Photosynthetic Fuel for Heterologous Enzymes: the Role of Electron Carrier Proteins

Home , Ferredoxin hydrogenase, Glutaredoxin, Thioredoxin reductase

Photosynth Res DOI 10.1007/s11120-017-0364-0

REVIEW

Photosynthetic fuel for heterologous enzymes: the role of electron carrier proteins

Silas Busck Mellor1 · Konstantinos Vavitsas1 · Agnieszka Zygadlo Nielsen1 · Poul Erik Jensen1

Received: 3 October 2016 / Accepted: 27 February 2017 © Springer Science+Business Media Dordrecht 2017

Abstract Plants, cyanobacteria, and algae generate a sur- Introduction plus of redox power through photosynthesis, which makes them attractive for biotechnological exploitations. While Oxygenic photosynthesis uses the energy from absorbed central metabolism consumes most of the energy, pathways photons for the synthesis of ATP and the generation of introduced through metabolic engineering can also tap into reducing power in the form of NADPH. Most of this this source of reducing power. Recent work on the meta- energy is subsequently used to incorporate CO2 into tri- bolic engineering of photosynthetic organisms has shown ose phosphate molecules, which form the basis of anabolic that the electron carriers such as ferredoxin and flavodoxin and catabolic reactions. The core of photosynthetic energy can be used to couple heterologous enzymes to photo- metabolism, the conversion of light energy into chemi- synthetic reducing power. Because these proteins have a cal energy, has attracted research efforts from many disci- plethora of interaction partners and rely on electrostatically plines, and consequently the chemical and physical princi- steered complex formation, they form productive electron ples are known in considerable detail (Witt 1996; Nelson transfer complexes with non-native enzymes. A hand- and Yocum 2006; Umena et al. 2011; Mazor et al. 2015; ful of examples demonstrate channeling of photosynthetic Ago et al. 2016). electrons to drive the activity of heterologous enzymes, The electrochemistry of photosynthesis is unique in and these focus mainly on hydrogenases and cytochrome employing the most extreme redox potentials recorded in P450s. However, competition from native pathways and biological systems: that of the photo-oxidized photosystem inefficient electron transfer rates present major obstacles, II [Em= +1120 mV (Diner and Rappaport 2002)] and of which limit the productivity of heterologous reactions cou- the photo-excited photosystem I [Em= −1000 mV (Brettel pled to photosynthesis. We discuss specific approaches to 1997)]. Furthermore, the redox state of the photosynthetic address these bottlenecks and ensure high productivity of apparatus is a major regulator of cell metabolism in plants, such enzymes in a photosynthetic context. algae, and cyanobacteria, e.g., by controlling the light activation of biosynthetic pathways via the ferredoxin/thiore- Keywords Ferredoxin · Flavodoxin · Synthetic biology · doxin, NADP/thioredoxin, and glutathione/glutaredoxin Metabolic engineering · Cytochrome P450 · Photosynthesis regulatory systems (Buchanan and Balmer 2005; Stenbaek and Jensen 2010; Yoshida and Hisabori 2016). Besides carbon fixation and redox regulation, cells channel photosynthetic reducing power towards a multitude of biological processes, but it can also be directed towards * Poul Erik Jensen non-native enzyme activities. This review focuses on how [email protected] to exploit photosynthesis as a reductive driving force for heterologous reactions. We first introduce and briefly 1 Copenhagen Plant Science Center, Center for Synthetic Biology ‘bioSYNergy’, Department of Plant describe the major electron carrier proteins involved in and Environmental Sciences, University of Copenhagen, photosynthetic reactions and their interactions with partner Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark enzymes. Next, we cover the work done using the electron

Vol.:(0123456789)1 3 Photosynth Res carrier ferredoxin to direct reducing power towards the bio- electrons from photosystem I to ferredoxin–NADP+ reduc- synthesis of hydrogen and a handful of target chemicals. tase (FNR). As ferredoxin is a one-electron carrier, reduc- Finally, we discuss how redox coupling may be improved, tion of NADP+ to NADPH requires two sequential electron and why photosynthetic electron transport holds an impor- transfers from ferredoxin to FNR (Hanke and Mulo 2013). tant place in metabolic engineering and synthetic biology Ferredoxin–FNR and ferredoxin–photosystem I interac- applications. tions have been studied extensively through structural and kinetic techniques coupled with site-specific mutagenesis and involve prominent contributions from electrostatic Characteristics of electron carrier proteins interactions (Fig. 1) (Hurley et al. 2006; Sétif 2006). involved in photosynthetic electron transport Midpoint potentials of reduced ferredoxins range from −300 to −430 mV (Cammack et al. 1977), and those of Ferredoxin ferredoxins in photosynthetic electron transfer typically fall in the more negative end of this range. This permits the Photosynthetic electron transport requires small soluble reduction of NADP+ to NADPH (−320 mV) and allows electron carrier proteins to transfer reducing power from them to act as a hub distributing electrons towards central the cytochrome b6f complex to photosystem I and from metabolic reactions in plants and cyanobacteria [reviewed photosystem I to various electron acceptors/acceptor pro- by (Hanke and Mulo 2013; Cassier-Chauvat and Chau- teins (Fig. 1). The latter is mostly carried out by ferre- vat 2014; Goss and Hanke 2014)]. Ferredoxin also plays doxins, which comprise a ubiquitous and highly diverse a key role in cyclic electron transport, which balances group of small, 6–13 kDa, iron–sulfur proteins, includ- the production of ATP and NADPH by recycling elec- ing 2Fe–2S, 3Fe–4S, 4Fe–4S, and single Fe (rubredoxin) trons through the plastoquinone pool and the cytochrome cluster configurations (Tagawa and Arnon 1968; Sticht b6f complex (Yamori and Shikanai 2016). Photosynthetic and Rösch 1998). Plant and cyanobacterial ferredoxins use organisms have several ferredoxin isoforms, with distinct a 2Fe–2S cluster coordinated by four conserved cysteines roles (Hanke et al. 2004; Cassier-Chauvat and Chauvat and act as the primary electron transfer protein for relaying 2014). Their central role in distributing reducing power

Positive charge + NADP FNR Fld Negative charge

NADPH Fd

Stroma

PQH2 PQ Lumen Photosystem I Cyt c Cytochrome b f 6 Photosystem II 6 H2O½O2

Fig. 1 Schematic illustration of the photosynthetic electron transfer tion sites. Crystal structures used are as follows: photosystem II from apparatus in and around the thylakoid membrane. Arrows indicate Thermosynechococcus vulcanus (PDB: 4UB6), Cytochrome b6f from linear electron transport during photosynthetic activity, starting with Chlamydomonas reinhardtii (PDB: 1Q90), Cytochrome (Cyt) c6 the oxidation of water and ending with the reduction of NADP+ to from Synechococcus elongatus (PDB: 1C6S), plastocyanin (PC) from NADPH. A dashed arrow indicates back-donation of electrons via Populus nigra (PDB: 4DP1), photosystem I and ferredoxin–NADP+ the cytochrome b6f complex during cyclic electron transport. Pro- reductase (FNR) from Pisum sativum (PDB: 4Y28 and 1QG0, respec- teins are shown by their electrostatic surface potentials, which were tively), flavodoxin (Fld) fromSynechococcus sp. PCC 6301 (PDB: calculated using the APBS plugin (Baker et al. 2001) and visualized 1CZN), and ferredoxin (Fd) from Spinacia oleracea (PDB: 1A70). in PyMol. Red or blue dashed lines show the polarities of interac- PQ/PQH2 plastoquinone/plastoquinol

1 3 Photosynth Res derived from the photosynthetic apparatus, together with is absolutely necessary for photosynthetic growth in their negative redox potentials and innate ability to transfer land plants (Weigel et al. 2003), but it can be replaced by electrons to many different acceptor proteins, makes ferre- cytochrome c6 in cyanobacteria (Zhang et al. 1994; Clarke doxins the most explored electron donors for heterologous and Campbell 1996). redox enzymes, as will be described below. Cytochrome c6 proteins are small, 9–12 kDa, and diverse heme-containing proteins, found in electron trans- Flavodoxin port chains in prokaryotic and eukaryotic organisms, with redox potentials similar to that of plastocyanin (+320 mV Flavodoxins are functional homologs of ferredoxins and for Synechocystis sp. PCC 6803 c6) (Kerfeld and Krog- use flavin mononucleotide (FMN) as their redox cofactor. mann 1998; Cho et al. 1999; Howe et al. 2006). While most The flavodoxins are larger than ferredoxins, usually around cyanobacteria and green algae produce both plastocyanin 15–20 kDa, and are absent in plants, probably because fla- and cytochrome c6 (De La Rosa et al. 2002), some cyano- vodoxin genes of the ancestors of land plants were lost dur- bacteria (such as Arthrospira platensis) use cytochrome ing adaptation to iron-rich coastal environments (Karlusich c6 instead of plastocyanin (Sandmann 1986). Like plasto- et al. 2014). That said, some enzymes, e.g., diflavin reduc- cyanin, cytochrome c6 relies on electrostatic interactions tases, possess FMN-binding domains structurally homolo- for electron transfer, although the polarity of these can dif- gous to flavodoxins (Medina 2009), and the flavodoxin fer between plants and cyanobacteria (De La Rosa et al. fold, like the ferredoxin fold, is ancient and figures among 2002). Although a cytochrome c6 isoform was discovered the nine most ancient protein folds (Caetano-Anollés et al. in plants (Gupta et al. 2002; Wastl et al. 2002), it does not 2007). Despite the fact that flavodoxin and ferredoxin share seem functionally homologous to plant plastocyanin and no sequence or structural homology, they possess many possesses a much more reducing midpoint potential of common biochemical characteristics, namely low isoelec- +140 mV (Molina-Heredia et al. 2003). tric points, comparable redox potentials, and similar electron transfer kinetics towards both photosystem I and FNR Role of electrostatics in electron transfer protein (Bottin and Lagoutte 1992; Hurley et al. 2006; Sétif 2006; interactions Goñi et al. 2008, 2009). Their electrostatic surface potentials can be closely aligned, which suggests that much of Electron transfer between proteins is very fast (10 6–1013 their functional equivalence stems from similar means of s−1) and requires that electron donor and acceptor pro- interaction (Fig. 1) (Ullmann et al. 2000). Although FMN teins form a transient complex bringing their redox cofac- is a 2-electron carrier, in flavodoxins they carry out one- tors within <14 Å (Page et al. 1999, 2003). A feature of electron transfers by transitioning between the 2-electron the small electron carrier proteins described above, and reduced hydroquinone and an unusually stable semiquinone particularly well described for ferredoxin and flavodoxin, is form (Hamdane et al. 2009; Medina 2009). Flavodoxins, the ability to transfer electrons to multiple partners, which like ferredoxins, interact promiscuously with many interac- requires them to make relatively unspecific interactions tion partners and appear to interact with even lower speci- (Crowley and Carrondo 2004). This is evident from the ficity (Goñi et al. 2008). Therefore, flavodoxins also make strength of their interactions compared to those found more promising electron donors for heterologously expressed generally in protein–protein complexes (Fig. 2a) (Kastritis redox enzymes, though they are comparatively less studied et al. 2011). All electron transfer complexes have dissocia- in this context. tion constants (KD) clustering in the µM–mM range and thus comprise some of the weakest known protein–protein Plastocyanin and cytochrome c interactions. A combination of electrostatic attraction, and structural complementarity allow close association and fast Plastocyanin is a small 10 kDa copper-binding protein, dissociation (Fig. 2b), which ensures fast electron trans- found in the thylakoid lumen, that mediates electron transfer in such weak complexes (Crowley and Ubbink 2003). fer from cytochrome b6f to photosystem I (Gross 1993; These interactions require a fine-tuned balance between Redinbo et al. 1994). It shows a relatively oxidizing redox hydrophobic and electrostatic contributions (Kinoshita potential of around +370 mV (Sanderson et al. 1986). et al. 2015). Plants encode two plastocyanin isoforms, the major one Electron transfer partners have distinct and oppo- of which constitutes 90% of the total plastocyanin pool sitely charged surfaces surrounding their interacting areas (Abdel-Ghany 2009). They are negatively charged, like (Figs. 1, 2b), which play key roles in the association. Ini- ferredoxins and flavodoxins, and interact with positively tially, they mediate long-range attraction between oppos- charged sites on the cytochrome b6f complex and photosys- ing protein dipole moments (analogous to the attraction of tem I (Fig. 1) (Gross 1993). The presence of plastocyanin opposite poles on two magnets), which orients the proteins

1 3 Photosynth Res

ab20 ionic attraction

e- e- ) 15

-1 Desolvation, encounter complex formation

10 Tight complex (kcal mole

g formation, electron transfer bindin

G Fd/Fld:Photosystem I

-Δ 5 Fd/Fld:FNR Fd/Fld:Cytochrome P450 Other e- e- Dissociation, 0 resolvation 10-14 10-12 10-10 10-8 10-6 10-4 10-2 1

KD (M)

Fig. 2 Electron transfer complexes involve distinct affinities and drogenase. The ΔG values for the 10 redox complexes were calcu- mechanisms of association. a Plot of free energy of binding (−ΔG) lated according to Kastritis et al. (2011). b The formation of an elec- as a function of dissociation constant (KD) for 154 characterized pro- tron transfer complex involves electrostatics, complementary surface tein–protein complexes. The dataset comprises 144 protein–protein shape, and hydrophobic interactions. Initially, long-range electrostatic complexes (Kastritis et al. 2011), 6 of which are electron transfer attractions between charged residues near the interacting surfaces complexes, publically available from the Protein–Protein Interaction (blue or red dots show positive or negative charges) pre-orient the Affinity Database 2.0 website (http://bmm.crick.ac.uk/~bmmadmin/ interacting partners. This leads to an encounter complex, in which Affinity/), and 10 additional electron transfer complexes (Holden the proteins share a common solvent shell (dotted lines). Formation et al. 1994; Furukawa and Morishima 2001; Sétif 2006; Goñi et al. of the final electron transfer complex involves sampling of possible 2009; Farooq and Roberts 2010). Gray crosses show non-redox com- interactions by surface diffusion and is stabilized mainly by interac- plexes, and colored circles depict the 16 electron transfer complexes. tions between small hydrophobic surface patches. Fast dissociation of The four electron transfer complexes not involving ferredoxin or fla- the complex occurs because resolvation of surface charges is thermo- vodoxin are cytochrome c:cytochrome c peroxidase (2 complexes), dynamically favorable thioredoxin:thioredoxin reductase, and amicyanin:methylamine dehy- for complex formation. They also increase the rate of asso- electron transfer complexes, and their small sizes make it ciation beyond what would result from diffusion alone possible to interact in a promiscuous manner with different (Northrup and Erickson 1992; Sheinerman et al. 2000). partners. Because these surface charges must desolvate, i.e., shed water molecules, when forming an electron transfer complex, their presence incurs a penalty in binding energy. Metabolic engineering by coupling enzyme activity Finally, charge resolvation provides a thermodynamic driv- to photosynthetic electron transport ing force that speeds up dissociation (Fig. 2b) (Crowley and Ubbink 2003). These effects are key to ensure rapid Photosynthesis supplies reducing power continuously, first electron transfer and lead to a characteristic bell-shaped in the form of ferredoxin, and subsequently NADPH during dependence of electron transfer rates on ionic strength light, and these cellular reducing currencies provide con- (Sétif 2006). Because electrostatic surfaces play such venient points to couple novel pathways to photosynthetic prominent roles in determining the connectivity of electron electron transport. Due to growing interest in using cyano- transfer proteins (Fig. 1), polarities and sizes of interac- bacteria for the production of commodity and high-value tion surfaces are key considerations when designing novel chemicals, engineering of NADPH- and NADH-depend- redox chains. In addition to electrostatic forces, hydropho- ent enzymes into cyanobacteria is relatively well stud- bic patches located centrally on interacting surfaces medi- ied (Nielsen et al. 2016). Several demonstrations of g L−1 ate apolar interactions, with effects opposite to the electro- scale titers by engineered pathways show that, with careful statics described above, i.e., thermodynamically favorable optimization, cyanobacteria are promising hosts for heter- desolvation and shielding upon complex formation (Reddy ologous NADPH-dependent metabolite production. These et al. 1998; Sheinerman et al. 2000; Crowley and Ubbink examples include the production of ethanol using alcohol 2003; Crowley and Carrondo 2004; Lee et al. 2011a, b). dehydrogenase (Gao et al. 2012), stereoselective reduc- These patches constitute the main stabilizing factor to the tion of various alkenes by an enoate reductase (Köninger

1 3 Photosynth Res et al. 2016), as well as complex multistep pathways like an and is oxygen sensitive. Further, its ability to carry out optimized methylerythritol phosphate pathway for isoprene reductive coupling of a wide variety of small alkenes, production (Gao et al. 2016) and a de novo engineered alkynes as well as N-, S-, and O-containing hydrocar- 2,3-butanediol biosynthetic pathway (Oliver et al. 2013). bons makes this enzyme highly interesting for metabolic However, some redox enzymes require dedicated reduc- engineering (Seefeldt et al. 2013). Yang and colleagues tases to supply reducing power for catalysis, which may expanded the scope of nitrogenase reactions by introducing complicate engineering because of a need to adjust both two amino acid substitutions in the substrate-binding pock- enzyme and reductase activity (Paddon and Keasling 2014). ets, conferring an ability to reduce CO2 to methane or cou- These enzymes may instead be coupled directly to the elec- ple it with acetylene to form the polymer precursor propyl- tron carrier proteins used by the photosynthetic apparatus, ene (Yang et al. 2012). Recently, this engineered enzyme and this is the main topic of the following discussion. was shown to produce methane in a light-dependent man- The idea of redirecting photosynthetic electrons towards ner upon expression in the purple non-sulfur bacterium products of biotechnological interest has mostly been Rhodopseudomonas palustris (Fixen et al. 2016). explored for biohydrogen production (Eq. 1), namely by + + + + e− → + + + N2 8H 16ATP 8 2NH3 H2 16ADP 16pi algal and cyanobacterial hydrogenases, which already depend on ferredoxin (Ghirardi et al. 2007). These hydro- (2) genases fall into two classes, containing [Fe–Fe] (algal) or Coupling heterologous enzymes to photosynthesis via [Ni–Fe] (cyanobacterial) redox centers. Different strategies reduced ferredoxin is a relatively new idea. It has been demonstrated for only a handful of enzymes (Table 1), all have been employed to address O2 inhibition of hydrogenase activity and improve their competitiveness towards of which are cytochrome P450s. Cytochrome P450s metab- reduced ferredoxin, ranging from gene fusion with ferre- olize drugs and toxins in humans (Nebert et al. 2013) and doxin to reconfigure the iron–sulfur clusters, to conver- feature prominently in the biosynthesis of plant special- sion of a hydrogen-consuming uptake hydrogenase into a ized metabolites of commercial interest such as terpenoids, hydrogen-generating one (Yacoby et al. 2011; Dubini and alkaloids, and polyphenols (Renault et al. 2014; Pateraki Ghirardi 2014; Eilenberg et al. 2016; Raleiras et al. 2016). et al. 2015). Consequently, they attract considerable atten- tion for use in biotechnology and are frequently expressed + + e− → 2H 2 H2 (1) in heterologous organisms and engineered for improved Nitrogenase enzymes convert atmospheric nitrogen to activity and recognition of new substrates (Fasan 2012; ammonia in a highly energy- and redox-dependent mecha- Lassen et al. 2014b). The canonical cytochrome P450 reac- nism (Eq. 2), which also produces hydrogen. Like hydroge- tion is the stereospecific hydroxylation on a substrate car- nase, nitrogenase obtains its redox power from ferredoxin bon atom (Eq. 3):

Table 1 Examples of biotechnological applications coupling enzymes to photosynthetic reducing power via ferredoxin or flavodoxin Enzyme Organism References

Heme enzymes CYP79A1/CYP71E1 Sorghum bicolor Jensen et al. (2011); Lassen et al. (2014a); Gangl et al. (2015); Wlodarczyk et al. (2016); Gnanasekaran et al. (2016); Mellor et al. (2016) CYP720B4 Picea sitchensis Gnanasekaran et al. (2015) CYP124 Mycobacterium tuberculosis Jensen et al. (2012) CYP106A2a Bacillus megaterium ATCC 13368 Goñi et al. (2009) CYP1A1 Rattus norvegicus Berepiki et al. (2016) CYP71AV1b Artemisia annua Saxena et al. (2014); Fuentes et al. (2016) Iron–sulfur cluster enzymes [FeFe]-hydrogenase C. reinhardtii Reviewed by Dubini and Ghirardi (2014) Methane-fixing nitrogenase Azotobacter vinelandii Fixen et al. (2016) Hydroxymethylbutenyl-diphosphate Thermosynechococcus elongatus Gao et al. (2016) synthase (IspG) Non-heme diiron enzymes Aldehyde-deformylating oxygenasea Synechococcus elongatus PCC7942 Zhang et al. (2013) a Ferredoxin reduced by the action of FNR and NADPH in vitro instead of photosystem I b Co-expressed in chloroplasts with the NADPH-dependent cognate reductase

1 3 Photosynth Res

+ + e− + + → + It may be possible to redirect reducing equivalents from RH O2 2 2H ROH H2O. (3) the photosynthetic electron transport chain at other points P450s may, however, catalyze many other reactions, than photosystem I/ferredoxin. However, since most pho- such as N- or S-hydroxylations, epoxidations, dealkyla- tosynthetic electron transfer steps occur within large mul- tions, and many more (Sono et al. 1996). Catalysis takes tiprotein complexes embedded in the thylakoid membrane, place by a complex cycle, wherein reductive activation only a few mobile electron carriers are accessible. Besides of O produces a highly reactive ferryl-oxo radical that 2 ferredoxin, these consist of plastocyanin or cytochrome c6 attacks the substrate R–H bond (Rittle and Green 2010; in the thylakoid lumen and the PQ/PQH2 pool in the thyla- Munro et al. 2013a, b). The required electrons are usu- koid membrane. The lumenal localization of plastocyanin ally provided by dedicated P450 reductases although some and cytochrome c6 insulates them from most of the bio- P450s obtain the necessary reducing power from H 2O2 or synthetic machinery in the stroma of chloroplasts or the by oxidizing NADH directly (Munro et al. 2007b). Eukary- cytoplasm of cyanobacteria, and their positive midpoint otic P450 reductases are diflavin enzymes that constitute potentials of +340 to +390 mV (Antal et al. 2013) render fused FAD/NADPH-binding (FNR-like) and FMN-binding them mostly unusable for metabolic engineering. However, (flavodoxin-like) domains, joined by a short linker peptide a mutant of photosystem II containing a single Lys-to-Glu and anchored to the ER membrane by an N-terminal trans- mutation near the QA site on the luminal surface of the PSII membrane domain (Porter and Kasper 1986; Porter 1991; complex was recently shown to pass electrons directly to Wang et al. 1997). Electron flow is reversed relative to that a (positively charged) mitochondrial cytochrome c (Larom + of NADP photoreduction in photosynthesis: first a 2-elec- et al. 2010, 2015). While cytochrome c is an unsuitable tron reduction of FAD by NAPDH, followed by a sequen- carrier to support many biosynthetic reactions, the fact tial 1-electron transfer via FMN onto the P450 heme (Ver- that this novel tapping point harbors a potential of −30 to milion et al. 1981; Murataliev et al. 2004). The midpoint −80 mV (Antal et al. 2013) and allows electron transfer potentials of the FMN semiquinone/hydroquinone couple to positively charged carrier proteins makes it possible to range from −250 to −130 mV (Das and Sligar 2009), con- assemble entirely novel redox chains from this point. The sistent with a reversed direction of electron flow compared consequences of introducing sinks so early in the photosyn- to flavodoxins and ferredoxins involved in photosynthesis. thetic electron transfer chain are still unclear, but uncou- Consequently, both ferredoxin and flavodoxin should be pling reducing power supply from the proton gradient thermodynamically adequate to drive P450 catalysis. might be advantageous for highly reductive but low ATP- Ferredoxin was already suggested 20 years ago to enable dependent pathways (Oliver and Atsumi 2014). coupling of cytochrome P450 activity to photosynthesis Plastoquinone is lipid soluble and resides within the thy- (Lacour and Ohkawa 1999), but the first demonstrations lakoid membrane leaflet. Although this localization makes of this idea were only recently realized in vitro (Jensen it relatively inaccessible, several different enzymatic pro- et al. 2011) and in vivo (Nielsen et al. 2013; Gnanasekaran cesses use it as either a reductant or an oxidant, and the et al. 2015, 2016; Wlodarczyk et al. 2016). Although PQ/PQH2 ratio also transmits the redox status of the pho- work on redirecting photosynthetic reducing equivalents tosynthetic apparatus (Rintamaki et al. 2000; Puthiyaveetil remains mostly proof of concept, light-driven P450 activ- et al. 2008). As the redox state of the plastoquinol pool ity has yielded reasonable titers. Expression of the bio- affects overall photosynthetic electron transport rates, sev- synthetic pathway consisting of two P450s (CYP79A1 eral enzymes are thought to regulate it. For example, the and CYP71E1) and a glucosyltransferase (UGT85B1) cytochrome bd oxidase of cyanobacteria and the plant plas- −1 −1 yielded 1–2 mg g DW and 3–5 mg L , respectively, of tid terminal oxidase oxidize plastoquinol to dissipate excess the cyanogenic glucoside dhurrin in N. tabacum chloro- reducing power (Carol and Kuntz 2001; Berry et al. 2002), plast (Gnanasekaran et al. 2016) or in Synechocystis PCC while PGR5/PGRL1 and NAD(P)H dehydrogenase reduce 6803 (Wlodarczyk et al. 2016). In another example, 40 µg it as part of cyclic electron transport to increase ATP pro- −1 g DW of the antimicrobial diterpenoid isopimaric acid duction (Iwai et al. 2010; Kramer and Evans 2011). The resulted from transiently expressing CYP720B4 from Sitka pool also participates in the biosynthesis of carotenoids spruce in tobacco (Gnanasekaran et al. 2015). While the and chlorophyll. Plastoquinone is reduced during turno- physiological consequences of coupling cytochrome P450 ver of the enzymes phytoene desaturase and ζ-carotene enzymes to photosynthetic reducing power are not yet fully desaturase, and plastoquinol oxidized in the conversion of explored, introduction of the mammalian CYP1A1 into Mg-protoporphyrin IX monomethyl ester to protochloro- Synechococcus elongatus PCC 7002 caused a 31% increase phyllide (Berthold and Stenmark 2003; Steccanella et al. in photosynthetic electron transfer rates (Berepiki et al. 2015). While few plastoquinol-dependent enzymes of inter- 2016), which suggests that the photosynthetic machinery est to the biotechnological community are known, both has the capacity to tolerate increased sink activity. aerobic cyclase and plastid terminal oxidase belong to the

1 3 Photosynth Res non-heme diiron oxygenase family (Berthold and Stenmark on ferredoxin interaction partners of Synechocystis and 2003). Another member of this family is the aldehyde- Chlamydomonas ferredoxins have identified ~200 pos- deformylating oxygenase of cyanobacteria, which can be sible ferredoxin-dependent proteins (Hanke et al. 2011; reduced by charged derivatives of hydrophobic phenazine. Peden et al. 2013; Cassier-Chauvat and Chauvat 2014). Since these compounds possess midpoint potentials (+63 This highlights the incompleteness of our knowledge to +155 mV) (Ksenzhek et al. 1977) similar to those of of ferredoxin-dependent processes and underlines the the plastoquinone/plastoquinol couple (+80 to +110 mV) importance of competitiveness for heterologous enzymes. (Antal et al. 2013), it raises the possibility that these Consistent with the pivotal importance of NADPH as a enzymes could also be reduced by plastoquinol. central reducing currency, FNR consumes the most electrons from ferredoxin (Fig. 3a) to provide continuous NADPH supply for CO2 fixation, which accounts for up Native competition for photosynthetic reducing to 80% of photosynthetic reducing power (Holfgrefe et al. power 1997; Backhausen et al. 2000). Reductive activation and inactivation of chloroplast enzymes of, e.g., the Calvin When attempting to redirect reducing power from ferre- cycle, starch biosynthesis, and nitrogen assimilation path- doxin towards heterologous enzymes, making these ways by the chloroplast thioredoxin system in response enzymes competitive against the many native processes to the redox status of the photosynthetic apparatus also that compete for electrons from reduced ferredoxin constitute a major electron sink (Fig. 3b) (Lemaire (Fig. 3) constitutes a major challenge. While the pri- et al. 2007; Meyer et al. 2009). Thioredoxins are small mary electron sinks—such as NADP + photoreduction— disulfide reductases that reduce protein disulfides by are relatively well understood, recent proteomic studies a thiol–disulfide exchange mechanism. Chloroplast thioredoxins are kept in a reduced state by the enzyme ferredoxin:thioredoxin reductase (Dai 2000). While the inner exact proportion of reducing power being consumed by envelope thioredoxin redox regulation is not well established, it is LUT1

CA probably substantial; protein thiols must be maintained in

O Pa O a reduced state to ensure continuous assimilatory activity NiR Fd- (Scheibe and Dietz 2012). SiR GOGAT e Nitrogen assimilation in chloroplasts and cyanobacteria d (Fig. 3c) involves two ferredoxin-dependent enzymes— c FTR nitrite reductase and glutamine oxoglutarate aminotrans- b Fd ferase (Knaff and Hirasawa1991 )—and consumes up to - a FNR f e 20% of photosynthetic reducing equivalents (Champigny stroma 1995; Holfgrefe et al. 1997). Reduction of sulfite to sulfide relies on a ferredoxin-dependent sulfite reductase to sup- PGR5/ NDH PSI ply sulfur for cysteine, methionine, and iron–sulfur clus- PGRL1 ter synthesis (Fig. 3d) (Yonekura-Sakakibara et al. 2000). thylakoid Biosynthesis and breakdown of chlorophyll and carot- lumen enoid pigments at the chloroplast inner envelope leaflet (Fig. 3e) are also known or thought to involve ferredoxin- Fig. 3 Diagram depicting selected native ferredoxin-dependent path- dependent enzymes such as chlorophyll a oxygenase and ways in chloroplasts and cyanobacteria. Arrows show electron flow, pheophorbide a oxygenase (Reinbothe et al. 2006), and dashed arrows according to Scheibe and Dietz (2012), indicate path- two bacterial-type carotenoid hydroxylating cytochromes ways of cyclic electron transport. a Photoreduction of NADP+ by ferredoxin:NADP+ reductase (FNR). b Redox regulation of enzyme P450s LUT1 and LUT5 (Tian et al. 2004; Kim and Del- activities through the reduction of cysteine thiols via the thioredoxin laPenna 2006; Kim et al. 2010). Likewise, biosynthesis of system and ferredoxin:thioredoxin reductase (FTR). c Fixation of phycobilisome bilin pigments in cyanobacteria requires nitrogen by nitrite reductase (NiR) and Fd-dependent glutamine oxo- ferredoxin-dependent heme oxygenase and bilin reductase glutarate aminotransferase (Fd-GOGAT). d Sulfate assimilation by sulfite reductase (SiR).e Biosynthesis of chlorophyll and carotenoid enzymes (Dammeyer and Frankenberg-Dinkel 2008). Such pigments, e.g., by chlorophyll a oxidase (CAO) and the carotene high abundance of enzymes that compete for reduced ferre- epsilon-monooxygenase CYP97C1 (LUT1) as well as degradation of doxin seriously limits the amount of reducing power that chlorophylls, such as by pheophorbide a oxygenase (PaO). f Cyclic can be redirected (Yacoby et al. 2011; Nielsen et al. 2013; electron transport through back-donation of electrons to the PQ pool, mediated by the enzymes NAD(P)H dehydrogenase (Fd-dependent) Mellor et al. 2016). Therefore, optimization of light-driven or the action of the proteins PGR5/PGRL1 biosynthesis requires improving the competitiveness of

1 3 Photosynth Res heterologous enzymes, strategies towards which will be the Another approach to direct more reducing power to het- topic of the final section. erologous pathways involves downregulating the competing pathways. This was demonstrated in Synechocystis PCC 6803, where nitrate and nitrite reductases were knocked down, and in Chlamydomonas strains carrying RuBisCo How can we improve coupling to photosynthetic mutants with reduced activity, which had 140-fold and electron transport? 10- to 15-fold increased hydrogen productivities, respectively (Baebprasert et al. 2011; Pinto et al. 2013). Similarly, The development of strategies that enable heterologous knocking out the genes coding for the proteins PGR5 and enzymes to compete with natural processes for reduced PGRL1, which mediate the back-donation of electrons from ferredoxin benefits from understanding how partition- ferredoxin into the PQ pool during cyclic electron flow ing of electrons occurs between these natural acceptors in (Fig. 3f), caused a 10-fold increase in hydrogen production photosynthetic organisms. Different acceptor enzymes are in Chlamydomonas (Steinbeck et al. 2015). While protec- arranged hierarchically (Backhausen et al. 2000). While tion of the hydrogenase due to higher oxygen consumption we do not know all the details of this hierarchy, NADP + partly explained this increase, removal of a pathway that photoreduction (and CO 2 fixation) sits at its top, followed dissipates excess reducing power may also have forced the by fixation of nitrogen and sulfur, and redox regula- cell to channel excess reducing power to the hydrogenase tion (Fig. 3). Affinity towards ferredoxin appears to be an as an alternative sink. Likewise, metabolic network analy- important positional determinant in this hierarchy. Evi- ses have suggested that the removal of electron-dissipating dence for this includes differing affinities of photosystem and NADPH-consuming pathways from cyanobacteria I, FNR, and sulfite reductase (listed in order of increas- could increase productivity for ethanol and other biofuels ing KM) towards the major leaf ferredoxin in Arabidop- (Erdrich et al. 2014; Shabestary and Hudson 2016). sis (Hanke et al. 2004), and also between ferredoxin and Improved competitiveness for reducing power may also FNR isoforms found in the roots and leaves of maize result from fusing enzymes with ferredoxin. This should (Matsumura et al. 1999; Onda et al. 2000). FNR probably reduce the likelihood that ferredoxin will interact with sequesters most reducing power, in part by being the most other proteins transferring electrons to the fusion partner abundant partner—its concentration is estimated to be and possibly accelerate overall association rates. The bac- 90–170 µM in cyanobacteria (Moal and Lagoutte 2012), terial cytochrome P450 BM3 (Fig. 4a) is a well-studied and because it relies on an abundant and readily available natural P450-reductase fusion, which contains both P450 substrate. In contrast to NADP+, which is constantly recy- and diflavin reductase moieties within a single polypep- cled during metabolic reactions, the availability of nitrate/ tide. It achieves the highest catalytic rates measured for nitrite is more variable. This requires the nitrogen assimila- a cytochrome P450 because of very fast electron trans- tion machinery to compete with constitutive sinks and be fer between the closely co-localized reductase and P450 poised for nitrogen assimilation upon availability. Consist- domains (Munro et al. 2002; Lewis and Arnold 2009). Con- ent with this, the addition of high concentrations of nitrite sequently, it has inspired many efforts to mimic its unique to isolated chloroplasts reduces CO 2 fixation, and ferre- domain organization (Munro et al. 2007a; Girhard et al. doxin-dependent nitrite reductase has a 100-fold lower KM 2015). Fusion with ferredoxin as a method of coupling for ferredoxin than FNR, which probably increases its com- enzymes to photosynthesis was first demonstrated in vitro petitiveness for reducing equivalents (Holfgrefe et al. 1997; on the Chlamydomonas hydrogenase HydA (Fig. 4b). This Backhausen et al. 2000; Hanke et al. 2004; Hirasawa et al. yielded 2.5-fold higher hydrogen productivities in the pres- 2009). Therefore, ferredoxins engineered towards increased ence of competing FNR and NADP+ in vitro (Yacoby et al. affinity towards heterologous enzymes should increase 2011) and also showed 4.5-fold increased hydrogenase the flow of reducing power into such pathways. Ferre- activity in vivo (Eilenberg et al. 2016). In a similar man- doxin variants must first and foremost retain high affinity ner, fusing ferredoxin with the first cytochrome P450 of towards photosystem I, which complicates any manipula- the dhurrin biosynthetic pathway, CYP79A1 (Fig. 4b), via tion of affinity. Directed evolution—the generation of ran- a flexible linker resulted in an enzyme less susceptible to dom or semi-random variants of an enzyme combined with competition by the presence of FNR, evident from a 1.5- screening of large variant libraries for desirable parameters fold improved light-driven activity in vivo (Mellor et al. (Dalby 2014)—might be well suited to tackle this task. 2016). This P450 was also fused directly to the M-subunit However, this approach is contingent on the development of photosystem I in the cyanobacterium Synechococcus of high-throughput methods to screen ferredoxin-depend- sp. PCC 7002 to place it close to the source of reduced ent enzyme activity, approaches to which were recently ferredoxin. This approach yielded active CYP79A1, but proposed (Atkinson et al. 2016). the effectiveness was not compared with that of non-fused

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ab+ CYP102A1 P450 Fld FNR NADPH NADP e- FNR + C NADPH C + e- + Fd NADPH NADP R-OH+H2O H2ase - - - e Fd e e Fd FAD FAD e- - 52 - FMN e FMN CYP e HEME HEME R-OH+H2O N N

52 5 DUDFKLGRQDWH NFDW PLQ Photosystem I

Fig. 4 Fusions between cytochrome P450 and reductases. a The the FNR-like domain to transfer electrons to the heme of the P450 natural fusion enzyme P450 BM3 (CYP102A1) from Bacillus meg- domain (redox cofactors shown in uppercase in the lower part of the aterium is the most active P450 characterized to date. It comprises diagram). b Fusing ferredoxin to either the P450 CYP79A1 (Mellor both an N-terminal heme-containing P450 domain and a C-terminal et al. 2016) or the hydrogenase HydA (Yacoby et al. 2011; Eilenberg CPR-like diflavin reductase domain in a single polypeptide (shown et al. 2016) improves their ability to sequester electrons in the pres- schematically on the top). Catalysis involves two sequential electron ence of competing FNR, demonstrating the viability of this strategy transfers via its flavodoxin-like FMN-binding domain flanked by for both soluble and thylakoid membrane-localized enzymes. Black linkers connecting it to the P450 and FNR-like FAD- and NADPH- wavy lines indicate flexible Gly–Ser-rich linkers, which were used in binding domains. This domain rapidly reorients upon reduction by both studies. Blue arrows indicate electron flow in both panels enzyme (Lassen et al. 2014a). While results from fusion some cases, by as much as +150 mV—through site- with ferredoxin in photosynthetic systems are still some- directed mutagenesis (Hoover et al. 1999; Hurley et al. what limited, examples from non-photosynthetic systems 2006). Electrons passed onto such an engineered ferre- suggest that the approach could become viable in photo- doxin would essentially be unavailable for NADP + pho- synthetic organisms. Particularly, noteworthy examples are toreduction, thereby increasing the likelihood of electron a 20-fold increase in in vivo activity of the soybean P450 transfer to heterologous enzymes. In this context, it is isoflavone synthase fused withCatharanthus roseus P450 also important to note that a highly negative redox poten- reductase (Leonard and Koffas 2007) and a >100-fold tial is not always desirable. Electron transfer reactions increase in activity of the bacterial P450cam anchored to a between donors (e.g., ferredoxin) and acceptors, which reductase system, consisting of a ferredoxin and ferredoxin differ greatly in redox potentials, risk falling within reductase via a trimeric protein scaffold (Hirakawa and the Marcus inverted region (Marcus 1992), resulting in Nagamune 2010). slower electron transfer rates than intuitively expected. Photosynthetic organisms express different ferredoxin Thus, the potential of ferredoxins notwithstanding, it isoforms involved in distinct physiological processes. may be advisable to select the optimal redox partner for These include a root isoform, which supports NADP + a given redox enzyme from a panel of carriers with suit- reduction less efficiently partly because it possesses a able redox potentials, and taking into consideration redox redox potential close to the NADP+/NADPH couple potentials of potential off-target enzymes. (Matsumura et al. 1997; Hanke et al. 2004; Terauchi et al. Photosynthetic organisms have many attractive fea- 2009). Because ferredoxins involved in photosynthesis tures for metabolic engineering, particularly in relation support the reduction of NADP+ to NADPH, they have to pathways involving cytochrome P450s and other redox redox potentials much more negative than those required enzymes, because they supply both the reducing power and for the reduction of, e.g., cytochrome P450s, whose the O2 required for turnover. The importance of electron reductases possess redox potentials of −200 to −270 mV carriers in biotechnological applications of photosynthetic (Das and Sligar 2009; Simtchouk et al. 2013). This sug- organisms is apparent from several recent examples where gests another avenue of optimization, namely control successful redox tuning increased the productivity of novel of electron partitioning by introducing ferredoxins with metabolic routes. Thus, there is much unexplored poten- more positive redox potentials, which would then be less tial for redirecting photosynthetic reducing power, and able to support NADP+ photoreduction. Redox potentials engineering of photosynthetic electron transfer chains to of both ferredoxin and flavodoxin have been modified—in accommodate heterologous enzymes could be an important

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