ShMks1) 10 µg/gDCW (SF) Sc [ •] Diacids (RBO, PpAlkBGT AcChnD AcChnE) 0.5 g/L C6-C10 diacids (SF) Ec [29] D c a ( elo3 ELO1,2 ACC1FR AtFAR 83.5 mg/L (SF) Sc [24] Compartmentalization PerMvFAS-AbAtfA) or ER-AbAtfA 15x- (ER) to 19x- (Per) Yl [12 ] ER-AbFAR and ER-PmADO 5.3xFAEE alkane titer titer Yl [12 ] Per-MaqFAR 2.2x fatty alcohol titer Yl [12 ] Per-MmarCAR, Per-SeADO, and Per-SeFd/FNR 1.9x alkanes titer Sc [31 ] Redox and cofactor engineering Increasing NADPH supply ( CaGapC McMCE2) 99 g/L lipid titer (FB, 98% Yl [19 ] The use of compatible Fd/FNR for ADO 1.7xTM) alkane titer Ec [46 ] JeOleT cofactor supply ( HEM3 ctt3 cta ccp) 1.2x alkene titer Sc [47] Note 1: Unless indicated by a two- e e e e ec e a e, e e e a e a e e . Note 2: Except for GLA and EPA, only examples using minimal medium were considered. The listed examples report thehighest titers for corresponding products within the e e ed b ca . S b a d e e : : e e e ; : d e a ; : de e ; c : c - ca ed; : truncated version. General abbreviations: CRISPRi: CRISPR-based interference; SF: shake-flask; FB: fed-batch bioreactor; DCW: dry-cell weight. Species abbreviations: Ab: Acinetobacter baylyi; Ac: Acinetobacter sp.; An: Aspergillus nidulans; Bm: Bacillus megaterium; Bs: Bacillus subtilis; Ca: Clostridium acetobutylicum; Ec: E. coli; Eg: Euglena gracilis; Fv: Fusarium verticillioides; Je: Jeotgalicoccus sp.; Mal: Mortierella alpina; Maq: Marinobacter aquaeolei; Mc: Mucor circinelloides; Mmar: Mycobacterium marinum; Mmus: Mus musculus; Mv: Mycobacterium vaccae; Pm: Prochlorococcus marinus; Pp: Pseudomonas putida; Rc: Ricinus communis; Rt: R. toruloides; Sci: Simmondsia chinensis; Se: Synechococcus elongatus; Sh: Solanum habrochaites; Yl: Y. lipolytica. Gene/Enzyme abbreviations: ACC: acetyl-CoA carboxylase; ADO: fatty-aldehyde deformylating oxygenase; AHR: aldehyde reductase; CAR: carboxylic ac d ed c a e; C16/C18E: e a e c e a a e (C16) ea a e (C18); d5, d8,d9, d12, d17: 5-, 8-, 9-, 12-de a a e, 17- desaturase, respectively; DGAT: diacylglycerol O-acyltransferase; FAR: fatty acyl-CoA- or fatty acyl-ACP reductase; FAS: fatty-acid synthase; Fd/FNR: ferredoxin and ferredoxin/NADP+ reductase; GPAT: glyceryl-3- a e ac a e a e; HTD: -hydroxyacyl-CoA dehydratase; KR: ß-ketoacyl-reductase; LPAT: lysophosphatidate acyltransferase; ME: malic enzyme; Sfp: phosphopantetheinyl
16 Chapter 2
transferase; TER: trans-ß-enoyl-CoA reductase; Tes: thioesterase; WS: wax-ester synthase. Product abbreviations: FAEE: fatty-acid ethyl esters; FFA: free fatty acids; HFA: hydroxylated fatty-acids, PK: phosphoketolase; VLC-WE: very-long-chain wax ester. To improve the stoichiometry, Qiao et al. [19 ] tested several alternative
strategies for NADPH generation in Y. lipolytica. The best-performing strain featured
expression of NADP-dependent glyceraldehyde 3-phosphate dehydrogenase from
Clostridium acetobutylicum and of malic enzyme from Mucor circinelloides. The strain
produced 99 g/L lipids with 1.2 g/L/h productivity, which to date, is the best
production performance of a lipid-accumulating microbial strain.
Combining increased NADPH production with upregulation of oxidative stress
defense pathways, improved the lipid yield by 82% and increased productivity by 5-
fold. This strategy also changed the cellular morphology from pseudohyphae to single
cells, which is beneficial for a large-scale fermentation process [20].
2.4. Controlling the chain length of products
Chain length is important for product properties, for example, gasoline-like and
diesel-like biofuels require short/medium-chain fatty acids (S/MCFAs), waxes require
very long chain (VLC) fatty acids and alcohols. Some specialty products such as lactone
fragrances and polyunsaturated fatty acids require a precise chain length. Several
methods to control the chain length of fatty acid products have been reported,
including engineering of fatty acid synthases, reverse beta-oxidation, and
compartmentalization.
2.4.1. Type I fatty acid synthases
Fungal type I fatty acid synthases (FAS I) are large multi- modular proteins,
releasing primarily palmitoyl-CoA. Due to the compact structure of fungal FAS I, the
shorter acyl-CoA intermediates remain trapped in the enzyme complex and are not
accessible for the cytosolic thioesterase. Various heterologous thioesterases have been
17 Chapter 2
overexpressed in Y. lipolytica and S. cerevisiae, and some smaller-sized thioesterases
had an influence on the fatty acyl-CoA profiles [21,22 ]. To obtain a better effect, Xu
et al. replaced the malonyl/palmitoyl transferase domain (MPT) in FAS1 of Y. lipolytica
with heterologous thioesterases [12 ]. The resulting hybrid FAS I resulted in a three-
fold increase of C14 acid, up to 29% of the total free fatty acids. Another approach was
undertaken by Gajewski et al. [23]. They introduced mutations in the active sites of the
beta-ketoacyl synthase (KS), acetyl-CoA:ACP transacetyltransferase (AT), and
malonyl/palmitoyl transferase (MPT) domains. The resulting mutated variants of
FASI produced up to $0.5 g/L of extracellular short/medium-chain fatty acids
(S/MCFA), predominantly hexanoate and octanoate, which do not occur in the wild
type. Zhu et al. also created chimeras of fungal FASI and thioesterases [22 ]. S.
cerevisiae strains expres- sing a chimera of FAS I of R. toruloides and a thioesterase of
Acinetobacter baylyi produced up to 1.7 mg/L of short/ medium chain fatty acids. This
strategy was found superior to the expression of thioesterase in free form or as a C-
terminal fusion to FAS I [22 ]. The type I FAS from mycobacteria produces fatty acids
of C16 C26 in length and has been used for the production of very long chain fatty
acids in S. cerevisiae [24].
2.4.2. Type II fatty acid synthases
Bacteria possess a type II fatty acid synthase (FAS II), which is a protein
complex assembled of multiple sub- units. Through the expression of heterologous
chain- specific thioesterases, the release of acyl-CoAs with a specific chain length can
be achieved. This has been demonstrated in several studies. Jawed et al. identified a
thioesterase from Bryantella formatexigens that enabled highly-specific butyric acid
production using the native FAS in E. coli [25]. To enable the specific production of
C7/9 alkanes, Sheppard et al. tested and optimized a thioesterase from Cuphea
18 Chapter 2
hookeriana and successfully produced octanoate and decanoate in E. coli without
detecting other free fatty acids [26].
The success with manipulating the chain-length specificity of type II FAS in
bacteria inspired the replacement of yeast FAS I with bacterial FAS II. In the study by
Fernandez-Moya et al. [27], nine genes encoding enzymes in E. coli type II FAS were
expressed in S. cerevisiae. The genes could partially compensate for the knock-out of
the native FAS2, albeit the growth was impaired. To alter the fatty acid chain-length
profile, a thioesterase from Ricinus communis was used instead of tesA. This shifted
chain-length profile of intracellular fatty acids from C18 to C14 fatty acids.
2.4.3. Reverse beta-oxidation
For each elongation cycle, which extends the acyl-CoA chain by two carbons,
one malonyl-CoA (C3) is required and a carbon dioxide molecule is released. The
biosynthesis of malonyl-CoA from acetyl-CoA requires an ATP and is hence
energetically costly. As a more energy-efficient route than FAS [28], a reverse beta-
oxidation (RBO) pathway that uses acetyl-CoA instead of malonyl-CoA has been
employed. Here, the initiation module, thiolase, and the last step, thioesterase, can be
varied to control the chain length. Clomburg et al. [29] produced C6 C10 fatty acids
by replacing native thiolase and thioesterase with the variants from Ralstonia eutropha
[29]. Sheppard et al. successfully designed an E. coli strain that specifically produced
pentane via reverse beta-oxidation [26]. A reverse beta-oxidation pathway has also
been implemented in S. cerevisiae [30].
2.5. Compartmentalization
Xu et al. [12 ] showed that the fatty acid ethyl ester (FAEE) titer increased 10
15-fold when the esterifying enzyme was targeted to either the endoplasmic reticulum
19 Chapter 2
or the peroxisomes although a certain extent of product degradation was observed
under peroxisomal localization. Localization of the alkane synthesis pathway into
endoplasmic reticulum [12 ] or peroxisomes [12 ,31 ] also improved the alkane titer,
partly because fatty aldehyde intermediates were isolated from the competing fatty
alcohol biosynthesis [31 ]. Sheng et al. [32] employed peroxisomal targeting of FAR to
increase the production of medium chain-length fatty alcohols due to the higher
abundance of acyl-CoA precursors in peroxisomes [32].
2.6. Functionalizing fatty acid intermediates into products
2.6.1. Lipids and specialty fatty acids
Oleaginous organisms can accumulate lipids at over 20% of the dry biomass
and this capacity can be further increased by metabolic engineering. Oleaginous yeast
Y. lipolytica was engineered by total to produce 85 g/L lipids with a productivity of 0.73
g/L/h. The relatively simple strategy comprised a careful selection of the parental
strain, overexpression of heterologous diacylglycerol acyltransferases type 1 and 2, and
deletion of the native lipase regulator TGL3 [33]. The cells can also be engineered to
change the profile of the acyl chains in lipids, for example, to produce lipids that
contain hydroxylated or desaturated fatty acids. Y. lipolytica has been engineered to
accumulate 12-hydroxy-9-cis-octadecenoic (ricinoleic) acid to 43% of total lipids with
a cellular content of 60 mg/g DW [34]. Ricinoleic acid is otherwise obtained from
castor seeds, which presents a safety problem due to the ricin toxin. Polyunsaturated
fatty acids (PUFAs) are attractive products due to their health and physiological
benefits. Sun et al. engineered Y. lipolytica to accumulate up to 71.6 mg/L of g-
linolenic acid (an omega-6 acid) in lipids [35]. DuPont developed Y. lipolytica strains,
capable of producing eicosapentaenoic acid (an omega-3 acid) at >25% DCW [36 ,37].
20 Chapter 2
Manipulation of the TAG synthesis pathway to produce TAGs with specific
combinations of acyl chains enabled the realization of cocoa-butter-like lipid
accumulation in yeast [38,39].
Hydroxy fatty acids can also be produced in free form, for example, up to 548
mg/L of mixed hydroxylated acids were produced in E. coli engineered for improved
fatty acid production and expressing the fatty acid hydroxylase (CYP102A1) from
Bacillus megaterium [40].
2.6.2. Fatty alcohols
Fatty alcohols can be generated from fatty acids, fatty acyl-ACPs or from acyl-
CoAs by fatty acid reductases, which carry out the reduction either to aldehydes or all
the way to alcohols. From fatty aldehydes, fatty alcohols can be produced relying solely
on the endogenous activity of aldehyde reductase [31 ,32,41 43] although
overexpressing either native [10 ] or heterologous aldehyde reductases [12 ] has also
been shown to enhance fatty alcohol production. Deletion of acyl-CoA synthases
[10 ,44], deletion of acyl-ACP thioesterases [43], and overexpression of acyl-CoA
synthases [12 ] were reported to improve fatty alcohol production when carboxylic
acid reductase (CAR), acyl-ACP-specific fatty acyl reductase (FAR), and acyl-CoA-
specific FAR were employed, respectively. Deletion of aldehyde reductase, catalyzing
reoxidation of fatty aldehydes into fatty acids, was shown to increase the production
of fatty alcohols [31 ].
2.6.3. Hydrocarbons
By combining fatty acyl reductases with aldehyde decarbonylases, alkanes can
be obtained [45]. Cao et al. produced 1.3 g/L of alkanes in an engineered E. coli [46 ].
In S. cerevisiae, peroxisomal localization of alkane biosynthesis improved the alkanes
titer ca. 7-fold, resulting in 3.5 mg/L alkanes [ 1•]. In Y. lipolytica, a 5-fold
21 Chapter 2
improvement was observed and the titer of alkanes was at 16.8 mg/L [12 ]. A proof-
of-concept production of alkenes at 3.7 mg/L, mainly C17 and C19, was also
demonstrated in S. cerevisiae with a heterologous cytochrome P450 fatty acid
decarboxylase [47].
Figure 2.1. Strategies for tuning the microbial metabolism towards fatty acid-derived products. FFA: free fatty acids, VLC: very long chain, EPA: eicosapentaenoic acid
2.6.4. Waxes
Microbial synthesis of very-long-chain fatty acids (VCFA) and their derivatives,
with applications ranging from food to cosmetics [24], has been demonstrated in S.
cerevisiae when using either endogenous long-chain fatty acid elongases or
mycobacterial type I FAS [24,48]. Expression of FAR capable of using VLCFA led to
the production of docosanol [24] and jojoba oil-like wax esters [48] (Figure 2.1).
22 Chapter 2
2.7. Perspectives
Oleochemicals produced by microbial fermentation have a prospect of contributing to the growth of the s18-billion global oleochemical market, and on a long run, they may also substitute many of the petrochemicals and fossil transportation fuels with a trillion-s-market. However, with the current favorable policies for the petrochemical industry, the fermentation-based low-value oleochemicals would result in low profit margins even if the technology for their production were mature. From the technological maturity standpoint, the Y. lipolytica-based single-cell oil process developed by the Stephanopoulos group features the best production metrics, with a titer of 100 g/L and a nearly theoretical yield [19 ]. Additionally, engineered utilization of unnatural mineral sources may allow fermentation at non-sterile conditions [49]. Con- version of Y. lipolytica lipids with a high content of oleic acid into biodiesel will, however, require hydrogenation. Other microbial processes for the production of low-value oleochemicals, such as hydrocarbons, fatty alcohols, and esters, are still very far from commercialization point (Figure 2.2).
23 Chapter 2
Figure 2.2. Reported titers of fatty acid-derived products from microbial synthesis. FFAs: free fatty acids, FAEEs: fatty acid ethyl esters, HFAs: hydroxylated fatty acids. *EPA titer is approximated by assuming a cell dry-weight of 50 g/L.
The commercial efforts in the next 5 10 years will likely focus on high-value specialty oleochemicals. Single cell oils enriched with polyunsaturated fatty acids
(gamma-linolenic, eicosapentaenoic, arachidonic, and docosahexaenoic acids) have been produced commercially since 90s using natural oleaginous algae and fungi [50], and recently also by engineered Y. lipolytica [36•] and this market is expected to grow further. Examples of other attractive high-value oleochemicals are specialty hydroxylated fatty acids for food and feed applications, waxes for cosmetics, lactone fragrances, and others. Improving the performance metrics of the strains remains a major hurdle for the emergence of the new processes and continuous research on metabolic engineering strategies for improved oleochemicals production is a pre- requisite for success.
24 Chapter 2
Conflict of interest
IB and CH are co-founders of BioPhero ApS. VS is co- founder of Biopetrolia.
Acknowledgements
The Novo Nordisk Foundation is acknowledged for the financial support.
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
of special interest
of outstanding interest
1. Pfleger BF, Gossing M, 30/03/2020 21:35:00Nielsen J: Metabolic engineering strategies for microbial synthesis of oleochemicals. Metab Eng 2015, 29:1 11. 2. Zhu L-H, Krens F, Smith MA, Li X, Qi W, Loo EN van, Iven T, Feussner I, Nazarenus TJ, Huai D, et al.: Dedicated Industrial Oilseed Crops as Metabolic Engineering Platforms for Sustainable Industrial Feedstock Production. Sci Rep 2016, 6:srep22181. 3. Bhuiya MMK, Rasul MG, Khan MMK, Ashwath N, Azad AK: Prospects of 2nd generation biodiesel as a sustainable fuel Part: 1 selection of feedstocks, oil extraction techniques and conversion technologies. Renew Sustain Energy Rev 2016, 55:1109 1128. 4. Schmidt JH: Life cycle assessment of five vegetable oils. J Clean Prod 2015, 87:130 138. 5. H. Isikgor F, Remzi Becer C: Lignocellulosic biomass: a sustainable platform for the production of bio-based chemicals and polymers. Polym Chem 2015, 6:4497 4559. 6. Jin M, Slininger PJ, Dien BS, Waghmode S, Moser BR, Orjuela A, Sousa L da C, Balan V: Microbial lipid-based lignocellulosic biorefinery: feasibility and challenges. Trends Biotechnol 2015, 33:43 54. 7. Wu J, Zhang X, Xia X, Dong M: A systematic optimization of medium chain fatty acid biosynthesis via the reverse beta-oxidation cycle in Escherichia coli. Metab Eng 2017, 41:115 124. 8. Krivoruchko A, Zhang Y, Siewers V, Chen Y, Nielsen J: Microbial acetyl-CoA metabolism and metabolic engineering. Metab Eng 2015, 28:28 42. 9. Jong BW de, Shi S, Siewers V, Nielsen J: Improved production of fatty acid ethyl esters in Saccharomyces cerevisiae through up-regulation of the ethanol degradation pathway and expression of the heterologous phosphoketolase pathway. Microb Cell Factories 2014, 13:39.
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10. Zhou YJ, Buijs NA, Zhu Z, Qin J, Siewers V, Nielsen J: Production of fatty acid-derived oleochemicals and biofuels by synthetic yeast cell factories. Nat Commun 2016, 7:ncomms11709. Achieved the highest so far free fatty acids titer (10 g/L) through optimization of cytosolic acetyl- CoA supply in S. cerevisiae. Investigated different combinations of expression of heterologous and native pathways as well as deletion of competing pathways for improved production of fatty alcohols and alkanes. 11. Ghosh A, Ando D, Gin J, Runguphan W, Denby C, Wang G, Baidoo EEK, Shymansky C, Keasling JD, García Martín H: 13C Metabolic Flux Analysis for Systematic Metabolic Engineering of S. cerevisiae for Overproduction of Fatty Acids. Front Bioeng Biotechnol 2016, 4. 12. Xu P, Qiao K, Ahn WS, Stephanopoulos G: Engineering Yarrowia lipolytica as a platform for synthesis of drop-in transportation fuels and oleochemicals. Proc Natl Acad Sci 2016, 113:10848 10853. First demonstration of fungal type I FAS engineering to obtain medium-chain-fatty acids. Presents various compartmentalization strategies, rewiring central carbon metabolism, and decoupling the product formation from growth. 13. Chen L, Zhang J, Lee J, Chen WN: Enhancement of free fatty acid production in Saccharomyces cerevisiae by control of fatty acyl-CoA metabolism. Appl Microbiol Biotechnol 2014, 98:6739 6750. 14. Qiao K, Imam Abidi SH, Liu H, Zhang H, Chakraborty S, Watson N, Kumaran Ajikumar P, Stephanopoulos G: Engineering lipid overproduction in the oleaginous yeast Yarrowia lipolytica. Metab Eng 2015, 29:56 65. The study presents a novel, elegant approach to avoid the repression of acetyl-CoA carboxylase by increasing fatty-acyl-CoA desaturation. The result is a Y. lipolytica strain with improved growth, increased tolerance to sugars, and high-level lipid production. 15. Liu L, Markham K, Blazeck J, Zhou N, Leon D, Otoupal P, Alper HS: Surveying the lipogenesis landscape in Yarrowia lipolytica through understanding the function of a Mga2p regulatory protein mutant. Metab Eng 2015, 31:102 111. 16. Xu P, Li L, Zhang F, Stephanopoulos G, Koffas M: Improving fatty acids production by engineering dynamic pathway regulation and metabolic control. Proc Natl Acad Sci 2014, 111:11299 11304. 17. Shin KS, Lee SK: Introduction of an acetyl-CoA carboxylation bypass into Escherichia coli for enhanced free fatty acid production. Bioresour Technol 2017, doi:10.1016/j.biortech.2017.05.169. 18. Wasylenko TM, Ahn WS, Stephanopoulos G: The oxidative pentose phosphate pathway is the primary source of NADPH for lipid overproduction from glucose in Yarrowia lipolytica. Metab Eng 2015, 30:27 39. 19. Qiao K, Wasylenko TM, Zhou K, Xu P, Stephanopoulos G: Lipid production in Yarrowia lipolytica is maximized by engineering cytosolic redox metabolism. Nat Biotechnol 2017, 35:173 177. This is the first demonstration that modulation of NADPH recovery pathways in Y. lipolytica can increase the theoretical maximum of lipid accumulation. 20. Xu P, Qiao K, Stephanopoulos G: Engineering oxidative stress defense pathways to build a robust lipid production platform in Yarrowia lipolytica. Biotechnol Bioeng 2017, 114:1521 1530. 21. Rutter CD, Zhang S, Rao CV: Engineering Yarrowia lipolytica for production of medium-chain fatty acids. Appl Microbiol Biotechnol 2015, 99:7359 7368. 22. Zhu Z, Zhou YJ, Krivoruchko A, Grininger M, Zhao ZK, Nielsen J: Expanding the product portfolio of fungal type I fatty acid synthases. Nat Chem Biol 2017, 13:360 362.
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Introduction of heterologous domains into reaction chamber of S. cerevisiae's fatty acid synthase enables production of short- and medium-chain fatty acids. 23. Gajewski J, Pavlovic R, Fischer M, Boles E, Grininger M: Engineering fungal de novo fatty acid synthesis for short chain fatty acid production. Nat Commun 2017, 8:ncomms14650. 24. Yu T, Zhou YJ, Wenning L, Liu Q, Krivoruchko A, Siewers V, Nielsen J, David F: Metabolic engineering of Saccharomyces cerevisiae for production of very long chain fatty acid-derived chemicals. Nat Commun 2017, 8:ncomms15587. 25. Jawed K, Mattam AJ, Fatma Z, Wajid S, Abdin MZ, Yazdani SS: Engineered Production of Short Chain Fatty Acid in Escherichia coli Using Fatty Acid Synthesis Pathway. PLOS ONE 2016, 11:e0160035. 26. Sheppard MJ, Kunjapur AM, Prather KLJ: Modular and selective biosynthesis of gasoline- range alkanes. Metab Eng 2016, 33:28 40. 27. Fernandez-Moya R, Leber C, Cardenas J, Da Silva NA: Functional replacement of the Saccharomyces cerevisiae fatty acid synthase with a bacterial type II system allows flexible product profiles. Biotechnol Bioeng 2015, 112:2618 2623. 28. Kim S, Clomburg JM, Gonzalez R: Synthesis of medium-chain length (C6 C10) fuels and chemical ia -oxidation reversal in Escherichia coli. J Ind Microbiol Biotechnol 2015, 42:465 475. 29. Clomburg JM, Blankschien MD, Vick JE, Chou A, Kim S, Gonzalez R: Integrated engineering of -o ida ion e e al and -oxidation pathways for the synthesis of medium chain -functionalized carboxylic acids. Metab Eng 2015, 28:202 212. 30. Lian J, Zhao H: Re e al of he -Oxidation Cycle in Saccharomyces cerevisiae for Production of Fuels and Chemicals. ACS Synth Biol 2015, 4:332 341. 31. Zhou YJ, Buijs NA, Zhu Z, Gómez DO, Boonsombuti A, Siewers V, Nielsen J: Harnessing Yeast Peroxisomes for Biosynthesis of Fatty-Acid-Derived Biofuels and Chemicals with Relieved Side-Pathway Competition. J Am Chem Soc 2016, 138:15368 15377. The authors show that localizing the pathway for alkane biosynthesis in peroxisomes significantly decreases the degradation of pathway intermediates, fatty aldehydes, and improves the overall alkane production. 32. Sheng J, Stevens J, Feng X: Pathway Compartmentalization in Peroxisome of Saccharomyces cerevisiae to Produce Versatile Medium Chain Fatty Alcohols. Sci Rep 2016, 6:srep26884. 33. Friedlander J, Tsakraklides V, Kamineni A, Greenhagen EH, Consiglio AL, MacEwen K, Crabtree DV, Afshar J, Nugent RL, Hamilton MA, et al.: Engineering of a high lipid producing Yarrowia lipolytica strain. Biotechnol Biofuels 2016, 9:77. 34. Beopoulos A, Verbeke J, Bordes F, Guicherd M, Bressy M, Marty A, Nicaud J-M: Metabolic engineering for ricinoleic acid production in the oleaginous yeast Yarrowia lipolytica. Appl Microbiol Biotechnol 2014, 98:251 262. 35. Sun M-L, Madzak C, Liu H-H, Song P, Ren L-J, Huang H, Ji X-J: Engineering Yarrowia lipolytica fo efficien -linolenic acid production. Biochem Eng J 2017, 117, Part A:172 180. 36. Xie D, Jackson EN, Zhu Q: Sustainable source of omega-3 eicosapentaenoic acid from metabolically engineered Yarrowia lipolytica: from fundamental research to commercial production. Appl Microbiol Biotechnol 2015, 99:1599 1610. Summarizes the process for developing Y. lipolytica strains for commercial production of an omega-3 acid. 37. Xue Z, Sharpe PL, Hong S-P, Yadav NS, Xie D, Short DR, Damude HG, Rupert RA, Seip JE, Wang J, et al.: Production of omega-3 eicosapentaenoic acid by metabolic engineering of Yarrowia lipolytica. Nat Biotechnol 2013, 31:734 740.
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38. Wei Y, Gossing M, Bergenholm D, Siewers V, Nielsen J: Increasing cocoa butter-like lipid production of Saccharomyces cerevisiae by expression of selected cocoa genes. AMB Express 2017, 7:34. 39. Wei Y, Siewers V, Nielsen J: Cocoa butter-like lipid production ability of non-oleaginous and oleaginous yeasts under nitrogen-limited culture conditions. Appl Microbiol Biotechnol 2017, 101:3577 3585. 40. Cao Y, Cheng T, Zhao G, Niu W, Guo J, Xian M, Liu H: Metabolic engineering of Escherichia coli for the production of hydroxy fatty acids from glucose. BMC Biotechnol 2016, 16:26. 41. Buijs NA, Zhou YJ, Siewers V, Nielsen J: Long-chain alkane production by the yeast Saccharomyces cerevisiae. Biotechnol Bioeng 2015, 112:1275 1279. 42. Rutter CD, Rao CV: Production of 1-decanol by metabolically engineered Yarrowia lipolytica. Metab Eng 2016, 38:139 147. 43. Liu Y, Chen S, Chen J, Zhou J, Wang Y, Yang M, Qi X, Xing J, Wang Q, Ma Y: High production of fatty alcohols in Escherichia coli with fatty acid starvation. Microb Cell Factories 2016, 15:129. 44. Teixeira PG, Ferreira R, Zhou YJ, Siewers V, Nielsen J: Dynamic regulation of fatty acid pools for improved production of fatty alcohols in Saccharomyces cerevisiae. Microb Cell Factories 2017, 16:45. 45. Kang M-K, Zhou YJ, Buijs NA, Nielsen J: Functional screening of aldehyde decarbonylases for long-chain alkane production by Saccharomyces cerevisiae. Microb Cell Factories 2017, 16:74. 46. Cao Y-X, Xiao W-H, Zhang J-L, Xie Z-X, Ding M-Z, Yuan Y-J: Heterologous biosynthesis and manipulation of alkanes in Escherichia coli. Metab Eng 2016, 38:19 28. The study reports so far the highest titer of alkanes obtained in a microbial process. The authors systematically apply a variety of metabolic engineering strategies to optimize an E. coli strain. 47. Chen B, Lee D-Y, Chang MW: Combinatorial metabolic engineering of Saccharomyces cerevisiae for terminal alkene production. Metab Eng 2015, 31:53 61. 48. Wenning L, Yu T, David F, Nielsen J, Siewers V: Establishing very long-chain fatty alcohol and wax ester biosynthesis in Saccharomyces cerevisiae. Biotechnol Bioeng 2017, 114:1025 1035. 49. Shaw AJ, Lam FH, Hamilton M, Consiglio A, MacEwen K, Brevnova EE, Greenhagen E, LaTouf WG, South CR, Dijken H van, et al.: Metabolic engineering of microbial competitive advantage for industrial fermentation processes. Science 2016, 353:583 586. 50. Ratledge C: Microbial oils: an introductory overview of current status and future prospects. OCL 2013, 20:D602. 51. Wang Y, Zhang S, Pötter M, Sun W, Li L, Yang X, Jiao X, Zhao ZK: O e e e ion of 12- Fatty Acid Desaturase in the Oleaginous Yeast Rhodosporidium toruloides for Production of Linoleic Acid-Rich Lipids. Appl Biochem Biotechnol 2016, 180:1497 1507.
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3
Advances in synthetic biology of oleaginous yeast Yarrowia lipolytica for
producing non-native chemicals
Farshad Darvishi, Mehdi Ariana, Eko Roy Marella, Irina Borodina
Reprinted by permission from Springer Nature GmbH: Springer Nature Applied Microbiology and Biotechnology Darvishi F, Ariana M, Marella ER, Borodina I. Advances in synthetic biology of oleaginous yeast Yarrowia lipolytica for producing non-native chemicals. Appl Microbiol Biotechnol. 2018; 102:5925 (doi: 10.1007/s00253-018-9099-x)
Chapter 3
Abstract
Abstract Oleaginous yeast Yarrowia lipolytica is an important industrial host for the production of enzymes, oils, fragrances, surfactants, cosmetics, and pharmaceuticals.
More recently, improved synthetic biology tools have allowed more extensive engineering of this yeast species, which lead to the production of non-native metabolites. In this review, we summarize the recent advances of genome editing tools for Y. lipolytica, including the application of CRISPR/Cas9 system and discuss case studies, where Y. lipolytica was engineered to produce various non-native chemicals: short-chain fatty alcohols and alkanes as biofuels, polyunsaturated fatty acids for nutritional and pharmaceutical applications, polyhydroxyalkanoates and dicarboxylic acids as precursors for biodegradable plastics, carotenoid-type pigments for food and feed, and campesterol as a precursor for steroid drugs.
3.1. Introduction
Yarrowia lipolytica is rapidly gaining popularity as an industrial host for production of recombinant proteins and other chemicals [1,2]. This oleaginous yeast species has an exceptional ability to accumulate lipids and to utilize hydrophobic compounds such as fats, oil, fatty acids, and n-alkanes [3,4]. It is generally regarded as safe (GRAS) by the Food and Drug Administration in the USA. Native strains of Y. lipolytica are used for the production of citric acid (Pfizer Inc. and ADM, USA), lipase
(Artechno, Belgium), erythritol (Baolingbao Biology Co., China), fodder yeast, and probiotics (Skotan SA, Poland). British Petroleum (UK) produced single-cell proteins from this yeast for livestock feeding (Toprina G) until 1978 [5,6]. Furthermore, genetically engineered strains of Y. lipolytica are used for the production of eicosapentaenoic acid (EPA)-rich single-cell oils (DuPont, USA), human lysosomal
30 Chapter 3 enzymes for the treatment of lysosomal storage diseases (Oxyrane, Belgium and UK), and lipase for the treatment of exocrine pancreatic insufficiency (Mayoly Spindler,
France) as enzyme replacement therapies[7].
Moreover, native strains of Y. lipolytica are applied for the bioremediation of environments contaminated with aliphatic and aromatic compounds, organic pollutants, 2,4,6-trinitrotoluene, and metals [6]. The freeze-dried Y. lipolytica cells and extracellular lipases are produced by Artechno (Belgium) for bioremediation of oily wastewaters [8].
Therefore, this non-conventional oleaginous yeast is a valuable candidate for industrial and environmental applications.
The genomes of several strains of Y. lipolytica have been sequenced and are publicly available from NCBI or GRYC databases (http://igenolevures.org/). The genome of Y. lipolytica type strain CLIB99 size is ca. 20.5 Mb with a GC content of
49% which is organized in six chromosomes ranging from 2.3 to 4.2 Mb in size. This yeast strain has 7357 genes which encode 6472 proteins [9,10,6,11,12].
In the past decade, the genetic toolbox for engineering yeasts has been significantly expanded, which opened up for opportunities to engineer strains for production of non-native metabolites [13 19].
In this review, we summarize the advances in engineering Y. lipolytica as a chassis for the production of bio-based chemicals. The review consists of two parts: one describing the recent developments in the genetic toolbox for engineering Y. lipolytica, including the CRISPR/Cas9 system and the second describing engineering of Y. lipolytica for the production of non-native metabolites for industrial, nutraceutical, and pharmaceutical applications.
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3.2. Synthetic biology tools for metabolic engineering of Yarrowia lipolytica
3.2.1. Promoters
Promoters are the key elements in controlling gene expression. The strength of a promoter is determined by several factors including core promoter, TATA box, proximal promoter sequence, and upstream activating sequences (UAS) [20].
Commonly used endogenous constitutive Y. lipolytica promoters are TEF1, EXP1,
GPD, GPAT, YAT1, FBA1, GPM1, TDH1, POX2, LEU2, and XPR2 [21]. Wong et al. [18] established a sensitive luciferase reporter to characterize a set of 12 native promoters to expand the Yarrowia genetic toolbox for transcriptional fine-tuning. They found that TEF1 promoter exhibited strongest expression levels, and other 11 promoters, derived from TCA cycle, glycolysis, pentose phosphate pathway, or lipid oxidation pathway, demonstrated relative transcriptional activity ranging from 0.7 to 29.7% of
TEF1 promoter activity.
Strong hybrid promoters were engineered by fusing a core promoter with UAS.
Specifically, the upstream region of the XPR2 promoter, containing four UAS tandem repeats, was fused to LEU2 core promoter to create a constitutive strong hybrid promoter HP4D [22].
A generalizable method was introduced for construction of new strong synthetic hybrid promoters. The fusion of between 1 and 32 tandem UAS sequences from minimal leucine promoter (pLEUm) and 8 16 copies of UAS from the core promoter of TEF1 increased the mRNA and protein expression levels. For example, mRNA level was 400-fold higher for hybrid promoter UAS1B24-LEUm in comparison with the core promoter pLEUm [23 25].
Many of the genes encoding the central metabolic enzymes in Y. lipolytica contain introns at the beginning of the gene. Including these introns was shown to
32 Chapter 3 improve the expression of heterologous genes. Thus, FBA1IN promoter, constructed from an FBA1 sequence of 826 to + 169 and an intron of + 64 to + 165, demonstrated five times higher expression level compared to the FBA1 promoter without the intron
[26]. Furthermore, an intron-containing translation elongation factor-1
(TEF1intron) promoter exhibited a 17- and 5-fold increase in gene expression over intron-less TEF1 and HP4D promoters, respectively [27]. Furthermore, promoter strength can be fine-tuned through the engineering of the TATA box sequence, core promoter, and upstream activating sequences [28].
Trassaert et al. [29] isolated and characterized the promoter of the EYK1 gene coding for an erythrulose kinase, which in the presence of erythritol or erythrulose is strongly induced. They constructed new hybrid promoters containing tandem repeats of either UAS1XPR2 or UAS1EYK1 showing higher expression levels than the native pEYK1 promoter. The promoter strength was improved in a strain carrying a deletion in the EYK1 gene, which prevented the catabolism of the inducers erythritol and erythrulose.
The comparative strengths of these promoters have been reported in several studies and are summarized in Table 4.1.
3.2.2. Terminators
Terminators influence net protein output by controlling mRNA half-life [31].
The native terminators XPR2t, LIP2t, PHO5t, and minimal XPR2t have been used since the early days of implementing heterologous gene expression in Y. lipolytica
[22]. Moreover, heterologous terminators such as CYC1t from Saccharomyces cerevisiae turned out to be functional in Y. lipolytica [23]. Native terminators are typically 100 450 bp long and thus need to be cloned. Short synthetic terminators
(35 75 bp) were developed to circumvent the necessity of cloning, because they can
33 Chapter 3 be simply included into the primer sequence. Seven short synthetic terminators previously developed for S. cerevisiae were evaluated in Y. lipolytica. Some terminators resulted in up to 60% improvement in gene expression compared to wild- type CYC1 terminator from S. cerevisiae. Some synthetic terminators, including
Tsynth2, Tsynth7, Tsynth8, Tsynth10, Tsynth22, Tsynth27, exhibited a similar performance in both Y. lipolytica and S. cerevisiae, which indicates the conformity of the synthetic terminators among yeast strains. Overall, the advantages of synthetic terminators are simplified cloning, improved functionality, and low homology with the inherent yeast genome that prevents unwanted events of homologous recombination between the integrated DNA elements [31].
Table 4.1. Comparison of strength of Y. lipolytica promoters
Relative ordering of promoters Comments Ref. based on their strength
EXP1 > TEF1 > GPD > GPAT > YAT1 > Reporter: Green fluorescent protein encoded by hrGFP. [23] XPR2 > FBA1 Analysis: Flow cytometry.
Reporter: -glucuronidase (GUS) protein encoded by TEF1> TDH1 > GPM1 gusA gene. Analysis: Fluorescence multiwell plate [26] reader.
Reporter: -galactosidase encoded by LacZ gene. TEF1intron >> HP4D > TEF1 [27] Analysis: Spectrophotometer.
TEF1> GPD> ACL2> ICL1> IDH2> FAS Reporter: Luciferase encoded by yNluc gene. > DGA1> FAS > POX4> ZWF> ACC1> [18] IDP2 Analysis: Microtiter plate reader.
TEF1intron >> GPD > EXP1 > TEF1 > Reporter: Green fluorescent protein encoded by hrGFP. ILV5 > YAT1 > ICL > FBA1 [30] GPAT, DGA1, GPM1 and FBA1intron = 0 Analysis: Flow cytometry. Abbreviations: hrGFP: Humanized renilla green fluorescent protein; GUS: -glucuronidase; yNluc: Cassettes with codon- optimized genes expressing yeast of 19 kDa luciferase NanoLuc (Nluc)
3.2.3. Selection markers
Different selection markers including auxotrophic and dominant markers have been used in Y. lipolytica. Auxotrophic markers complement a mutation in a cell line and allow growth on selective medium, lacking an essential nutrient. LEU2 and URA3,
34 Chapter 3 which repair auxotrophy for leucine and uracil correspondingly in the strains with mutations in the same genes, are the most commonly used auxotrophic selection markers in Y. lipolytica. Two URA3 alleles were used for selection of transformants in yeast: a wild-type ura3d1 allele and a mutant ura3d4 allele. The ura3d1 as non- defective allele is used for the selection of single integration event. The ura3d4 is a defective allele with promoter truncated to only 6 bp upstream the ATG codon. The ura3d4 is used for the selection of multiple integrations. The dominant markers are related to antibiotic resistance or a new metabolic property. For example, some Y. lipolytica strains are sensitive to bleomycin/phleomycin and hygromycin B and the genes conferring resistance to these antibiotics are used as dominant selection markers [32]. Another type of dominant selection markers are markers that allow new metabolic capabilities, such as D-serine deaminase encoding gene dsdA and giving the cell an ability to grow on D-serine [30,33].
If repeated rounds of genome editing are required, then it is necessary to remove the selection markers from the genome, so they can be re-used. The Cre-lox system is an efficient method to remove the marker genes from Y. lipolytica genome by recombination of loxP sites that are placed up- and downstream of the selection marker. This system requires the introduction of Cre-expression vector into the cell; then, Cre-protein intracellularly conducts recombination procedure between two loxP sites at both termini of the selected marker. However, scars remain in the genome after marker excision by the Cre-lox system [34].
More recently, Wagner et al. [35] adapted the piggyBac transposon system to enable efficient generation of genome-wide insertional mutagenesis libraries and the introduction of scarless, footprint-free genomic modifications in Y. lipolytica. They also developed a variety of piggyBac compatible selection markers and subsequent marker excision, including a novel dominant marker cassette (Escherichia coli guaB)
35 Chapter 3 for effective Y. lipolytica selection using mycophenolic acid as well as auxotrophic selection markers (uracil or tryptophan) or dominant selection markers (hygromycin, nourseothricin, chlorimuron ethyl, and mycophenolic acid resistance). The new dominant selection marker for mycophenolic acid resistance increases the options for engineering Y. lipolytica strains without relying on auxotrophic markers.
3.2.4. Integrative vectors
Integrative vectors are more stable than replicative vectors and also result in more homogeneous expression of genes within a clonal population [36]. Integrative vectors contain one or two regions homologous to the genomic DNA for respectively single or double-crossover recombination. In the first case, the whole plasmid gets integrated; in the second case, only the region surrounded by homologous sequences gets integrated. In Y. lipolytica, it is necessary to use rather long homologous regions of 0.5 1 kb length to achieve good efficiencies of recombination [2]. Moreover, deletion of non-homologous end-joining responsible genes ( k 70 and k 80) improves the success rate of homologous recombination [37].
The integrative vectors can be classified into three categories: (i) single integrative vectors with selection markers, (ii) multiple integrative vectors with weakened or defective selection markers, and (iii) single integrative vectors without selection markers for integration via CRISPR/Cas9.
Single integrative vectors contain sequences homologous to the Y. lipolytica genome for integration. Holkenbrink et al. [30] constructed 26 vectors for 11 specific intergenic integration sites with Nat (Nourseothricin), Hph (hygromycin B), or URA3 selection markers as part of synthetic toolbox EasyCloneYALI. The integrative vectors can be cloned via USER® assembly to express one or two genes. The integration efficiencies vary between different sites and are at 30 100%.
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Multiple integration vectors have been designed that target multiple rDNA or
Ylt1-retrotransposon (zeta-elements) re-gions. Vectors carrying the ura3d4 allele are not able to confer an Ura+ phenotype when present in a single copy, but they are able to restore growth on selective medium in multiple integrations [32].
Marker-less integrative vectors for insertion via CRISPR/ Cas9 system have been created by Holkenbrink et al. [30]. Five vectors targeting different intergenic regions integrate with over 80% efficiency.
3.2.5. Replicative vectors
Replicative vectors as shuttle vectors are also used for introducing DNA into Y. lipolytica. No inherent episomal plasmids have been found in Yarrowia strains so far.
Therefore, replicative vectors have been designed using chromosomal autonomously replicating sequence and centromere (ARS/CEN). These vectors are maintained at one to three copies per cell. The vectors require selection pressure for maintenance and are commonly used for a transient expression like Cre recombinase expression for efficient marker rescue [2]. The stability and copy number per cell can be improved by the fusion of various promoters upstream of the centromeric region [38].
Bredeweg et al. [39] constructed a multipurpose expression vector pYL15 to enable rapid analysis of protein localization by expression of GFP fusion proteins in Y. lipolytica. De Pourcq et al. [34] constructed a vector for gene knock-outs and marker rescue using Cre-lox recombination system.
Different types of replicative and integrative vectors, as well as surface display and gene knockout vectors, and their characteristics for Yarrowia are summarized in
Table 4.2.
37 Chapter 3
Table 3.2. Shuttle vectors for Y. lipolytica Selection Secretion Characteristics / Type of vector Promoter Terminator Integration site Example Ref marker signal Availability
Integrative vectors for gene expression
No signal pINA1269 Homologous integration with a pHP4D XPR2t LEU2 - XPR2 pre pINA1296 high transformation efficiency / [32,40] On request XPR2 prepro pINA1267
No signal pINA1312 pHP4D XPR2t XPR2 prepro pINA1314 XPR2 pre pINA1317 No signal pINA1311 [32,41] pHP4D LIP2t LIP2 prepro pINA1313 Non-homologous integration Single integrative pPOX2 URA3d1 - No signal JMP62 into the genome of Ylt1-free Y. vectors with selection Inducible LIP2t lipolytica strains / On request markers promoter LIP2 prepro JMP61
pTEF1 LIP2t No signal pKOV96 [42]
pICL1 Inducible XPR2t No signal p66IP [43] promoter 11 different 26 different Integration and expression of up Standardized PEX20t or Nat, Hph integration vectors of to two genes simultaneously via - promoters LIP2t or URA3 defined sites in EasyClone standardized USER® cloning / the genome YALI toolbox Addgene [30] IntB Hph - pCfB4906 The integrative Cas9 pTEF1 TEFt (Chromosome B) expression vector / Addgene DsdA ku70 locus pCfB6364
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Table 3.2. Shuttle vectors for Y. lipolytica (continued) Selection Secretion Type of vector Promoter Terminator Integration site Example Characteristics / Availability Ref marker signal No signal pINA1292 pHP4D XPR2t XPR2 prepro pINA1294 XPR2 pre pINA1297 Multiple integrative No signal pINA1291 Zeta The vectors present multiple [32,41] vectors with pHP4D LIP2t LIP2 prepro pINA1293 integrations with low weakened or defective URA3d4 transformation selection markers pPOX2 No signal JMP64 efficiency / On request Inducible LIP2t promoter LIP2 prepro JMP63 pICL1 No signal p67IP Inducible XPR2t rDNA [43] promoter No signal p64IP The pCASyl-based CRISPR-Cas9 system enabled efficient, scarless, pTEF1in CYCt - - - pCAS1yl [44] single or multigene editing through Single integrative NHEJ and HR / On request vectors via 7 different 7 different Marker-less integration and CRISPR/Cas9 Standardized PEX20t or integration vectors of expression of up to two genes - - [30] promoters LIP2t defined sites in EasyClone simultaneously via standardized the genome YALI toolbox USER® cloning / Addgene Replicative vectors for gene expression High transformation efficiency / On Replicative vector pHP4D XPR2t LEU2 - - pRRQ1 [45] request Improvement of plasmid Engineered CEN Standardized Promoter- - - - - copy number and expression level / [38] vector promoters CEN Plasmid On request Contains CRISPR/Cas9 system for pUAS1B8- Centromeric vector CYCt LEU2 - - pCRISPRyl marker-less gene disruption and [46] TEF1 integration / On request Multipurpose vector pEXP1 - LEU2 - - pYL15 High-level expression / On request [39] It used to modular pathway YaliBricks vector pTEF1 XPR2t LEU2 - - pYaliA1 engineering and facile genetic [18] operation / On request Episomal vector of High transformation efficiency for EasyCloneYALI pPOT1 RPR1t Nat - - pCfB3405 [30] gRNA expression / Addgene toolbox
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Table 3.2. Shuttle vectors for Y. lipolytica (continued) Selection Secretion Type of vector Promoter Terminator Integration site Example Characteristics / Availability Ref marker signal Surface display vector
XPR2 pre / pINA1317- Surface display on 100 % cells / On Surface display vector pHP4D XPR2t URA3d1 Zeta YlCWP1 GPI [47] YlCWP110 request anchor
Vectors for gene knock-outs Cre-lox recombination system was Knock-out vector pYlMNN9- - - URA3 - - used to gene knock-out with [34] using Cre-lox system PUT efficient marker rescue / On request YaliBricks vector for pYaliA1- The CRISPR/Cas9 system was used pTEF1 XPR2t LEU2 - - [18] gene knock-out hCas9-gRNA to gene knock-out / On request pCfB3405 To obtain gene knock-outs via the Vectors for gene (gRNA) CRISPR/Cas9, cells were knock-out of pPOT1 / Nat, Hph pCfB4906 RPR1t / TEFt - - transformed with a [30] EasyCloneYALI pTEF1 or DsdA (Cas9) 90 bp double-stranded DNA repair toolbox pCfB6364 template / Addgene (Cas9)
40 Chapter 3
3.2.6. CRISPR-Cas9 system for genome editing
Clustered regularly interspaced short palindromic repeat-associated protein-9 nuclease (CRISPR-Cas9) has been widely used to genome editing of various organisms since 2012.
CRISPR was applied for genome editing of S. cerevisiae in 2013 [48] and since then was extended to multiple yeast species, including Y. lipolytica [16,46,49].
The CRISPR/Cas9 system for genome editing combines three parts: (1) the
Cas9 endonuclease which is capable of creating dsDNA breaks, (2) a guide RNA
(gRNA) consisting of CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA) which binds Cas9 endonuclease (often combined into a single guiding RNA gRNA), and (3) a dsDNA repair template which is used by the homologous recombination pathway to repair the dsDNA break.
The main advantages of using CRISPR for genome editing in comparison to classic engineering with selective plasmids are the following (Figure 3.1): (i) shortened time of each editing round, because there is no need to remove selective markers from the genome, while the loss of replicative gRNA-vector is a fast process; (ii) possibility to edit multiple gene alleles by targeting a common homology region; and (iii) possibility to multiplex.
Several studies demonstrated the potential of the CRISPR/Cas9 system in Y. lipolytica. Schwartz et al. [46] constructed a centromeric vector, pCRISPRyl, containing Yarrowia codon-optimized Cas9 from Streptococcus pyogenes and a gRNA under hybrid SCR1 -tRNA promoter for disruption of MFE1 gene. The disruption rate reached 100% after 2- or 4-day outgrowth in Y. lipolytica PO1f with disrupted KU70.
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Figure 3.1. Comparison of traditional and CRISPR-based genome editing in Y. lipolytica
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Gao et al. [44] used a single vector to simultaneously introduce Yarrowia codon-optimized Cas9 from S. pyogenes and relevant guide RNA expression cassettes in wild-type, ku70-deficient, and ku70/ku80 double-deficient strains of Y. lipolytica.
CAS1yl vector without donor DNA induced DSBs and NHEJ repair, whereas CAS2yl vector with donor DNA induced DSBs and HR repair. Two Cas9 target genes, TRP1 and PEX10, were repaired by NHEJ or HR with maximal efficiencies of 85.6% for the wild-type strain and 94.1% for the ku70/ku80 double-deficient strain within 4 days.
Simultaneous double and triple multigene editing was achieved with pCAS1yl by
NHEJ with efficiencies of 36.7 or 19.3%, respectively.
CRISPR/Cas9 system has successfully been used for insertion of expression constructs and gene knockout after 2 4 days of recovery phase. The cells divide through multiple generations during the long recovery phase which is imperative to use replicating repair templates. Holkenbrink et al. [30] have recently developed a
CRISPR/Cas9 system to achieve high genome editing efficiency using non-replicating
DNA repair templates. They have used synthetic double-stranded oligos as repair templates for gene deletions/mutations and linearized non-replicating vectors for gene insertions. The genome engineering toolbox EasyCloneYALI comprises a set of
CRISPR integrative expression vectors which allow expression of one or two genes per vector and integration into highly expressed intergenic genome sites. The vectors are assembled by USER cloning to ease and accelerate vector construction, and all vectors in this toolbox are standardized and allow recycling of biobricks. The toolbox also provided vectors for a knockout of up to two genes simultaneously using 90 bp double- stranded oligos as DNA repair templates with efficiencies of 90% for individual knockouts and 6 66% for double knock-outs [30].
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3.3. Engineering of Yarrowia lipolytica to produce non- native chemicals
3.3.1. Short-chain fatty alcohols for biofuels
Fatty alcohols, with carbon chain lengths ranging from 6 to 22, are used as solvents, surfactants, lubricants, etc. Furthermore, they can be used as fuels. Y. lipolytica has been engineered to produce fatty alcohols by integration of fatty-CoA reductase. The engineered strain of Y. lipolytica produced 1-decanol up to 500 mg/L; the strain expressed a fatty acyl-CoA reductase from Arabidopsis thaliana and had a deletion of the major peroxisome biogenesis factor Pex10p [50]. Wang et al. [51] obtained 660 mg/L 1-hexadecanol using Y. lipolytica expressing codon-optimized fatty acyl-CoA reductase from Tyto alba (TaFAR1) and deleting DGA1 and FAO1 genes.
3.3.2. Volatile aldehydes
Volatile aldehydes (hexanal, cis-3-hexenal, and trans-2-hexenal) and their corresponding alcohols (hexanol, cis-3-hexenol, and trans-2-hexenol) are responsible for the fresh green odor of many vegetables and fruits. Formation of these compounds occurs by the lipoxygenase pathway, which was first demonstrated in tea (Thea sinensis) leaves. The precursors of green odor are linoleic and -linolenic acids. These free fatty acids are oxygenated to form their hydroperoxides by the lipoxygenase and then the fatty acid chains of the hydroperoxides are cleaved between C-12 and C-13 by fatty acid hydroperoxide lyase (HPL) to form C6-aldehyde and C12-oxo-acid. The HPL from green bell pepper fruit was expressed in Y. lipolytica. The recombinant strain produced 6 mM (600 mg/L) of hexanal in the biocatalytic medium containing 50 mM
44 Chapter 3 of dithiothreitol and 119 mM of 13-hydroperoxides (37 g/L) under oxido-reducing conditions [52].
Current biofuel technology is limited by the lack of pathways to produce short- chain n-alkanes, such as the C5 n-alkane, pentane, to use in variable mixtures of gasoline and jet fuel. Blazeck et al. [53] designed a pathway for microbial production of pentane by Y. lipolytica. This pathway utilizes a soybean lipoxygenase enzyme to cleave linoleic acid to pentane and a tridecadienoic acid as a by-product. The resulting strain produced proof-of-concept amounts of pentane (ca. 5 mg/L).
3.3.3. Polyunsaturated fatty acids for nutritional and pharmaceutical
applications
Many polyunsaturated fatty acids (PUFAs) are used as food and feed supplements, through systematic data on health benefits of these acids is inconclusive.
Y. lipolytica produces conjugated linoleic acids (CLA), which is a mix of linoleic acid isomers (18:2, n9, n12). Zhang et al. [54] co-expressed the fatty acid delta 12 desaturase gene (FADS12, d12) from Mortierella alpina and the codon-optimized linoleic acid isomerase (opai) gene from Propionibacterium acnes in Y. lipolytica under the control of promoter hp16d modified by fusing 12 copies of UAS1B to the original promoter HP4D. The engineered strain successfully produced trans-10, cis-
12-CLA at 4 g/l. Imatoukene et al. [55] constructed a mutant strain of Y. lipolytica with various genetic modifications to produce CLA, including the elimination of - oxidation (pox1 6 ) and the inability to store lipids as triglycerides (dga1 dga2 a e1 l o1 ). They further expressed PAI gene-encoding linoleic acid isomerase in
Propionibacterium acnes and overexpressed Y. lipolytica 12-desaturase gene
(YlFAD2). The strain produced 302 mg/L of CLA in bioconversion medium containing soybean oil (2% v/v; 62% purity) in a bioreactor culture.
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-Linolenic acid (GLA) has various beneficial physiological effects and pharmaceutical and nutraceutical applications. Sun et al. [56] established a biotechnological process for GLA production by Yarrowia lipolytica. The codon- optimized △6-desaturase from Mortierella alpina was introduced into this yeast under the control of the strong HP4D promoter. The recombinant strain produced 71.6 mg/L of GLA in the bioreactor.
To construct eicosapentaenoic acid (EPA) producing Yarrowia strain, Xue et al. [57] inserted an integration cassette containing one copy of a -12 desaturase gene, two copies of a -9 elongase gene, and one copy of a C16 elongase into the LEU2 locus in Y. lipolytica ATCC 20362. Then, they expressed -5, -8, and -17 desaturases as well as disrupted peroxisome biogenesis gene PEX10, LIP1 gene-encoding lipase 1, and
SCP2 gene-encoding sterol carrier protein. The resulting strain produced 56.6% EPA of total fatty acid from 80 g/L glucose.
3.3.4. Polyhydroxyalkanoates for biodegradable plastics
Polyhydroxyalkanoates (PHAs) can be used for packaging and for medical applications [58]. Short-chain (SC) hydroxyalkanoates are three to five carbon chain length, while medium chain (MC) are six to 16 carbon chain length. The MC-PHA synthase, encoded by phaC1 from Pseudomonas aeruginosa PAO1, was expressed under recombinant HP4D promoter with a peroxisomal targeting signal in Y. lipolytica. The resulting strain accumulated up to 1.11 g/L of MC-PHA, or 5% MC-PHA per dry cell weight, on a medium containing triolein [58]. Recently, Li et al. [59] reported polyhydroxybutyrate (PHB) production in Y. lipolytica Po1g strain by expressing ß-ketothiolase, acetoacetyl-CoA and PHB synthase from Ralstonia eutropha. This strain produced 7.35 g/L of PHB through fed-batch cultivation with acetate as the sole carbon source.
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3.3.5. Dicarboxylic acids as precursors of plastics
Itaconic acid
The natural producers of itaconic acid, such as Aspergillus terreus, present challenges for industrial scale-fermentations due to poor growth and low productivity in agitated bioreactors. On the other hand, Y. lipolytica is well amenable for large- scale fermentation and can be engineered to produce itaconic acid by expression of a single heterologous enzyme, cis-aconitase decarboxylase (CAD) [60]. Blazeck et al.
[60] obtained 33 mg/L itaconic acid through the episomal expression of CAD from A. terreus (AtCAD) in Y. lipolytica Po1f strain. The strain was further engineered for increased precursor supply, resulting in the final itaconic acid titer of 5.8 from 20 g/L dextrose [60].
Dodecanedioic acid
Dodecanedioic acid (DDDA) is a monomer of bio-based plastics and can also be used in lubricants, adhesives, and cosmetics. Y. lipolytica strains were developed that can shorten fatty acids of variable length to 12 carbons-length via -oxidation and oxidize them by -oxidation to obtain DDDA from plant oils and animal fats [61].
3.3.6. Ricinoleic acid
Ricinoleic acid (12-hydroxy-octadec-cis-9-enoic acid, C18:1-OH, RA) and its derivatives find applications as pharmaceuticals, food additive, surfactants and pigment wetting agents, and others. It is naturally synthesized in castor seeds (Ricinus communis), but the presence of ricin toxin in the seeds restricts certain applications.
B opoulos et al. [62] engineered Y. lipolytica by removing peroxisomal oxidases
(Pox1 6p) and native diacylglycerol acyltransferases (Dga1p and Dga2p) as well as overexpressing phospholipid/diacylglycerol acyltransferase (Lro1p) and Claviceps
47 Chapter 3 purpurea fatty acid 12-hydroxylase (CpFAH12). The resulting strain accumulated RA at 6% of its dry weight on glucose as the substrate.
3.3.7. -Decalactone
-Decalactone is a lactone with a peach-like aroma. It is widely used in dairy products, beverages, and other products. Some strains of Y. lipolytica can naturally convert ricinoleic acid into -decalactone [63]. The strains perform b-oxidation of ricinoleic acid into -hydroxydecanoic acid, which spontaneously lactonizes into - decalactone. The strain can be improved by disrupting some and upregulating other peroxisomal oxidases encoded by POX genes (disruption of POX2 5 genes and overexpression of POX2). The resulting strain produces 7 g/L of -decalactone from castor oil as a substrate [64].
3.3.8. Pigments
Carotenoids are organic pigments used commercially as food and feed additives and in the pharmaceutical and cosmetics industries. There are over 600 known carotenoids, and they are all produced from isoprenoid pathway intermediate phytoene. Lycopene is the first colored carotenoid in the pathway, requiring three enzymatic steps: geranylgeranyl diphosphate synthase (encoded by crtE), phytoene synthase (encoded by crtY), and phytoene desaturase (encoded by crtI). The chromosomal integration of these three genes was performed for lycopene production in Y. lipolytica. Expression of codon-optimized genes of lycopene cyclase (crtB) and crtI from Pantoea ananatis under TEF1 promoter led to the production of 142 g/g lycopene by Y. lipolytica after 96 h in YPD shake flask cultures. The additional overexpression of geranylgeranyl diphosphate synthase (GGS1) and 3-hydroxy-3- methylglutaryl-coenzyme A reductase (HMG1) led to 2.6-fold (370 g/g) and 6.9-fold
(980 g/g) increases in lycopene content, respectively. Both modifications together
48 Chapter 3 increased the yield 10.8-fold to 1540 g/g. Furthermore, POX1 6 and GUT2 deletions increased the lycopene formation up to 16 mg/g DW in a bioreactor culture [65].
In another study, the bi-functional enzymes phytoene synthase/lycopene cyclase (carRP) and phytoene dehydrogenase (carB) from Mucor circinelloides under control of strong constitutive EXP1 and GPD promoters were integrated into the
MYA2613 k 70 strain. Subsequently, truncated HMGR1 (tHmgR) and GGS1 were overexpressed and ERG9 was downregulated, and also seven genes in the acetyl-CoA
FPP synthesis pathway, ERG8,10, 12, 13, 19, 20, and IDI, were overexpressed. The resulting strain could produce 4 g/ L of -carotene from ~ 233 g/L glucose in the fed- batch fermentation [66].
Larroude et al. [19] engineered a lipid overproducer Y. lipolytica strain in order to maximize -carotene production. They developed a combinatorial synthetic biology approach based on Golden Gate DNA assembly to screen the best combination of promoters for each gene of the -carotene pathway. The best strain reached a production titer of 6.5 g/L of -carotene and 90 mg/g DCW with a concomitant production of 42.6 g/L of lipids using a fed-batch fermentation.
Astaxanthin is a red-colored carotenoid used as food and feed additives.
Kildegaard et al. [67] engineered Y. lipolytica for astaxanthin production. First, they introduced bi-functional phytoene synthase/lycopene cyclase (crtYB) and phytoene desaturase 7 (crtI) from the red yeast Xanthophyllomyces dendrorhous into Y. lipolytica genome. The activities of HMG1 and GGS1/crtE were optimized, and the competing squalene synthase SQS1 was downregulated. Finally, they introduced - carotene ketolase (crtW) from Paracoccus sp. N81106 and hydroxylase (crtZ) from
Pantoea ananatis to convert -carotene into astaxanthin. The engineered strain accumulated 10.4 ± 0.5 mg/L of astaxanthin and its intermediates (5.7 ± 0.5 mg/L canthaxanthin and 35.3 ± 1.8 mg/L echinenone). After optimization of crtZ and crtW
49 Chapter 3 copy numbers, the production of astaxanthin reached to 54.6 mg/L in a microtiter plate cultivation.
Violacein is a bis-indole pigment with antibacterial, anti-cancer, antiviral, trypanocidal, and antioxidant properties. The gene cluster vioABCDE of
Chromobacterium violaceum is responsible for converting the precursor tryptophan to violacein via a combination of enzymatic and non-enzymatic steps. Wong et al. [18] constructed the 12 kb five-gene pathway for violacein production in the standardized pYaliBrick vectors. After transformation of the vectors into the Y. lipolytica Po1g, 31.5 mg/L violacein was produced.
3.3.9. Isoprenoids
-Farnesene, one of the simplest acyclic sesquiterpenes, is a compound with high-functional performance in plant defense, an alarm pheromone to signal danger in aphids and termites, and as a molecule implicated in the orientation of worker fire ants. -Farnesene is being developed as a precursor for biofuel as well as a potential biojet fuel candidate. The overexpression vectors harboring combinations of tHMG1,
IDI, ERG20, and codon-optimized -farnesene synthase (OptFS) genes were integrated into the genome of Y. lipolytica Po1h. The recombinant strain produced ~
57 and ~ 260 mg/L of -farnesene in shake flask and bioreactor cultures, respectively
[68].
Limonene, a monocyclic monoterpene, is an important precursor of many flavors, pharmaceutical, and biodiesel products. Two genes encoding neryl diphosphate synthase (NDPS1) and limonene synthase (LS) were codon-optimized and heterologously expressed in Y. lipolytica to construct the limonene synthetic pathway. The engineered strain Po1f-LN-051 produced a maximum limonene titer of
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~ 24 mg/L in the optimized medium containing glucose and pyruvic acid in flask
culture [69].
3.3.10. Precursor for steroid drugs
Campesterol is an important precursor for the production of steroid drugs such
as progesterone and hydrocortisone, which are used in pharmaceutical industry. The
yeast Y. lipolytica produced 453 mg/L of campesterol on sunflower seed oil-based
medium after disruption of C-22 desaturase and expression of 7-dehydrocholesterol
reductase (DHCR7) from Xenopus laevis [70]. Furthermore, Zhang et al. [71]
overexpressed POX2 gene in Y. lipolytica ATCC201249 beside expression of DHCR7
from Danio rerio and disruption of C-22 desaturase. The strain produced 942 mg/L
of campesterol from sunflower seed oil in the bioreactor.
Metabolic pathways towards fatty acid-derived chemicals in Y. lipolytica are
shown on Figure 3.2. Furthermore, some examples of non-native chemicals produced
in engineered Y. lipolytica strains are summarized in Table 4.3.
3.4. Perspectives
Y. lipolytica is a promising biorefinery platform strain for the production of
fatty acid-based compounds from low-cost substrates. Recent developments in
metabolic engineering and synthetic biology led to the production of a variety of value-
added oleochemicals by this yeast. Already, several products have reached
commercialization or are at pilot scale. More research is needed to better understand
the physiology and regulation of fatty metabolism in Y. lipolytica to fully utilize its
potential as a cell factory for oleochemicals.
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Figure 3.2. Examples of synthetic metabolic pathways towards high-value chemicals in Y. lipolytica. Abbreviations: ADH fatty-alcohol dehydrogenase, ALK1 12 cytochrome P450, CPR1 NADPH-cytochrome P450 reductase, FADS12, d12 fatty acid delta 12-desaturase, FAH12 fatty acid 12-hydroxylase, FALDH1 4 fatty-aldehyde dehydrogenase, FAO fatty alcohol oxidase, FAS1,2 fatty acid synthase, GmhpI1 soybean hydroperoxide lyase, Gmlox1 soybean lipoxygenase I, HPO fatty acid hydroperoxide lyase, LAHPO 13-hydroperoxide of linoleic acid, LPO lipoxygenase, MFE1 2-enoyl-CoA hydratase and 3-OH acyl-CoA dehy- drogenase, opai codon-optimized linoleic acid isomerase, POT1 3- ketoacyl-CoA thiolase, POX1 6 acyl-CoA oxidase
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Table 3.3. Some examples of non-native chemicals produced in Y. lipolytica Carbon Parental Compounds Modification Titer Ref source Strain Fatty alcohols, fatty aldehydes, fatty esters, and fatty alkanes/alkenes pex10 AtFAR 550 mg/L (shake- 1-Decanol 50 g/L glucose W29 [50] CpFAT flask) 660.8 mg/L Hexadecanol 160 g/L glucose Po1f fao dga1 TaFAR [51] (shake-flask) 9.67 g/L Fatty alcohols 100 g/L glucose Po1g Maqu220 EcfadD [73] (bioreactor) Fatty acid ethyl 142.5 mg/L 100 g/L glucose Po1g AbAtfA ScCAT2 [73] esters (shake flask) Fatty MmCAR BsSfp 23.3 mg/L (shake 100 g/L glucose Po1g [73] alkanes/alkenes PmADO flask) C12 and C14 fatty hFAS-EcTesA fusion 9.67 g/L 100 g/L glucose Po1g [73] acids protein (bioreactor)
Volatile aldehydes 37 g/L of 13- Hexanal Po1d CaCYP74 600 mg/L [52] HPOD Pentane 160 g/L glucose Po1f mfe1 GmLox1 4.98 mg/L [24]
Polyunsaturated and hydroxylated fatty acids 18.7 g/L CLA Po1h Paopai MaFADS12 4 g/L (bioreactor) [54] soybean oil 10 g/L glucose pox1-6 dga1 302 mg/L CLA and 20 g/L Po1d dga2 are1 lro1 [55] (bioreactor) soybean oil fad2 FAD2 oPAI
71.6 mg/L -linolenic acid 80 g/L glucose Po1f Ma 6D [56] (bioereactor) MaC16E Fm 12D Eg 9E Et 9E 15% of dry-cell ATCC weight or 56.6% of EPA 80 g/L glucose Eg 8D Eg 5D [57] 20362 Pe 8D, Pa 17D total fatty acid pex10 leu2 lip1 (shake-flask) scp2 pox1-6 dga1,2 Ricinoleic acid 50 g/L glucose W29 fad2 l o1 ~60 mg/gCDW [62] CpFAH12 LRO1 PHAs and organic acids 1.11 g/L (shake- PHA 20 g/L triolein Po1h ParPhaC1 [58] flask) 7.35 g/L PHB Acetate Po1g RePhaCAB [59] (bioreactor) Po1f leu+ 5.8 g/L Itaconic acid 20 g/L dextrose AtACD cytosolic- [60] ura+ ACO1 (bioreactor) 15 g/L 11 g/L Dodecanedioic acid H222 pox1-6 FAO1 [61] dodecane (bioreactor) Lactone 120 g/L castor -decalactone W29 pox2,3,4,5 POX2 7 g/L (bioreactor) [64] oil
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Table 3.3. Some examples of non-native chemicals produced in Y. lipolytica (continued) Carbon Parental Compounds Modification Titer Ref source Strain
Pigments PaCrtB PaCrtI 16 mg/g Lycopene Glucose H222 [65] GGS1 HMG1 (bioreactor) o 1-6 g 2 CarB CarRP ERG8,10,12,13,19.2 ~233 g/L ß-carotene MYA2613 4 g/L (bioreactor) [66] glucose 0 GGS1 tHMGR IDI g 2 o 3,4,5,6 crtYB crtI HMG1 YP+8% 54.6 mg/L Astaxanthin GB20 GGS1/crtE crtW [67] glucose (microtiter plate) crtZ 31.5 mg/L(shake- Violacein YNB-Leu Po1g vioABCDE [18] flask)
Isoprenoids
Glucose and Sc-tHMG IDI 260 mg/L -farnesene Po1h [68] fructose ERG20 MdFS (bioreactor) Glucose and ArtLS SltNDPS1 23.6 mg/L Limonene Po1f [69] pyruvic acid HMG1 ERG12 (shake-flask) Sterol ~58 g/L ATCC 453 mg/L Campesterol sunflower 201249 erg5 XlDHCR7 [70] (bioreactor) seed oil ~55 g/L ATCC erg5 DrDHCR7 942 mg/L sunflower 201249 [71] (bioreactor) seed oil POX2 Abbreviations: CLA: conjugated linolenic acid; EPA: eicosapentaenoic acid; PHA: polyhydroxyalkanoate PHB: polyhydroxybutyrate. Species: Ab: Acinetobacter baylyi; Ar: Agastache rugose; At: Arabidopsis thaliana; Ca: Capsicum annuum; Dr: Danio rerio; Ec: E. coli; Eg: Euglena gracilis; Et: Eutreptiella sp.; Fm: Fusarium moniliforme; Gm: Glycine max; Ma: Mortierella alpine; Mm: Mycobacterium marinum; Pan: Pantoea ananatis; Par: Pseudomonas aeruginosa; Pe: Peridinum sp.; Pm: Prochlorococcus marinus; Re: Ralstonia eutropha; Sl: Solanum lycopersicum; Ta: Tyto alba; Xl: Xenapus laevis. Genes abbreviations: DHCR7: 7- dehydrocholesterol reductase; FAH12: oleate 12-hydroxylase FAR: fatty acyl-CoA or fatty acyl-ACP reductase; FAT: fatty acyl-ACP thioesterase; Maqu220: fatty acyl-CoA reductase from Marinobacter aquaeolei; FADS12: 12- desaturase; FS: farnesene synthase; tLS: limonene synthase without transit peptides; tNDPS1: neryl diphosphate synthase 1 without transit peptides; 5,8,9,15,17D: 5-, 8-, 9-, 15-, and 17-desaturase, respectively.
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Acknowledgments The authors would like to thank Dr. Ali Abghari, Dr. Carina
Holkenbrink, and Hamideh Moradi for their valuable comments on this review.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.
Ethical statement This article does not contain any studies with human participants or animals performed by any of the authors.
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4
A single-host fermentation process for the production of flavor lactones from non- hydroxylated fatty acids
Eko Roy Marella, Jonathan Dahlin, Marie Inger Dam, Jolanda ter Horst, Hanne Bjerre Christensen, Suresh Sudarsan, Guokun Wang, Carina Holkenbrink, Irina Borodina
This chapter is a published article in Metabolic Engineering.
Marella ER, Dahlin J, Dam MI, ter Horst J, Christensen HB, Sudarsan S, Wang G, Holkenbrink C, Borodina I. A single-host fermentation process for the production of flavor lactones from non-hydroxylated fatty acids. Metab. Eng. 2019. (doi:10.1016/j.ymben.2019.08.009)
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Highlights
Oleaginous yeast Yarrowia lipolytica was engineered to produce flavor lactones.
The yeast expressed heterologous hydratases to obtain hydroxylated fatty acids.
-Oxidation was engineered for preferential fatty acid chain shortening to 10/12
carbons.
-Dodecalactone as produced from oleic acid and -decalactone from linoleic
acid.
-Dodecalactone titer reached 282 mg/L in a fed-batch process.
Abstract
Lactone flavors with fruity, milky, coconut, and other aromas are widely used in the food and fragrance industries. Lactones are produced by chemical synthesis or by biotransformation of plant-sourced hydroxy fatty acids. We established a novel method to produce flavor lactones from abundant non-hydroxylated fatty acids using yeast cell factories. Oleaginous yeast Yarrowia lipolytica was engineered to perform hydroxylation of fatty acids and chain-shortening via -oxidation to preferentially t elve or ten carbons. The strains could produce -dodecalactone from oleic acid and
-decalactone from linoleic acid. Through metabolic engineering, the titer was improved 4-fold, and the final strain produced 282 mg/L -dodecalactone in a fed- batch bioreactor. The study paves the way for the production of lactones by fermentation of abundant fatty feedstocks.
Keywords: Flavor lactone, Beta-oxidation, Yarrowia lipolytica, Hydroxy fatty acids,
Gamma-dodecalactone, Delta-decalactone
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4.1. Introduction
Lactones are c clic carbo lic esters. Lactones ith five( )- or si ( )-membered rings and eight-to sixteen-carbons have fruity and milky aromas and are used in food and fragrance industry [1]. The rising consumer demand for natural flavors and changes in legislations (EU Flavouring Regulation (EC) No 1334/2008, United States
Code of Federal Regulation Title 21) [2,3] made biological processes for lactone production more attractive. Due to the low abundance (ppm levels [1,4,5]) of lactones in natural sources, extraction of natural lactones is not economically feasible.
Commercial flavor lactones are produced by chemical synthesis or by enzymatic or microbial conversion of hydroxy fatty acids (HFA). The HFAs are extracted from plants [1,3,6], which poses challenges as low yields, supply instability, and the presence of plant toxins in some plant materials [6,7]. As an e ample, -decalactone can be made from ricinoleic acid, derived from castor seed oil. The content of ricinoleic acid in castor oil is up to 90%, translating into a yield of up to 1200 kg ricinoleic acid per ha [7]. Despite low production cost compared to other plant oils [8], for farmers in many areas, profitability of castor oil production is still low and supply stability is affected by climate change [7]. Furthermore, castor seeds contain up to 32 mg of ricin, a lethal phytotoxin, per gram of seeds [9]. The production of lactones from castor oil requires meticulous detoxification.
Microbial hosts capable of lactone biosynthesis from fatty acids, sugars or glycerol, would enable a cheaper and more sustainable lactone production. Several studies have demonstrated the production of lactones on complex medium with fungi
Ashbya gossypii [10] and Aureobasidium pullulans [11]. A. gossypii produces a broad-spectrum of lactones. Due to the similar physical properties of these lactones, it may be difficult and costly to isolate individual lactones. A. pullulans has been
62 Chapter 4 reported to produce over 5 g/L massoia lactone in the patent literature [12,13]. The lactone is derived from the secreted polyol lipids containing several hydroxy acid moeieties [14]. However, production of lactone other than massoia lactone has not been reported using this fungus. Another alternative is to produce hydroxylated fatty acids using microbes [15,16] and then convert them into lactones with the aid of enzymes or other microbes in a two-step or single-pot process (Farbood et al., 1994;
An et al., 2013; Kang et al., 2016) [17 19]. This would require the development and operation of two processes, resulting in extra capital and running costs.
We propose a method for single-step lactone production from non- hydroxylated fatty acid using oleaginous yeast Yarrowia lipolytica. Y. lipolytica is a
GRAS organism, well suited for large-scale fermentation and genetic manipulation
[20 22]. Oleic acid and linoleic acid were selected as example substrates because they are major constituents in various renewable low-value fatty feedstocks, such as used cooking oil, oil press cakes, olive oil distillates, and animal fats [23,24]. The proposed process enables lactone production from cheap feedstocks by a single microbial host.
4.2. Material and methods
4.2.1. Strain construction and cultivation
All strains in this study were derived from Y. lipolytica Y-63746 (MatA, Y. lipolytica W29, ATCC® 20460TM). Y. lipolytica Y-63746 was a kind gift from ARS
Culture Collection, NCAUR, USA. Complete list of strains, plasmids, biobricks (DNA fragments for cloning), and primers is provided in the supplementary information
(Tables S1 S5). Unless mentioned otherwise, cells were grown in YPD medium
(20 g/L yeast extract, 10 g/L peptone, 20 g/L glucose) at 30 °C and 250 rpm
(ThermoFisher Scientific MaxQ8000) for preculture and cryostock preparation. YPD
63 Chapter 4 agar plates contained 15 g/L agar (Sigma 05040). To enable CRISPR-Cas9 mediated strain engineering, Y. lipolytica W29 was transformed with linearized vector pCfB3634, which inserted Cas9 expression cassette into the KU70 locus [20], resulting in Δku70 strain ST6512. ST6512 was used as a reference and parental strain for the creation of other strains in this study. Plasmid constructions, gene insertions, and gene deletions were performed as described previously [20]. Heterologous genes were codon optimized for Y. lipolytica and obtained as synthetic DNA fragments (GeneArt,
Invitrogen). Deletions of POX1-6 genes were performed using 90-bp double-stranded oligos (IDT DNA) as repair templates, while other deletions were performed using
DNA fragment containing 250 500 bp upstream and downstream homology arms
(500 1000 bp in total). Guide RNA (gRNA) vectors and integrative vectors for gene expression were sequence-verified using Sanger sequencing (Eurofins). Yeast transformations were performed according to PEG/ssDNA/LiAc transformation protocol as described previously [20] using nourseothricin resistance (NatMX) as a selection marker. NatMX selection plate was prepared with standard YPD agar supplemented with 250 mg/L nourseothricin (Jena Bioscience, AB-101).
4.2.2. Spot assay
For pre-cultures, cells from cryostocks were streaked in YPD agar for 2 days and further propagated in 14 mL tubes (Greiner 187262) ith 1 mL YNBD-AA medium
(6.7 g/L Yeast Nitrogen Base without Amino Acids (Sigma, Y0626), 20 g/L glucose,
12 g/L KH2PO4, adjusted to pH 6) at 30 °C, 250 rpm overnight. The cells were centrifuged at 3000 g for 5 min and washed with 1 mL of sterile deionized water twice to remove residual glucose. The cells were resuspended in sterile deionized water to obtain an OD600 of 10, and used to prepare ten-fold serial dilutions. 10 L of diluted cells were spotted on fatty acid agar plates. Fatty acid agar plates contained 6.7 g/L
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YNB-AA, 15 g/L bacto agar, and 5 g/L of a fatty acid compound as the sole carbon source: oleic acid (Sigma, 364525), ethyl palmitate (Et-C16, Sigma, W245100), methyl myristate (Me-C14, Sigma, W272205), methyl laurate (Me-C12, Sigma, W271500), methyl decanoate (Me-C10, Sigma, W505501), and methyl octanoate (Me-C8, Sigma,
W272809).
4.2.3. Preparation of cell extracts for enzymatic assay
The cell extracts were prepared as following. The cells from cryostock were propagated in 14 mL tubes with 1 mL YPD at 30 °C, 250 rpm overnight. The whole overnight culture was transferred into 25 mL YNBD-AA medium in 250 mL shake- flasks and incubated at 30 °C and 250 rpm overnight. Cells were washed 2 times and inoculated at OD 0.5 into 50 mL YNBD-AA medium in 500 mL shake-flasks, incubated at 30 °C, and harvested at mid-exponential phase (OD600 between 8 to 12, Implen
NanoPhotometer P300). Following this, ST6512 and ST6852 strains were centrifuged at 3000 x g for 5 min and resuspended in 50 mL sterile water. This step was repeated one more time to wash away residual glucose. Washed cells were resuspended in YNB-
AA medium supplemented with 1 g/L ethyl palmitate (Sigma W245100) and 1 g/L
Methyl Oleate (Sigma 311111) and incubated in 500 mL shake flask at 30 °C, 250 rpm for induction. Cells were harvested after 6 h. For other strains, cells from YNBD-AA were harvested without induction since heterologous acyl-CoA oxidases were expressed under a constitutive promoter. The harvested cells were washed twice with
Wash Buffer (10 mM KH2PO4, pH 7.2). In each washing step, cells were first centrifuged at 6000 g, 4 °C for 5 min, resuspended in 50 mL Wash Buffer, and finally centrifuged again. Cells were then resuspended in 10 mL ice-cold Wash Buffer, and stored as two 5 mL-aliquots at 20 °C.
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For the preparation of the cell extract, 5 mL cells were thawed at room temperature, washed two times with 5 mL ice-cold Enzyme Assay Buffer (50 mM
KH2PO4, pH 7.4)25 and resuspended in 750 L Enzyme Assay Buffer. The suspension was transferred into 2-mL-microtubes (Sarsted, 72.694.006) pre-filled with 700 mg of glass beads (Sigma, G1277), and homogenized in Precellys® 24 (Bertin
Instruments) for five cycles of 20 s shaking at 5000 rpm with 5-min pauses between cycles, where the samples were placed on ice. Cell debris was removed by centrifugation at 21,000 x g for 10 min at 4 °C. If needed, the centrifugation step was repeated or extended up to 20 min until a clear supernatant was obtained.
4.2.4. Acyl-CoA oxidase enzymatic assay
Acyl-CoA oxidase enzymatic assay mixture was prepared as described previously [25,26]. Enzyme assay mixture contained 50 mM KH2PO4, 0.825 mM 4- aminoantipyrine (Sigma A4283), 10.6 mM phenol (Sigma P1037), 0.01 mM FAD
(Sigma F2665), 0.1 mM acyl-CoA, and 30 IU/mL horseradish peroxidase (Sigma
P8250), and cell extracts (0.07 0.1 mg total-protein/mL assay mix). The following substrates were used: oleoyl-CoA (Sigma O1012), palmitoyl-CoA (Sigma P9716), myristoyl-CoA (Sigma M4414), lauroyl-CoA (Sigma D5269), decanoyl-CoA (Sigma
L2659), and octanoyl-CoA (Sigma O6877). All chemicals were dissolved in Enzyme
Assay Buffer as separate reagents and warmed up to 30 C before mi ing. 200 L assa mix in 96- ell plate (Greiner 655101) as incubated at 30 °C in a plate reader
(BioTe S nerg M ), and absorbance at 500 nm was monitored. The activity was determined by using an extinction coefficient of 12.78 mM 1 cm 1 and pathway length of 5.9 mm (mathematically calculated). Reported numbers are means of two biological replicates measured with at least two technical replicates.
4.2.5. Lactone production in tube-cultures
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Strains from cryostock or agar plates were grown in 2 mL YPD for 48 h in a 24 deep-well plate (VWR AXYGP-DW10ML24C) at 30 °C and 250 rpm shaking. This culture was used to inoculate 5 mL YPD in a Corning mini bioreactor (Sigma,
CLS431720) with an initial OD600 between 0.1 to 0.5 and cultured (30 °C, 250 rpm) until reaching exponential phase (OD600 of 10 70). The cells were washed two times with 25 mL sterile deionized water (centrifugation at 3000 g for 5 min) and used to inoculate 2 mL of lactone production medium to an OD600 of 1 in rimless glass tubes
(VWR, 212 0326) closed with Labocap lid (VWR, 391 590). The lactone production
(LP) medium was mineral medium without glucose (Jensen et al, 2014) supplemented with 10 g/L yeast extract and 30 g/L of 90% oleic acid (Sigma, 364525) or 97% linoleic acid (TCI Europe, L0124) as carbon source. The fatty acids were dissolved in 99% ethanol to 60% w/v and then added to the medium. The tubes were incubated at 30 °C with 250 rpm shaking for 48 h.
4.2.6. Bright-field and GFP fluorescence image acquisition
The cells from cryostock were propagated in 14 mL tubes with 1 mL YPD at
30 °C, 250 rpm overnight. Cells were washed two times with 2 mL of 10 mM KH2PO4 pH 6 (centrifugation at 3000 g and 250 rpm). Cells were resuspended in 1 mL of
10 mM KH2PO4 pH 6, and loaded onto microscope slides. Bright-field and fluorescence images were taken at 100× magnification in a Leica DFC300 FX microscope equipped with Leica EL600 external light source. All images were taken with the same acquisition settings.
4.2.7. Fed-batch bioreactor cultivation
Cells from cryostock were inoculated into 1 mL YPD in a 10 mL preculture tube followed by inoculation in 25 mL YPD in a sterile 250 mL baffled shake-flask overnight. 10 mL inoculum was transferred into two 1-L bioreactors (Biostat Q Plus,
67 Chapter 4
Sartorius, Gottingen, Germany) each contained 400 mL starter medium. Starter medium was mineral medium (Jensen et al, 2014) supplemented with 10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose, 20 g/L glycerol, and 5 mL of Antifoam 204
(Sigma A6426). Cells were grown in the starter medium until carbon source depletion.
During the growth phase, dissolved oxygen level (DO) was maintained above 40% with minimum stirring and aeration speed of 300 rpm and 0.5 standard-liter per min
(SPLM), respectively.
After the growth phase in starter media, 30, 60, and 80 mL of 800 g/L oleic acid solution in ethanol was fed at three time points for biotransformation. Equal volumes of concentrated YP solution (67 g/L yeast extract, 133 g/L peptone) and 5, 20, and 20 mL of Antifoam 204 were fed along with the oleic acid solution. Stirring speed and aeration rate were adjusted to 600 rpm and 1.3 SPLM, respectively, at the start of biotransformation and increased at each feeding step. The temperature was kept at
30 °C and pH was kept at 6.0 by addition of 10 M KOH solution at all times of the cultivation. Data from two bioreactors is presented.
4.2.8. Cell dry-weight measurement
To remove the oil phase, 1 mL (CDW < 100 g/L) or 0.5 mL (CDW 100 g/L) bioreactor sample was washed two times by pelleting at 18,000 x g for 5 min and resuspending the pellet with 1 mL deionized water. The suspension was loaded into a
Whatman cellulose nitrate membrane filters disc ith 0.45 m pore si e (VWR
7184 004) and washed with 1 mL deionized water through vacuum. Before use, filters were dried in a microwave at 390 Watt for 10 min and weighed. After loading the cells, the filters were microwaved for 20 min at 390 Watt. Dry weight was determined by the difference of dry filter weight before and after loading the sample. Dry weight for each
68 Chapter 4 bioreactor was measured in duplicates. The presented data is average values from two bioreactors.
4.2.9. Lactone extraction
For quantification of lactones produced in tube-cultures, 200 L of absolute ethanol containing 1 g/L of internal standard (IS) -undecalactone (Sigma, 89985) was added directly into the cultivation tube (final IS concentration 100 mg/L). For quantification of lactones in bioreactors, 2 mL sample was transferred into a rimless glass tube, and 1 mL of an ethanol containing internal standard at 4.75 g/L concentration was added (final IS concentration 237.5 mg/L). The tube was vortexed in a multi-vortexer (VWR, DVX-2500) in pulse-mode at 1500 rpm for 45 s. Then, three mL of n-hexane (Merck Life Science, 1.04391.1000) was added and the tube was vortexed in the multi-vortexer in pulse-mode at 1500 rpm for 5 min to extract lactones into the hexane phase. Water and hexane phase were separated by centrifugation at
1000 x g for 5 min (room temperature). The hexane phase was transferred to a 4 mL glass vial (Mikrolab Aarhus, ML33134) filled with a small amount of Na2SO4 (Sigma,
798592) to remove residual moisture. 120 L of the he ane phase as loaded into 1 mL
HPLC glass vial with 200 L insert (Mikrolab Aarhus ML 33117) and sent for GC-MS analysis.
4.2.10. GC-MS analysis
The GC-MS analysis was performed on a Bruker Daltronics Scion GC436-MS instrument equipped with Agilent HP-Innowax column (30m × 0.250mm × 0.25- m).
One microliter of the sample was injected in Split/splitless injector set at 250 °C initially in splitless mode and after 2 min with a split ratio of 50. The helium gas flow was 1.0 mL/min. The temperature of the column oven was set to the following: 50 °C for 1 min, ramp 10 °C/min to 210 °C, 210 °C for 10 min, ramp 10 °C/min to 230 °C,
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230 °C for 5 min. Samples were ionized with EI (70 eV) in full scan mode from 50-
350 Da. Transfer line and ion source were set to 250 °C and 200 °C, respectively, and the collection delay was set to 4 min. Ion-85 count and the sum of ion-99 and 71 counts
ere used to quantif -dodecalactone and -decalactone, respectively. A calibration analysis using lactone standards was performed to determine correlations between peak areas of these quantifying ions with an area of ion-85 of -undecalactone. The obtained correlations were used to determine lactone concentration based on internal standard concentration. Lactone standards ere mi tures of -undecalactone, - dodecalactone (Sigma, 77991), and -decalactone (Sigma, 74026) at 6.25 400 mg/L concentration in n-hexane.
4.3. Results
4.3.1. Controlled chain-shortening through engineering of β-oxidation
pathway
Production of lactones from abundant fatty acids required the engineering of controlled chain-shortening and of fatty acid hydroxylation (Fig. 1a). Controlled chain- shortening was achieved by manipulating the substrate specificity of peroxisomal acyl-
CoA o idase (POX), hich catal es the first step of -o idation c cle. During - o idation, the -carbon of an acyl-CoA is oxidized in four steps, which give off one acetyl CoA and one acyl-CoA with 2-carbon shorter. Different chain-length specificities of several oxidases have been reported, especially in plants and yeasts
[27 31].
We aimed to establish a chain-shortening module that terminates peroxisomal fatt acid -oxidation at 10- (C10) and 12-carbons (C12), which are the most common chain-lengths of commercial flavor lactones (Figure 4.1b and c). Y. lipolytica has six
70 Chapter 4
POX enzymes encoded by genes POX1-6. These POXes have different chain-length specificities [31], and deletion of POX genes with high activity on short-chain acyl-CoA improved the production of -decalactone from ricinoleate [32].
Figure 4.1. The concept of a single-step lactone production from non-hydroxylated fatty acids. a) Free fatty acid is hydroxylated at positions that generate 4- or 5-hydroxy fatty acid after several -o idation c cles, hich spontaneousl converts into - or -lactone, respectively. Further degradation of these hydroxy fatty acids is prevented by replacing the native acyl-CoA oxidases with long chain-specific oxidases. Transformation of b) oleic acid into -dodecalactone and c) linoleic acid into -lactone.
We started by deleting the six acyl-CoA oxidase coding genes (POX1-6) in Y. lipolytica strain ST6512 (ku70Δ::Cas9), resulting in strain ST6852 (Δpox1-6). Next, we selected several long-chain-specific POXes (LCPOXes) that may be suitable for obtaining our targeted chain-lengths of C10 and C12. We chose a putative long-chain
POX from moth Agrotis segetum (AsePOX). The transcript of AsePOX was present in
A. segetum pheromone glands [33]. As the major constituent of this moth's pheromone is derived from C12 C14 fatty acid [34], we selected this POX as a candidate. We also chose two plant acyl-CoA oxidases, ACX2 from Arabidopsis
71 Chapter 4 thaliana (AtACX2) and a long-chain POX from a cucurbit cultivar (CuLACO), which have been previously characterized in vitro and showed specificity towards long-chain fatty acids [28,35]. By performing BLASTp search [36] using protein sequence of
AtACX2 and CuLACO as separate queries, putative long-chain acyl-CoA oxidase from
Rhinolophus sinicus [37] (RsAcox2) was identified. Lastly, we included POX2 from Y. lipolytica (YlPOX2) as its long-chain specificity for C10 fatty acid or longer has been previously demonstrated [32]. The long-chain POXes used in this study are summarized in Table 1 and Supplementary Information 2.
Codon-optimized genes encoding selected LCPOXes were expressed under the control of a strong constitutive TEF promoter with intron [20,38] in ST6852 (Δpox1-
6) strain. The native sequence was used for YlPOX2. Although all of the four heterologous LCPOXes contained the N-terminal peroxisome-targeting signal 1 (PTS1,
Supplementary Information 1), we still appended the yeast PTS1 (SKL) at the C- terminal of the proteins. No PTS1 sequence was added to YlPOX2. To determine the cellular localization of the LCPOXes, hrGFP-tag was attached to the N-termini of heterologous LCPOX and inserted into ST6851 (Δpox1,3,4,5,6). The fluorescence pattern was typical for peroxisomal localization for all the LCPOXes, with exception of
AsePOX, for which the fluorescence signal was low (Figure 4.2a d). The low fluorescence may be due to low expression level or protein misfolding. Notably, the
GFP intensities of AtACX2 and CuLACO strains were considerably higher than that of
RsAcox2 (Figure 4.2b d).
To assess the chain-length specificity of five LCPOXes in vivo, the LCPOX genes were individually expressed without a GFP tag under TEFintron promoter in ST6852
(Δpox1-6) strain. The strains were plated on oleic acid or fatty acid methyl esters
(FAMEs) of different chain-lengths as sole carbon source (Figure 4.3a). After 5 days of incubation, the control strain ST6512 (POX1-6) grew well on all tested substrates
72 Chapter 4 except methyl octanoate. Because ST6512 has a significant enzymatic activity on octanoyl-CoA (Figure 4.3b), the lack of growth of ST6512 in octanoate plate might be the result of either toxicity of octanoic acid or insufficient induction of POXes by octanoic acid. ST6852 (Δpox1-6) did not grow on any of the tested substrates except for C16, where it grew poorly. Nevertheless, growth on C16 may be attributed to the activity of four putative mitochondrial acyl-CoA dehydrogenases (ACADs), identified by BLAST search with Rhodosporidium toruloides ACAD protein sequence [42] as query (Supplementary Information 3).
Table 4.1. Heterologous genes used in this study. Name Origin Genbank Accession # Ref AsePOX Long-chain acyl-CoA oxidase KJ622094.1 [39] AtACX2 Long-chain acyl-CoA oxidase AF057043.1 [29] CuLACO Long-chain acyl-CoA oxidase AF002016.1 [35] RsAcox2 Long-chain acyl-CoA oxidase XP019576058.1 [37] SmOHY Oleate 10-hydratase AM743169.1 [40] LaLHY Linoleate 13-hydratase AHW98239.1 [41]
Figure 4.2. Localization of heterologous proteins expressed in Y. lipolytica. Bright-field and corresponding fluorescence images are shown on the left and right panel of each figure, respectively. The text below each figure indicates the gene of interest and the relative position of GFP. Bottom-right bar in each image represents 5 m. SKL: pero isomal targeting signal 1 (PTS1, serine-lysine-leucine).
73 Chapter 4
Figure 4.3. Engineering of controlled chain-shortening module. a) Spot assays of strains with different LCPOX genes. Agar plates contained 5 g/L of the indicated free fatty acids or fatty acid methyl esters as the sole carbon source. Blue arrows indicate the chain-length, where the strains no longer grow or grow poorly. The image is representative of three biological replicates. b) and c) Acyl-CoA oxidase activity on various acyl-CoA substrates. Enzymatic assay was performed at 30 °C using crude cell-e tracts. Activit unit of mU is equal to mol- substrate/min. Results are obtained from at least two technical replicates.
ST7384 (Δpox1-6 AsePOX) showed only a slightly better growth than ST6852
(Δpox1-6) on oleic acid and on C14 substrate, which is consistent with the low expression of AsePOX (Figure 4.2a). ST7386 (Δpox1-6 CuLACO) grew on C16 and only slightl on C14. ST7385 ( po 1-6 AtACX2) and ST7581 ( po 1-6 YlPOX2) grew on
C18, C16, C14, and C12 fatty acid, but not on C10, which makes these strains applicable for the production of C10 lactones. ST7387 (Δpox1-6 RsAcox2) grew well on C18, C16, and C14 fatty acids, but only weakly on C12 fatty acid, suggesting selectivity towards
C14 fatty acids and higher. This makes ST7387 (Δpox1-6 RsAcox2) a suitable chassis for the production of C12 lactones.
74 Chapter 4
4.3.2. Analysis of chain-length specificity of heterologous POXes by
enzymatic assay
We measured the oxidase activities of the crude extracts of engineered strains on different acyl-CoA substrates (Figure 4.3b and c). For the preparation of extracts, strains ST6512 and ST6852 (Δpox1-6) were cultivated with supplementation of ethyl palmitate and methyl oleate to induce expression of POXes. The rest of the strains did not require induction, because LCPOXes were expressed from the constitutive
TEFintron promoter.
Oxidase activity of ST6852 (Δpox1-6) was below the detection limit for all acyl-
CoA tested, which correlated with the lack of growth of this strain on fatty substrates.
The activities of heterologous LCPOXes (Figure 4.3c) were about two orders of magnitude lower than YlPOX2 in ST7581 (Δpox1-6 YlPOX2) (Figure 4.3b). This low oxidase activity was nevertheless sufficient to support the growth of the strains on fatty substrates (Figure 4.3a), indicating that the growth may be limited by other factors, e.g., the uptake and transport of the fatty substrates in the cells. It could also be that the in vitro enzymatic assay does not reproduce the in vivo conditions well. It is possible that in vivo, the concentration of oxidase substrates are well below the Km values and the native enzymes have much lower activities than in in vitro assays with high concentrations of substrates.
AsePOX activity was only measurable on methyl hexadecanoate (C16), which also correlates that the strain could only grow on C16 substrate (Figure 4.3a). The oxidase activity profiles of ST7581 (Δpox1-6 YlPOX2) and control strain ST6512
(POX1-6) were in agreement with the previous reports [31,43]. Notably, there was a discrepancy between the in vitro activity and in vivo growth for oxidases RsAcox2,
AtACX2, and YlPOX2. ST7387 (Δpox1-6 RsAcox2) extracts had a high activity on C12
75 Chapter 4 substrate, but the strain only grew on substrates longer than C12. Similarly, AtACX2 and YlPOX2 were active on C10 in vitro, but the cells did not grow on C10 substrates.
A possible explanation could be that liberation of a single acetyl-CoA molecule per substrate molecule cannot provide the cell with sufficient energy for substrate uptake and activation, transport into peroxisomes, the export of chain-shortened product, and for the growth. While if more than two acetyl-CoA molecules are released per molecule of substrate, then enough energy can be generated and the growth becomes feasible. Furthermore, based on the low activities on C8-CoA, it is likely that this and shorter acyl-CoAs are not processed effectively and may accumulate in CoA form or as free fatty acids and this can elicit some toxicity effects [44] as suggested by our spot assay result on Me C8 (Figre 4.3a). This could explain why despite the high in vitro activity on C10 and C12, strains ST7581 (Δpox1-6 YlPOX2) and ST7387 (Δpox1-
6 RsAcox2) did not grow on C10 and 12 subtrates, respectively.
Based on the in vivo and in vitro results, we selected YlPOX2 oxidases for the production of C10 lactones, and RsAcox2 oxidase for C12 lactones. While the strain with CuLACO ST7386 (Δpox1-6 CuLACO) grew well on C14 and C16 substrates, its growth on oleic acid was poor. As we intended to use oleic acid as one of the substrates for lactone production, we did not choose CuLACO oxidase for the further work.
4.3.3. Lactone production through fatty acid hydratases and controlled
chain-shortening
To obtain - and -lactones with 10- to 12-carbons via chain-shortening, h dro group needs to be located in 8 and 11 positions of C16-fatt acid or in 10 and 13 positions of C18-fatty acids. Hydroxylation can be achieved by the action of hydratase, hydroxylase, lipoxygenase, or epoxy-hydrolase (Tressl et al., 1996; Schwab et al., 2008) [45,46]. Compared to alternative pathways such as fatty acid (oleate)
76 Chapter 4 hydroxylase in fungi and plants or fatty acid lipoxygenase and peroxygenase in fruits, fatty hydratases route takes only one step towards fatty acid hydroxylation [47,48].
Fatty hydroxylase requires incorporation of fatty acid through the long Kennedy pathway prior to hydroxylation (Lin et al., 1998) [49]. Lipoxygenase and epoxy- hydrolase routes require at least two steps from free fatty-acid and form unstable intermediates [46,50]. Conversely, the known hydratases act on free fatty acids as substrates [47]. We chose hydratases because free fatty acids are easier to generate in the cell in comparison to the substrates of other hydroxylating enzymes.
Oleate 10-hydratase (OHY) generates 10-hydroxystearic acid from oleate. We chose the variant from Strenotrophomonas maltophilia [40] (SmOHY, Table 4.1).
Linoleate 13-hydratase (LHY) makes 13-hydroxyoleic acid from linoleate. LHY gene was sourced from Lactobacillus acidophilus [41] (LaLHY, Table 4.1). We designed to e press OHY in the strain that performs chain shortening to 12 carbons to obtain - dodecalactone from oleic acid (Figure 4.1b). LHY was to be expressed in the strain with chain shortening to 10 carbon atoms, so -decalactone is produced from linoleic acid
(Figure 4.1c). The SmOHY and LaLHY genes were codon-optimized for Y. lipolytica and expressed with C-terminal hrGFP tags from TEFintron promoter to check the expression and localization. Both enzymes were expressed in the cytosol (Figure 4.2e and g). We also wanted to express these enzymes in peroxisomes. To achieve this, we expressed them with a C-terminal PTS1 signal (SKL) and an N-terminal hrGFP tag.
The PTS1 signal was either fused directly to the protein or was spaced by a 2xGGGS linker. The variants with the linker localized into peroxisomes as expected (Figure 4.2f and h), while SmOHY variant without the linker showed in the cytosolic localization
(Supplementary Infromation 4).
Next, we expressed heterologous hydroxylases in a suitable chain-shortening yeast chassis (Figure 4.4). Specifically, we inserted SmOHY and LaLHY without
77 Chapter 4 hrGFP tag into strain ST6512 (POX1-6), resulting in strains ST6759 (POX1-6 SmOHY) and ST6760 (POX1-6 LaLHY) and cultivated them on mineral medium supplemented with 10 g/L yeast extract and 30 g/L of either oleic acid or linoleic acid. We observed production of 1.9 ± 0.8 mg/L of -dodecalactone in ST6759 with oleic acid as substrate
(Figure 4.5a) and 1.2 ± 0.4 mg/L of -decalactone in ST6760 with linoleic acid as substrate (Figure 4.5b). When the hydratases were inserted into strain ST6852
( po 1-6) lacking the pero isomal -oxidation capacity, no lactone production was detected (Figure 4.5a and b). We further generated strain ST7417 (Δpox1-6 SmOHY
RsAcox2) to specifically terminate -oxidation at C12 and thereby avoid degradation
4-h dro dodecanoic acid, hich is the precursor of -dodecalactone. This strain produced 12 ± 6.4 mg/L of -dodecalactone, which proved the effectiveness of replacing the native POXes with RsAcox2 to control chain-shortening. Analogously, we constructed strains ST7584 (Δpox1-6 LaLHY YlPOX2) expressing YlPOX2 for chain shortening to ten carbons. The strains, however, did not produce detectable levels of -decalactone from linoleic acid (Figure 4.5b). It could be that the oxidase activity was too high, when the oxidases were expressed from a very strong promoter and fine-tuning this could improve -decalactone production.
4.3.4. Metabolic engineering to improve lactone production
It has been reported previously that oleate 10-hydratase from Streptococcus pyogenes uses free oleic acid as the substrate [47]. In Y. lipolytica, imported free fatty acids are activated into acyl-CoAs by the action of cytosolic fatty acyl-CoA synthase encoded by FAA1 gene [51]. We speculated that deletion of FAA1 gene would reduce the activation of oleic acid, resulting in more substrate for the hydroxylation reaction.
At the same time, the fatty acid would not get incorporated into storage lipids that use
78 Chapter 4 acyl-CoAs as substrates (Figure 4.4). The strain with FAA1 deletion ST8276 (Δpox1-6
SmOHY RsAcox2 Δfaa1) produced 19.3 ± 8.9 mg/L -dodecalactone (Figure 4.5a).
We tried to further increase free fatty acid pool by expressing mutated Fat1p from Saccharomyces cerevisiae (ScFAT1D508A). Disruption of this gene in S. cerevisiae has been shown to reduce the oleate uptake [52]. While the native form of ScFat1p simultaneously transports and activates free fatty acids, ScFat1D508Ap has little activating function but maintains a considerable transport activity (Zou et al., 2002).
Introduction of this gene into ST8276 did not increase -dodecalactone production and therefore this strategy was not included in the following new strain designs.
Figure 4.4. Metabolic engineering strategies for lactone production. Abbreviations:FFA: free fatty acids, FHFA: free hydroxyfatty acids, FHY: fatty hydratases, ei-therSmOHY orLaLHY, FHYper: peroxisome-targeted FHY, LCPOX: long-chainspecific acyl-CoA oxidase, eitherRsAcox2orYlPOX2,FAA1: acyl-CoA synthase.
79 Chapter 4
Figure 4.5. a) -dodecalactone and b) -decalactone titer after different gene deletions and insertions. 30 g/L oleic acid ( -dodecalactone) or linoleic acid ( -decalactone) was present at the start of cultivation. - and + symbols indicate absence or presence of the corresponding genetic change, respectivel . ++ indicates 2 copies of the corresponding gene are present. Results were obtained from at least three biological replicates. Error bars represent standard deviation. Statistical analysis was performed using Student's t-test (two-tailed; *P 0.05, **P 0.01, ***P 0.001, n.s: not significant; t o-sample unequal variance).
We continued by examining whether expressing SmOHYp in peroxisomes would result in lactone production. We tested this by comparing production in a strain expressing a cytosolic SmOHY variant (SmOHYcyt, ST8276) or peroxisomal SmOHY variant (SmOHYper, ST8822) or both (SmOHYcyt+per, ST8824) in Δpox1-6 Δfaa1
RsAcox2 background. Peroxisomal variant resulted in less lactone than the cytosolic
80 Chapter 4 variant. The addition of peroxisomal variant to the strain expressing a cytosolic variant did not significantly improve the titer (Figure 4.5a). This implies that peroxisomal expression provided little to no benefit. Furthermore, there is also a possibility that lactone production in ST8822 occurred due to the presence of SmOHYp in the cytosol before the protein got imported into peroxisomes.
Following the above findings, we hypothesized that lactone production was limited by the low SmOHY activity. We expressed an additional copy of cytosolic
SmOHY resulting in strain ST8896 (Δpox1-6 Δfaa1 RsAcox2 2xSmOHYcyt
SmOHYper). ST8896 produced 74.6 ± 11.0 mg/L -dodecalactone, which is 3.6-fold higher than the parental strain (Figure 4.5a).
We applied analogous engineering approaches to the strain ST7584 (Δpox1-6
LaLHY YlPOX2). Deletion of FAA1 resulted in -decalactone production at
0.17 ± 0.03 mg/L (Figure 4.5b). ScFAT1D508A and peroxisomal expression strategies did not give improvement, as as the case ith -dodecalactone production.
Additional copy of cytosolic LaLHY improved the -decalactone titer 10-fold to
1.74 ± 0.3 mg/L, indicating that low hydratase activity was limiting the flux towards lactone.
Lastly, we tried to use commercial olive oil instead of oleic acid and we obtained
1.62 ± 0.45 mg/L -dodecalactone with strain ST8896. Although the titer was inferior to that obtained on pure oleic acid, this result suggests that lactone production from plant oils is possible.
4.3.5. Lactone production in fed-batch bioreactor
The highest -dodecalactone-producer strain ST8896 (Δpox1-6 Δfaa1 RsAcox2
2xSmOHYcyt SmOHYper) was tested in fed-batch bioreactors. Previous studies have provided valuable insights on optimizing stirred-bioreactor conditions for -
81 Chapter 4 decalactone production from castor oil and meth l ricinoleate. Production of - decalactone from methyl ricinoleate was better at high aeration rate (600 rpm stirring,
3 vvm aeration rate), high methyl ricinoleate concentration in the medium (50 g/L)
[53], and at high cell densities [54]. Comparison of media used by Moradi et al. (2013)
[55] and Braga and Belo (2015) [54] suggests that higher C/N ratio and repeated feeding could also benefit lactone production. We, therefore, designed our bioreactor cultivation conditions accordingly.
Figure 4.6. Fed-batch bioreactor results for bio-transformation of oleic acid to -dodeca- lactone. a) Cell dry-weight (orange, left axis), -dodecalactone titer (blue, right axis), and accumulated oleate (green, second right-axis). Cell dry-weight was measured two times for each reactor. Cumulative oleic acid was calculated from the total of oleic acid fed divided by the culture volume at the corresponding time point (calculated). Error bars represent standard deviation from two bioreactors. b) Stirring speed (blue, left axis), aeration rate (orange, right axis), and dis-solved oxygen level (right, second right-axis) from one of the two bioreactors showing similar profile. Time zero defined as the time of inoculation. The black arrows indicate the timingof oleic acid pulses. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
82 Chapter 4
During the growth on the starter media, 12.9 ± 2.2 g/L of cell dry-weight (CDW) was measured. Oleic acid was then added in three pulses marked with black arrows on
Figure 4.6a. Upon the addition of oleic acid, the dissolved oxygen dropped to near zero and remained low in spite that we increased the agitation rate during the cultivation.
Lactone concentration peaked at 109 mg/L ± 28 mg/L at 16 h and at 282 ± 75 g/L at
88 h (Figure 4.6a). There was a sharp increase in lactone concentration between 62 and 83 h, which was likely due to the higher cell density. In fact, specific productivity in the 62-83-h period (0.10 gr-lactone/L/h per average CDW) was still lower than in the 0-16-h period (0.25 gr-lactone/L/h per average CDW).
Some degradation of lactone clearly occurred during the fermentation (e.g., between 20 and 60 h and after 83 h). This was not due to the lack of substrate, because oleic acid was present at all the tested time points (visible as a distinct top layer in the centrifuged samples). It has been previousl reported that -decalactone produced from castor oil was degraded by strains that had intact POXes [56]. The observed degradation suggests that our strain could benefit from a more tight chain-shortening control in the future. Furthermore, significant improvements in lactone production could be achieved by optimizing the fermentation media, feeding profile, and fermentation conditions.
4.4. Discussion
We report, for the first time, a single-host microbial process for lactone production from abundant non-hydroxylated fatty acids. The host was oleaginous yeast Y. lipolytica, engineered to carry out fatty acid hydroxylation and a controlled chain shortening. We employed bacterial fatty acid hydratases, which hydroxylate
83 Chapter 4 fatty acids in a single step in contrast to alternative pathways, such as fatty acid (oleate) hydroxylase, lipoxygenase, or peroxygenase.
Controlled -oxidation is the key step in lactone formation from hydroxy fatty acids [1]. Previous studies in Y. lipolytica showed that by substituting all POXes with only the long-chain specific POX2, -decalactone production was improved. In the current study, we tested four heterologous acyl-CoA oxidases in addition to the native
YlPOX2. The RsAcox2, which originates from a Chinese rufous horsehoe bat
Rhinolophus sinicus, enabled us to improve production of -dodecalactone. Two different chain-length characterization methods were tested in selecting suitable
POXes, namely growth assay and enzymatic assay. The combination of these methods guided the selection of suitable POXes.
We explored several metabolic engineering strategies for improving the lactone titer. We showed that increasing the copy number of hydratase genes eventually yielded the best improvement. This might be explained by the poor kinetic properties of fatty acid hydratases. The measured kcat and Km of SmOhyp are, respectively, 118 min 1 and 38.9 M on oleic acid [57]. For LaLhyp, the kcat and Km values on linoleic acid are 553 min 1 and 7 mM, respectively (Kim et al., 2015) [58]. As comparison, the reported values of kcat and Km of purified YlPox2p are 22.51 s 1 (1350 min 1) and 18 M, respectively [43]. These values reflect how the kinetic performances of the hydratases are inferiror relative to that of YlPox2p. Poor kinetics of LaLhyp could also explain the lo titer -decalactone titer we obtained.
Although it has been shown that fatty acid hydratases cannot accept acyl-CoA as substrate, deletion of FAA1, which was expected to increase the pool of free fatty acids, did not yield a significant improvement. Since free fatty-acids are more hydrophobic than acyl-CoAs, their availability to the hydratases may be limited by the transport events. Therefore, instead of deleting FAA1, fusing the hydratase with a
84 Chapter 4 thioesterase might help tackling the limited free fatty-acids availability due to their hydrophobic nature. In this manner, the thioesterase would act like a shuttle that will provide the substrate from the cytosolic acyl-CoAs. Peroxisomal localization of hydratases was not beneficial either. This could be due to the highly-oxidative peroxisomal environment or due to some inhibitors, such as hydrogen peroxide and reactive oxygen species.
The fed-batch fermentation of the engineered -dodecalactone producing strain resulted in lactone titer of 282 ± 75 g/L at 88 h, however some degradation was observed during the fermentation process, indicating that further process optimization is needed.
4.5. Conclusion
We have established strains to produce -dodecalactone and -decalactone from non-hydroxylated fatty acids by engineering of controlled chain-shortening and hydroxylation modules in oleaginous yeast Y. lipolytica. By testing various metabolic engineering strategies, we concluded that boosting the expression and activity of fatty acid hydratases would result in further strain improvement.
Acknowledgements
This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sk odo ska-Curie grant agreement No 722287 (PAcMEN). IB acknowledges the financial support from the
Novo Nordisk Foundation (Grant agreement NNF15OC0016592 and
NNF10CC1016517) and from the European Research Council under the European
Union's Horizon 2020 research and innovation programme (YEAST-TRANS project,
85 Chapter 4
Grant Agreement No 757384). We thank NRRL Agricultural Research Service (ARS)
Culture Collection for the gift of Yarrowia lipolytica Y-63746. We thank Matej Rusnák,
Steven Axel van der Hoek, Mahsa Babaei, and Jacqueline Medina, for assistance during experimental works.
Author contributions
ERM and IB conceived this project. ERM, JD, GW, CH, and IB designed the metabolic engineering strategies and lactone production experiments. ERM and JD performed the experiments. ERM, MID, and HBC developed, validated, and performed lactone extraction and analysis. ERM, JD, JtH, SS, and IB designed the bioreactor experiments. ERM and JtH carried out the bioreactor experiments. ERM,
JD, GW, CH, and IB were involved in analyzing and interpreting the data.
Competing interest
IB and CH have financial interest in BioPhero ApS.
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Supplementary Information
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Supplementary information 1 Strain, primers, plasmids, and DNA fragments used in this study
Table S1. Strains used in this study.
Parent gRNA Repair oligo/ No Strain name Genotype Source Strain vector vector ST4842 (Y. lipolytica ARS, NCAUR, 1 W29, Y-63746, ATCC MATa N/A N/A N/A USA 20460) ST6512 (ku70::cas9- MATa 70 ::P TEF1-cas9-TTef12::PrGPD- 2 dsdAMX-TLip2 ST4842 N/A pCfB6364 This study dsdAMX) (MATa 70 ::SpCas9-EcDsdAMX4) PR-19088, 3 ST6717 ( 5,6) MATa 70 ::SpCas9-EcDsdAMX4 5,6 ST6512 pCfB7049 This study PR-19089
MATa 70 ::SpCas9-EcDsdAMX4 PR-19084, 4 ST6757 ( 1,4,5,6) ST6717 pCfB7050 This study 1,4,5,6 PR-18069
ST6759 (IntC_3- MATa 70 ::SpCas9-EcDsdAMX4 5 ST6512 pCfB6630 pCfB7057 This study >PrTEF-SmOhy-TLip2) IntC_3::PrTEFintron-SmOHY-TLip2
ST6760 (IntC_3- MATa 70 ::SpCas9-EcDsdAMX4 6 ST6512 pCfB6630 pCfB7058 This study >PrTEF-LaLhy-TLip2) IntC_3::PrTEFintron-LaLHY-TLip2
MATa 70 ::SpCas9-EcDsdAMX4 7 ST6850 ( 1,2,4,5,6) 1,2,4,5,6 ST6757 pCfB7059 PR-19085 This study
MATa 70 ::SpCas9-EcDsdAMX4 8 ST6851 ( 1,3,4,5,6) 1,3,4,5,6 ST6757 pCfB7052 PR-19086 This study
ST6852 MATa 70 ::SpCas9-EcDsdAMX4 9 ST6850 pCfB7052 PR-19086 This study ( 1,2,3,4,5,6) 1,2,3,4,5,6
ST7018 ( 1,2,4,5,6 MATa 70 ::SpCas9-EcDsdAMX4 10 ST6850 pCfB6627 pCfB7076 This study AsPOX) 1,2,4,5,6 I C_2::P TEF -AsPox-TLip2
ST7019 ( 1,2,4,5,6 MATa 70 ::SpCas9-EcDsdAMX4 11 ST6850 pCfB6627 pCfB7077 This study AtAcx2) 1,2,4,5,6 I C_2::P TEF -AtAcx2-TLip2
ST7020 ( 1,2,4,5,6 MATa 70 ::SpCas9-EcDsdAMX4 12 1,2,4,5,6 I C_2::P TEF -CuLAco- ST6850 pCfB6627 pCfB7078 This study CuLACO) TLip2 ST7021 ( 1,2,4,5,6 MATa 70 ::SpCas9-EcDsdAMX4 13 1,2,4,5,6 I C_2::P TEF -RsAcox2- ST6850 pCfB6627 pCfB7079 This study RsAcox2) TLip2 ST7379 ( 1,3,4,5,6 MATa 70 ::SpCas9-EcDsdAMX4 14 1,3,4,5,6 I C_3::P TEF -SmOHY- ST6851 pCfB6630 pCfB7057 This study SmOhy) TLip2 ST7380 ( 1,3,4,5,6 MATa 70 ::SpCas9-EcDsdAMX4 15 1,3,4,5,6 I C_3::P TEF -LaLHY- ST6851 pCfB6630 pCfB7058 This study LaLhy) TLip2 ST7382 ( 1-6 MATa 70 ::SpCas9-EcDsdAMX4 16 1,2,3,4,5,6 I C_3::P TEF - ST6852 pCfB6630 pCfB7057 This study SmOhy) SmOHY-TLip2 ST7383 ( 1-6 MATa 70 ::SpCas9-EcDsdAMX4 17 1,2,3,4,5,6 I C_3::P TEF -LaLHY- ST6852 pCfB6630 pCfB7058 This study LaLhy) TLip2 ST7384 ( 1-6 MATa 70 ::SpCas9-EcDsdAMX4 pox1-6 18 ST7018 pCfB7052 PR-19086 This study AsPox) IntC2:PrTEFin-AsPox-TL 2 3
ST7385 ( 1-6 MATa 70 ::SpCas9-EcDsdAMX4 pox1-6 19 ST7019 pCfB7052 PR-19086 This study AtAcx2) IntC_2::PrTEFin-AtAcx2-TL 2 3
ST7386 ( 1-6 MATa 70 ::SpCas9-EcDsdAMX4 pox1-6 20 ST7020 pCfB7052 PR-19086 This study CuLAco) IntC_2::PrTEFin-CuLAco-TL 2 3
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ST7387 ( 1-6 21 MATa 70 ::SpCas9-EcDsdAMX4 pox1-6 ST7021 pCfB7052 PR-19086 This study RsAcox2) IntC_2::PrTEFin-RsAcox2-TL 2 3
ST7389 ( 1,3,4,5,6 MATa 70 ::SpCas9-EcDsdAMX4 22 1,3,4,5,6 I D_1:P TEF -SmOHY- ST6851 pCfB6631 pCfB7136 This study SmOhy-PTS1) PTS1-TLip2 ST7390 ( 1,3,4,5,6 MATa 70 ::SpCas9-EcDsdAMX4 23 1,3,4,5,6 I D_1:P TEF -LaLHY-PTS1- ST6851 pCfB6631 pCfB7137 This study LaLhy-PTS1) TLip2 ST7392 ( 1,3,4,5,6 MATa 70 ::SpCas9-EcDsdAMX4 24 1,3,4,5,6 I D_1:P TEF -hrGFP- ST6851 pCfB6631 pCfB7142 This study hrGFP-SmOhy-PTS1) SmOHY-PTS1-TLip2 ST7393 ( 1,3,4,5,6 MATa 70 ::SpCas9-EcDsdAMX4 25 1,3,4,5,6 I D_1:P TEF -hrGFP- ST6851 pCfB6631 pCfB7143 This study hrGFP-LaLhy-PTS1) LaLHY-PTS1-TLip2 MATa ku70 ::SpCas9-EcDsdAMX4 ST7417 ( 1-6 1,2,3,4,5,6 I C_2::P TEF - 26 ST7384 pCfB6630 pCfB7057 This study RsAcox2 SmOHY) RsAcox2-TLip2 IntC_3::PrTEFintron- SmOHY-TLip2 ST7419 ( 1,2,4,5,6 MATa 70 ::SpCas9-EcDsdAMX4 27 1,2,3,4,5,6 I C_2::P TEF -hrGFP- ST6850 pCfB6627 pCfB7144 This study AsPox-GFP) AsPOX-TLip2 ST7420 ( 1,2,4,5,6 MATa 70 ::SpCas9-EcDsdAMX4 28 1,2,3,4,5,6 I C_2::P TEF -hrGFP- ST6850 pCfB6627 pCfB7145 This study AsPox-GFP) AsPOX-TLip2 ST7421 ( 1,2,4,5,6 MATa 70 ::SpCas9-EcDsdAMX4 29 1,2,3,4,5,6 I C_2::P TEF -hrGFP- ST6850 pCfB6627 pCfB7146 This study AsPox-GFP) AsPOX-TLip2 ST7422 ( 1,2,4,5,6 MATa 70 ::SpCas9-EcDsdAMX4 30 1,2,3,4,5,6 I C_2::P TEF -hrGFP- ST6850 pCfB6627 pCfB7147 This study AsPox-GFP) AsPOX-TLip2 ST7424 ( 1,3,4,5,6 MATa 70 ::SpCas9-EcDsdAMX4 31 1,3,4,5,6 I C_3::P TEF -SmOHY- ST6851 pCfB6630 pCfB7139 This study SmOHY-GFP) hrGFP-TLip2 ST7425 ( 1,3,4,5,6 MATa 70 ::SpCas9-EcDsdAMX4 32 1,3,4,5,6 I C_3::P TEF -LaLHY- ST6851 pCfB6630 pCfB7140 This study LaLHY-GFP) hrGFP-TLip2 ST7581 ( 1-6 MATa 70 ::SpCas9-EcDsdAMX4 33 1,2,3,4,5,6 I C_2::P TEF - ST6852 pCfB6627 pCfB7683 This study YlPOX2) YlPOX2-TLip2 MATa 70 ::SpCas9-EcDsdAMX4 ST7583 ( 1-6 1,2,3,4,5,6 I C_3::P TEF - 34 ST7382 pCfB6627 pCfB7683 This study SmOHY YlPOX2) SmOHY-TLip2 IntC_2::PrTEFintron-YlPOX2- TLip2 ST7584 ( 1-6 MATa 70 ::SpCas9-EcDsdAMX4 35 1,2,3,4,5,6 I C_3::P TEF -LaLHY- ST7383 pCfB6627 pCfB7683 This study LaLHY YlPOX2) TLip2 IntC_2::PrTEFintron-YlPOX2-TLip2 ST7597 ( 1-6 MATa 70 ::SpCas9-EcDsdAMX4 pox1-6 36 ST7419 pCfB7052 PR-19086 This study AsPox-GFP) IntC_2::PrTEFintron-hrGFP-AsPOX-TLip2
ST7598 ( 1-6 MATa 70 ::SpCas9-EcDsdAMX4 pox1-6 37 ST7420 pCfB7052 PR-19086 This study AtAcx2-GFP) IntC_2::PrTEFintron-hrGFP-AtAcx2-TLip2
ST7599 ( 1-6 MATa 70 ::SpCas9-EcDsdAMX4 pox1-6 38 ST7421 pCfB7052 PR-19086 This study CuLACO-GFP) IntC_2::PrTEFintron-hrGFP-CuLACO-TLip2
ST7600 ( 1-6 MATa 70 ::SpCas9-EcDsdAMX4 pox1-6 39 ST7422 pCfB7052 PR-19086 This study RsAcox2-GFP) IntC_2::PrTEFintron-hrGFP-RsAcox2-TLip2
ST7602 ( 1-6 MATa 70 ::SpCas9-EcDsdAMX4 pox1-6 40 IntD_1::PrTEFintron-SmOHY-linker-PTS1- ST6852 pCfB6631 pCfB7722 This study SmOHY-linker-per) TLip2 ST7603 ( 1-6 MATa 70 ::SpCas9-EcDsdAMX4 pox1-6 41 IntD_1::PrTEFintron-LaLHY-linker-PTS1- ST6852 pCfB6631 pCfB7723 This study LaLHY-linker-per) TLip2 ST7605 ( 1-6 GFP- MATa 70 ::SpCas9-EcDsdAMX4 pox1-6 42 IntD_1::PrTEFintron-hrGFP-SmOHY-linker- ST6852 pCfB6631 pCfB7748 This study SmOHY-linker-per) PTS1-TLip2 ST7606 ( 1-6 GFP- MATa 70 ::SpCas9-EcDsdAMX4 pox1-6 43 IntD_1::PrTEFintron-hrGFP-LaLHY-linker- ST6852 pCfB6631 pCfB7749 This study LaLHY-linker-per) PTS1-TLip2 ST7914 ( 1-6 MATa 70 ::SpCas9-EcDsdAMX4 pox1-6 44 RsAcox2 SmOHY-linker- IntC_2::PrTEFintron-RsAcox2-TLip2 ST7387 pCfB6631 pCfB7722 This study IntD_1::PrTEFintron-SmOHY-linker-PTS1- per) TLip2
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ST7921 ( 1-6 MATa 70 ::SpCas9-EcDsdAMX4 pox1-6 IntC_2::PrTEFintron-YlPOX2-TLip2 45 YlPOX2 LaLHY-linker- IntD_1::PrTEFintron-LaLHY-linker-PTS1- ST7581 pCfB6631 pCfB7723 This study per) TLip2
46 ST8274 ( 1-6 aa1) MATa 70 ::SpCas9-EcDsdAMX4 pox1-6 ST6852 pCfB8043 BB2676 This study aa1
ST8275 ( 1-6 aa1 47 MATa 70 ::SpCas9-EcDsdAMX4 pox1-6 ST7382 pCfB8043 BB2676 This study SmOHY) aa1 I C_3::P TEF -SmOHY-TLip2
ST8276 ( 1-6 aa1 MATa 70 ::SpCas9-EcDsdAMX4 pox1-6 48 aa1 I C_2::P TEF -RsAcox2-TLip2 ST7417 pCfB8043 BB2676 This study SmOHY RsAcox2) IntC_3::PrTEFintron-SmOHY-TLip2 ST8277 ( 1-6 aa1 49 MATa 70 ::SpCas9-EcDsdAMX4 pox1-6 ST7383 pCfB8043 BB2676 This study LaLHY) aa1 I C_3::P TEF -LaLHY-TLip2
ST8278 ( 1-6 aa1 MATa 70 ::SpCas9-EcDsdAMX4 pox1-6 50 aa1 I C_3::P TEF -LaLHY-TLip2 ST7584 pCfB8043 BB2676 This study LaLHY YlPOX2) IntC_2::PrTEFintron-YlPOX2-TLip2 ST8610 ( 1-6 MATa 70 ::SpCas9-EcDsdAMX4 pox1-6 54 IntC_3::PrTEFin-SmOHY IntD_1::PrTEFin- ST7382 pCfB6631 pCfB8353 This study SmOHYScFAT1*) ScFAT1(D508A) IKL ST8611 ( 1-6 MATa 70 ::SpCas9-EcDsdAMX4 pox1-6 55 SmOHYScFAT1*- IntC_3::PrTEFin-SmOHY IntD_1::PrTEFin- ST7382 pCfB6631 pCfB8354 This study SmOHY) ScFAT1(D508A) IKL-SmOHY ST8620 ( 1-6 aa1 MATa 70 ::SpCas9-EcDsdAMX4 pox1-6 aa1 I C_3::P TEF -SmOHY 56 SmOHY RsAcox2 ST8276 pCfB6631 pCfB8353 This study IntC_2::PrTEFin-RsAcox2 IntD_1::PrTEFin- ScFAT1*) ScFAT1(D508A) IKL ST8621 ( 1-6 aa1 MATa 70 ::SpCas9-EcDsdAMX4 pox1-6 aa1 I C_3::P TEF -SmOHY 57 SmOHY RsAcox2 IntC_2::PrTEFin-RsAcox2 IntD_1::PrTEFin- ST8276 pCfB6631 pCfB8354 This study ScFAT1*-SmOHY) ScFAT1(D508A) IKL-SmOHY ST8625 ( 1-6 aa1 MATa 70 ::SpCas9-EcDsdAMX4 pox1-6 aa1 I C_3::P TEF -LaLHY 58 LaLHY YlPOX2 IntC_2::PrTEFin-YlPOX2 IntD_1::PrTEFin- ST8278 pCfB6631 pCfB8353 This study ScFAT1*) ScFAT1(D508A) IKL ST8626 ( 1-6 aa1 MATa 70 ::SpCas9-EcDsdAMX4 pox1-6 59 LaLHY YlPOX2 aa1 I C_3::P TEF -LaLHY ST8278 pCfB6631 pCfB8355 This study IntC_2::PrTEFin-YlPOX2 IntD_1::PrTEFin- ScFAT1*-LaLHY) ScFAT1(D508A) IKL-LaLHY ST8822 ( 1-6 aa1 MATa 70 ::SpCas9-EcDsdAMX4 pox1-6 60 RsAcox2 SmOHY-linker- aa1 I C_2::P TEF -RsAcox2-TLip2 ST7914 pCfB8043 BB2676 This study IntD_1::PrTEFintron-SmOHY-linker-PTS1- per) TLip2 ST8823 ( 1-6 aa1 MATa 70 ::SpCas9-EcDsdAMX4 pox1-6 aa1 I C_2::P TEF -YlPOX2-TLip2 61 YlPOX2 LaLHY-linker- ST7921 pCfB8043 BB2676 This study IntD_1::PrTEFintron-LaLHY-linker-PTS1- per) TLip2 MATa 70 ::SpCas9-EcDsdAMX4 pox1-6 ST8824 ( 1-6 aa1 aa1 I C_2::P TEFintron-RsAcox2-TLip2 62 SmOHY RsAcox2 IntC_3::PrTEFintron-SmOHY-TLip2 ST8276 pCfB6631 pCfB7722 This study SmOHY-linker-per) IntD_1::PrTEFintron-SmOHY-linker-PTS1- TLip2 MATa 70 ::SpCas9-EcDsdAMX4 pox1-6 ST8825 ( 1-6 aa1 aa1 I C_2::P TEF -YlPOX2-TLip2 63 LaLHY YlPOX2 LaLHY- IntC_3::PrTEFintron-LaLHY-TLip2 ST8278 pCfB6631 pCfB7723 This study linker-per) IntD_1::PrTEFintron-LaLHY-linker-PTS1- TLip2 MATa 70 ::SpCas9-EcDsdAMX4 pox1-6 ST8896 ( 1-6 aa1 aa1 I C_2::P TEF -RsAcox2-TLip2 64 RsAcox2 2xSmOHYcyt IntC_3::PrTEFintron-SmOHY-TLip2 ST8824 pCfB6638 pCfB8614 This study SmOHYper) IntD_1::PrTEFintron-SmOHY-linker-PTS1- TLip2 IntE_4::PrTEFin-SmOHY-TPex20 MATa 70 ::SpCas9-EcDsdAMX4 pox1-6 ST8897 ( 1-6 aa1 aa1 I C_2::P TEF -YlPOX2-TLip2 65 YlPOX2 2xLaLHYcyt IntC_3::PrTEFintron-LaLHY-TLip2 ST8825 pCfB6638 pCfB8615 This study LaLHYper) IntD_1::PrTEFintron-LaLHY-linker-PTS1- TLip2 IntE_4::PrTEFintron-LaLHY-TPex20
94 Chapter 4
Table S2. Plasmids used in this study
Plasmid Parent Biobricks Reference Plasmid
pCfB3405 (pNat, CEN1) See ref. See ref. 19
pCfB5124 (pIntB-HphMx-PrTEFin-hrGFP) See ref. See ref. 19 C B6364 ( 70 ::SpCas9-EcDsdAMX4) See ref. See ref. 19 pCfB6371 (pIntC_3-TPex20-TLip2) See ref. See ref. 19 pCfB6627 (pNAT_YLgRNA2_IntC_2) See ref. See ref. 19 pCfB6630 (pNAT_YLgRNA3_IntC_3) See ref. See ref. 19 pCfB6631 (pNAT_YLgRNA2_IntD_1) See ref. See ref. 19 pCfB6633 (pNAT_YLgRNA2_IntE_1) See ref. See ref. 19 pCfB6638 (pNAT_YLgRNA2_IntE_4) See ref. See ref. 19 pCfB6677 (pIntE_1-TPex20-TLip2) See ref. See ref. 19 pCfB6679 (pIntE_4-TPex20-TLip2) See ref. See ref. 19 pCfB6682 (pIntC_2-TPex20-TLip2) See ref. See ref. 19 pCfB6684 (pIntE_4-TPex20-TLip2) See ref. See ref. 19 pCfB7049 (pNat_YLgRNA_POX5_POX6) pCfB3405 BB2226, BB2227 This study pCfB7050 (pNat_YLgRNA_POX1_POX4) pCfB3405 BB2222, BB2225 This study pCfB7051 (pNat_YLgRNA_POX2_POX3) pCfB3405 BB2223, BB2224 This study pCfB7052 (pNat_YLgRNA_POX3) pCfB6371 BB2232 This study pCfB7057 (pIntC_3-PrTEFintron-SmOhy-TLip2) pCfB6371 BB2194, BB2229 This study pCfB7058 (pIntC_3-PrTEFintron-LaLhy-TLip2) pCfB3405 BB2230, BB2195 This study pCfB7059 (pNat_YLgRNA_POX2) pCfB6682 BB2231 This study pCfB7076 (pIntC_2-PrTEFint-AsPOX-TLip2) pCfB6682 BB2242 This study pCfB7077 (pIntC_2-PrTEFint-AtAcx2-TLip2) pCfB6682 BB2243, BB2249 This study pCfB7078 (pIntC_2-PrTEFint-CuLAco-TLip2) pCfB6682 BB2244, BB2249 This study pCfB7079 (pIntC_2-PrTEFint-RsAcox2-TLip2) pCfB6684 BB2250, BB2245 This study pCfB7136 (pIntD_1-PrTEFin-SmOhy-PTS1-TLip2) pCfB6684 BB2229, BB2247 This study pCfB7137 (pIntD_1-PrTEFin-LaLhy-PTS1-TLip2) pCfB6371 BB2230, BB2248 This study pCfB7139 (pIntC_3-PrTEFin-SmOHY-hrGFP-TLip2) pCfB6371 BB2262, BB2264 This study pCfB7140 (pIntC_3-PrTEFin-LaLHY-hrGFP-TLip2) pCfB6684 BB2263, BB2264 This study pCfB7142 (pIntD_1-PrTEFin-hrGFP-SmOhy-PTS1- pCfB6684 BB2249, BB2266, BB2272 This study TLip2) pCfB7143 (pIntD_1-PrTEFin-hrGFP-LaLhy-PTS1- pCfB6682 BB2249, BB2267, BB2272 This study TLip2) pCfB7144 (pIntC_2-PrTEFin-hrGFP-AsPox-TLip2) pCfB6682 BB2249, BB2272, BB2268 This study pCfB7145 (pIntC_2-PrTEFin-hrGFP-AtAcx2-TLip2) pCfB6682 BB2249, BB2272, BB2269 This study pCfB7146 (pIntC_2-PrTEFin-hrGFP-CuLAco-TLip2) pCfB6682 BB2249, BB2272, BB2270 This study pCfB7147 (pIntC_2-PrTEFin-hrGFP-RsAcox2-TLip2) pCfB6682 BB2249, BB2272, BB2271 This study pCfB7683 (pIntC_2-PrTEFin-YlPOX2-TLip2) pCfB6684 BB2419, BB2249 This study pCfB7722 (pIntD_1-PrTEFint-SmOhy-linker-PTS1- pCfB6684 BB2518 This study TLip2) pCfB7723 (pIntD_1-PrTEFint-LaLhy-linker-PTS1- pCfB6684 BB2519 This study TLip2) pCfB7748 (pIntD_1-PrTEFin-hrGFP-SmOhy-linker- pCfB6684 BB2521 This study PTS1-TLip2) pCfB7749 (pIntD_1-PrTEFin-hrGFP-LaLhy-linker- pCfB6677 BB2272, BB2522 This study PTS1-TLip2) pCfB8043 (pNat_YLgRNA_FAA1) pCfB3405 BB2694 This study pCfB8353 (pIntD_1-ScFAT1-D508A- IKL) pCfB6677 BB2249, BB3100, BB3101 This study pCfB8354 (pIntD_1-ScFAT1-D508A- IKL-SmOHY) pCfB6677 BB2249, BB3100, BB3105, BB2279 This study pCfB8355 (pIntD_1-ScFAT1-D508A- IKL-LaLHY) pCfB6677 BB2249, BB3100, BB3105, BB2282 This study pCfB8614 (pIntE_4-PrTEFin-SmOHY-TPex20) pCfB6679 BB3700, BB3704 This study pCfB8615 (pIntE_4-PrTEFin-LaLHY-TPex20) pCfB6679 BB3701, BB3705 This study
95 Chapter 4
Table S3. Synthetic genes used in this study
Name Nucleotide sequence
ATGCCCATCTTCATCTGCATCATCACCTCTCAGGCCATCATCCGATCTAACGTCGAGCGAGTGGCCGTGATCCTGAACATCAACATGGGCAAGGT
GAACGAGGACCTGGTGCGAGAGCGAGCCAAGTGCACCTTCAACATCGAGGAACTGACCTACTTCCTGGACGGCGGCAAGGACAAGACCCTCGAGC
GAAAGGAAACCGAGCGAGCTATGCTGACCAAGCGAGAGGAACTGTTCGGCGGCGTGCCCGACGAGTACCTGTCTCACAAGGAAAAGTACGAGAAC
TCTATGCGAAAGGCCGTGATTCTGTTCGGCATCCTGCGAAAGATCCAGAAGGACAACAACACCGACCTGACCAACTACCGAAACCTGCTGTCTGG
CGTGCTGTCTGTGTCTATCTCTCAGGACGGCTCTCCCTTCGGCCTGCACTACATCATGTTCATGCCCGTGCTGCTGTCTCAGGCCGACGAGAAGC
AGCAAGAGAAGTGGCTGAAGCGAGCCATGAACTGCGAGATCATCGGCTCTTACGCCCAGACCGAGCTTGGCCACGGCACCTTCATCCGAGGCCTC
GAGACTACCGCCACTTACGACCCCGCCACTCAAGAGTTCGTGCTGCACTCTCCCGCTCTGTCCTCTTACAAGTGGTGGCCCGGTGGCCTGGGCAA
CACCGTGAACTACTGCATCGTGATCGCCCAGCTGTACTCTAAGGGCGTGTGCCACGGCATCCACTCGTTCATCGTGCAGGTCCGAGATGAGGACA
CCCACATGCCTCTGCCTGGCATCAAGGTGGGCGAGATCGGCGTGAAGATGGGCCTGAACTCTGTGAACAACGGCTTCCTGGGCTTCGAGAACGTG
CGAATTCCCCGAGTGAACATGCTGATGAAGCACGCCAAGATCCTCGAGGACGGCACCTACGTGAAGTCTAAGAACAACAAGCTGATCTACGGCGC
AsePOX CATGGTGTTCGTGCGAGTGGTGATCGTGTTCGACTCTGTCAACTACCTGGCCAAGGCCATCACTATCGGAGCCCGATACTCTCTGGTGCGACGAC
AGTCTCAGCTGAAGGCCGGCGAGCCCGAGCGACAGATCCTGGACTACGTGACCCAGCAGCACAAGATTCTGCCCGCCATTGCCGGCTGCTACGCC
ATGAAGATGAACGCCTGGCGACTGTGGGACACCTTTAACCTGATCAACGGCCAGCTGCACCAGGGCAACATGGAACGACTGGGCGAGCTGCACGC
CCTGGCCTGCTGCCTGAAGGCCATCTCTACCACCGACGCCGCTATGTTCACCTCTCTGTGCCGACTCGGCTGTGGCGGCCACGGCTACATGACCT
CTTCTAACCTGCCTCCTACCTACGCTCTGACCTCTGCCTCTTGCACCTACGAGGGCGACAACACTGTGCTGCTGCTGCAGACCGCTCGATTCCTG
CTCAAGACCTGGCGACAGATTGACACCCATCCTCTGACTCGAACCGTGGCCTACCTCAAGACCGTGTCTGCTCCCGGCTTCTCTGACCGATGGGA
GTCCTCTGTCGAGGGCATCATTCGAGGCTTCCAGACCGTGGCTATGAAGAAGATCTCTTCTTGCCTGGACATCATGACCTCCAAGGTGATGTCTG
GCATGTCTCAAGAGGACGCCTGGAACGCCATCTCCATCCAGCTGGTGTCTGCCGCCGAGTCTCACTCTCGAGGCACCGTGATCTCTACCTTCTAC
GAGGACATGTCTAAGGCCATGCGATCTATGACCGCTCCTCTGGCTAAGGTGATGGGCCAGCTGGTCGAGCTGTACGCTGTGTACTGGACTCTCGA
GCGACTGGGAGACATGCTGCAGTACACCTCTATCTCCCACACCGACGTGGTGGACCTGCGATCTTGGTACGAGGAACTCCTCCGAAAGATTCGAC
CCAACACCATCGGCCTGGTGGACGCCTTCGACATCATCGACGAGCTGCTCCAGTCTACCCTGGGCGCCTACGACGGCCGAGTGTACGAGCGACTC
ATGGAAGAGGCCCTGAAGTCTCCCCTGAACGCTGAGCCCGTGAACCAGTCTTTCCACAAGTACCTGAAGCCTTTCATGCAGTCTAAGCTGTAA
ATGTACTACTCTTCTGGCAACTACGAGGCCTTCGCTCGACCCCGAAAGCCTGCCGGCGTGGACGGCAAGCGAGCCTGGTTCGTCGGCTCTGGCCT
GGCCTCTCTGGCCGGTGCCGCCTTCCTGATCCGAGATGGCCGAATGGCCGGCGAGCGAATCACCATCCTCGAGCAGCAGCACATTCCCGGCGGAG
CCCTGGACGGCCTGAAGGTGCCCGAGAAGGGCTTCGTGATTCGAGGCGGCCGAGAGATGGAAGATCACTTCGAGTGCCTGTGGGACCTGTTCCGA
TCTATCCCCTCGCTCGAGATCGAGGACGCCTCTGTGCTGGACGAGTTCTACTGGCTGAACAAGGACGACCCCAACTACTCTCTGCAGCGAGCCAC
CATCAACCGAGGCGAGGACGCTCACACCGACGGCCTGTTCACCCTGACCGAGCAGGCCCAGAAGGATATTATCGCCCTGTTCCTGGCTACCCGAC
SmOHY AAGAAATGGAAAACAAGCGAATCAACGAGGTGCTGGGCCGAGACTTCCTGGACTCTAACTTCTGGCTGTACTGGCGAACCATGTTCGCTTTCGAG
GAATGGCACTCTGCCCTCGAGATGAAGCTGTACCTGCACCGATTCATCCACCACATCGGCGGACTGCCCGACTTCTCTGCCCTGAAGTTCACCAA
GTACAACCAGTACGAGTCTCTGGTGCTGCCCCTGGTGAAGTGGCTGCAGGACCAGGGCGTCGTGTTCCAGTACGGCACCGAGGTGACCGACGTGG
ACTTCGACCTGCAGCCTGACCGAAAGCAGGCTACTCGAATCCACTGGATGCACGACGGCGTGGCTGGCGGAGTTGACCTGGGCGCCGACGACCTG
CTGTTCATGACCATCGGATCTCTGACCGAGAACTCTGACAACGGCGACCACCACACCGCCGCTCGACTGAACGAGGGACCCGCTCCTGCCTGGGA
CCTCTGGCGACGAATCGCCGCTAAGGACGACGCCTTCGGACGACCCGACGTGTTCGGCGCTCACATCCCCGAGACTAAGTGGGAGTCTGCCACCG
TGACCACTCTGGACGCTCGAATCCCCGCCTACATCCAGAAGATCGCCAAGCGAGATCCATTCTCTGGCAAGGTGGTGACCGGCGGCATCGTGTCT
96 Chapter 4
GTGCGAGACTCTCGATGGCTGATGTCTTGGACCGTGAACCGACAGCCTCACTTCAAGAACCAGCCTAAGGACCAGATCGTGGTGTGGGTGTACTC
TCTGTTCGTGGACACCCCTGGCGACTACGTGAAGAAGCCCATGCAGGACTGCACCGGCGAAGAGATCACCCGAGAGTGGCTGTACCACCTGGGCG
TGCCCGTGGAAGAGATCGACGAGCTGGCCGCCACCGGCGCCAAGACCGTGCCTGTGATGATGCCCTACATCACCGCTTTCTTCATGCCCCGACAG
GCCGGCGACCGACCTGATGTGGTGCCCGACGGCGCTGTGAACTTCGCCTTCATCGGCCAGTTCGCCGAGTCTAAGCAGCGAGACTGCATCTTCAC
CACCGAGTACTCTGTGCGAACCCCTATGGAAGCCGTGTACACCCTGCTGGACATTGAGCGAGGCGTCCCCGAGGTGTTCAACTCTACCTACGACG
TGCGATCTCTGCTGGCTGCTACCGGCCGACTGCGAGATGGCAAGGAACTGGGAATCCCCGGACCTGTGTTCCTGCGAAACCTGCTGATGAACAAG
CTGGACAAGACCCAGATCGGCGGCCTGCTGCGAGAGTTCAAGCTGGTGCAAGAGGACTAA
ATGCACTACTCTTCCGGCAACTACGAGGCCTTCGTGAACGCCTCTAAGCCCAAGGACGTGGACCAAAAGTCTGCCTACCTGGTCGGCTCTGGCCT
GGCCTCTCTGGCCTCCGCCGTGTTCCTGATCCGAGATGGCCACATGAAGGGCGACCGAATCCACATTCTCGAGGAACTGTCTCTGCCCGGTGGCT
CTATGGACGGCATCTACAACAAGCAGAAGGAATCTTACATCATCCGAGGCGGCCGAGAGATGGAAGCCCACTTCGAGTGCCTGTGGGACCTGTTC
CGATCTATCCCCTCTGCCGAGAACAAGGACGAGTCTGTGCTGGACGAGTTCTACCGACTGAACCGAAAGGACCCCTCGTTCGCTAAGACCCGAGT
GATCGTGAACCGAGGCCACGAGCTGCCCACCGACGGCCAGCTGCTGCTGACCCCTAAGGCCGTGAAGGAAATCATCGACCTCTGTCTGACCCCTG
AGAAGGACCTGCAGAACAAGAAGATCAACGAGGTTTTCTCTAAGGAATTCTTCGAGTCTAACTTCTGGCTGTACTGGTCTACCATGTTCGCCTTC
GAGCCCTGGGCCTCTGCCATGGAAATGCGACGATACCTGATGCGATTCGTGCAGCACGTGTCTACCCTGAAGAACCTGTCCTCTCTGCGATTCAC
CAAGTACAACCAGTACGAGTCTCTGATTCTGCCCATGGTGAAGTACCTGAAGGACCGAGGCGTGCAGTTCCACTACAACACCGTGGTGGACAACA
TCTTCGTCAACCGATCTAACGGCGAGAAGATCGCCAAGCAGATCCTGCTGACCGAGAACGGTGAGAAGAAGTCTATCGACCTGACTGAGAACGAC LaLHY CTCGTGTTCGTGACCAACGGCTCTATCACCGAGTCTACCACCTACGGCGACAACCTGCATCCTGCCTCTGAGGAACACAAGCTGGGCGCCACCTG
GAAGCTGTGGCAGAACCTGGCCGCTCAGGACGACGACTTCGGACACCCCGACGTGTTCTGCAAGGACATCCCCAAGGCCAACTGGGTGATGTCTG
CCACCATCACCTTCAAGAACAACGACATCGTGCCCTTCATCGAGGCCGTCAACAAGAAGGACCCTCACTCTGGCTCTATCGTGACCTCTGGACCC
ACCACCATCAAGGACTCTAACTGGCTGCTGGGCTACTCTATCTCTCGACAGCCCCACTTTGAGGCTCAGAAGCCCAACGAGCTGATCGTGTGGCT
GTACGGCCTGTTCTCTGACACCAAGGGAAACTACGTGGAAAAGACCATGCCTGACTGCAACGGCATCGAGCTGTGCGAGGAATGGCTGTACCACA
TGGGCGTGCCCGAGGAACGAATCCCTGAGATGGCCTCTGCTGCCACCACTATTCCCGCTCACATGCCCTACATCACCTCTTACTTCATGCCCCGA
GCACTGGGTGACCGACCTAAGGTGGTGCCCGACCACTCTAAGAACCTCGCCTTCATCGGCAACTTCGCCGAGACTCCCCGAGACACCGTGTTCAC
CACCGAGTACTCTGTGCGAACCGCCATGGAAGCCGTGTACACCCTGCTGAACATCGACCGAGGTGTCCCCGAGGTGTTCGCCTCTGCCTTCGACG
TGCGAATGCTGATGAACGCCATGTACTACCTGAACGACCAGAAGAAGCTCGAGGACCTGGACCTGCCTATCGCCGAGAAGCTGGCCATCAAGGGC
ATGCTGAAGAAGGTGAAGGGAACCTACATTGAGGAACTGCTCAAGAAGTACAAGCTGGTCCTGTAA
ATGGAATCTCGACGAGAGAAGAACCCCATGACCGAGGAAGAGTCTGACGGCCTGATCGCCGCTCGACGAATCCAGCGACTGTCTCTGCACCTGTC
GCCTTCTCTGACCCCTTCTCCTTCGCTGCCCCTGGTGCAGACCGAGACTTGCTCTGCCCGATCTAAGAAGCTGGACGTCAACGGCGAGGCCCTGT
CTCTGTACATGCGAGGCAAGCACATCGACATCCAAGAGAAGATTTTCGACTTCTTCAACTCTCGACCCGACCTGCAGACCCCTATCGAGATCTCT
AAGGACGACCACCGAGAGCTGTGCATGAACCAGCTGATCGGCCTGGTGCGAGAGGCCGGCGTGCGACCCTTCCGATACGTGGCTGACGACCCTGA
GAAGTACTTTGCCATCATGGAAGCCGTGGGCTCTGTGGACATGTCTCTGGGCATCAAGATGGGCGTGCAGTACTCTCTGTGGGGCGGCTCTGTGA
AtACX2 TCAACCTGGGCACCAAGAAGCACCGAGACAAGTACTTCGACGGCATCGACAACCTGGACTACACCGGCTGCTTCGCTATGACCGAGCTGCACCAC
GGCTCTAACGTGCAGGGACTGCAGACCACCGCCACTTTCGACCCTCTGAAGGACGAGTTCGTGATCGACACCCCTAACGACGGCGCCATCAAGTG
GTGGATCGGCAACGCCGCCGTCCACGGCAAGTTCGCCACCGTGTTCGCCCGACTGATTCTGCCCACTCACGACTCTAAGGGCGTGTCTGACATGG
GAGTGCACGCCTTCATCGTGCCCATCCGAGACATGAAGACCCACCAGACTCTGCCCGGCGTCGAGATCCAGGACTGCGGCCACAAGGTGGGCCTG
AACGGCGTGGACAACGGCGCCCTGCGATTCCGATCTGTGCGAATTCCCCGAGACAACCTGCTGAACCGATTCGGCGACGTGTCTCGAGATGGCAC
CTACACCTCTTCTCTGCCCACCATCAACAAGCGATTCGGAGCTACCCTGGGCGAGCTGGTCGGCGGACGAGTCGGCCTGGCCTACGCCTCTGTGG
GCGTGCTGAAGATCTCCGCCACTATCGCCATCCGATACTCCCTGCTGCGACAGCAGTTCGGCCCTCCTAAGCAGCCCGAGGTGTCTATTCTGGAC
97 Chapter 4
TACCAGTCTCAGCAGCACAAGCTGATGCCCATGCTGGCCTCTACCTACGCCTACCACTTCGCTACCGTGTACCTGGTGGAAAAGTACTCTGAGAT
GAAGAAGACTCACGACGAGCAGCTGGTGGCCGACGTGCACGCCCTGTCTGCCGGACTGAAGTCTTACGTGACCTCTTACACCGCCAAGGCTCTGT
CTGTGTGCCGAGAGGCCTGTGGCGGCCACGGCTACGCCGCTGTCAACCGATTTGGCTCTCTGCGAAACGACCACGACATCTTCCAGACCTTCGAG
GGCGACAACACCGTGCTGCTCCAGCAGGTCGCCGCCGACCTCCTGAAGCGATACAAGGAAAAGTTCCAAGGCGGCACCCTGACCGTCACCTGGTC
TTACCTGCGAGAGTCTATGAACACCTACCTCTCGCAGCCCAACCCTGTGACCGCTCGATGGGAAGGCGAGGACCATCTGCGAGATCCCAAGTTTC
AGCTGGACGCTTTCCGATACCGAACCTCTCGACTGCTGCAGAACGTGGCTGCCCGACTGCAGAAGCACTCTAAGACCCTCGGCGGCTTCGGCGCC
TGGAACCGATGCCTGAACCATCTGCTGACCCTGGCCGAGTCTCACATCGAGACTGTGATCCTGGCCAAGTTCATCGAGGCCGTGAAGAACTGCCC
CGATCCTTCTGCCAAGGCCGCTCTGAAGCTGGCCTGCGACCTGTACGCCCTGGACCGAATCTGGAAGGACATCGGCACCTACCGAAACGTGGACT
ACGTGGCTCCCAACAAGGCCAAGGCCATCCACAAGCTCACCGAGTACCTGTCTTTCCAGGTGCGAAACGTCGCCAAGGAACTGGTGGACGCCTTC
GAGCTGCCTGACCACGTGACTCGAGCCCCTATTGCCATGCAGTCTGACGCCTACTCTCAGTACACCCAGGTGGTGGGCTTCGCCAAGATCTAA
ATGGCTTCTCCCGGCGAGCCCAACCGAACCGCCGAGGACGAGTCTCAGGCCGCTGCTCGACGAATCGAGCGACTGTCTCTGCATCTGACCCCTAT
TCCTCTGGACGACTCCCAGGGCGTCGAGATGGAAACCTGTGCCGCCGGAAAGGCCAAGGCCAAGATCGAGGTGGACATGGGATCTCTGTCTCTGT
ACATGCGAGGCAAGCACCGAGAGATCCAAGAGCGAGTGTTCGAGTACTTCAACTCTCGACCCGAGCTGCAGACCCCTGTGGGCATCTCTATGGCC
GACCACCGAGAGCTGTGCATGAAGCAGCTGGTCGGCCTGGTGCGAGAGGCCGGCATTCGACCCTTCCGATTCGTGAACGAGGACCCCGCCAAGTA
CTTCGCCATCATGGAAGCCGTGGGCTCTGTGGACGTGTCTCTGGCCATCAAGATGGGCGTGCAGTTCTCTCTGTGGGGCGGCTCTGTGATCAACC
TGGGCACCAAGAAGCACCGGGACCGATTCTTCGACGGCATCGACAACGTGGACTACCCCGGCTGCTTCGCCATGACTGAGCTGCACCACGGCTCT
AACGTGCAGGGCCTGCAGACCACCGCCACTTTCGACCCCATCACCGACGAGTTCATCATCAACACCCCTAACGACGGCGCCATCAAGTGGTGGAT
CGGCAACGCCGCCGTCCACGGCAAGTTCGCCACCGTGTTCGCCAAGCTGGTGCTGCCCACTCACGACTCTCGAAAGACCGCCGACATGGGAGTGC
ACGCCTTCATCGTGCCCATCCGAGATCTGAAGTCTCACAAGACCCTGCCTGGCATCGAGATCCACGACTGCGGCCACAAGGTGGGCCTGAACGGC
GTGGACAACGGCGCCCTGCGATTCCGATCTGTGCGAATTCCCCGAGACAACCTGCTGAACCGATTCGGCGAGGTGTCTCGAGATGGCAAGTACAA
CuLACO GTCCTCTCTGCCCTCTATCAACAAGCGATTCGCCGCCACTCTGGGCGAGCTGGTTGGCGGCCGAGTCGGACTGGCCTACTCTTCTGCCTCTGTGC
TGAAGATCGCCTCTACTATCGCCATCCGATACTCCCTGCTGCGACAGCAGTTCGGCCCTCCTAAGCAGCCCGAGGTGTCCATCCTGGACTACCAG
TCTCAGCAGCACAAGCTGATGCCCATGCTGGCCTCTACCTACGCCTTCCACTTCTCTACCATGCAGCTCGTCGAGAAGTACGCCCAGATGAAGAA
GACCCACGACGAGGAACTGGTGGGCGACGTGCACGCCCTGTCTGCCGGCCTGAAGGCCTACGTGACCTCTTACACCGCCAAGTCTCTGTCTACCT
GCCGAGAGGCCTGTGGCGGCCACGGATACGCCGTGGTCAACCGATTTGGCACCCTGCGAAACGACCACGACATCTTCCAGACCTTCGAGGGCGAC
AACACCGTGCTGCTCCAGCAGGTCGCCGCCTACCTGCTCAAGCAGTACCAAGAGAAGTTCCAAGGCGGCACCCTGGCCGTGACCTGGAACTACCT
GCGAGAATCTATGAACACCTACCTCTCGCAGCCCAACCCTGTGACCGCTCGATGGGAGTCTGCCGACCATCTGCGAGATCCCAAGTTTCAGCTGG
ACGCTTTCCAGTACCGAACCTCTCGACTGCTGCAGTCTGTGGCCGTGCGACTGCGAAAGCACACCAAGAACCTGGGATCTTTCGGCGCCTGGAAC
CGATGCCTGAACCATCTGCTGACCCTGGCTGAGTCTCACATCGAGTCTGTGATTCTGGCCCAGTTCATCGAGTCCGTGCAGAGATGTCCCAACGC
TAACACCCAGGCTACCCTGAAGCTGGTGTGCGACCTGTACGCTCTGGACCGAATCTGGAACGACATCGGCACCTACCGAAACGTCGACTACGTGG
CTCCCAACAAGGCTAAGGCCATCCACAAGCTCACCGAGTACCTGTGCTTCCAGGTGCGAAACATTGCCCAAGAGCTGGTGGACGCCTTCGACCTG
CCTGACCACGTGACTCGAGCCCCTATTGCCATGAAGTCTAACGCCTACTCTCAGTACACCCAGTACATCGGCTTCGCCAAGATCTAA
ATGGAATCTCGACGAGGCAACCAGATGGCCGAAGAGGAATTCTCTGAGGTGGCCGCTGCTCGACGAATCCAGCGACTGACCTCTCACATCTCTCC
CGCCGCTACCGCTCCTTCGCAGCTGCAGCGAGAGGCCTGTTCTTCTCGATCTAAGAAGCTCGAGGTCGACTCTGAGGCCCTGTCTGTGTACATGC
RsAcox2 GAGGCAAGCACATGGACATCCAAGAGAAGGTGTTCGAGTTCTACAACTCTCGACCCGACCTGCAGACCCCTATCGAGATCTCTAAGGACGACCAC
CGAGAGCTGTGTATGCGACAGCTGTACGGCCTGGTCCGAGAGGCCGGCATTCGACCCTTCCGATACGTGGCTGAGGACCCCGAGAAGTACTTTGC
CATCATGGAAGCCGTGGGCTCTGTGGACATGTCTCTGGGCATCAAGATGGGCGTGCAGTACTCTCTGTGGGGCGGCTCTGTGATCAACCTGGGCA
CCAAGAAGCACCGAGACAAGTACTTCGACGGCATCGACAACCTGGACTACACCGGCTGCTTCGCCATGACCGAGCTGCACCACGGCTCTAACGTG
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CAGGGACTGCAGACCACCGCCACTTTCGACCCCATCACCGACGAGTTCGTGATCGACACCCCTCACGACGGCGCCATCAAGTGGTGGATCGGCAA
CGCCGCCGTCCACGGCAAGTTCGCCACCGTGTTCGCCCGACTGATTCTGCCCACTCACGACACCAAGGGCGTGTCTGACATGGGAGTGCACGCCT
TCATCGTGCCCATCCGAGACATGAAGACCCACCAGACTCTGCCCGGCGTCGAGATCCAGGACTGCGGCCACAAGGTGGGCCTGAACGGCGTGGAC
AACGGCGCCCTGCGATTCCGATCTGTGCGAATTCCCCGAGACAACCTGCTGAACCGATTCGGCGACGTGTCTCGAGATGGCAAGTACACCTCTTC
TCTGCCCACCATCAACAAGCGATTCGGAGCTACCCTGGGCGAGCTGGTCGGCGGACGAGTCGGCCTGGCCTACGCCTCTGTGGGCGTGCTGAAGA
TCTCCGCCACTATCGCCATCCGATACTCCCTGCTGCGACAGCAGTTCGGACCTCCTAAGCAGCCCGAGGTGTCTATTCTGGACTACCAGTCTCAG
CAGCACAAGCTGATGCCCATGCTGGCCTCTACCTACGCCTACCACTTCGCTACCGTGTACCTGGTGGAAAAGTACTCTGAGATGAAGAAGACTCA
CGAGGAACAGCTGGTGGCCGACGTGCACGCCCTGTCCGCCGGACTGAAGTCTTACGTGACCTCTTACACCGCCAAGGCTCTGTCCGTGTGCCGAG
AGGCTTGTGGCGGCCACGGCTACGCCGCTGTCAACCGATTTGGCTCTCTGCGAAACGACCACGACATCTTCCAGACCTTCGAGGGCGACAACACC
GTGCTGCTCCAGCAGGTCGCCGCCGATCTGCTGAAGCAGTACAAGGAAAAGTTCCAAGGCGGCACCCTGACCGTCACCTGGTCTTACCTGCGAGA
GTCTATGAACACCTACCTGGCTCAGCCCAACCCTGTGACCGCTCGATGGGAAGGCGAGGACCATCTGCGAGATCCCAAGTTTCAGCTGGACGCTT
TCCGATACCGAACCTCTCGACTGCTGCAGTCTGTGGCCATGCGACTGAAGAAGCACTCTAAGACCCTGGGAACCTTCGGCGCCTGGAACCGATGC
CTGAACCATCTGCTGACCCTGGCCGAGTCTCACATCGAGTCTGTGATCCTGGCCAAGTTCATCGAGGCCGTGCAGAACTGCTCTGACCCCTCTGC
TCGAGCCGGCCTGAAGCTGGCCTGCGACCTGTACGCTCTGGACCGAATCTGGAAGGACATCGGCACCTACCGAAACGTGGACTACGTGGCTCCCA
ACAAGGCCAAGGCCATCCACAAGCTCACCGAGTACCTGTCTTTCCAGGTGCGAAACGTCGCCAAGGAACTGGTGGACGCCTTCGACCTGCCTGAC
CACGTGACTCGAGCCCCTATTGCCATGCAGTCTGACGCCTACGCTCAGTACACCCAGGTGGTGGGCTTCGCCAAGATCTAA
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Table S4. Biobricks used in this study
Biobricks Template for PCR Fw Primer Rv Primer BB2194 (SmOhy-forPrTEFin_gene2) pCfB7054 (DNA Fragment SmOHY) PR-19301 PR-19303 BB2195 (LaLhy-forPrTEFin_gene2) pCfB7055 (DNA Fragment LaLHY) PR-19304 PR-20767 BB2222 (gRNAYL_POX1_cass1) BB1635, BB1636, PR-19076, PR-19077 PR-15791 PR-10607 BB2223 (gRNAYL_POX2_cass2) BB1635, BB1636, PR-19078, PR-19079 PR-15790 PR-10604 BB2224 (gRNAYL_POX3_cass1) BB1635, BB1636, PR-19080, PR-19081 PR-15791 PR-10607 BB2225 (gRNAYL_POX4_cass2) BB1635, BB1636, PR-19083, PR-19082 PR-15790 PR-10604 BB2226 (gRNAYL_POX5_cass1) BB1636, BB1635, PR-19020, PR-19021 PR-15791 PR-10607 BB2227 (gRNAYL_POX6_cass2) BB1635, BB1636, PR-19022, PR-19023 PR-15790 PR-10604 BB2229 (PrTEFintron for SmOhy) pCfB5124 PR-10595 PR-19302 BB2230 (PrTEFintron for LaLhy) pCfB5124 PR-10595 PR-19305 BB2231 (gRNAYL_POX2_single) PR-19078, PR-19079, BB1635, BB1636 PR-10607 PR-10604 BB2232 (gRNAYL_POX3_single) PR-19081, PR-19080, BB1635, BB1636 PR-10607 PR-10604 BB2242 (AsPox_codoptYL) pCfB6584 (DNA Fragment AsePOX) PR-20681 PR-20682 BB2243 (AtAcx2_codoptYL) pCfB7073 (DNA Fragment AtAcx2) PR-20683 PR-20684 BB2244 (CuLAco_codoptYL) pCfB7074 (DNA Fragment CuLACO) PR-20685 PR-20686 BB2245 (RsAcox2_codoptYL) pCfB7075 (DNA Fragment YlPOX2) PR-20687 PR-20689 BB2247 (SmOhy_YL-PTS1) pCfB7057 PR-19301 PR-20707 BB2248 (LaLhy_YL-PTS1) pCfB7058 PR-19304 PR-20708 BB2249 (PrTEFintron_Gene2) BB2229 PR-10595 PR-18214 BB2250 (PrTEFintron for RsAcox2) BB2229 PR-10595 PR-20688 BB2262 (PrTEFin-SmOhy_Clinker-hrGFP) pCfB7057 PR-10595 PR-20770 BB2263 (PrTEFin-LaLhy_Clinker-hrGFP) pCfB7058 PR-10595 PR-20771 BB2264 (hrGFP_C-terminal-fusion) pCfB5124 PR-20772 PR-15506 BB2266 (SmOhy-PTS1_N-hrGFP) pCfB7057 PR-20774 PR-20707 BB2267 (LaLhy-PTS1_N-hrGFP) pCfB7058 PR-20775 PR-20768 BB2269 (AtAcx2-PTS1_N-hrGFP) pCfB7077 PR-20779 PR-20684 BB2270 (CuLAco-PTS1_N-hrGFP) pCfB7078 PR-20780 PR-20686 BB2271 (RsAcox2-PTS1_N-hrGFP) pCfB7079 PR-20781 PR-20689 BB2272 (hrGFP_N-terminal-fusion) pCfB5124 PR-20777 PR-20776 BB2419 (YlPox2_Gene2) Genomic DNA Y. lipolytica W29 PR-21083 PR-21084 BB2518 (PrTEFin-SmOhy-linker-PTS1) pCfB7139 PR-10595 PR-21251 BB2519 (PrTEFin-LaLhy-linker-PTS1) pCfB7140 PR-10595 PR-21251 BB2521 (linker-SmOhy-linker-PTS1) pCfB7139 PR-20774 PR-21251 BB2522 (linker-LaLhy-linker-PTS1) pCfB7140 PR-20775 PR-21251 BB2674 (YlFAA1_repair-up) Genomic DNA Y. lipolytica W29 PR-21638 PR-21639 BB2675 (YlFAA1_repair-down) Genomic DNA Y. lipolytica W29 PR-21640 PR-21641 BB2676 (YlFAA1_repair-fragment) BB2674, BB2675 PR-21638 PR-21641 BB2694 (YlFAA1_single-gRNA) BB2690 PR-10607 PR-10604 BB2698 (YlFAA1_double-gRNA_gene1) BB2690 PR-10607 PR-15791 BB3100 (ScFAT-D508A- IKL_ PrTEFin_up) Genomic DNA S. cerevisiae CEN.PK113-7D PR-22357 PR-22359
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BB3101 (ScFAT-D508A- IKL_ PrTEFin_down) Genomic DNA S. cerevisiae CEN.PK113-7D PR-22358 PR-22656 BB3103 (ScFAT-D508A- IKL_ P TEF ) BB3100, BB3101 PR-22357 PR-22359 BB3105 (ScFAT-D508A- IKL_ C-terminal fusion) BB3103 PR-22357 PR-22657 BB3700 (SmOHY_forPrTEFin_gene1) pCfB7057 PR-19301 PR-23256 BB3701 (LaLHY_forPrTEFin_gene1) pCfB7057 PR-19304 PR-23257 BB3704 (PrTEFin_forSmOHY_gene1) pCfB7058 PR-18928 PR-19032 BB3705 (PrTEFin_forLaLHY_gene1) pCfB7058 PR-18928 PR-19305
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Table S5. Primers used in this study
Primer/Oligo Sequence 5´to 3´ PR-10595 (PrTefYL _fw) cgtgcgaUAGAGACCGGGTTGG PR-10604 (tracrRNA _rev) cacgcgaUaccgtacccacacaaaaaaagcaccaccgactc PR-10607 (PrtRNAGly _fw) cgtgcgaUagtgaatcattgctaacagatc PR-15506 (hrGFP_U2_rev) CACGCGAUttacacccactcgtgca PR-15790 (gRNA_cass2_fw) AGTGCAGGUagtgaatcattgctaacagatc PR-15791 (gRNA_cass2_rev) ACCTGCACUaccgtacccacacaaaaaaagcac PR-15792 (gRNA_cass3_fw) ATCTGTCAUagtgaatcattgctaacagatc PR-15793 (gRNA_cass3_rev) ATGACAGAUaccgtacccacacaaaaaaagcac ccaaatcataaaaaacgttaatacttactaaaacctacaaaatcatgttgtgggtcgtttca PR-18069 (dsOLIGO_POX4_KO) atgaaagagcatgccactgcaactatat PR-18214 AGTACTGCAAAAAGUGCTG (PTEFintron_USER_rv) PR-18928 (PrTEF1 <-_U1_fw) CACGCGAUAGAGACCGGGTTGG PR-19032 (SmOhyA GP2F) atctgtcaUATGTACTACTCTTCTGGCAAC PR-19078 (Pox2_gRNA_sense) AGCAGAGCCAGGTTAAGCAGgttttagagct PR-19079 CTGCTTAACCTGGCTCTGCTtaaccaacct (Pox2_gRNA_antisense) PR-19080 (Pox3_gRNA_sense) AAGCAGACGAGCAAAGCAGGgttttagagct PR-19081 CCTGCTTTGCTCGTCTGCTTtaaccaacct (Pox3_grRNA_antisense) ctcctacaaaagagagcaggtggaataacacactattgaccgacatgttagtaattatga PR-19084 (Pox1_repair) gatacattctttgcaatgatgagggctctt ataccaaagggatgggtcctcaaaaatcacacaagcaacgacgccacaagcgggtat PR-19085 (Pox2_repair) ttattgtatgaataaagattatgtattgattgc cagtcgccctgtggacaacacgtcactacctctacgatacacacaatggagcgtgtgttct PR-19086 (Pox3_repair) gagtcgatgttttctatggagttgtgagt aattgattctacacttacactaccaattcttacatcaaaccaaacgttgttgtaatactatgatt PR-19088 (Pox5_repair) tattgtgtttatatgttattgatac cataaataaaacagttcattagtaacaatatatccttcaatccatgttttggtacacgagata PR-19089 (Pox6_repair) gaagggggttttagggtggacagtagc PR-19301 aaccgcagTACUACTCTTCTGGCAAC (SmOhy_PrTEFintron_fw) PR-19302 agtaCTGCGGTUAGTACTGCAAAAAGTG (PrTEFintron_SmOhy_rv) PR-19303 (SmOhy_USER_rv) cacgcgaUTTAGTCCTCTTGCACCAGCTTGA PR-19304 aaccgcagCACUACTCTTCCGGC (LaLhy_PrTEFintron_fw) PR-19305 agtgCTGCGGTUAGTACTGCAAAAAGTG (PrTEFintron_LaLhy_rv) PR-20681 actttttgcagtacuaaccgcagTTACAGCTTAGACTGCATGAAAG (AsPox_PrTEFintron_USER_fw) PR-20682 (AsPox_USER_rv) cacgcgaUCCCATCTTCATCTGCATCATCACCTCTCA PR-20683 actttttgcagtacuaaccgcagGAATCTCGACGAGAGAAGAACCCCAT (AtAcx2_PrTEFintron_USER_fw) G PR-20684 cacgcgaUttacagcttagaGAAGCCCACCACCTGGGTGTA (AtAcx2_SKL_USER_rv)
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PR-20685 actttttgcagtacuaaccgcagGCTTCTCCCGGCGAGCCCAAC (CuLAco_PrTEFintron_USER_fw) PR-20686 cacgcgaUttacagcttagaGAAGCCGATGTACTGGGTGTAC (CuLAco_SKL_USER_rv) PR-20687 aaccgcagGAAUCTCGACGAGGCAACCAGATGGCCGAAGAGG (RsAco2_PrTEFintron_USER_fw) A PR-20688 attcCTGCGGTUAGTACTGCAAAAAGTG (PrTEFintron_RsAco2_rv) PR-20689 cacgcgaUttacagcttagaGAAGCCCACCACCTGGGTG (RsAco2_SKL_USER_rv) PR-20771 (<-LaLhy-hrGFP_C- atcctcctccUccCAGGACCAGCTTGTACTTCTTGAG linker_rv) PR-20767 (LaLhy_USER_rv2) cacgcgaUTTACAGGACCAGCTTGTACTTCTTGAG PR-20768 cacgcgaUTTAcagcttagaCAGGACCAGCTTGTACTTCTTGAG (LaLhy_PTS1_gene2_rv) PR-20770 (<-SmOhy-hrGFP_C- atcctcctccUccGTCCTCTTGCACCAGCTTGA linker_rv) PR-20771 (<-LaLhy-hrGFP_C- atcctcctccUccCAGGACCAGCTTGTACTTCTTGAG linker_rv) PR-20772 (C-linker_hrGFP_fw->) aggaggaggaUctggaggaggaggatctATGGTGAGCAAGCAGATC PR-20774 (hrGFP_N-linker- aggaggatcUATGTACTACTCTTCTGGCAACTACG >SmOhy_fw) PR-20775 (hrGFP_N-linker- aggaggatcUATGCACTACTCTTCCGGCAACTAC >LaLhy_fw)
PR-20776 (<-N_linker-hrGFP_rv) agatcctccUcctccagatcctcctcctccCACCCACTCGTGCAGGCTG
PR-20777 actttttgcagtacuaaccgcagGTGAGCAAGCAGATCCTGAAGA (hrGFP_PrTEFintron_USER_fw) PR-20779 (hrGFP_N-linker- aggaggatcUATGGAATCTCGACGAGAGA >AtAcx2_fw) PR-20780 (hrGFP_N-linker- aggaggatcUATGGCTTCTCCCGGCGAG >CuLAco_fw) PR-20781 (hrGFP_N-linker- aggaggatcUATGGAATCTCGACGAGGC >RsAcox2_fw) PR-21083 actttttgcagtacuaaccgcagAACCCCAACAACACTGGCA (YlPox2_PrTEFintron_USER_fw) PR-21084 (YlPox2_USER_rv) cacgcgaUCTATTCCTCATCAAGCTCGCA PR-21251 (<-3xGGGGS- cacgcgaUTTACAGCTTAGAagatcctcctcctccAGATCCTCCTCCTC SKL_linker) CAGA PR-21636 (YlFAA1_sense) GGAGTACTACAAGAACGAGGgttttagagct PR-21637 (YlFAA1_antisense) CCTCGTTCTTGTAGTACTCCtaaccaacct PR-21638 (YlFAA1_repair-up_fw) CTCTTCAACCACCTCTGGG PR-21639 (YlFAA1_repair-up_rv) aggccacUAGTGTTTAAACCCTCTGACAG PR-21640 (YlFAA1_repair- agtggccUGGAGGGGGTTGGTATAGAGAG down_fw) PR-21641 (YlFAA1_repair- CTCTGTTTTCTGGCTCCC down_rv) PR-22357 (ScFAT1_TEFin_fw) actttttgcagtacUaaccgcagTCTCCCATACAGGTTGTTG PR-22358 (ScFAT1_D508A_fw) ATGTGGAgccUTATTAAAAGCGGACGAATATGG PR-22359 (ScFAT1_D508A_rv) AggcTCCACAUCTATACCAAGCATCGCCA
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PR-22656 (ScFAT1- cacgcgaUTCATGTTTGTGCATCGATGGC IKL_ _ e e2) PR-22657 (ScFAT1- IKL_C- AgatcctccUcctccagatcctcctcctccagatcctcctcctccTGTTTGTGCATC fusion_rv) GAT PR-23256 (SmOHY_U1_rv) cgtgcgaUTTAGTCCTCTTGCACCAGC PR-23257 (LaLHY_U1_rv) cgtgcgaUTTACAGGACCAGCTTGTACTTC
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Supplementary Information 2 Alignment native-sequence of YlPOX2 and heterologous LCPOXes used in this study. All of the
LCPOXes contain domains belonging to Acyl-CoA oxidases family (shown in red box), which were identified through Pfam motif search. Except for YlPOX2, peroxisome localization signal 1 (PTS1) is present in all of the LCPOXes (shown in blue box). The alignments were prepared in CLC Main
Workbench 8.
Acyl-CoA oxidase family
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PTS1 signal
Conservation:
White: less than or equal to 50%
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Supplementary Information 3 BLASTp (https://blast.ncbi.nlm.nih.gov/) result of R. toruloides acyl-CoA dehydrogenase protein (Query ID
EMS25969.1) on Y. lipolytica CLIB122 genome (taxid:284591). ACAD superfamily domain (shown as pink bars) was predicted in all of the four hits.
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Supplementary Information 4 Fluorescence microscopy image of SmOHY and LaLHY tagged with peroxisomal targeting signal 1
(PTS1, -SKL) without linker in between SmOHY or LaLHY ORF and PTS1. hrGFP protein was tagged at the N-terminal. Without linker, GFP image from LaLHY-SKL indicates peroxisomal localization was achieved. On the other hand, SmOHY-SKL showed cytosolic localization pattern. Since SmOHY-SKL with linker (3xGGGGS) showed peroxisomal localization pattern (Figure 2f, main text) and LaLHY -SKL with linker still maintained peroxisomal localization as well, the variants with linker were used for the production test.
hrGFP-SmOHY-SKL
hrGFP-LaLHY-SKL
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5
Engineering of acyl-editing pathway for an enhanced production of linoleic acid in the oleaginous yeast Yarrowia lipolytica
Eko Roy Marella, Jonathan Dahlin, Javier Sáez Sáez, Hanne Bjerre Christensen, Guokun Wang, Irina Borodina
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet 220, 2800 Kongen Lyngby, Denmark
Manuscript in preparation
Chapter 5
Abstract
Polyunsaturated fatty acids (PUFA) offer many health benefits for humans and are used as feed supplement in aquaculture. To supply PUFAs more sustainably, microbial hosts are increasingly employed for PUFA production, especially the oleaginous yeast Yarrowia lipolytica. Although multiple strategies to increase lipid production have been reported, metabolic engineering approaches for accumulating specific fatty acids remain limited. In this study, we aimed at refining fatty acid profile towards accumulation of linoleic acid as a model of PUFA. In particular, we introduced acyl-editing enzymes to increase the conversion of oleoyl-CoA into linoleic acid. We first engineered Y. lipolytica ST6512 to contain genetic modifications reported previously, which are deletion of POX1-6, deletion of DGA1, expression of an elongase,
e e e i n f 9-desaturase, and expression of heterologous DGAT. This strain accumulated fatty acids at 27% of its cell dry weight, 39% of which was linoleic acid.
Subsequently, castor LPCAT2 (RcLPCAT2), which encodes a lysophopshatidylcholine acyltransferase (LPCAT) was introduced into this strain. In plants, LPCAT is involved in acyl editing which results in substantial accumulation of seed oil PUFA. After introduction of RcLPCAT2, 19% increase in linoleic acid content was obtained, yielding a strain with linoleic acid comprising 47% of its total fatty acids.
The described approach is based on the recycling of the acyl chains of phosphatidylcholines, effectively resulting in a higher degree of fatty acid desaturation by the desaturases that act on phosphatidylcholines. The same approach may be applicable for the production of other PUFAs than linoleic acid as well.
Keywords: polyunsaturated fatty acid, oleaginous yeast, metabolic engineering, acyl- editing, acyl-remodeling
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5.1. Introduction
Polyunsaturated fatty acids (PUFAs) are fatty acids containing two or more double bonds within their acyl chain. Linoleic acid (18:2n-6) and alpha-linolenic acid
(18:3n-3) are two essential fatty acids that humans must obtain from the diet [1]. The alpha-linolenic acid can be elongated into omega-3 eicosapentaenoic acid (EPA) and further into docosahexaenoic acid (DHA), however this conversion is inefficient in the human body, therefore EPA and DHA are primarily obtained from foods, such as fish and some algal products. Eicosanoids exert influence on regulation of immune system, cholesterol metabolism, and neurotransmission [2]. DHA makes up the most of structural fatty acids in the central nervous system and therefore is a critical nutrient for infant and child brain development [3]. A meta-analysis of publications studying the effects of PUFAs on depression concluded that there has been sufficient evidence to support positive benefits of PUFAs [4]. Furthermore, benefits of PUFA on cardiovascular health such as by lowering triglycerides level have been documented
[5].
For higher PUFAs such as eicosapentaenoic acids (EPA) and DHA, marine fish oils remain as main sources while there has been increasing efforts to top up the supply via transgenic plants [2]. Alternative production methods for PUFAs aside from marine fish aquaculture offer economic and environmental advantages [6,7].
Notably, the yeast Yarrowia lipolytica has been successfully engineered previously to produce over 56% of EPA of its total fatty acid (TFA) content, with TFA accounting 30% of the cell dry weight [6]. To achieve de novo biosynthesis of EPA, heterologous desaturases and elongases were expressed. Then the general biosynthesis and accumulation of fatty acids was improved by deletion of peroxisomal
111 Chapter 5 biogenesis factor 10 (YlPEX10), overexpression of diacylglycerol acyltransferase 2
(DGAT2, YlDGA1), and overexpression of choline phosphotransferase (CPT, YlCPT1).
Overexpression of DGAT and disruption of peroxisomal biogenesis factors are common strategies employed to increase PUFA and unusual fatty acids accumulation in microbes, along with expression of the biosynthetic pathways towards a specific fatty acid [8,9,6,10 13].
With a few exceptions, desaturation of unsaturated fatty acids in plants, yeasts, and cyanobacteria are mediated by membrane-bound desaturases acting on acyl chain at sn-2 position of phospholipids [14 16]. Land c cle c n i f ec ing de-acylation and re-acylation of phospholipids and is one of the main mechanism to remodel fatty acids composition of the phospholipids [17 19]. In de-acylation reaction, free fatty acid is released through the action of phospholipase A2 (PLA2) leaving a lysophospholipid (LP). The free fatty acids are then activated to acyl-CoA by acyl-CoA synthetase (ACS). Re-acylation of LP is then facilitated by lysophospholipid acyltransferase with acyl-CoA as acyl donor, yielding diacyl phospholipid.[20] In theory, phospholipid desaturation and Land cycle will result in a net reaction of acyl-
CoA desaturation. As a consequence, this net reaction would enrich the cellular acyl-
CoA pool with more desaturated acyl-CoAs. Because biosynthesis of triacylglycerols
(TAG) relies on acyl-CoA as acyl donor, higher desaturation of acyl-CoA can be exploited to increase the fraction of PUFAs in the storage lipids that are mainly composed of TAGs. The use of acyl remodelling enzymes have been mentioned in scientific and patent literatures [7,21,22]. However, how they changed the profile of fatty acid composition have not been described.
In this study, multiple strategies to increase the content of polyunsaturated fatty acids in Yarrowia lipolytica were evaluated with linoleic acid as a model product.
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5.2. Material and methods
5.2.1. Strain construction
All strains in this study were derived from Y. lipolytica ST6512 (a
Y. lipolytica w29 derivative) [23]. Complete list of strains, plasmids, biobricks (DNA fragments for cloning), and primers is provided in the Table S1-S4. Cryostock cultures were prepared in YPD medium (20 g/L yeast extract, 10 g/L peptone, 20 g/L glucose) at 30°C and 250 rpm (ThermoFisher Scientific MaxQ8000) and stored as 25% glycerol solution in -80oC. Gene integration and knock-outs were performed according to
EasyCloneYALI protocol using NatMX as marker for guide-RNA plasmids [24].
Heterologous genes were codon optimized for Y. lipolytica using GeneGenie online tool (http://g.gene-genie.appspot.com/) and obtained as linear DNA fragments
(Geneart, Invitrogen). Codon-optimized gene sequence are presented in
Supplementary Information 1.
5.2.2. Cultivation for fatty acid content and fatty acid profile analysis
The cells from the cryostocks were plated on YPD agar consisting 10 g/L yeast extract (Sigma, 70161), 20 g/L bacto peptone (Ansell, 211677), 20 g/L glucose
(Sigma, G7021), and 20 g/L agar (Sigma, 05040) and incubated at 30oC for 24-48 hours. Individual colonies were inoculated into 500 µL of mineral medium in a 96 deep-well plate and cultivated for 48 hours at 30oC and 500 rpm in a shaking incubator (Ne B n ick , Innova® 44). The mineral medium was prepared as described previously [25].The preculture was used to inoculate 2 mL CN100 mineral medium at OD600 of 0.1 in 24 deep-well plates (Enzyscreen, CR1424) closed with sandwich covers (Enzyscreen, CR1224). The cultures were incubated for 96 hours in a shaking incubator set at 30oC and 500 rpm shaking. CN100 mineral medium differed
113 Chapter 5 from the mineral medium above in the concentrations of nitrogen and carbon, 1.1 g/L
(NH2)2SO4 and 50 g/L D-glucose, resulting carbon to nitrogen ratio (C/N ratio) of 100
[26]. Cultivations were performed with three biological replicates for each strain.
5.2.3. Extraction and derivatization for total fatty acid analysis
Extraction and derivatization protocol was developed based on the previous studies [26 28]. Acid-catalyzed methanolysis was chosen to cover different fatty acid species in the cells. For extraction, 200 µL of whole broth from 96-hour cultures (1.5-
2 mg dry cells) were harvested and transferred to 4 mL glass vials (Mikrolab Aarhus,
ML33134). 50 µL of 2 g/L methyl cis-10-heptadecenoate (Sigma, H9021) in absolute ethanol was added to each vial as internal standard. Glass beads (Sigma, G1277) were added (~60 mg) to each vial to aid cell disruption in subsequent steps. For cell disruption and lipid solubilization, 500 µL 2:1 chloroform:methanol was added and the mixes were vortexed on a multi-vortexer (VWR, DVX-2500) at 2000 rpm pulse mode for 30 minutes. The solvent was then evaporated using nitrogen stream. For derivatization, 500 µL 1.2 M HCl in methanol was added to the dried samples and vortexed for 1 minute at 1000 rpm, pulse mode. The HCl solution in methanol was prepared by mixing 37% HCl (Sigma, 30721) and methanol (VWR, 20864.290P) at 1:9 volume ratio. For derivatization, the vials were closed with PP screw caps with butyl/PTFE seal (Mikrolab Aarhus, ML33144) and incubated at 85oC for 2 hours with
2000 rpm-vortexing pulses of 45 seconds every 30-45 minutes. The samples were cooled down to room temperature and neutralized with 1.2 M NaOH in methanol. To extract methyl esters, 2 mL n-hexane (LiChrosolv, Merck Life Science, 1.04391.1000) was added to the samples followed by vortexing at 2000 rpm for 1 minute. Hexane phase was separated from methanol phase by centrifugation at 1,000 g for 10 seconds.
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Up to 1.5 mL of hexane phase was transferred into a new vial and diluted with n-hexane for GC-MS analysis.
5.2.4. GC-MS analysis
Analysis was performed in a Bruker Daltronics Scion GC436-MS instrument equipped with an Agilent HP-Innowax column (30m x 0.250mm x 0.25- m). One microliter of sample was injected in Split/splitless injector set at 250°C initially in splitless mode and after 2 minutes with a split ratio of 50. The gas flow was set to constant at 1.0 ml/min, using helium as carrier gas. The temperature programming of the column oven was set to the following: Initial set at 80°C hold for 1 min, ramp
10°C/min to 210°C hold for 10 min., followed by a second ramp with 10°C/min to a final temperature of 230°C, which was held for 10 min, with a total run time of 31 min.
Samples were ionized with EI (70 eV) in full scan mode from 50-350 Da. Transfer line and ion source were set to 250°C and 200°C, respectively, and the collection delay was set for 4 min. Peaks were identified using standards: methyl-palmitate (Sigma, 76159), methyl stearate (Sigma, S5376), methyl oleate (Sigma, 75160), and methyl linoleate
(Sigma, L1876). Peak area was used as quantifier. Concentration of FAMEs were determined from correlation obtained from internal standard calibration.
5.3. Results
This study aimed to understand the effect of fatty acyl chain remodeling on accumulation of linoleic acid as a model for polyunsaturated fatty acid production.
Lin leic acid i n he i ed b 12 de a a e Fad2 , hich ac n he phosphatidylcholine (PC)-bound oleate (Figure 5.1) [11,29]. In this study, native FAD2 was expressed under strong, constitutive TEF1-intron promoter [30] to avoid possible interference of expression arising from various genetic modifications. FAD2
115 Chapter 5 overexpression cassette was introduced into background strains containing different genetic modifications (Table 5.1). The control strain ST6512 expressing FAD2 gene(ST9742) accumulated fatty acids at 24% of cell dry-weight (CDW) (Figure 5.2b), of which 33% was linoleate (Figure 5.3).
Figure 5.1. Pathway illustration and metabolic engineering strategies for accumulation of linoleic acid. Gene names in black, red, and blue colors represent native, heterologously expressed or overexpressed, and deleted genes, respectively. ACL: ATP citrate lyase, DAG: diacylglycerol, G3P: glycerol-3-phosphate, PA: phosphatidic acid, PC: phosphatidylcholine, PLA: phospholipase A, PLB: phospholipase B, TAG: triacylglycerol.
5.3.1. Deletion of acyl-CoA oxidases significantly increased lipid
accumulation
Y. lipolytica has six genes POX1-POX6 that encode peroxisomal acyl-CoA oxidases [31]. Deletion of POX1-6 yielded an undetectable acyl-CoA oxidase activity
116 Chapter 5
[23]. When FAD2 was overexpressed in the pox1-6 strain background (ST9743), total fatty acid content relative to CDW (TFA content) increased to 33% of total CDW. The
TFA fraction of stearate (C18:0) increased by 21 %, while fraction of unsaturated fatty acids (oleate and linoleate) decreased by 5% as compared to ST9742 (Figure 5.3).
Furthermore, although total fatty acid titer became higher, the CDW of ST9743 was
13% lower than that of ST9742.
Table 5.1. Strains used for characterization of fatty acid accumulation. FAD2 overexpression cassette was introduced into the parental strains containing different genetic backgrounds. Complete list of strains and vectors for strain construction is provided in Table S1 and S2.
Strain Parent Genetic features of parent strain Note 1 tested strain ST9742 ST6512 70 ::PrTEF1-SpCas9-TTef1 ST9743 ST6852 pox1-6 ST9744 ST8997 dga1 ST9745 ST9000 fad2 ST9746 ST9001 PrTEFin-CpFAH12-TLip2 ST9747 ST9045 TPex20-MaC16E<-PrGPD-PrEXP->OLE1-TLip2 ST9748 ST9131 PrTEFin->RcDGAT2-TLip2 ST9749 ST9132 PrGPD->RcACS2-TLip2 ST9750 ST9133 PrTEFin->RcLPCAT2-TLip2 ST9751 ST9134 TPex20-RcsPLA2 <-PrEXP-PrTEFin->RcLPCAT2-TLip2 ST9752 ST9135 ST9133 + TPex20-RcGPAT9<-PrEXP-PrGPD->RcLPAT2-TLip2 ST9753 ST9136 ST9134 + TPex20-RcGPAT9<-PrEXP-PrGPD->RcLPAT2-TLip2 Note 1: Unless mentioned otherwise, the parent strains were constructed with the strain above it by introducing genetic modification stated on the column. Description of (over-)expressed genes is provided in Table 5.2.
5.3.2. Deletion of DGA1 increased linoleate content
Dga1p, along with Lro1p, make up the most triacylglycerol synthesis activity in Y. lipolytica [32] (Figure 5.1). Dga1p, a diacylglycerol acyltransferase (DGAT), utilizes acyl-CoA as acyl donor, whereas Lro1p, a phospholipid:diacylglycerol acyltransferase (PDAT), takes acyl chain from phospholipid to synthesize TAG. In a d a1 Y. lipolytica strain, therefore, TAG would be synthesized mainly by Lro1p.
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Table 5.2. Heterologous genes used in this study. DNA sequence codon-optimized for Y. lipolytica is presented in Supplementary Information 1. Gene Enzyme product Origin Genbank Ref Accession
FAH12 Oleate 12-hydroxylase Claviceps purpurea CDG23429.1 [11]
C16E Palmitoyl-CoA elongase Mortierella alpina BAI40363 [6]
DGAT2 Diacylglycerol acyltransferase 2 Ricinus communis NP_001310616 [40]
ACS2 Acyl-CoA synthetase 2 Ricinus communis ABC02881 [42]
LPCAT2 Lyso-PC acyltransferase Ricinus communis AGO14581.1 [54] sPLA2 Secretory phospholipase A2 Ricinus communis XP_002523659 [47]
GPAT9 G3P acyltransferase Ricinus communis ACB30546.1 [46]
LPAT2 Lyso-PA acyltransferase Ricinus communis AFR42413.1 [55] Abbreviation: G3P: glycerol-3-phosphate, PA: phosphatidic acid, PC: phosphatidylcholine.
DGA1 deletion in ST9744 (pox1-6 d a1 ) yielded a decreased TFA content from 34% in ST9743 to 29% in ST9744 (Figure 5.2b). CDW also further decreased in
ST9744, half of which could be explained by drop in TFA (Figure 5.2a). Interestingly, decrease in TFA content was coincided with higher linoleate (C18:2)- but lower oleate
(C8:1) fraction in TFA (Figure 5.3). This is in-line with the implication of DGA1 deletion, where Lro1p was left as the major TGA producer. Since Lro1p takes acyl chain f m he ame i e he e 12-desaturation occur (sn-2 acyl group of PC), the probability of incorporating linoleate into TAG should be higher than compared to when Dga1p was present. The replacement of linoleate from PC by Lro1p and blocked acyl-CoA incorporation via DGAT in ST9744 should, in theory, re-route the path of major acyl-CoAs to TAG via PC (Figure 5.1). In turn, this PC by-pass would increase the probability of incorporating desaturated fatty acids and eventually resulted in higher linoleate content.
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Figure 5.2. Cell dry weight, CDW (a) and total fatty acids, TFA titer (b) of strains after 96 hours cultivation in CN100 mineral medium containing 50 g/L glucose and 1.1 g/L ammonium sulfate. The bar chart showed TFA (green) as a part of CDW (orange + green). Error bars represent standard deviation from three biological replicates. Statistical analysis was performed using Student's t-test (two-tailed; * p < 0.05, ** p < 0.01, *** p < 0.001, n.s.: not significant; two-sample unequal variance).
5.3.3. The native expression of FAD2 was significantly lower than from
the TEF1-intron promoter
To evaluate the contribution of the overall FAD2 expression coming from its native promoter in FAD2-overexpressing strain, the whole open reading frame (ORF)
119 Chapter 5 of FAD2 was completely deleted (ST9745). Neither the TFA content (Figure 5.2a) nor the level of linoleate (Figure 5.3) were affected by the deletion. This suggests that the transcription of FAD2 gene under its native promoter was almost negligible compared to when the FAD2 was expressed under TEF1-intron promoter. This result could therefore support the motivation of expressing multiple copies of membrane desaturases which has been a common strategy for maximizing PUFA production
[6,13].
Figure 5.3. Relative amount of C16 and C18 fatty acids (FAs) to the total fatty acids. The content of other fatty acids such as C14 and C20 FAs and other C16 and C18 FAs are not shown as these FAMEs make very little proportion of the total FAME (< 5% in total). Error bars represent standard deviation from three biological replicates. Statistical analysis was performed using Student's t-test (two-tailed; * p < 0.05, ** p < 0.01, *** p < 0.001, n.s.: not significant; two-sample unequal variance).
5.3.4. The expression of fatty acid 12-hydroxylase from Claviceps
purpurea did not result in production of ricinoleate
Production of ricinoleic acid in Y. lipolytica has been demonstrated before using fatty acid 12-hydroxylase from Claviceps purpurea (CpFAH12, Table 5.2) [11].
120 Chapter 5
To evaluate the effect of a competing pathway on the biosynthesis of linoleate, we expressed CpFAH12 under TEF1-intron promoter in the pox1-6 d a1 ad2 strain background, resulting in strain ST9746. However, in contrast to the previous studies, we observed no production of ricinoleic acid (Figure S1) and, consequently, no effect on the fatty acid content or profile (Figure 5.2, 3). Absence of linoleate could probably due to substrate depletion by overexpressed Fad2p because Fad2p and CpFah12p utilize the same substrate [33]. To test this, FAME profile of a CpFAH12-expressing strain before FAD2 overexpression was measured (ST9001, Table 5.1). No ricinoleate production was observed in this strain (Figure S1).
To validate the expression of CpFAH12, it was tagged with a C-terminal hrGFP (humanized GFP from Renilla reniformis) [34]. While the expression was low in the cells where the fluorescent signal was visible, the pattern was as common for
ER-localized proteins (Figure S2).
We further tested whether the expression of CpFAH12 could be improved by replacing its N-terminus with the N-terminus of the native Fad2p. Swapped domain was determined based on alignment of the CpFah12p sequence with that of Fad2p
(Figure S3). 26 N-terminal amino acids of Fad2p was used to replace 57 amino acids of CpFah12p. DeepLoc predicted ER to be most-likely localization (99%) for CpFah12p with this new signal peptide (Figure S4). However, this swapping also did not result in ricinoleate production (Figure S1).
5.3.5. Overexpression of C16-elongase and 9-desaturase shifted fatty
acid profile
Oleoyl-CoA (C18:1-CoA) is the initial substrate for PUFA biosynthesis.
Oleoyl-CoA is synthesized from stearoyl-CoA (C18:0-CoA) via desaturation by 9-
121 Chapter 5 desaturase ( 9D). Stearoyl-CoA is either produced directly by fatty acid synthetase
(FAS) [35] or by the action of elongase activity on palmitoyl-CoA (C16:0-CoA) [36]. To increase the flux towards oleoyl-CoA, C16 elongase from Mortierella alpina (MaC16E)
[6] and native 9D (YlOLE1, YALI1_C07638g) [35] were expressed, resulting in strain
ST9747 (Table 5.1, Figure 5.1). When fatty acid profile of the strain ST9747 was compared to that of ST9746, 19% decrease in fraction of palmitate (C16:0) in TFA coincided with 14% and 17% increase in oleate and linoleate fraction in TFA, respectively (Figure 5.3). Such effect was expected and in agreement with previous studies.[6,35] Unexpectedly, however, lipid content became lower while cell dry weight remained similar, implying an increase in the non-lipid biomass (Figure 5.2).
Such effect was expected and in agreement with previous studies [6,35]. Fatty acid profile was not affected by expression of ricinolate-specific DGAT2 (ST9748) and
ACS2 (ST9749) from castor plant.
Diacylglycerol acyltransferase (DGAT) catalyzes the terminal steps in TAG biosynthesis (Figure 5.1). DGAT introduces the third acyl chain into glycerol backbone of diacylglycerol (DAG) by using acyl-CoA as acyl donor [37]. DGAT with distinctive specificity towards certain acyl-CoAs have been exploited for accumulating preferred fatty acids.[38] DGAT2 from castor plant Ricinus communis (RcDGAT2) exhibit high specificity not only towards ricinoleyl-CoA as acyl donor, but also diricinoleyl-DAG
(1,2-sn-diricinoleylglycerol) as acyl acceptor when tested in vitro [39,40].
Such strict specificity prompted us to ask whether RcDGAT2 would exert any influence in a strain without its preferred substrates. In ST9748, RcDGAT2 was expressed under the strong and constitutive EXP1 promoter [41]. CDW, fatty acid content, and fatty acid profile were not different compared to ST9747 which did not express RcDGAT2. Since slight CpFah12p activity could still probably be present in
122 Chapter 5
ST9748, there was a possibility that ricinoleic acid was present in free fatty acid form and therefore were not accessible by RcDGAT2. To check whether this was the case,
ACS2 from castor (RcACS2) was expressed (ST9749, Table 5.1). RcACS2 was shown in previous study to possess high activity towards ricinoleic acid [42]. After introduction of RcACS2, there was still no change in total fatty acid amount and composition.
5.3.6. Introduction of phosphatidylcholine-editing enzyme improved
linoleate production
To further expand metabolic engineering strategies for accumulation of linoleate, an acyl-editing strategy was adopted. In plants, it has been suggested that de novo acyl-CoAs transit via PC before being assimilated to the glycerol backbone for
TAG synthesis [19]. This transit was suggested to occur at a faster rate than acyl-CoA incorporation to TAG [19]. Lysophosphatidylcholine acyltransferase (LPCAT) was demonstrated as a key player on this process [43]. LPCAT synthesizes PC from lyso-
PC (1-acyl-sn-glycerol-3-phosphocholine) with acyl-CoA as the acyl donor. This reaction is a igned a he f a d eac i n f LPCAT [44]. The reverse reaction, in contrast, releases the sn-2 acyl chain from PC in form of acyl-CoA [44] (Figure 5.1).
An LPCAT from castor plant (RcLPCAT2) was selected for this study as it showed high activity for acylating oleyl-CoA (forward reaction) but low activity in releasing oleate from PC (reverse reaction). With this property, it was expected that oleate was better maintained on PC as such providing bigger substrate pool for Fad2p.
In agreement to this hypothesis, the linoleate and oleate fraction in TFA were higher and lower, respectively, in strain ST9750 (expressing RcLPCAT2) compared to
ST9749. The fraction of linoleate in ST9750 reached 47% of the TFA, which is 19% higher than in ST9749 (Figure 5.3). With a TFA titer of 2.11 g/L, ST9750 produced an
123 Chapter 5 equivalent of 0.99 g/L linoleic acid (Figure 5.2). This linoleate content in TFA and the titer were 14% and 39% higher, respectively, than that of the initial strain ST9742
(Figure 5.2, 3). Palmitic acid was the second most abundant fatty acid after linoleate, comprising 26% of the TFA, followed by stearate at 13%.
5.3.7. Introduction of GPAT9 and LPAT2 from castor did not reduce
saturated fatty acid content
Since saturated fatty acids remained abundant in ST9750, we hypothesized that the specificity of glycerol-3-phosphate acyltransferase (GPAT) and lysophosphatidate acyltransferase (LPAT) could play a role in maintaining saturated fatty acids. GPAT and LPAT transfer acyl chains into sn-1 and sn-2 of glycerol backbone during TAG synthesis (Figure 5.1). Swapping native GPAT with heterologous
GPAT was shown to reduce palmitate content in Y. lipolytica with an increase in oleate.[28] On a different take, a study on Arabidopsis thaliana demonstrated that palmitate content at sn-2 position of TAG could be enhanced by targeting palmitate- specific, chloroplast-resident Lpat1p into the ER [45].
To test whether a GPAT and an LPAT, which are able to discriminate against
C16:0-CoA, could divert a portion of palmitate into linoleate, we introduced castor
GPAT9 (RcGPAT9) and LPAT2 (RcLPAT2) [46] (Table 5.1). Nonetheless, there was no difference in fatty acid profile and amount was observed between ST9752 (expressing
RcGPAT9 and RcLPAT2) and ST9750 (Figure 5.3, Table 5.1). Since fatty acid titers were similar between the two strains, this was probably caused by low activity of the two enzymes relative to the native GPAT and LPAT.
124 Chapter 5
5.3.8. Insertion of phospholipase A2 gene did not change fatty acid
profile
We next asked the question whether releasing more fatty acid from PC could enrich acyl-CoA pool with linoleate, which would eventually translate in higher linoleate content (Figure 5.1). In an attempt to enhance PLA2 activity, we employed castor secretory PLA2 (RcsPLA2 ) [47]. The gene was tagged the protein with yeast
ER retention signal HDEL at the C-terminal (supp info) based on the information that other types of plant PLA2s localized in ER or golgi [48] as well as the access for substrate and Ca2+ ion for activity [49]. Nonetheless, there was no change in lipid profile observed after addition of PLA2 (ST9750 vs ST9751).
We had expected that PLA2 would enrich acyl-CoA pool with PC-derived linoleate (Figure 5.1). Following this, we introduced RcGPAT9 and RcLPAT2 into
RcsPLA2 -expressing strain (ST9753, Table 5.1). However, any difference on lipid profile between the two corresponding strains (ST9751 vs ST9753) was within the measurement error and thus there is not enough evidence to support the hypothesis.
5.4. Discussion
We have demonstrated various metabolic engineering strategies for increasing linoleic acid content in Y. lipolytica. Besides previously reported strategies, we presented here an acyl-editing strategy which successfully increase linoleic acid production. Our data reinforced previous findings that deleting acyl-CoA oxidase genes POX1-6 improved lipid accumulation. Deletion of DGA1 also showed negative impact on fatty acid titer. However, when accumulation of specific fatty acid is desired, high fatty acid content is only important once a refined lipid profile is obtained. In our case, DGA1 deletion reduced lipid titer but improved linoleate content.
125 Chapter 5
When overexpressing OLE1, we did not observe increased fatty acid content as might be expected based on study by Qiao et al. [35]. However, the authors also observed that overexpression of OLE1 did not result in lipid overproduction without overexpression of both acetyl-CoA carboxylase (ACC1) and DGA1. One explanation could be because palmitoyl-CoA (C16:0-CoA) not only inhibits ACC1, but likely also
ATP:citrate lyase (ACL) as demonstrated in filamentous fungi [50]. ACL converts citrate to acetyl-CoA which cells use for biomass production.
We did not obtain increase in fatty acid content after expression of
RcDGAT2. RcDGAT2 has specificity on both its acyl donor and acceptor. If the lack of change in total fatty acid composition after RcDGAT2 insertion was indeed due to its duplexed specificity, this observation suggests that such specificity could be an excellent strategy for accumulating highly specific TAG. Indeed, the majority of TAG in castor seed oil is triricinolein (sn-1,2,3-triricinoleylglycerol) [40].
In our current study, web showed that the implementation of acyl-editing resulted in higher linoleate production. De novo synthesis of Fad1p substrate (sn-2- oleoyl-PC) relies on the multienzyme, tightly regulated Kennedy pathway (Figure 5.1)
[51]. LPCAT could provide a bypass for oleate to PC (Figure 5.1) [51]. The shifted proportion of oleate content into linoleate in ST9750 suggests that there was a faster oleate conversion to linoleate. Neither acceleration of Kennedy pathway nor increase in Fad2p activity were likely to be responsible for this as no meaningful genetic modification had targeted them. This increase, therefore, can very well be attributed to the acyl-editing activity of LPCAT.
Al h gh ini ial g al i enhance Land c cle (LPCAT, ACS, and PLA2 cycle), only LPCAT showed positive effect. It is unclear if LPCAT also contributed
126 Chapter 5 significantly on liberating fatty acids from PC and providing lyso-PC (Figure 5.1).
Endogenous phospholipase B activity [52] as well as Lro1p could largely contribute for those activites (Figure 5.1). Furthermore, beneficial effect form LPCAT also suggests that substrate availability and/or diffusivity seemed to be limiting in the FAD2- overexpressing strain.
The high amount of palmitate remained in ST9750, ST9751, ST9752, and
ST9753 could be attributed to the activity of native GPAT encoded by
YALI1_C00230g, the homolog of S. cerevisiae SCT1. In S. cerevisiae, SCT1 was suggested to be the key enzyme for incorporating palmitate to lipid.[53] Further improvement could utilize multiple copies of C16-el nga e and 9-desaturase [6] or by replacing native GPAT with heterologous variants [28]. Further bioprocess engineering such as lowering temperature [13] could also be deployed to complement metabolic engineering.
5.5. Concluding remarks
In this study, we demonstrated that acyl-editing strategy could be employed for increasing production of polyunsaturated fatty acid. Our results further showed that acyl-editing complementary, rather than redundant, for existing metabolic engineering strategies in polyunsaturated fatty acid production.
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5.6. Acknowledgements
This project has received funding from the European Union's Horizon 2020 research and inn a i n g amme nde he Ma ie Sk d ka-Curie grant agreement No
722287 (PAcMEN). IB acknowledges the financial support from the Novo Nordisk
Foundation (Grant agreement NNF15OC0016592 and NNF10CC1016517) and from the European Research Council under the European Union's Horizon 2020 research and innovation programme (YEAST-TRANS project, Grant Agreement No 757384).
We thank Dr. Kanchana R Kildegaard (Biophero ApS, Denmark) for kindly providing pBP8003. We thank Larissa R. Tramontin for providing pCfB8092.
ERM and IB conceived this project. ERM, JD, and GW designed the experiments.
ERM, JD, and JSS performed the strain constructions. ERM performed strain cultivations and fatty acid extraction and derivatization. HBC developed GC-MS methods and performed GC-MS analysis. ERM, JD, HBC, GW, and IB were involved in analyzing and interpreting the data.
5.7. Conflict-of-interest statement
IB has financial interest in Biophero ApS.
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39. Kroon JTM, Wei W, Simon WJ, Slabas AR: Identification and functional expression of a type 2 acyl-CoA:diacylglycerol acyltransferase (DGAT2) in developing castor bean seeds which has high homology to the major triglyceride biosynthetic enzyme of fungi and animals. Phytochemistry 2006, 67:2541 2549.
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45. Erp H van, Bryant FM, Martin-Moreno J, Michaelson LV, Bhutada G, Eastmond PJ: Engineering the stereoisomeric structure of seed oil to mimic human milk fat. Proc Natl Acad Sci 2019, doi:10.1073/pnas.1907915116.
46. Brown AP, Kroon JTM, Swarbreck D, Febrer M, Larson TR, Graham IA, Caccamo M, Slabas AR: Tissue-Specific Whole Transcriptome Sequencing in Castor, Directed at Understanding Triacylglycerol Lipid Biosynthetic Pathways. PLOS ONE 2012, 7:e30100.
47. Bayon S, Chen G, Weselake RJ, Browse J: A Small Phospholipase A2- from Castor Catalyzes the Removal of Hydroxy Fatty Acids from Phosphatidylcholine in Transgenic Arabidopsis Seeds. Plant Physiol 2015, 167:1259 1270.
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49. Ståhl U, Ek B, Stymne S: Purification and Characterization of a Low-Molecular-Weight Phospholipase A2 from Developing Seeds of Elm. Plant Physiol 1998, 117:197 205.
50. Pfitzner A, Kubicek CP, Röhr M: Presence and regulation of ATP:citrate lyase from the citric acid producing fungus Aspergillus niger. Arch Microbiol 1987, 147:88 91.
51. Henry SA, Kohlwein SD, Carman GM: Metabolism and Regulation of Glycerolipids in the Yeast Saccharomyces cerevisiae. Genetics 2012, 190:317 349.
52. De Smet CH, Cox R, Brouwers JF, de Kroon AIPM: Yeast cells accumulate excess endogenous palmitate in phosphatidylcholine by acyl chain remodeling involving the phospholipase B Plb1p. Biochim Biophys Acta 2013, 1831:1167 1176.
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55. Arroyo-Caro JM, Chileh T, Kazachkov M, Zou J, Alonso DL, García-Maroto F: The multigene family of lysophosphatidate acyltransferase (LPAT)-related enzymes in Ricinus communis. Cloning and molecular characterization of two LPAT genes that are expressed in castor seeds. Plant Sci 2013, 199 200:29 40.
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Supplementary Information
133 Chapter 5
Supplementary material
Supplementary information 1 DNA sequence of heterologous genes codon-optimized for Y. lipolytica
>CpFAH12 (pCfB7053)
ATGATGGCCTCTGCCACCCCCGCCATGTCTGAGAACGCCGTGCTGCGACACAAGGCCGCCTCTACCACCGGAATCGACTACGA GTCCTCTGCCGCCGTGTCTCCCGCCGAGTCTCCCCGAACCTCTGCCTCTTCTACCTCGCTGTCCTCTCTGTCCTCCCTGGACG CCAACGAGAAGAAGGACGAGTACGCCGGCCTGCTGGACACCTACGGCAACGCCTTCACCCCCCCTGACTTCTCTATCAAGGAC ATCCGAGCCGCCATCCCCAAGCACTGCTACGAGCGATCTACCATCAAGTCTTACGCCTACGTGCTGCGAGATCTGCTGTGCCT GTCTACCACCTTCTACCTGTTCCACAACTTCGTGACCCCCGAGAACATCCCCTCTAACCCCCTGCGATTCGTGCTGTGGTCTA TCTACACCGTGCTGCAGGGCCTGTTCGCCACCGGCCTGTGGGTGATCGGCCACGAGTGCGGCCACTGCGCCTTCTCTCCCTCT CCCTTCATCTCTGACCTGACCGGCTGGGTGATCCACTCTGCCCTGCTGGTGCCCTACTTCTCTTGGAAGTTCTCTCACTCTGC CCACCACAAGGGCATCGGCAACATGGAGCGAGACATGGTGTTTCTGCCCCGAACCCGAGAGCAGCAGGCCACCCGACTGGGCC GAGCCGTCGAGGAGCTGGGCGACCTGTGCGAGGAGACTCCCATCTACACCGCCCTGCACCTGGTGGGCAAGCAGCTGATCGGC TGGCCCTCTTACCTGATGACCAACGCTACCGGCCACAACTTCCACGAGCGACAGCGAGAGGGCCGAGGCAAGGGCAAGAAGAA CGGCTTCGGCGGAGGCGTGAACCACTTCGACCCCCGATCTCCCATCTTCGAGGCCCGACAGGCCAAGTACATCGTGCTGTCTG ACATCGGCCTGGGCCTGGCCATTGCCGCCCTGGTGTACCTGGGCAACCGATTCGGCTGGGCCAACATGGCCGTGTGGTACTTT CTGCCCTACCTGTGGGTGAACCACTGGCTGGTGGCTATCACCTTCCTGCAGCACACCGACCCCACCCTGCCCCACTACAACCG AGAGGAGTGGAACTTCGTGCGAGGCGGAGCCTGCACCATCGACCGAGATCTGGGCTTCATCGGCCGACACCTGTTCCACGGAA TCGCCGACACCCACGTGGTGCATCACTACGTGTCTCGAATCCCCTTCTACAACGCCGACGAGGCCTCTGAGGCTATCAAGCCC ATCATGGGCAAGCACTACCGATCTGACACCGCTCACGGCCCCGTGGGCTTTCTGCACGCCCTGTGGAAGACCGCCCGATGGTG CCAGTGGGTCGAGCCCTCTGCCGACGCTCAGGGCGCTGGCAAGGGCATCCTGTTCTACCGAAACCGAAACAAGCTGGGCACCA AGCCCATCTCTATGAAGACCCAGTAA
>MaC16E (in pCfB8092)
ATGGAATCTGGCCCTATGCCTGCCGGAATTCCATTTCCTGAGTACTACGACTTCTTCATGGACTGGAAGACCCCTCTGGCCAT TGCCGCCACCTACACCGTGGCCGTGGGCCTGTTCAACCCCAAGGTCGGCAAGGTGTCTCGAGTGGTGGCCAAGTCTGCCAACG CTAAGCCCGCCGAGCGAACCCAGTCTGGCGCCGCTATGACCGCCTTCGTGTTCGTGCACAACCTGATCCTGTGCGTGTACTCT GGCATCACCTTCTACCACATGTTCCCCGCCATGGTGAAGAACTTCCGAACTCACACCCTGCACGAGGCCTACTGCGACACCGA CCAGTCTCTGTGGAACAACGCCCTCGGCTACTGGGGCTACCTGTTCTACCTGTCTAAGTTCTACGAGGTGATCGACACCATCA TCATCATTCTGAAGGGCCGACGATCTTCTCTGCTGCAGACCTACCACCACGCTGGCGCCATGATCACCATGTGGTCTGGAATC AACTACCAGGCTACCCCTATCTGGATCTTCGTGGTGTTCAACTCTTTCATCCACACCATTATGTACTGCTACTACGCCTTCAC CTCTATCGGCTTTCACCCTCCTGGCAAGAAGTACCTGACCTCTATGCAGATTACCCAGTTCCTGGTGGGCATCACCATTGCCG TGTCTTACCTGTTCGTGCCCGGCTGCATTCGAACCCCTGGCGCTCAGATGGCCGTGTGGATCAACGTGGGATACCTGTTTCCT CTGACCTACCTGTTTGTGGACTTCGCCAAGCGAACCTACTCTAAGCGAACCGCCATTGCTGCCCAGAAGAAGGCCCAGTAA
>RcDGAT2 (pCfB8159)
ATGGGCGAAGAGGCCAACCATAACAACAACAACAACAACATCAACTCCAACGACGAGAAGAACGAGGAAAAGTCTAACTACAC CGTGGTGAACTCCCGAGAGCTGTACCCCACCAACATCTTCCACGCTCTGCTGGCCCTGTCTATCTGGATCGGCTCTATCCACT TCAACCTGTTTCTGCTGTTTATCTCTTACCTGTTCCTGTCTTTCCCCACCTTCCTGCTGATCGTGGGCTTCTTCGTGGTGCTG ATGTTCATCCCCATCGACGAGCACTCTAAGCTGGGCCGACGACTGTGCCGATACGTGTGCCGACACGCCTGCTCTCACTTCCC CGTGACTCTGCACGTCGAGGACATGAACGCCTTCCACTCTGACCGAGCCTACGTGTTCGGCTACGAGCCCCACTCTGTGTTCC CTCTGGGCGTGTCTGTGCTGTCTGACCACTTCGCCGTGCTGCCCCTGCCTAAGATGAAGGTGCTGGCCTCTAACGCCGTGTTT CGAACCCCTGTGCTGCGACACATCTGGACCTGGTGCGGCCTGACCTCTGCCACCAAGAAGAACTTCACTGCCCTGCTGGCTTC CGGCTACTCTTGCATCGTGATTCCCGGCGGAGTGCAAGAGACTTTCTACATGAAGCACGGCTCTGAGATCGCCTTCCTGAAGG CCCGACGAGGCTTCGTGCGAGTGGCCATGGAAATGGGCAAGCCCCTGGTGCCTGTGTTCTGCTTCGGCCAGTCTAACGTGTAC AAGTGGTGGAAGCCCGACGGCGAGCTGTTCATGAAGATCGCCCGAGCCATCAAGTTCTCTCCCATCGTGTTCTGGGGCGTGCT GGGCTCTCATCTGCCTCTGCAGCGACCCATGCACGTGGTGGTGGGAAAGCCCATCGAGGTCAAGCAGAACCCTCAGCCTACCG TGGAAGAGGTGTCTGAGGTGCAGGGCCAGTTCGTGGCCGCTCTGAAGGACCTGTTCGAGCGACACAAGGCCCGAGTGGGCTAC GCCGACCTGACTCTCGAGATCCTGTAA
134 Chapter 5
>RcACS2 (pCfB8789)
ATGGACATGGACTCTGCCCAGCGACGAATCCAGGCCATCCACGGCCATCTGCTGGCCGCTCGAGACTGCTCTCCCTCTCACCT CCGACTGAACCCCACCGCTGGCGAGTTCTTCTCGGAGCAGGGCTACTCTGTGGTGCTGCCCGAGAAGCTGCAGACCGGCAAGT GGAACGTGTACCGATCTGCTCGATCTCCCCTGCGACTGGTGTCTCGATTCCCCGACCATCCTGACATCGGCACCCTGCACGAC AACTTCGCCCGATCTGTGGACACCTTCCGAGACTACAAGTACCTGGGCACCCGAATCCGAGTGGACGGCACCGTGGGCGAGTA CAAGTGGATGACCTACGGCGAGGCCGGCACCGCTCGAACCGCCATCGGCTCTGGCCTGATGCACCACGGAATCCCCAAGGGAT CTTCTGTGGGCCTGTACTTCATCAACCGACCTGAGTGGCTGATCGTGGACCACGCCTGCTCTGCCTACTCTTACATCTCTGTG CCCCTGTACGACACCCTGGGACCTGACGCCGTGAAGTTCATTGTGAACCACGCCGACGTGCAGGCCATCTTCTGCGTGCCCCA GACTCTGACCCCTCTGCTGTCTTTCCTGTCTGAGATCTCTTCTGTGCGACTGATTGTGGTGGTCGGCGGCATGGACGACCAGA TGCCTTCTCTGCCCTCTTCGACCGGCGTGCAGGTCGTGACCTACTCTAAGCTGCTGTCTCAGGGCCACTCTAACCTGCAGCCT TTCTGTCCTCCTAAGCCTGAGGACGTGGCCACCATCTGCTACACCTCTGGCACCACCGGCACTCCCAAGGGCGCTGCTCTGAC CCACGGCAACCTGATCGCCAACGTGGCTGGCGCTACCCTGGCCACCAAGTTCTACCCCTCTGACATCTACATCTCTTACCTGC CTCTGGCTCACATCTACGAGCGAGCCAACCAGGTGCTGACCGTGTACTACGGCGTCGCCGTGGGCTTCTACCAGGGCGACAAC CTGAAGCTGATGGACGACATGGCCGCTCTGCGACCCACCATTTTCTGCTCTGTGCCTCGACTGTACAACCGAATCTACGCCGG CATCACCAACGCCGTCAAGACCTCTGGCGGCCTGCGAGAGCGACTGTTCAACGCCGCCTACAACGCCAAGAAGCAGGCCATTC TGAACGGACGATCTCCCTCGCCTATGTGGGACCGACTCGTGTTCGACAAGATCAAGGCCAAGCTCGGCGGACGAGTGCGATTC ATTGCCTCCGGCGCCTCGCCTCTGTCTCCCGACGTGATGGAATTCCTGAAGATCTGCTTCGGAGGCCGAGTGTCTGAAGGCTA CGGAATGACCGAGACTTCTTGCGTGATCTCTGCCATGGAAGAGGGTGACAACCTCACCGGCCACGTGGGCTCTCCCAACCCTG CCTGTGAGATCAAGCTGGTGGACGTGCCCGAGATGTCTTACACCTCCGATGACCAGCCTTATCCTCGAGGCGAGATCTGCGTG CGAGGCCCCATCGTGTTCCAGGGCTACCACAAGGACGAGGCTCAGACTCGAGATGTGATCGACGAGGACGGCTGGCTGCACAC CGGCGACATCGGCCTGTGGCTGCCTGAGGGCCGACTGAAGATCATCGACCGAAAGAAGAACATCTTCAAGCTGGCCCAGGGTG AGTACATTGCCCCTGAGAAGATCGAGAACGTCTACGCCAAGTGCCGGTTCATTGCCCAGTGCTTCGTGTACGGCGACTCTCTG AACTCTGCCCTGGTGGCCATCGTGGCCGTCGACCAGGACACCCTGAAGGCCTGGGCCGCCTCTGAGGGCATCAAGTACGAGAA CCTGGGACAGCTGTGCAACGACCCTCGAGCACGAGCCGCCGTGCTGGCCGACATGGACGCCGTGGGACGAGAGGCTAAGCTGC GAGGCTTCGAGTTCGCCAAGGCTGTGACCCTGGTGCTCGAGCCCTTCACCATGGAAAACGGCCTGCTGACCCCTACCTTCAAG ATTAAGCGACCCCAGGCCAAGGCCTACTTCCAGAACACCATCTCTAAGATGTACGAGGAACTGGCCACCTCTGATCCTTCGCC TAAGCTGTAA
>RcLPCAT2 (pCfB8211)
ATGGACCTGGATCTGGAGTCTATGGCTTCCGCCATTGGCGTCTCTATCCCCGTTCTGCGATTCCTGCTCTGCTTCGTTGCCAC CATTCCCGTTTCTTTCATGCACCGACTGGCACCTGGTTCCCTGGGTAAGCACTTGTACGCCGCTCTTACTGGTGCTTTTCTTT CCTACCTGTCCTTCGGCTTCTCCTCCAATCTGCACTTCCTTGTCCCCATGCTCCTGGGCTACGCCAGCATGGTGCTGTTCCGA TCGCACTGCGGCATTCTGGTTTTCCTGCTGGGTTTCGGTTACCTGATTGGTTGTCATGTTTACTACATGTCTGGCGATGCTTG GAAGGAGGGTGGTATCGACGCCACCGGCGCCCTGATGGTGCTGACCCTAAAGGTGATCTCCTGTGCCATTAACTACAAGGACG GTCTGCTGAAGGAGGAGGAGCTGCAGGGCTCCCAGAAGAAGAACCGACTGATCAAGCTGCCTTCCCTGATTGAGTACTTCGGA TACTGCCTCTGCTGCGGCTCGCATTTTGCTGGCCCCGTTTACGAGATGAAAGATTACCTTGAATGGACTGAGCGAAAGGGTAT CTGGGCCGGTACTGAGAAAGGTCCCTCTCCCTCCCCTTTCGGTGCTACCATCCGGGCCATCCTGCAGGCTGCCATCTGCATGG TTATTCATCTGTACCTCGTGCCCCACTACCCTCTGTCCCGATTCACCGATCCCGTGTACCAGGAGTGGGGCTTCTGGAAGCGA CTCACCTACCAGTACATGTCCGGTCTGACCGCTCGATGGAAGTACTACTTCATCTGGTCGATCTCCGAGGCTTCTATCATCAT CTCGGGTCTGGGCTTCTCTGGCTGGACTGATACCTCCCCTCCCAAGCCCCAGTGGGATCGCGCTCGAAACGTCGACATCCTGG GTGTCGAGTTCGCTAAGAGCGCAGCTGAGCTGCCCCTGGTCTGGAACATTCAGGTCTCTACCTGGTTGCGACACTACGTGTAC GATCGACTGGTGCCCAAGGGCAAGAAGGCCGGCTTTCTCCAGCTGCTTGCCACTCAGACCACATCTGCCGTTTGGCACGGCCT GTATCCCGGTTACATCATCTTCTTCGTCCAGTCTGCTCTGATGATTGCCGGATCCAAGGTCATCTACCGATGGCAGCAGGCTA TTCCCTCTAACAAGGCCCTGGAGAAGAAGATTCTGGTCTTCATGAACTTCGCCTACACCGTTCTGGTCCTTAACTACTCCTGT GTCGGTTTCATGGTCCTGTCTCTGCACGAGACCATTGCCGCCTACGGTTCTGTCTACTTTATCGGCACCATCGTCCCTGTCGT TTTCTTTCTCCTGGGTTTTATTATCAAGCCCGCCCGACCTTCCCGATCCAAGACTCGAAAGGACGAGTAA
>RcsPLA2a (pCfB8158) + linker + HDEL
ATGGCCAACCTGTCTCAGCCCCTGAACCTGGTGGCCTACCTGTTCTTCTTCTGCTTCCTGCTGTCTCTGTCTTTCCCCTCTAC TCCCGTGCACGCCCTGAACATCGGCGTGCAGACCGCCAACTCTGCTATCACCCTGTCTAAGGAATGCTCTCGAAAGTGCGAGT CTGAGTTCTGCTCTGTGCCTCCTTTCCTGCGATACGGCAAGTACTGCGGCCTGCTGTACTCCGGCTGTCCCGGCGAGAAGCCC TGCGACGGCCTGGACGCCTGCTGCATGAAGCACGACTCTTGCGTGCAGGCCAAGAACAACGACTACCTGTCGCAAGAGTGCTC TCAGAACTTCATCAACTGCATGAACGACTTCAAGAACAAGGGCGGCCACACCTTCAAGGGCTCTAAGTGCCAGGTGGACGAGG TGATCGACGTGATCTCTGTGGTGATGGAAGCCGCTCTGATCGCCGGACGATACCTGCACAAGCCCggtggtggaggcggctct ggcggcggcggtggttccCACGATGAGCTGTAAA
135 Chapter 5
>RcGPAT9 (pCfB8219)
ATGTCTACCGCTGGCAAGCTGAACTCCAGTTCTTCTGAGCTTGATCTGGACAGACCCAACATCGAGGACTACCTGCCCTCGGG CTCCTCCATTCACGAGCCTCACGGTAAGCTACGACTTCGAGATCTGCTGGACATCTCTCCCGCCCTGACCGAAGCCGCCGGTG CCATTGTCGACGACTCTTTCACCCGCTGTTTCAAGTCTAACCCTCCCGAGCCCTGGAATTGGAACATTTACCTCTTTCCTCTG TGGTGCTGCGGTGTCGTGATTAGATACGGCATCCTGTTCCCCGTTAGAGTGCTCGTCCTGACTATCGGTTGGATCATCTTCCT GTCTGCCTACATTCCCGTCCACCTCCTGCTCAAGGGCCACGAGAAACTCCGAAAGAAGCTGGAGCGATGCCTTGTCGAACTTA TTTGTTCCTTCTTCGTCGCTTCTTGGACTGGCGTGGTTAAGTACCACGGCCCTCGACCCTCCATTCGACCTAAGCAGGTGTTC GTCGCCAACCACACCTCCATGATCGACTTCATCGTCCTGGAGCAGATGACAGCCTTCGCTGTCATCATGCAGAAGCACCCCGG CTGGGTCGGTCTTCTGCAGTCCACCATCCTGGAGTCTGTCGGCTGCATTTGGTTCAACCGATCCGAGGCCAAGGACCGAGAGA TCGTTGCCAAGAAGCTCCGAGACCACGTCCAGGGTGCCGACAACAACCCCTTACTGATCTTCCCCGAGGGTACCTGTGTCAAC AACCACTACACCGTCATGTTCAAGAAGGGTGCTTTCGAACTGGGCTGCACCGTCTGCCCCATCGCTATCAAGTACAACAAGAT TTTCGTCGACGCCTTCTGGAACTCCCGAAAGCAGTCCTTCACCACCCACCTGCTTCAACTGATGACTTCCTGGGCCGTCGTCT GTGACGTGTGGTACCTCGAGCCCCAGAACCTGCGACCCGGTGAAACTCCCATTGAGTTCGCCGAGCGAGTCCGAGACATTATC TCCGTCCGAGCCGGCCTCAAGAAGGTTCCTTGGGACGGTTATCTGAAGTACTCCAGACCCTCTCCCAAACACAGAGAGCGAAA GCAGCAGTCTTTCGCTGAGTCCGTTCTGAGACGACTGGAGGAGAAGTAA
>RcLPAT2 (pCfB8161)
ATGGCCGTGGCCGCCGTCGCCGTGATTCTGCCCCTGGGCGTGCTGTTCTTCCTGTCTGGCCTGGTGGTGAACCTGTTCCAGGC CATCTGCTTCGTGCTGATCCGACCTATCTCTAAGTCCTCTTACCGAATGATCAACAGAGCCCTGGCCGAGCTGCTGTGGCTCG AGCTGGTCTGGCTGGTCGACTGGTGGGCCAAGATCAAGATCCAGGTCTACACCGACCGAGAGACTCTGTGCCTGATGGGCAAG GAACACGCCCTGATCCTGGCCAACCACCGATCTGACATCGACTGGCTGGTTGGATGGATGCTGGCCCAGCGAGCCGGCTGCCT GGGCTCTGCCCTGGCTGTGATGAAGAAGTCCTCTAAGTTCCTGCCTGTGATCGGCTGGTCTATGTGGTTCTCTGAGTACCTGT TCCTCGAGCGATCTTGGGCCAAGGACGAGTCTACCCTGAAGTCTGGCATCCAGCGACTGAAGGACTTCCCTCGACCTTTCTGG CTGGGCCTGTTCGTCGAGGGCACCCGATTCACCAAGGCCAAGCTGCTGGCCGCTCAGCAGTACGCCGCCTCTCAGGGACTGCC CATTCCTCGAAACGTGCTGATTCCCCGAACTAAGGGCTTCGTGTCTGCCGTGTCTAACATGCGATCTTTCGTGCCCGCCATCT ACGACGTGACCGTGGCTATCCCCAAGAACTCTCCCCAGCCTACCATGCTGCGACTGTTCAAGGGCCAGTCCTCTGTGGTGCAC GTCCACATCAAGCGACACCTGATGAAGGACCTGCCTGAGACTGACGACGCCGTGGCTCAGTGGTGCAAGGACCTGTTCGTGGC TAAGGACGCCCTGCTGGACAAGCACATTGCCGAGGACACCTTCTCTGAGCAAGAGCTGCAGGACATCGGACGACCCAAGAAGT CTCTGGTCGTCGTCACCCTGTGGTCTTGCCTGCTGATCTTCGGAACCCTCAAGTTTCTGCAGTGGTCCTCTCTGCTGTCCTCT TGGAAGGGAATCGCCCTGTCTATGTCTGCTCTGGCCATTGTGACCGTGCTGATGCACATCCTGATCCGATTCTCTCAGTCTGA GCACTCTACCCCTGCTCAGGTGGCTCCCGCCAACCACGCTCAGGTCGCCCCTGAGAACCCTCAGAACGGCGGCGAGCCCTCTG AGAAGGCCGAGAACAAGCAGGACTAA
136 Chapter 5
Supplementary information 2
Sanger sequencing result for integrated CpFAH12 expression cassette in ST9001.
IntC_3 integration site was amplified from ST9001 genomic DNA extract using primer PR-14838 and PR-14588, resulting in a DNA band of 4.2 kb. Purified band was used as template for Sanger sequencing. PR-10595, PR-14619, PR-19299, PR-20692, and PR-22844 were used as sequencing primers (Table S4).
Alignment of sequencing reads and their trace date into DNA sequence of the expression cassette is shown below. Sequencing primers, TEF1intron promoter, CpFAH12 ORF, and LIP2 terminator are indicated as blue, green, yellow, and red boxes on the top of reference sequence.
137 Chapter 5
780 800 820 840 860 880 900 920 940 960
Pr TEFintron pCfB7056 (pIntC_3 _PrTEFint- CpFAH- TLip2G) T T G A A T C C A T C C A T C T T GGA T T GC C A A T T GT GC A C A C A GA A C CGGG C A C T C A C T T C CC C A T C C A C A C T T C A A CGGA A T G c g t g c g a t A G A G A C CGGG T T GG CGGCGC A T T T GT GT C C C A A A A A A C A GC C C C A A T T GC C C C A A T T G A C C C C A A A T T GA C C C A GT A GCGGGC C C A A C C C CGGCGA G A G C C C C C T T C
Consensus A A A C A GC C C C A A T T GC C C C A A T T G A C C C C A A A T T GA C C C A GT A GCGGGC C C A A C C C CGGCGA G A G C C C C C T T C 4 4 Coverage 0 0
EF30776726_PR- 10595 T C C CGGC T T GT GC C A A A C A GC C C C A A T T GC C C C A A T T G A C C C C A A A T T GA C C C A GT A GCGGGC C C A A C C C CGGCGA G A G C C C C C T T C
Trace data
EF31206216_PR- 20691
Trace data
EF31206215_PR- 19299
Trace data
EF31206218_PR- 14619
Trace data
EF31206214_PR- 20692
Trace data
960 980 1,000 1,020 1,040 1,060 1,080 1,100 1,120 1,140 1,160
pCfB7056 (pIntC_3 _PrTEFint- CpFAH- TLip2T) C T C C C C A C A T A T C A A A C C T C C C C CGGT T C C C A C A C T T GC CGT T A A GGG CG T A GGG T A C T GC A GT C T GGA A T C T A CGC T T GT T C A GA C T T T G T A C T A G T T T C T T T GT C T GGC C A T C CGGGT A A C C C A T GC CGGA CGC A A A A T A G A C T A C T G A A A A T T T T T T T GC T T T GT GGT T GGGA C T T T A GC C A A GGG T A T A A
ConsensusT C T C C C C A C A T A T C A A A C C T C C C C CGGT T C C C A C A C T T GC CGT T A A GGG CG T A GGG T A C T GC A GT C T GGA A T C T A CGC T T GT T C A GA C T T T G T A C T A G T T T C T T T GT C T GGC C A T C CGGGT A A C C C A T GC CGGA CGC A A A A T A G A C T A C T G A A A A T T T T T T T GC T T T GT GGT T GGGA C T T T A GC C A A GGG T A T A A 4 4 Coverage 0 0
EF30776726_PR- 10595T C T C C C C A C A T A T C A A A C C T C C C C CGGT T C C C A C A C T T GC CGT T A A GGG CG T A GGG T A C T GC A GT C T GGA A T C T A CGC T T GT T C A GA C T T T G T A C T A G T T T C T T T GT C T GGC C A T C CGGGT A A C C C A T GC CGGA CGC A A A A T A G A C T A C T G A A A A T T T T T T T GC T T T GT GGT T GGGA C T T T A GC C A A GGG T A T A A
Trace data
EF31206216_PR- 20691
Trace data
EF31206215_PR- 19299
Trace data
EF31206218_PR- 14619
Trace data
EF31206214_PR- 20692 Trace data
3
138 Chapter 5
1,160 1,180 1,200 1,220 1,240 1,260 1,280 1,300 1,320 1,340
Intron TEFint_seq_f w pCfB7056 (pIntC_3 _PrTEFint- CpFAH- TLip2A) A A A G A C C A C CG T C C CCGA A T T A C C T T T C C T C T T C T T T T C T C T C T C T C C T T G T C A A C T C A C A C CCGA A A T CGT T A A GC A T T T C C T T C T GA GT A T A A G A A T C A T T C A A A A T GGT GA GT T T C A GA GGC A GC A GC A A T T GC C A CGGG C T T T G A G C A C A CGG CCGGGT GT GGT C C C A T T C C C A T CGA C A C A A GA CG C C A
ConsensusA A A A G A C C A C CG T C C CCGA A T T A C C T T T C C T C T T C T T T T C T C T C T C T C C T T G T C A A C T C A C A C CCGA A A T CGT T A A GC A T T T C C T T C T GA GT A T A A G A A T C A T T C A A A A T GGT GA GT T T C A GA GGC A GC A GC A A T T GC C A CGGG C T T T G A G C A C A CGG CCGGGT GT GGT C C C A T T C C C A T CGA C A C A A GA CG C C A 4 4 Coverage 0 0
EF30776726_PR- 10595A A A A G A C C A C CG T C C CCGA A T T A C C T T T C C T C T T C T T T T C T C T C T C T C C T T G T C A A C T C A C A C CCGA A A T CGT T A A GC A T T T C C T T C T GA GT A T A A G A A T C A T T C A A A A T GGT GA GT T T C A GA GGC A GC A GC A A T T GC C A CGGG C T T T G A G C A C A CGG CCGGGT GT GGT C C C A T T C C C A T CGA C A C A A GA CG C C A
Trace data
EF31206216_PR- 20691
Trace data
EF31206215_PR- 19299
Trace data
EF31206218_PR- 14619
Trace data
EF31206214_PR- 20692
Trace data
1,360 1,380 1,400 1,420 1,440 1,460 1,480 1,500 1,520 1,540
TEFint_seq_f w CpFAH12_codoptYL pCfB7056 (pIntC_3 _PrTEFint- CpFAH- TLip2C) A CG T C A T C CG A C C A GC A C T T T T T GC A GT A C T a a c c g c a g A T GGC C T C T G C T A C C C C T G C C A T GT C T GA GA A CGC CGT GC T GCGA C A C A A GGC CGC C T C T A C C A C CGG A A T CGA C T A CGA GT C C T C T GC CGC CGT GT C T C C CGC T G A G T C T C C C CG A A CC T C T GC C T C T T C T A C C T CGC T GT C C T C T C T GT C C T C
ConsensusC A CG T C A T C CG A C C A GC A C T T T T T GC A GT A C T A A C CGC A GA T GGC C T C T G C T A C C C C T G C C A T GT C T GA GA A CGC CGT GC T GCGA C A C A A GGC CGC C T C T A C C A C CGG A A T CGA C T A CGA GT C C T C T GC CGC CGT GT C T C C CGC T G A G T C T C C C CG A A CC T C T GC C T C T T C T A C C T CGC T GT C C T C T C T GT C C T C 4 4 Coverage 0 0
EF30776726_PR- 10595C A CG T C A T C CG A C C A GC A C T T T T T GC A GT A C T A A C CGC A GA T GGC C T C T G C T A C C C C T G C C A T GT C T GA GA A CGC CGT GC T GCGA C A C A A GGC CGC C T C T A C C A C CGG A A T CGA C T A CGA GT C C T C T GC CGC CGT GT C T C C CGC T G A G T C T C C C CG A A CC T C T GC C T C T T C T A C C T CGC T GT C C T C T C T GT C C T C
Trace data
EF31206216_PR- 20691 C C T C C A A T CG A C A GGC A C T T T T T GC A GT A C T A A C CGC A GA T GGC C T C T G C T A C C C C T G C C A T GT C T GA GA A CGC CGT GC T GCGA C A C A A GGC CGC C T C T A C C A C CGG A A T CGA C T A CGA GT C C T C T GC CGC CGT GT C T C C CGC T G A G T C T C C C CG A A CC T C T GC C T C T T C T A C C T CGC T GT C C T C T C T GT C C T C
Trace data
EF31206215_PR- 19299 C C C CG T CGT C T GA A A A CGC CGT GC T GCGA C C A A GGC CGC C T C T A C C A C CGG A A T CGA C T A CGA GT C C T C T GC CGC CGT GT C T C C CGC T G A G T C T C C C CG A A CC T C T GC C T C T T C T A C C T CGC T GT C C T C T C T GT C C T C
Trace data
EF31206218_PR- 14619
Trace data
EF31206214_PR- 20692 Trace data
4 139 Chapter 5
1,540 1,560 1,580 1,600 1,620 1,640 1,660 1,680 1,700 1,720 1,740
pCfB7056 (pIntC_3 _PrTEFint- CpFAH- TLip2T) CGC T GGA CG C C A A CG A G A A G A A GGA CGA GT A CGC CGGC C T GC T GGA C A C C T A CGGC A A CG C T T T C A C C C C T C C T GA C T T C T C T A T C A A GGA C A T C CGA GC CGC T A T T C C C A A G C A C T GC T A CGA GCGA T C T A C C A T C A A GT C T T A CGC C T A CG T C C T G CG A G A T C T GC T GT GC C T GT C T A C C A C C T T C T A C C T G
ConsensusT CGC T GGA CG C C A A CG A G A A G A A GGA CGA GT A CGC CGGC C T GC T GGA C A C C T A CGGC A A CG C T T T C A C C C C T C C T GA C T T C T C T A T C A A GGA C A T C CGA GC CGC T A T T C C C A A G C A C T GC T A CGA GCGA T C T A C C A T C A A GT C T T A CGC C T A CG T C C T G CG A G A T C T GC T GT GC C T GT C T A C C A C C T T C T A C C T G 4 4 Coverage 0 0
EF30776726_PR- 10595T CGC T GGA CG C C A A CG A G A A G A A GGA CGA GT A CGC CGGC C T GC T GGA C A C C T A CGGC A A CG C T T T C A C C C C T C C T GA C T T C T C T A T C A A GGA C A T C CGA GC CGC T A T T C C C A A G C A C T GC T A CGA GCGA T C T A C C A T C A A GT C T T A CGC C T A CG T C C T G CG A G A T C T GC T GT GC C T GT C T A C C A C C T T C T A C C T G
Trace data
EF31206216_PR- 20691T CGC T GGA CG C C A A CG A G A A G A A GGA CGA GT A CGC CGGC C T GC T GGA C A C C T A CGGC A A CG C T T T C A C C C C T C C T GA C T T C T C T A T C A A GGA C A T C CGA GC CGC T A T T C C C A A G C A C T GC T A CGA GCGA T C T A C C A T C A A GT C T T A CGC C T A CG T C C T G CG A G A T C T GC T GT GC C T GT C T A C C A C C T T C T A C C T G
Trace data
EF31206215_PR- 19299T CGC T GGA CG C C A A CG A G A A G A A GGA CGA GT A CGC CGGC C T GC T GGA C A C C T A CGGC A A CG C T T T C A C C C C T C C T GA C T T C T C T A T C A A GGA C A T C CGA GC CGC T A T T C C C A A G C A C T GC T A CGA GCGA T C T A C C A T C A A GT C T T A CGC C T A CG T C C T G CG A G A T C T GC T GT GC C T GT C T A C C A C C T T C T A C C T G
Trace data
EF31206218_PR- 14619
Trace data
EF31206214_PR- 20692
Trace data
1,740 1,760 1,780 1,800 1,820 1,840 1,860 1,880 1,900 1,920
pCfB7056 (pIntC_3 _PrTEFint- CpFAH- TLip2T) GT T C C A C A A C T T CG T G A C C C C T G A GA A C A T C C C T T C T A A C C C T C T GCGA T T CGT GC T G T GG T C T A T C T A C A C CGT GC T GC A GGGC C T GT T CGC C A C CGGC C T GT GGG T G A T CGG C C A CGA GT GCGGC C A C T GCGC C T T C T C T C C C T C T C C T T T C A T C T C T G A C C T GA C CGGC T GGGT GA T C C A C T C T GC C C T GC
ConsensusT GT T C C A C A A C T T CG T G A C C C C T G A GA A C A T C C C T T C T A A C C C T C T GCGA T T CGT GC T G T GG T C T A T C T A C A C CGT GC T GC A GGGC C T GT T CGC C A C CGGC C T GT GGG T G A T CGG C C A CGA GT GCGGC C A C T GCGC C T T C T C T C C C T C T C C T T T C A T C T C T G A C C T GA C CGGC T GGGT GA T C C A C T C T GC C C T GC 4 4 Coverage 0 0
EF30776726_PR- 10595T GT T C C A A A A C T T CG T G A C C C C T G A GA A C A T C C C T T C T A A C C C T C T GCGA T T CGGGC T GGGG T C T A T C T A C A C CGGGC T GA A GGGC C T GT T CGC A C C CGGC C CGGGG T GG A T CGG C A C A A A T GGGGC C CGGGC C C T T C T C C C C C C C C C T T T T T C T T T A C A C T A A A A GGA GGGGA A A A A A A T C A GC CGCGGGGGGG
Trace data
EF31206216_PR- 20691T GT T C C A C A A C T T CG T G A C C C C T G A GA A C A T C C C T T C T A A C C C T C T GCGA T T CGT GC T G T GG T C T A T C T A C A C CGT GC T GC A GGGC C T GT T CGC C A C CGGC C T GT GGG T G A T CGG C C A CGA GT GCGGC C A C T GCGC C T T C T C T C C C T C T C C T T T C A T C T C T G A C C T GA C CGGC T GGGT GA T C C A C T C T GC C C T GC
Trace data
EF31206215_PR- 19299T GT T C C A C A A C T T CG T G A C C C C T G A GA A C A T C C C T T C T A A C C C T C T GCGA T T CGT GC T G T GG T C T A T C T A C A C CGT GC T GC A GGGC C T GT T CGC C A C CGGC C T GT GGG T G A T CGG C C A CGA GT GCGGC C A C T GCGC C T T C T C T C C C T C T C C T T T C A T C T C T G A C C T GA C CGGC T GGGT GA T C C A C T C T GC C C T GC
Trace data
EF31206218_PR- 14619 A A A GGGG A T T A A T T A C A T T T T C C T T T A T T C A GC C T T T T T C A GA T CGG C C C A G A A A A T T GGGGA GGC C A C C T T GGGGT T C C A A T GT T C C C T T CG C T T G C C T T T C CG C C T A T CGGC A GGGT T T GC A A A A T T CGCGA GA
Trace data
EF31206214_PR- 20692 Trace data
5
140 Chapter 5
1,940 1,960 1,980 2,000 2,020 2,040 2,060 2,080 2,100 2,120
CpFAH_seq_f w pCfB7056 (pIntC_3 _PrTEFint- CpFAH- TLip2G) C T GGT GC C C T A C T T C T C T T GG A A G T T C T CT C A C T C T GC T C A C C A C A A GGGC A T CGGC A A C A T GG A A CG A G A C A T GG T GT T T C T GC C C CGA A C T CGA GA GC A GC A GGC T A C C CG A C T GGG C CG A GC CGT CGA GGA A C T GGGCGA C C T GT GCGA GGA A A C T C C C A T C T A C A C T GC C C T GC A C C T GGT GGGC A A GC A
ConsensusGC T GGT GC C C T A C T T C T C T T GG A A G T T C T CT C A C T C T GC T C A C C A C A A GGGC A T CGGC A A C A T GG A A CG A G A C A T GG T GT T T C T GC C C CGA A C T CGA GA GC A GC A GGC T A C C CG A C T GGG C CG A GC CGT CGA GGA A C T GGGCGA C C T GT GCGA GGA A A C T C C C A T C T A C A C T GC C C T GC A C C T GGT GGGC A A GC A 4 4 Coverage 0 0
EF30776726_PR- 10595GGGC C T T T T T T T GGA A T T T T T T T T T T T T T CT C C A A A A T C T T T C C T A A A GA A A A T A A A A A A A T A T A G A T T
Trace data
EF31206216_PR- 20691GC T GGT GC C C T A C T T C T C T T GG A A G T T C T CT C A C T C T GC T C A C C A C A A GGGC A T CGGC A A C A T GG A A CG A G A C A T GG T GT T T C T GC C C CGA A C T CGA GA GC A GC A GGC T A C C CG A C T GGG C CG A GC CGT CGA GGA A C T GGGCGA C C T GT GCGA GGA A A C T C C C A T C T A C A C T GC C C T GC A C C T GGT GGGC A A GC A
Trace data
EF31206215_PR- 19299GC T GGT GC C C T A C T T C T C T T GG A A G T T C T CT C A C T C T GC T C A C C A C A A GGGC A T CGGC A A C A T GG A A CG A G A C A T GG T GT T T C T GC C C CGA A C T CGA GA GC A GC A GGC T A C C CG A C T GGG C CG A GC CGT CGA GGA A C T GGGCGA C C T GT GCGA GGA A A C T C C C A T C T A C A C T GC C C T GC A C C T GGT GGGC A A GC A
Trace data
EF31206218_PR- 14619GA GGT C C C CGA GA T A T G C T CGG C A G A A G T T C A C T T A GA T CGGC A C A A GGGC A T CGGC A T C T T GG A T CG A GG A C A G CG T GGGT C C A A GC T GA A C T CGA GA T C A GC A GT C T A C C CG A T C T G A C CG A GC CGT CGA GGA A C T GGGCGA C C T GT GCGA GGA A A C T C C C A T C T A C A C T GC C C T GC A C C T GGT GGGC A A GC A
Trace data
EF31206214_PR- 20692
Trace data
2,120 2,140 2,160 2,180 2,200 2,220 2,240 2,260 2,280 2,300 2,320
pCfB7056 (pIntC_3 _PrTEFint- CpFAH- TLip2C) A GC T GA T CGGC T GGC C C T C T T A C C T G A T GA C C A A CGC C A C CGGA C A C A A C T T C C A CGA GCGA C A G CG A G A A GG C CG A GGC A A GGGC A A GA A GA A CGGC T T CGGCGGA GG CG T G A A C C A C T T CG A C C C T CGA T C T C C C A T C T T CGA GGC C CGA C A GGC C A A G T A C A T CG T G C T GT C T GA C A T CGGC C T GGGC C T C
ConsensusC A GC T GA T CGGC T GGC C C T C T T A C C T G A T GA C C A A CGC C A C CGGA C A C A A C T T C C A CGA GCGA C A G CG A G A A GG C CG A GGC A A GGGC A A GA A GA A CGGC T T CGGCGGA GG CG T G A A C C A C T T CG A C C C T CGA T C T C C C A T C T T CGA GGC C CGA C A GGC C A A G T A C A T CG T G C T GT C T GA C A T CGGC C T GGGC C T C 4 4 Coverage 0 0
EF30776726_PR- 10595
Trace data
EF31206216_PR- 20691C A GC T GA T CGGC T GGC C C T C T T A C C T G A T GA C C A A CGC C A C CGGA C A C A A C T T C C A CGA GCGA C A G CG A G A A GG C CG A GGC A A GGGC A A GA A GA A CGGC T T CGGCGGA GG CG T G A A C C A C T T CG A C C C T CGA T C T C C C A T C T T CGA GGC C CGA C A GGC C A A G T A C A T CG T G C T GT C T GA C A T CGGC C T GGGC C T C
Trace data
EF31206215_PR- 19299C A GC T GA T CGGC T GGC C C T C T T A C C T G A T GA C C A A CGC C A C CGGA C A C A A C T T C C A CGA GCGA C A G CG A G A A GG C CG A GGC A A GGGC A A GA A GA A CGGC T T CGGCGGA GG CG T G A A C C A C T T CG A C C C T CGA T C T C C C A T C T T CGA GGC C CGA C A GGC C A A G T A C A T CG T G C T GT C T GA C A T CGGC C T GGGC C T C
Trace data
EF31206218_PR- 14619C A GC T GA T CGGC T GGC C C T C T T A C C T G A T GA C C A A CGC C A C CGGA C A C A A C T T C C A CGA GCGA C A G CG A G A A GG C CG A GGC A A GGGC A A GA A GA A CGGC T T CGGCGGA GG CG T G A A C C A C T T CG A C C C T CGA T C T C C C A T C T T CGA GGC C CGA C A GGC C A A G T A C A T CG T G C T GT C T GA C A T CGGC C T GGGC C T C
Trace data
EF31206214_PR- 20692 C T CGC A GG C C T C T T A A C T G A T GA C A A CGC C A C CGGGA C A C A A C T T C C A CGA GCGA C A G CG A G A A GG C CG A GGC A A GGGC A A GA A GA A CGGC T T CGGCGGA GG CG T G A A C C A C T T CG A C C C T CGA T C T C C C A T C T T CGA GGC C CGA C A GGC C A A G T A C A T CG T G C T GT C T GA C A T CGGC C T GGGC C T C
Trace data
6 141 Chapter 5
2,320 2,340 2,360 2,380 2,400 2,420 2,440 2,460 2,480 2,500
pCfB7056 (pIntC_3 _PrTEFint- CpFAH- TLip2T) CG C C A T T G C CGC T C T GGT GT A C C T GGGC A A C CGA T T CGGC T GGG C C A A C A T GG C CG T GT GGT A C T T T C T GC C C T A C C T GT GGGT C A A C C A C T GG C T GG T GG C T A T CA C C T T T C T GC A GC A C A C T GA C C C T A C T C T GC C C C A C T A C A A C CG A G A GGA A T GGA A C T T CGT GCGA GGCGGA GC C T GC A C C A T CG A C C
ConsensusT CG C C A T T G C CGC T C T GGT GT A C C T GGGC A A C CGA T T CGGC T GGG C C A A C A T GG C CG T GT GGT A C T T T C T GC C C T A C C T GT GGGT C A A C C A C T GG C T GG T GG C T A T CA C C T T T C T GC A GC A C A C T GA C C C T A C T C T GC C C C A C T A C A A C CG A G A GGA A T GGA A C T T CGT GCGA GGCGGA GC C T GC A C C A T CG A C C 4 4 Coverage 0 0
EF30776726_PR- 10595
Trace data
EF31206216_PR- 20691T CG C C A T T G C CGC T C T GGT GT A C C T GGGC A A C CGA T T CGGC T GGG C C A A C A T GG C CG T GT GGT A C T T T C T GC C C T A C C T GT GGGT C A A C C A C T GG C T GG T GG C T A T CA C C T T T C T GC A GC A A A C T GA A C C C T A C T C T GC C C C A C T A C A A C CG A GA GGA A T GGA A C T T CGT GCGA GGCGGA GC C T GC A C C A T CG A A
Trace data
EF31206215_PR- 19299T CG C C A T T G C CGC T C T GGT GT A C C T GGGC A A C CGA T T CGGC T GGG C C A A C A T GG C CG T GT GGT A C T T T C T GC C C T A C C T GT GGGT C A A C C A C T GG C T GG T GG C T A T CA C C T T T C T GC A GC A C A C T GA C C C T A C T C T GC C C C A C T A C A A C CG A G A GGA A T GGA A C T T CGT GCGA GGCGGA GC C T GC A C C A T CG A C C
Trace data
EF31206218_PR- 14619T CG C C A T T G C CGC T C T GGT GT A C C T GGGC A A C CGA T T CGGC T GGG C C A A C A T GG C CG T GT GGT A C T T T C T GC C C T A C C T GT GGGT C A A C C A C T GG C T GG T GG C T A T CA C C T T T C T GC A GC A C A C T GA C C C T A C T C T GC C C C A C T A C A A C CG A G A GGA A T GGA A C T T CGT GCGA GGCGGA GC C T GC A C C A T CG A C C
Trace data
EF31206214_PR- 20692T CG C C A T T G C CGC T C T GGT GT A C C T GGGC A A C CGA T T CGGC T GGG C C A A C A T GG C CG T GT GGT A C T T T C T GC C C T A C C T GT GGGT C A A C C A C T GG C T GG T GG C T A T CA C C T T T C T GC A GC A C A C T GA C C C T A C T C T GC C C C A C T A C A A C CG A G A GGA A T GGA A C T T CGT GCGA GGCGGA GC C T GC A C C A T CG A C C
Trace data
2,520 2,540 2,560 2,580 2,600 2,620 2,640 2,660 2,680 2,700
CpFAH_T 2570_f w CpFAH_2 570T_r v pCfB7056 (pIntC_3 _PrTEFint- CpFAH- TLip2C) CG A G A T C T GGGC T T C A T CGGC CGA C A C C T GT T T C A CGGA A T CGC T G A C A C C C A CG T GGT C C A C C A C T A CGT GT C T CGA A T T C C C T T C T A C A A CG C CG A CG A GG C C T C T GA GGC C A T T A A GC C C A T C A T GGGA A A GC A C T A C CG A T C T G A C A C CGC T C A CGGC C C CGT GGGC T T T C T GC A CGC C C T G T GG A A G A C
ConsensusC CG A G A T C T GGGC T T C A T CGGC CGA C A C C T GT T T C A CGGA A T CGC T G A C A C C C A CG T GGT C C A C C A C T A CGT GT C T CGA A T T C C C T T C T A C A A CG C CG A CG A GG C C T C T GA GGC C A T T A A GC C C A T C A T GGGA A A GC A C T A C CG A T C T G A C A C CGC T C A CGGC C C CGT GGGC T T T C T GC A CGC C C T G T GG A A G A C 4 4 Coverage 0 0
EF30776726_PR- 10595
Trace data
EF31206216_PR- 20691A A CG A G A T C T GGGC T T C A T CGGC CGA A C C C T GT T T C C CGGA A T CG C T G A A C C C C C CGG T GGT C C A C C C A T A CGGGGC C CGA A A T T C C C T T C T A A A A A CG C C A A A G A GGC C C C T GGA GGGC T T T T A A C C C C C T T T GGGGA A A A A C C A A C C CG A A T T GA A A C C C C C C C CGGC C C CGGGGGGT T T T C T GC C C C C C CG T
Trace data
EF31206215_PR- 19299C CG A G A T C T GGGC T T C A T CGGC CGA A A C C T GT T T C A CGGA A T CGC T G A A A C C C A CG T GGT C C A C C A C T A CGT GT C T CGA A T T C C C T T C T A C A A CG C CG A A G A A GG C CT C T GA A GGC C T T T A A GC C C A T C T T GGGA A A GC C T A A C CG A T T T G A A A C C CGC T C CGGGC C C C CGGGGGT T T T T T GC C C C C C CG T GGG A
Trace data
EF31206218_PR- 14619C CG A G A T C T GGGC T T C A T CGGC CGA C A C C T GT T T C A CGGA A T CGC T G A C A C C C A CG T GGT C C A C C A C T A CGT GT C T CGA A T T C C C T T C T A C A A CG C CG A CG A GG C C T C T GA GGC C A T T A A GC C C A T C A T GGGA A A GC A C T A C CG A T C T G A C A C CGC T C A CGGC C C CGT GGGC T T T C T GC A CGC C C T G T GG A A G A C
Trace data
EF31206214_PR- 20692C CG A G A T C T GGGC T T C A T CGGC CGA C A C C T GT T T C A CGGA A T CGC T G A C A C C C A CG T GGT C C A C C A C T A CGT GT C T CGA A T T C C C T T C T A C A A CG C CG A CG A GG C C T C T GA GGC C A T T A A GC C C A T C A T GGGA A A GC A C T A C CG A T C T G A C A C CGC T C A CGGC C C CGT GGGC T T T C T GC A CGC C C T G T GG A A G A C Trace data
7 142 Chapter 5
2,700 2,720 2,740 2,760 2,780 2,800 2,820 2,840 2,860 2,880 2,900
pCfB7056 (pIntC_3 _PrTEFint- CpFAH- TLip2A) C CG C T CG A T GG T G T C A GT GGGT CGA GC C C T C T GC T GA CGC C C A A GG CG C CGG A A A GGG C A T C C T GT T C T A C CGA A A C CGA A A C A A GC T GGGC A C C A A G C C T A T C T C T A T GA A GA C C C A GT A A a t c g c g t g C A T T C C T T C T G T T CGG A A T C A A C C T CA A GGT T A A CGGC C A CGA T C C C C T CGT T GT T A C T C T T GG
ConsensusA C CG C T CG A T GG T G T C A GT GGGT CGA GC C C T C T GC T GA CGC C C A A GG CG C CGG A A A GGG C A T C C T GT T C T A C CGA A A C CGA A A C A A GC T GGGC A C C A A G C C T A T C T C T A T GA A GA C C C A GT A A A T CGCGT GC A T T C C T T C T G T T CGG A A T C A A C C T CA A GGT T A A CGGC C A CGA T C C C C T CGT T GT T A C T C T T GG 4 4 Coverage 0 0
EF30776726_PR- 10595
Trace data
EF31206216_PR- 20691G T GG A A A A A C C C C C CGA GGGGGT T C A GGGGT C C A C C C C C C CGT GGA A C C C C C A GGG C C C CG A A A A GGGT T T CGT T T T C C A A A A A C A A A A A A A A A GGG T GG C C C C C C C C T T T T T T T T T A GA A C A
Trace data
EF31206215_PR- 19299G A A A A A C C C C C C A GGGGGT C T GGGGGT C C A A C C C T C T GGGA C C C C CGGGG C CG A A A GG C T C C T GT T T T C C C A A A A C A A A A A A A GGGT GGGC C A C A C C C T T T T T T T T T T A A A A A A A C C C A A A A A A A CGGGGGGT T T T T T T T T G T G T GG A A A A A C C C C CGGGGA T A A A GGC C C A A A A C C C C C T GT T T T T T T T T G T GG
Trace data
EF31206218_PR- 14619A C CG C T CG A T GG T G T C A GT GGGT CGA GC C C T C T GC T GA CGC C C A A GG CG C CGG A A A GGG C A T C C T GT T C T A C CGA A A C CGA A A C A A GC T GGGC A C C A A G C C T A T C T C T A T GA A GA C C C A GT A A A T CGCGT GC A T T C C T T C T G T T CGG A A T C A A C C T CA A GGT T A A CGGC C A CGA T C C C C T CGT T GT T A C T C T T GG
Trace data
EF31206214_PR- 20692A C CG C T CG A T GG T G T C A GT GGGT CGA GC C C T C T GC T GA CGC C C A A GG CG C CGG A A A GGG C A T C C T GT T C T A C CGA A A C CGA A A C A A GC T GGGC A C C A A G C C T A T C T C T A T GA A GA C C C A GT A A A T CGCGT GC A T T C C T T C T G T T CGG A A T C A A C C T CA A GGT T A A CGGC C A CGA T C C C C T CGT T GT T A C T C T T GG
Trace data
2,900 2,920 2,940 2,960 2,980 3,000 3,020 3,040 3,060 3,080
PR- 14619 pCfB7056 (pIntC_3 _PrTEFint- CpFAH- TLip2G) G T C A G C C C A T T G T CGGT A A CGC T GGC T T T GC T A A C T GGGT CGA T A A A C T C T T C T T T GG C C A GGA GA A C C C CGA T GT C T C C A A GGT GT C C A A A GA C CG A A A G C T C T A C CGA A T C A C C C A C CGA GGA GA T A T CGT C C C T C A A G T G C C C T T C T GGG A CGGT T A C C A GC A C T GC T C T GGT GA GGT C T T T A T T G A C T GG
ConsensusGG T C A G C C C A T T G T CGGT A A CGC T GGC T T T GC T A A C T GGGT CGA T A A A C T C T T C T T T GG C C A GGA GA A C C C CGA T GT C T C C A A GGT GT C C A A A GA C CG A A A G C T C T A C CGA A T C A C C C A C CGA GGA GA T A T CGT C C C T C A A G T G C C C T T C T GGG A CGGT T A C C A GC A C T GC T C T GGT GA GGT C T T T A T T G A C T GG 4 4 Coverage 0 0
EF30776726_PR- 10595
Trace data
EF31206216_PR- 20691
Trace data
EF31206215_PR- 19299GG C C C C C C T GGGGGGA A GGGGT T T T T T T T A GGGGGGA A A A T C T T T T G T G T GG A G A A A C A A T T C C CGGGGC
Trace data
EF31206218_PR- 14619GG T C A G C C C T T T CGGCGC T
Trace data
EF31206214_PR- 20692GG T C A G C C C A T T G T CGGT A A CGC T GGC T T T GC T A A C T GGGT CGA T A A A C T C T T C T T T GG C C A GGA GA A C C C CGA T GT C T C C A A GGT GT C C A A A GA C CG A A A G C T C T A C CGA A T C A C C C A C CGA GGA GA T A T CGT C C C T C A A G T G C C C T T C T GGG A CGGT T A C C A GC A C T GC T C T GGT GA GGT C T T T A T T G A C T GG Trace data
8
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Table S1. Constructed strains in study Strain Parent gRNA Integration Expression cassette/Deletion vector vector/ KO fragment
ST8997 ST6852 pCfB8040 BB2679 DGA1 deletion ST9000 ST8997 pCfB8036 BB2673 FAD2 deletion ST9001 ST9000 pCfB6630 pCfB7056 IntC_3::PrTEFin->CpFAH-TLip2 ST9002 ST9000 pCfB6630 pCfB8766 IntC_3::PrTEFin->CpFAH_N-swap- TLip2 ST9045 ST9001 pCfB6627 pCfB8765 IntE_4::TPex20-MaC16E<-PrEXP- PrGPD->YlOLE1-TLip2 ST9131 ST9045 pCfB6627 pCfB8972 IntC_2::PrEXP->RcDGAT2-TLip2 ST9132 ST9131 pCfB6633 pCfB8864 IntE_1::PrGPD->RcACS2-TLip2 ST9133 ST9132 pCfB6684 pCfB8973 IntD1::PrTEFin->RcLPCAT2-TLip2 ST9134 ST9132 pCfB6631 pCfB8974 IntD_1::RcsPLA2�_HDEL<-PrEXP- PrTEFin->RcLPCAT2-TLip2 ST9135 ST9133 pBP8003 pCfB8867 IntF_3::TPex20<-RcGPAT9-PrEXP- PrGPD->RcLPAT2-TLip2 ST9136 ST9134 pBP8003 pCfB8867 IntF_3::TPex20<-RcGPAT9-PrEXP- PrGPD->RcLPAT2-TLip2 ST9742 ST6512 pCfB6637 pCfB9724 IntE_3::PrTEFin->YlFAD2-TLip2 ST9743 ST6852 pCfB6637 pCfB9724 IntE_3::PrTEFin->YlFAD2-TLip2 ST9744 ST8997 pCfB6637 pCfB9724 IntE_3::PrTEFin->YlFAD2-TLip2 ST9745 ST9000 pCfB6637 pCfB9724 IntE_3::PrTEFin->YlFAD2-TLip2 ST9746 ST9001 pCfB6637 pCfB9724 IntE_3::PrTEFin->YlFAD2-TLip2 ST9747 ST9045 pCfB6637 pCfB9724 IntE_3::PrTEFin->YlFAD2-TLip2 ST9748 ST9131 pCfB6637 pCfB9724 IntE_3::PrTEFin->YlFAD2-TLip2 ST9749 ST9132 pCfB6637 pCfB9724 IntE_3::PrTEFin->YlFAD2-TLip2 ST9750 ST9133 pCfB6637 pCfB9724 IntE_3::PrTEFin->YlFAD2-TLip2 ST9751 ST9134 pCfB6637 pCfB9724 IntE_3::PrTEFin->YlFAD2-TLip2 ST9752 ST9135 pCfB6637 pCfB9724 IntE_3::PrTEFin->YlFAD2-TLip2 ST9753 ST9136 pCfB6637 pCfB9724 IntE_3::PrTEFin->YlFAD2-TLip2
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Table S2. Plasmids used in this study Name Parent vector Biobricks Ref. Dr. K.R. Kildegaard, pBP8003 - - Biophero ApS, Denmark L.R.R.Tramontin, DTU CfB, pCfB8092 - - Denmark pCfB3405 - - [1] pCfB4785 - - [1] pCfB6367 - - [1] pCfB6371 - - [1] pCfB6627 - - [1] pCfB6630 - - [1] pCfB6631 - - [1] pCfB6633 - - [1] pCfB6637 - - [1] pCfB6637 - - [1] pCfB6684 - - [1] pCfB7056 pCfB6371 BB2193, BB2228 This study pCfB8036 pCfB3405 BB2693 This study pCfB8040 pCfB3405 BB2695 This study pCfB8765 pCfB6679 BB3781 This study pCfB8766 pCfB6371 BB3782, BB3784 This study pCfB8864 pCfB6367 BB1562, BB3800 This study pCfB8867 pCfB4785 BB3062, BB2174, BB3759 This study pCfB8972 pCfB6682 BB3708, BB2942 This study pCfB8973 pCfB6684 BB2249, BB3093 This study pCfB8974 pCfB6684 BB3938, BB2813, BB3093 This study
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Table S3. Biobricks used in this study Biobricks Primers for PCR Template for PCR BB1562 PR-15527, PR-15528 pCfB8092 BB1621 PR-15605, PR-15606 pCfB8092 BB1635 PR-10607, PR-15788 pCfB6630 BB1636 PR-15789, PR-10604 pCfB6630 BB2174 PR-15606, PR-15528 pCfB8092 BB2193 PR-19307, PR-19308 pCfB7053 (Note 1) BB2211 PR-18931, PR-18214 pCfB8092 BB2228 PR-10595, PR-19300 pCfB8092 BB2249 PR-10595, PR-18214 pCfB8092 BB2671 PR-21610, PR-21611 ST6512 genomic DNA BB2672 PR-21612, PR-21613 ST6512 genomic DNA BB2673 PR-21610, PR-21613 BB2671, BB2672 (Note 2) BB2677 PR-21791, PR-21792 ST6512 genomic DNA BB2678 PR-21793, PR-21794 ST6512 genomic DNA BB2679 PR-21791, PR-21794 BB2677, BB2678 (Note 2) BB2689 PR-15790, PR-15791 BB1635, BB1636, PR-21608, PR-21609 (Note 2) BB2691 PR-15790, PR-15791 BB1635, BB1636, PR-21789, PR-21790 (Note 2) BB2693 PR-10604, PR-10607 BB2689 BB2695 PR-10604, PR-10607 BB2691 BB2813 PR-15606, PR-15606 BB2211, BB1621 (Note 2) BB2942 PR-22430, PR-22431 pCfB8159 (Note 1) BB3062 PR-22651, PR-22652 pCfB8210 (Note 1) BB3093 PR-22686, PR-22687 pCfB8211 (Note 1) BB3708 PR-15521, PR-15522 pCfB8092 BB3759 PR-22566, PR-23731 pCfB8161 (Note 1) BB3781 PR-21931, PR-21886 pCfB8092 BB3782 PR-23769, PR-19031 pCfB7056 BB3784 PR-10595, PR-23770 pCfB8092 BB3800 PR-23778, PR-23779 pCfB8789 (Note 1) PR-22646, PR-24231, BB3938 pCfB8158 (Note 1) PR-24232 Note 1: The template was synthetic gene fragment (refer to Supplementary information 1). Note 2: Biobricks were first digested with USERTM enzyme (New England Biolabs) then ligated with T4 DNA ligase (ThermoFisher Scientific). Ligation products were used as PCR template.
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Table S4. Primers used in this study Primers Sequence PR-10595 cgtgcgaUAGAGACCGGGTTGG PR-10604 cacgcgaUaccgtacccacacaaaaaaagcaccaccgactc PR-10607 cgtgcgaUagtgaatcattgctaacagatc PR-14619 tatcgacccagttagc PR-15521 cgtgcgaUaaggagtttggcgcccgtt PR-15522 atgacagaUtgctgtagatatgtcttgt PR-15527 CGTGCGAUGACGCAGTAGGATGTCCTGCACGG PR-15528 ATGACAGAUTGTTGATGTGTGTTTAATTCAAGAATG PR-15605 ATCAGTAGCUAAGGAGTTTGGCGCCCGTT PR-15606 ACCTGCACUTGCTGTAGATATGTCTTGT PR-15788 taaccaaccUgcgccgacccggaatcgaac PR-15789 gttttagagcUagaaatagcaagttaaaataag PR-15790 AGTGCAGGUagtgaatcattgctaacagatc PR-15791 ACCTGCACUaccgtacccacacaaaaaaagcac PR-18214 AGTACTGCAAAAAGUGCTG PR-18931 AGCTACTGAU AGAGACCGGGTTGG PR-19031 cacgcgaUTTACTGGGTCTTCATAGAGATAGG PR-19299 aaccgcagAUGGCCTCTGCTACCC PR-19300 atCTGCGGTUAGTACTGCAAAAAGTG PR-19307 aaccgcagAUGGCCTCTGCTACCCCTGCCATGTCTGAGAACGC PR-19308 cacgcgaUTTACTGGGTCTTCATAGAGATAGGCTTGGTGCCCAGCTT PR-20692 TGCACCTGGTGGGCAAG PR-21610 CCGGTTTCGGCGAGCTCG PR-21611 aggccacUAACTGAACAGCACCGAGC PR-21612 agtggccUCTATGGTCGGTCTGTTGG PR-21613 TGTAACTGGTTCTTTCACTC PR-21791 GAATCGCACACAAACCGGG PR-21792 aggccacUGTGTGACTTGTCTGTTGCC PR-21793 agtggccUTCGCCCTTGTTCCCATCA PR-21794 ACAATCCCCTCTTCAACAGT PR-21886 cacgcgaUCTAAGCAGCCATGCCAGAC PR-21931 ACTTTTTGCAGTACU AACCGCAG GATTCGACCACGCAGACC PR-22430 atctgtcaUgccacaATGGGCGAAGAGGCCAAC PR-22431 cacgcgaUTTACAGGATCTCGAGAGTCAGG PR-22566 atctgtcaUgccacaATGGCCGTGGCCGCCGTC PR-22646 agtgcaggUgccacaATGGCCAACCTGTCTCAGCC PR-22651 agtgcaggUgccacaATGTCTACCGCTGGCAAG PR-22652 cgtgcgaUTTACTTCTCCTCCAGTCGTC PR-22686 actttttgcagtacUaaccgcagGACCTGGATCTGGAGT PR-22687 cacgcgaUTTACTCGTCCTTTCGAGTCT PR-23731 cacgcgaUTTAGTCCTGCTTGTTCTCGGCC PR-23769 AAGCCCATUGAGAAGAAGGACGAGTACGC PR-23770 AATGGGCTUAATGAAGGCCG PR-23778 atctgtcaUgccacaATGGACATGGACTCTGCC PR-23779 cacgcgaUTTACAGCTTAGGCGAAGGATC PR-24231 ccgccgccagagccgcctccaccaccGGGCTTGTGCAGGTATCGTCCGGC PR-24232 cgtgcgaUTTACAGCTCATCGTGggaaccaccgccgccgccagagccgcct
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Figure S1. Lack of ricinoleate peak in CpFAH12-expressing strain
For detection of methyl ricinoleate (Me-ricinoleate), undiluted derivatization extract was analysed with GC-MS. GC-MS method was modified to allow Me-ricinoleate detection (80°C for 1 min; 6°C/min ramp to 210°C and hold for 10 min; 6°C/min ramp to 230°C and hold for 10 min).
FAME standards consisted of Me-C16, Me-C17:1, Me-C18:0, Me-C18:1, Me-C18:2, and Me- ricinoleate. Chromatograms show samples for ST6512 (POX1-6, FAD2, no CpFAH12), ST9001 (∆pox1-6 ∆fad2 CpFAH12), and ST9002 (∆pox1-6 ∆fad2, CpFAH12 with swapped N-terminus). Me-ricinoleate was not detected in the strains tested.
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Figure S2. GFP pattern presented by strain expressing CpFAH12 with hrGFP tagged at C-terminal Fluorescent image acquisition was performed according to previous study [2]. The cells from cryostock were propagated in 14 mL tubes with 1 mL YPD at 30 °C, 250 rpm overnight. Cells were washed two times with 2 mL of 10 mM KH2PO4 pH 6 (centrifugation at 3000 g and 250 rpm). Cells were resuspended in 1 mL of 10 mM KH2PO4 pH 6 and loaded onto microscope slides. Fluorescence images were taken at 100× magnification in a Leica DFC300 FX microscope equipped with Leica EL600 external light source. All images were taken with the same acquisition settings. Scale bar corresponds to 5 µm.
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Figure S3. Swapping the N-terminus of CpFah12p. a) Alignment of CpFah12p, YlFad2p, and RcFah12p. Light and dark red shading indicates 50% and 100% consensus among the three sequences, respectively. TMHMM[3] prediction for CpFah12p b) before and c) after swapping the domain. RcFAH12 sequence (Genbank XP_002528127) was included in the alignment to ensure important domains were not cleaved.
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Figure S4. DeepLoc [4] result for CpFah12p after swapping the N-terminus.
Supplementary material references
1. Holkenbrink C, Dam MI, Kildegaard KR, Beder J, Dahlin J, Belda DD, Borodina I: EasyCloneYALI: CRISPR/Cas9-Based Synthetic Toolbox for Engineering of the Yeast Yarrowia lipolytica. Biotechnol J 2018, 13:1700543.
2. Marella ER, Holkenbrink C, Siewers V, Borodina I: Engineering microbial fatty acid metabolism for biofuels and biochemicals. Curr Opin Biotechnol 2017, 50:39–46.
3. Krogh A, Larsson B, von Heijne G, Sonnhammer EL: Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 2001, 305:567–580.
4. Almagro Armenteros JJ, Sønderby CK, Sønderby SK, Nielsen H, Winther O: DeepLoc: prediction of protein subcellular localization using deep learning. Bioinformatics 2017, 33:3387–3395.
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6
Perspectives
Eko Roy Marella, Irina Borodina
Chapter 6
6.1. Introduction
Y. lipolytica has become more popular in recent years. With accumulated data
and insights from past studies, research in the engineering of Y. lipolytica is
addressing more advance questions and problems, making the need for more efficient
tools for strain engineering more apparent. In particular, tools that enable faster and
high-throughput strain engineering, from strain construction to strain screening, are
desired. In this chapter, these challenges are discussed and potential solutions are
proposed.
6.2. Whole-genome sequence & genome-scale model
6.2.1. Whole-genome sequence: completing annotation and accounting
diversity
The first genome sequence of Y. lipolytica was published in 2004 for the strain
CLIB122 (E105) [1], a highly inbred strain originated from CLIB9 (W29) wild-type
parent [2]. The genome annotation was manually curated following an automated
identification of ORFs, revealing in total 6,703 CDS encoded in six chromosomes of a
total of 20.5 Mb genome. As many as 1,513 (22.5%) of the identified CDS were not
assigned to any protein family. In comparison, 5,807 CDS were identified in
Saccharomyces cerevisiae from a 12.1 Mb genome, where only 426 of the CDS (7.3%)
were not assigned to any protein family [1]. This implies the need for annotation of the
current genome sequence.
This first high-quality genome sequence of CLIB122 and its annotation were
then used as the reference for annotating the draft genome sequences of the wild-type
strain W29 [3,4] as well as the lab strain Po1f [5]. Therefore, these genome sequences
do not cover the diversity of Y. lipolytica as they were established using the wild-type
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W29 strain and its descendants, which are largely used for studying and engineering of lipid production [6 8]. Different Y. lipolytica strains, such as the H222 and WSH-
Z06 strains, have been the strains of choice for the production of organic acids [9,10].
These strains secrete large amounts of organic acids as an alternative to lipid accumulation under nitrogen limitation [2]. Indeed, the karyotyping and whole- genome sequence of H222 strain revealed a significant difference in chromosomal architecture between H222 and W29 strains [11,12], which might partially explain the citrate-producing nature of H222 strain and the lipid-accumulating tendency of W29 strain.
It is then possible that certain Y. lipolytica strains are better suited than the other for the production of specific compounds. Such a strain-product match could reduce strain engineering time and costs. Therefore, it would be useful to explore the genetic and phenotypic diversity of Y. lipolytica.
6.2.2. A genome-scale model with better utility
There are at least five available genome-scale models (GSMs) of Y. lipolytica
[13 17]. Among these models, iYali4 is arguably the model with the highest quality as it covers more reactions than other models and the reconstruction involved the annotation of unknown protein sequences predicted from the whole genome sequence. [15]. Furthermore, based on testing on Memote tool [18], iYali4 model scores at 64% (1-100% scale), while the rest of Y. lipolytica models score lower than
30%. The models other than iYali4 scored very low (0-25%) in some- or all of these basic parameters: stoichiometric consistency, mass balance, and charge balance. On the other hand, iYali4 scores higher than 70% for those parameters.
One drawback of the iYali4, however, is that the naming system of metabolites
(e.g. s_0002, s_0015) makes it not possible to cross-annotate them with databases such as PubChem or KEGG. Furthermore, any visualization tool of this model is not
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yet available and the available tools (such as from https://caffeine.dd-decaf.eu/) could
not generate a visual representation of this model. As visualization aids in designing
strategies and interpreting experimental data, model visualization would boost the
utility of this model.
Secondly, in the current model, lipid biosynthesis reactions are lumped.
Consequently, the model could not capture diversity of reactions and compounds in
lipid metabolism. These compounds and reactions are particularly of interest when
the accumulation of specific fatty acids or lipids is desired, or when fatty acid pathway
metabolites are the precursors for a pathway of interest. In this regard, the model
could be improved by accounting different classes of lipids and composition of acyl
chains, such as in SLIMEr (Split Lipids Into Measurable Entities), which was
previously applied to the GSM of S. cerevisiae [19]. With SLIMEr, the GSM contains a
higher resolution of lipid species and classes and thereby enabling GSM to account for
changes in lipid profiles in different conditions.
Thirdly, the GSM could also be improved by incorporating proteomics and
lipidomics data. Introducing enzyme-level constrains for setting maximum allowable
fluxes such as in GECKO (Genome-scale model to account for Enzyme Constraints,
using Kinetics and Omics) has improved a S. cerevisiae model conformity with
experimental data [20]. Lipidomics is relevant as it has a direct consequence on
biomass composition, especially in Y. lipolytica as lipid content could reach up to 20%
in wild-type [15] and higher than 50% in engineered strains [6] under nitrogen
limitation. Furthermore, as Y. lipolytica also uses its membrane as a lipid sink [15],
accounting different acyl chains and lipid classes in GSM could refine the model s
accuracy and its predictive power.
6.3. Physiological studies
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6.3.1. Improving the lipid production
It has been observed that under nitrogen limitation, there is a higher lipid accumulation in Y. lipolytica. Different studies have revealed that the flux through
Acc1p as well as the supply of NADPH poses the bottlenecks for fatty acid synthesis
[15,21,22]. Furthermore, it has been demonstrated that overexpressions of ACC1 and
DGA1 as well as increasing NADPH supply are sufficient to obtain lipid production at the maximum stoichiometric yield [6].
While these have addressed thermodynamic limitations for high lipid titer and yield, the next task would be to optimize the productivity. To date, the highest productivity reported for lipid accumulation in Y. lipolytica is 1.3 g/L/h [6].
One possible way is to boost substrate uptake rate by, for example, overexpressing native glucose or glycerol transporters and catabolic enzymes as described by Qiao et al. [6]. In this way, one could examine the maximum capacity of the central carbon metabolism and increase the flux towards the product. Following that, different copies of the lipid production pathway are added until lipid production plateaued. Such iterative overexpression of upstream- (glucose or glycerol to acetyl-
CoA) and downstream (acetyl-CoA to triacylglycerol) pathways, combined with omics studies (e.g. fluxomics and phospho-proteomics), could elucidate the pathways where flux should be increased.
Another possibility is to survey various media compositions to identify an optimized composition, which can be applied for different products other than lipid as well. A study demonstrated that double limitation of magnesium and nitrogen could yield up to 24.7% lipid content (g-lipid/g-biomass) in a wild-type strain, compared to only 10.9% with only limited nitrogen [23]. Phosphate limitation has also been shown to improve lipid production in R. toruloides [24].
6.3.2. Physiology at different carbon-to-nitrogen ratios
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The understanding that the respiro-fermentative lifestyle of S. cerevisiae
correlates with growth rate [25,26] has shaped the metabolic- and biochemical
engineering strategies in S. cerevisiae [27] and enable improvements of GSMs [20].
In Y. lipolytica and oleaginous yeast and fungi in general, it is known that the
channelling of carbon towards lipid (or citrate) is driven by nitrogen limitation [15,21].
Therefore, the nitrogen limitation is a subject of interest in engineering oleaginous
microorganisms. Carbon-to-nitrogen (C/N) ratio is a parameter that reflects nitrogen
limitation in cultivation media, especially in mineral media where the C/N ratio can
be directly computed from the amount of carbon source and nitrogen source. In
reported experiments so far, C/N ratios of 5-10 and >100 are employed to establish
carbon- and nitrogen limitation, respectively [6,15,21].
Although studies comparing low and high C/N ratios have elucidated
important insights, questions remain on how the metabolism behaves between these
extreme values. This could be useful for products other than lipids such as stilbenoids,
where the precursors come from different pathways [28]. By using a correct C/N ratio,
an optimum supply of each precursor might be achieved without the need for
sophisticated metabolic engineering strategies. Furthermore, data on carbon flux and
proteomics distribution at different C/N ratios could be used to build a GSM that
allows better prediction for different media with varying C/N ratios.
6.4. Enabling multiplexed and high-throughput strain
construction
Metabolic engineering involves screening of enzyme variants with desired
properties and manipulating the hosts metabolism via gene overexpression and
157 Chapter 6 knock-outs [29]. Such pursuit implies the need for efficient molecular biology methods to enable fast, accurate, and scalable strain construction. The following subchapters discuss the relevant barriers and potential solutions in achieving highly efficient engineering of Y. lipolytica.
6.4.1. Improving the efficiency of transformation method
High transformation efficiency is desired to avoid time losses due to the need for repeating experiments [30]. It is also particularly critical for library screening where non-biased transformation and high library coverage are desired [31].
An efficient PEG/LiAc transformation method of plasmid DNA for Y. lipolytica was developed by Chen et al. (1997) which gave transformation efficiency of up to 6 105 transformants/µg-DNA [32]. The study highlighted that efficiency was improved by using PEG 4000 instead of PEG 3350, cells from solid media instead of liquid culture, and the addition of 100 mM dithiothreitol (DTT) in the transformation mix. As a comparison, the efficiency of widely used transformation protocol for S. cerevisiae is around 106 transformants/µg-DNA [33], which is about two times higher.
This suggests that the available transformation method of Y. lipolytica is comparable with that of S. cerevisiae. Interestingly, one study found that a plasmid library transformation with a commercial kit gave higher efficiency (up to 4.35 104 transformants/µg-DNA) than the PEG/LiAc method [34].
The Chen et al. (1997) protocol described above was initially developed because plasmid transformation efficiency was lower using the preceding method by
Xuan et al. (1988), which were optimized for linearized DNA transformation [35]
(complete protocol in [2]). The method yielded up to 106 transformants/µg-DNA for the integration of a linearized plasmid [35]. The method requires exponentially growing cells in liquid culture and without the use of DTT. This suggests that
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homologous recombination might be more efficient using cells from liquid culture and
without the addition of DTT.
Electroporation serves as an alternative to achieve higher efficiency. However
the reported efficiency for electroporation of Y. lipolytica so far (2.1 to 2.8 104
transformants/µg-DNA [36,30]) was not superior to chemical-competence
transformation methods. Furthermore, electroporation is more difficult to scale-up for
a large number of transformations, as it requires specific equipment and very sensitive
regarding handling time and salt contamination. On the other hand, PEG/LiAc
transformation can be conveniently performed in a thermal cycler and less affected by
variations on the timing and reagent concentrations.
6.4.2. Improving homologous recombination
It is desired to integrate genes in specific loci. Furthermore, for screening
multiple gene variants, screening combinations of gene overexpressions or deletions,
and whole-pathway insertion, enabling the integration of more than one DNA
fragment could greatly accelerate strain engineering.
6.4.2.1. KU70 deletion and downregulation
Unlike in S. cerevisiae, a double-stranded break (DSB) in Y. lipolytica is
resolved mainly by NHEJ instead of homologous recombination (HR) [37,38]. The
homologous recombination efficiency is also dependent on the length of homology
arms [37,38]. Fragments with homology arms shorter than 500 bp almost certainly
integrated randomly via NHEJ [38]. By using 1 kb homology arms, the frequency of
transformants with HR repair could reach up to 11% [37].
By disrupting the KU70 gene, the frequency of transformants with
homologous recombination was increased to 43-50% and 63-85% for homology arms
of shorter than 500 bp and longer than 750 bp, respectively, with 0% frequency using
159 Chapter 6 fragment with 25 bp [37,38]. The deletion of KU80 in the ku70 mutant did not further increase the homologous recombination [37,38]. Notably, these results were achieved without inducing a double-stranded break. As an alternative to KU70 deletion, a CRISPRi method for KU70 transcriptional inhibition could also achieve higher HR frequency [31]. While the number of total transformants usually dropped
6-30 times after disruption of KU70 [37,38], the downregulation of KU70 with
CRISPRi did not result in a decrease in transformants [31]. It is important to note that these studies targeted different loci, so it cannot be concluded yet whether KU70 downregulation is better than disruption in terms of the number of transformants.
Nonetheless, the downregulation of KU70 showed promise for a more efficient homologous recombination without sacrificing the number of transformants. Based on the data by Schwartz et al. (2017) [31], the number of transformants and HR frequency could probably still be improved by using longer homology arms. This does not seem to be the case with the KU70 deletion [37].
6.4.2.2. Further improvement for homologous recombination
Even if it is still possible to increase HR frequency of one DNA fragment above
1 kb arms, this is still far less efficient than that of S. cerevisiae where with only 60 bp arms, multiple fragments can be joined and integrated efficiently with the aid of a nuclease [39]. This suggests that native Y. lipolytica machinery might still not be efficient enough, either because Y. lipolytica HR proteins are not abundant enough, not active enough, or their expression is not strongly induced by DSB as to that of S. cerevisiae.
One intuitive solution is to test whether implanting the HR proteins of S. cerevisiae could help. According to a review, there are at least 12 recombination factors in S. cerevisiae [40]. One could examine whether introducing some or all of these
160 Chapter 6 proteins in Y. lipolytica could result in higher HR efficiency. The expression of RAD52 from S. cerevisiae (ScRAD52) was shown to increase a targeted integration by up to
37-fold in human cells [41]. Furthermore, although S. cerevisiae does have KU70 and
KU80 proteins, they are dissociated from the DSB site through a mechanism dependent on the MRX complex (ScMre11p-ScRad50p-ScXrs2p) [42]. BLASTp search
[43] for the homology of these proteins in Y. lipolytica returned YALI0D15246p and
YALI0B14553p as the homologs of ScRAD50 and ScMRE11, respectively, but none for
ScXRS2. Since the MRX complex is also important for HR in S. cerevisiae [44],
ScXRS2 expression in Y. lipolytica might also be useful. It is therefore recommended to express ScRAD52, ScXRS2, and other S. cerevisiae recombination proteins to elevate HR efficiency in Y. lipolytica.
6.4.3. Integration sites
The next requirement for strain engineering is the availability of several integration sites for gene (over-)expressions. Desired integration sites should be stable, allow high expression, do not contain any ORF, and do not cause growth impairment when they are disrupted [45]. EasyCloneYALI toolbox [46] presented 11 of such sites. However, it was also shown that targeting efficiencies for different integration sites are different for both marker-based integration and Cas9-mediated integration [46]. As failure in obtaining correct integration will slow down strain engineering process, integration sites also need to allow a high success rate. This is achievable when high-efficiency guide-RNAs (gRNAs) for the sites can be identified and the sites allow for efficient homologous recombination.
A study by Schwartz et al. (2019) presented a method for large-scale screening of gRNAs with high efficiency in Y. lipolytica [47], which can be applied to search for efficient gRNAs from candidate sites. This can be done for example by transforming a pool of gRNA candidates that target a set of integration sites to a strain and its ku70
161 Chapter 6
mutant. This is then followed by outgrowing the transformants in selective liquid
media to allow enrichment of cells bearing inefficient gRNA sequences, as
transformants containing the more efficient gRNAs have higher death probability due
to more frequent DSB. The gRNA plasmids from the outgrowth culture are then
extracted and sequenced via the next-generation sequencing. Efficient gRNA
sequences can be identified by looking for sequences with lower abundance in the ku70
mutant population relative to that of in the parent strain. Once the gRNA sequences
for efficient cutting are identified, sites with high HR probability can be screened by
using short homology arms (e.g. 100-250 bp).
6.5. Perspective on fatty acid analysis
Analysis of fatty-acid derived compounds today is frequently done with
methods based on gas chromatography (GC) [48 51]. They require either or both lipid
extraction and derivatization into fatty-acid methyl esters (FAMEs) which are
laborious and time-consuming and difficult to be used for screening many strains. A
solution to shorten the derivatization time was presented by Khoomrung et al. (2012)
using microwave-assisted heating. The method could shorten derivatization time from
1-2 hours into only as short as 11 minutes [52]. Although these methods have enabled
accurate and precise quantification for fatty acid species, they could not distinguish
between lipid classes (e.g. triacylglycerol, phosphatidylcholine, etc) and therefore
provide limited insights.
Avoiding derivatization step altogether and quantifying different lipid species
can be both addressed by a shotgun lipidomics method described by Ejsing et al.
(2008) [53]. The method employed quadrupole time-of-flight mass spectrometry and
linear ion trap-orbitrap mass spectrometry to quantify unique lipid species belonging
to broad lipid classes [53]. However, as this method require two different mass
162 Chapter 6
spectrometry equipment, it might not be easily applied in many research labs due to
huge investments for purchasing the equipment as well as the costs for service and
maintenance.
A method by Khoomrung et al. (2013) seems to bridge the trade-offs between
low cost and high-resolution in lipid analysis [54]. In addition to fast FAME analysis
described by Khoomrung et al. (2012), the method also includes quantification of up
to 10 lipid classes, which comprises rapid microwave-assisted lipid extraction followed
by hydrophilic interaction liquid chromatography with charged aerosol detector
(HILIC-CAD). In this method, unlike the shotgun-lipidomics method, each lipid class
is quantified as a whole, as their constituents elute as a single peak during HILIC-CAD
analysis.
Lastly, fluorophore lipid-probes (such as BODIPY) [55] could be used when a
lipid-probe that preferentially bound to the product of interest could be identified.
This can be particularly useful for the first-round screening of a large number of
enzyme variants, where the purpose is to identify distinguish top-performing enzymes
rather than quantification.
6.6. Towards better lactone production in Y. lipolytica
6.6.1. Optimizing hydroxylation based on bacterial hydratases
In Chapter 4, bacterial hydratases were chosen as they catalyze the
hydroxylation of double bonds in fatty acids. The challenge, however, is the
accessibility of free fatty acids (FFA) for the hydratases since the hydratases localized
in the cytosol while the hydrophobic nature of FFA might make it not abundant in the
cytosol. It is further complicated by the yet unresolved mechanisms of activation of
exogenous FFA in Y. lipolytica [8]. As has been elaborated in Chapter 4, the bacterial
163 Chapter 6 hydratases have poor kinetic performance. This implies the need for screening hydratases with better activities or protein engineering to improve the turnover and the affinity of the enzymes.
Another possible solution is to fuse the hydratase with a thioesterase. The thioesterase will catalyze the hydrolysis of acyl-CoAs into FFAs [56], which are then hydroxylated by the hydratase. Since acyl-CoAs are soluble, they are theoretically more accessible in the cytosol than the free fatty acids. The fusion should increase the probability of the resulting FFA to be hydroxylated by hydratase. Another possible solution is overexpression of phospholipases, such as phospholipase B (PLB), and triacylglycerol lipase (TGL), which liberate free fatty acids from membrane phospholipids and the lipid body. The hydratase could also be tethered with the lipases for better proximity with the FFAs.
6.6.2. Tighter control of -oxidation
Deletion of POX1-6 abolished the acyl-CoA oxidase activity in vitro assays
(Figure 4.3b). The insertion of specific acyl-CoA oxidases into pox1-6 background changed the acyl-CoA oxidase activity towards different acyl-CoAs, with decreased activities towards acyl-CoAs shorter than 12 carbons (Figure 4.3c). This strategy was employed as described in Chapter 4 to prevent the degradation of the lactones.
However, degradation of -dodecalactone in the bioreactor culture was still observed
(Figure 4.6a), suggesting that the acyl-CoA oxidase activity towards C12-CoA was still too high.
This can be tackled by tuning down the expression of acyl-CoA oxidase by using a weaker promoter or transcriptional inhibition via CRISPRi. Furthermore, a large screening of acyl-CoA oxidase (POX) variants could be useful to find enzymes with a higher substrate specificity.
164 Chapter 6
6.6.3. Lactone production with triacylglycerol as substrate
The overarching goal of producing lactones in Y. lipolytica is to enable the production of lactone from industrial side-streams such as from oil-milling factories, food processing industry, or oleochemical factories. The fatty residues of these industries are mainly in the form of triacylglycerols (TAG) [57,58]. Although overproducing extracellular lipase is an intuitive approach to boost TAG utilization, - decalactone production from castor oil was not improved [59]. An alternative metabolic engineering approach would be to optimize the availability of intracellular free fatty acids, as described in section 6.6.1 above. In this strategy, TAGs will be utilized and assimilated via native pathways into membrane lipid or lipid bodies, where they will be subsequently cleaved into FFAs via the action of PLB or TGL.
6.6.4. Lactone production de novo
Some fungi such as Ashbya gossypii [60] and Aureobasidium pullulans [61] can naturally produce lactones. However, their complete biosynthetic pathway has not been elucidated and A. pullulans secreted various lactone compounds [62], which could complicate downstream processing. Y. lipolytica can utilize glycerol efficiently
[63], which is a by-product in biodiesel and oleochemical industries. Therefore, it is interesting to explore the possibilities for producing lactones from glycerol.
The first option is by using polyketide synthase (PKS) pathways. It is proposed that massoia lactone production in A. pullulans originated from PKS-derived hydroxy fatty acids [64]. A recent study identified the gene encoding the PKS that could be responsible for massoia lactone production in Aureobasidium melanogenum [65].
Since previous studies showed that heterologous fungal PKS can be functionally expressed in S. cerevisiae [66,67], expression of the A. melanogenum PKS in Y. lipolytica might result in massoia lactone production.
165 Chapter 6
Another alternative is by using bacterial hydratase coupled with thioesterase
or lipases (PLB or TGL). The idea is similar to what has been described above, where
FFAs are made available through hydrolysis of acyl-CoA, phospholipids, and TAG by
thioesterase, PLB, and TAG, respectively. The required acyl backbone for the
hydratase reactions, such as oleate and linoleate, can be made more abundant by
overexpression of ∆9-desaturase and ∆12-desaturase.
6.7. Utilizing lipid remodeling pathways and enzymes for
optimizing polyunsaturated fatty acids
One potential use of Y. lipolytica in the future is to realize the microbial
production of oils rich in certain fatty acids [68]. Chapter 5, along with other studies
[48,69,70] demonstrated how unusual- and polyunsaturated fatty acids could be
accumulated at a high level in Y. lipolytica, with the aim to reduce dependency on their
natural sources. To be economically feasible, obtaining the desired lipid profile alone
is not sufficient as these oils have been available at low prices due to their well-
established supply chains and production methods [71]. Higher lipid content and
productivity are therefore required.
Such pursuit requires not only the expression of acyl-modifying enzymes (e.g.
desaturases, elongases, hydroxylases) and diacylglycerol acyltransferases (DGATs),
but also enzymes that promotes remodeling of lipids (e.g. O-acyltransferases other
than DGATs and phospholipases) [72,73,69]. The latter group of enzymes, which will
subsequently be referred to as acyl-remodeling enzymes, enhance the substrate
availability for the acyl-modifying enzymes through the exchange of acyl chains or
head groups of membrane lipids [72]. Acyl-remodeling pathways provide a shortcut
for the incorporation of acyl-CoA into membrane lipids (illustrated in Figure 5.1.) and
166 Chapter 6
increase the proportion of diacylglycerols (DAGs) with modified acyl chains [73].
Some of these enzymes, such as phospholipase A2 and phospholipase C, are substrate-
specific [74,75].
Relative to DGATs, acyl-editing enzymes are relatively underexploited in
engineering yeast for the production of lipids, as most of the existing studies have
focused largely on increasing lipid titer and proof-of-concept of unusual fatty acid
production. Therefore, characterizing multiple acyl-editing enzymes and their
combinations could pave the way for realizing the productions of unusual and poly-
unsaturated fatty acids.
Lastly, de novo synthesis of glycerolipids is initiated by glycerol-3-phosphate
acyltransferase (GPAT) and lysophosphatidylglycerol acyltransferase (LPAT) (Figure
5.1). Variants of these enzymes indeed showed preferences for certain acyl-CoAs
(reviewed in [76]). It is therefore advantageous to express GPAT and LPAT that
preferentially incorporate the desired acyl chain.
6.8. Conclusion
There are many possible solutions for the challenges in realizing more efficient
engineering Y. lipolytica. The tools and methods developed in the past could serve as
starting points to develop better tools. Expanding the search of solutions into studies
for S. cerevisiae and other species has resulted in useful tools and methods and should
be continued.
6.9. Acknowledgments
The authors have received funding from the European Union's Horizon 2020
research and innovation programme under the Marie Sk odowska-Curie grant
167 Chapter 6
agreement No 722287 (PAcMEN) and the financial support from the Novo Nordisk
Foundation. We thank Javier Sáez Sáez for providing the Memote output for Y.
lipolytica GSMs, Jonathan Dahlin for inputs on the physiology and strain construction
sections, and David Romero Suarez for the discussion regarding expression of fungal
PKS in yeast.
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