Photosynthetic fuel for heterologous enzymes: the role of electron carrier proteins
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Plants, cyanobacteria, and algae generate a surplus of redox power through photosynthesis, which makes them attractive for biotechnological exploitations. While central metabolism consumes most of the energy, pathways introduced through metabolic engineering can also tap into this source of reducing power. Recent work on the metabolic engineering of photosynthetic organisms has shown that the electron carriers such as ferredoxin and flavodoxin can be used to couple heterologous enzymes to photosynthetic reducing power. Because these proteins have a plethora of interaction partners and rely on electrostatically steered complex formation, they form productive electron transfer complexes with non-native enzymes. A handful of examples demonstrate channeling of photosynthetic electrons to drive the activity of heterologous enzymes, and these focus mainly on hydrogenases and cytochrome P450s. However, competition from native pathways and inefficient electron transfer rates present major obstacles, which limit the productivity of heterologous reactions coupled to photosynthesis. We discuss specific approaches to address these bottlenecks and ensure high productivity of such enzymes in a photosynthetic context.
KeywordsFerredoxin Flavodoxin Synthetic biology Metabolic engineering Cytochrome P450 Photosynthesis
Oxygenic photosynthesis uses the energy from absorbed photons for the synthesis of ATP and the generation of reducing power in the form of NADPH. Most of this energy is subsequently used to incorporate CO2 into triose phosphate molecules, which form the basis of anabolic and catabolic reactions. The core of photosynthetic energy metabolism, the conversion of light energy into chemical energy, has attracted research efforts from many disciplines, and consequently the chemical and physical principles are known in considerable detail (Witt 1996; Nelson and Yocum 2006; Umena et al. 2011; Mazor et al. 2015; Ago et al. 2016).
The electrochemistry of photosynthesis is unique in employing the most extreme redox potentials recorded in biological systems: that of the photo-oxidized photosystem II [E m= +1120 mV (Diner and Rappaport 2002)] and of the photo-excited photosystem I [E m= −1000 mV (Brettel 1997)]. Furthermore, the redox state of the photosynthetic apparatus is a major regulator of cell metabolism in plants, algae, and cyanobacteria, e.g., by controlling the light activation of biosynthetic pathways via the ferredoxin/thioredoxin, NADP/thioredoxin, and glutathione/glutaredoxin 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 non-native enzyme activities. This review focuses on how to exploit photosynthesis as a reductive driving force for heterologous reactions. We first introduce and briefly describe the major electron carrier proteins involved in photosynthetic reactions and their interactions with partner enzymes. Next, we cover the work done using the electron carrier ferredoxin to direct reducing power towards the biosynthesis of hydrogen and a handful of target chemicals. Finally, we discuss how redox coupling may be improved, and why photosynthetic electron transport holds an important place in metabolic engineering and synthetic biology applications.
Characteristics of electron carrier proteins involved in photosynthetic electron transport
Photosynthetic electron transport requires small soluble electron carrier proteins to transfer reducing power from the cytochrome b6f complex to photosystem I and from photosystem I to various electron acceptors/acceptor proteins (Fig. 1). The latter is mostly carried out by ferredoxins, which comprise a ubiquitous and highly diverse group of small, 6–13 kDa, iron–sulfur proteins, including 2Fe–2S, 3Fe–4S, 4Fe–4S, and single Fe (rubredoxin) cluster configurations (Tagawa and Arnon 1968; Sticht and Rösch 1998). Plant and cyanobacterial ferredoxins use a 2Fe–2S cluster coordinated by four conserved cysteines and act as the primary electron transfer protein for relaying electrons from photosystem I to ferredoxin–NADP+ reductase (FNR). As ferredoxin is a one-electron carrier, reduction of NADP+ to NADPH requires two sequential electron transfers from ferredoxin to FNR (Hanke and Mulo 2013). Ferredoxin–FNR and ferredoxin–photosystem I interactions have been studied extensively through structural and kinetic techniques coupled with site-specific mutagenesis and involve prominent contributions from electrostatic interactions (Fig. 1) (Hurley et al. 2006; Sétif 2006).
Midpoint potentials of reduced ferredoxins range from −300 to −430 mV (Cammack et al. 1977), and those of ferredoxins in photosynthetic electron transfer typically fall in the more negative end of this range. This permits the reduction of NADP+ to NADPH (−320 mV) and allows them to act as a hub distributing electrons towards central metabolic reactions in plants and cyanobacteria [reviewed by (Hanke and Mulo 2013; Cassier-Chauvat and Chauvat 2014; Goss and Hanke 2014)]. Ferredoxin also plays a key role in cyclic electron transport, which balances the production of ATP and NADPH by recycling electrons through the plastoquinone pool and the cytochrome b6f complex (Yamori and Shikanai 2016). Photosynthetic organisms have several ferredoxin isoforms, with distinct roles (Hanke et al. 2004; Cassier-Chauvat and Chauvat 2014). Their central role in distributing reducing power derived from the photosynthetic apparatus, together with their negative redox potentials and innate ability to transfer electrons to many different acceptor proteins, makes ferredoxins the most explored electron donors for heterologous redox enzymes, as will be described below.
Flavodoxins are functional homologs of ferredoxins and use flavin mononucleotide (FMN) as their redox cofactor. The flavodoxins are larger than ferredoxins, usually around 15–20 kDa, and are absent in plants, probably because flavodoxin genes of the ancestors of land plants were lost during adaptation to iron-rich coastal environments (Karlusich et al. 2014). That said, some enzymes, e.g., diflavin reductases, possess FMN-binding domains structurally homologous to flavodoxins (Medina 2009), and the flavodoxin fold, like the ferredoxin fold, is ancient and figures among the nine most ancient protein folds (Caetano-Anollés et al. 2007). Despite the fact that flavodoxin and ferredoxin share no sequence or structural homology, they possess many common biochemical characteristics, namely low isoelectric points, comparable redox potentials, and similar electron transfer kinetics towards both photosystem I and FNR (Bottin and Lagoutte 1992; Hurley et al. 2006; Sétif 2006; Goñi et al. 2008, 2009). Their electrostatic surface potentials can be closely aligned, which suggests that much of their functional equivalence stems from similar means of interaction (Fig. 1) (Ullmann et al. 2000). Although FMN is a 2-electron carrier, in flavodoxins they carry out one-electron transfers by transitioning between the 2-electron reduced hydroquinone and an unusually stable semiquinone form (Hamdane et al. 2009; Medina 2009). Flavodoxins, like ferredoxins, interact promiscuously with many interaction partners and appear to interact with even lower specificity (Goñi et al. 2008). Therefore, flavodoxins also make promising electron donors for heterologously expressed redox enzymes, though they are comparatively less studied in this context.
Plastocyanin and cytochrome c
Plastocyanin is a small 10 kDa copper-binding protein, found in the thylakoid lumen, that mediates electron transfer from cytochrome b6f to photosystem I (Gross 1993; Redinbo et al. 1994). It shows a relatively oxidizing redox potential of around +370 mV (Sanderson et al. 1986). Plants encode two plastocyanin isoforms, the major one of which constitutes 90% of the total plastocyanin pool (Abdel-Ghany 2009). They are negatively charged, like ferredoxins and flavodoxins, and interact with positively charged sites on the cytochrome b6f complex and photosystem I (Fig. 1) (Gross 1993). The presence of plastocyanin is absolutely necessary for photosynthetic growth in land plants (Weigel et al. 2003), but it can be replaced by cytochrome c6 in cyanobacteria (Zhang et al. 1994; Clarke and Campbell 1996).
Cytochrome c6 proteins are small, 9–12 kDa, and diverse heme-containing proteins, found in electron transport chains in prokaryotic and eukaryotic organisms, with redox potentials similar to that of plastocyanin (+320 mV for Synechocystis sp. PCC 6803 c6) (Kerfeld and Krogmann 1998; Cho et al. 1999; Howe et al. 2006). While most cyanobacteria and green algae produce both plastocyanin and cytochrome c6 (De La Rosa et al. 2002), some cyanobacteria (such as Arthrospira platensis) use cytochrome c6 instead of plastocyanin (Sandmann 1986). Like plastocyanin, cytochrome c6 relies on electrostatic interactions for electron transfer, although the polarity of these can differ between plants and cyanobacteria (De La Rosa et al. 2002). Although a cytochrome c6 isoform was discovered in plants (Gupta et al. 2002; Wastl et al. 2002), it does not seem functionally homologous to plant plastocyanin and possesses a much more reducing midpoint potential of +140 mV (Molina-Heredia et al. 2003).
Role of electrostatics in electron transfer protein interactions
Electron transfer between proteins is very fast (106–1013 s−1) and requires that electron donor and acceptor proteins form a transient complex bringing their redox cofactors within <14 Å (Page et al. 1999, 2003). A feature of the small electron carrier proteins described above, and particularly well described for ferredoxin and flavodoxin, is the ability to transfer electrons to multiple partners, which requires them to make relatively unspecific interactions (Crowley and Carrondo 2004). This is evident from the strength of their interactions compared to those found more generally in protein–protein complexes (Fig. 2a) (Kastritis et al. 2011). All electron transfer complexes have dissociation constants (K D) clustering in the µM–mM range and thus comprise some of the weakest known protein–protein interactions. A combination of electrostatic attraction, and structural complementarity allow close association and fast dissociation (Fig. 2b), which ensures fast electron transfer in such weak complexes (Crowley and Ubbink 2003). These interactions require a fine-tuned balance between hydrophobic and electrostatic contributions (Kinoshita et al. 2015).
Electron transfer partners have distinct and oppositely charged surfaces surrounding their interacting areas (Figs. 1, 2b), which play key roles in the association. Initially, they mediate long-range attraction between opposing protein dipole moments (analogous to the attraction of opposite poles on two magnets), which orients the proteins for complex formation. They also increase the rate of association beyond what would result from diffusion alone (Northrup and Erickson 1992; Sheinerman et al. 2000). 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. Finally, charge resolvation provides a thermodynamic driving force that speeds up dissociation (Fig. 2b) (Crowley and Ubbink 2003). These effects are key to ensure rapid electron transfer and lead to a characteristic bell-shaped dependence of electron transfer rates on ionic strength (Sétif 2006). Because electrostatic surfaces play such prominent roles in determining the connectivity of electron transfer proteins (Fig. 1), polarities and sizes of interaction surfaces are key considerations when designing novel redox chains. In addition to electrostatic forces, hydrophobic patches located centrally on interacting surfaces mediate apolar interactions, with effects opposite to the electrostatics described above, i.e., thermodynamically favorable desolvation and shielding upon complex formation (Reddy et al. 1998; Sheinerman et al. 2000; Crowley and Ubbink 2003; Crowley and Carrondo 2004; Lee et al. 2011a, b). These patches constitute the main stabilizing factor to the electron transfer complexes, and their small sizes make it possible to interact in a promiscuous manner with different partners.
Metabolic engineering by coupling enzyme activity to photosynthetic electron transport
Photosynthesis supplies reducing power continuously, first in the form of ferredoxin, and subsequently NADPH during light, and these cellular reducing currencies provide convenient points to couple novel pathways to photosynthetic electron transport. Due to growing interest in using cyanobacteria for the production of commodity and high-value chemicals, engineering of NADPH- and NADH-dependent enzymes into cyanobacteria is relatively well studied (Nielsen et al. 2016). Several demonstrations of g L−1 scale titers by engineered pathways show that, with careful optimization, cyanobacteria are promising hosts for heterologous NADPH-dependent metabolite production. These examples include the production of ethanol using alcohol dehydrogenase (Gao et al. 2012), stereoselective reduction of various alkenes by an enoate reductase (Köninger et al. 2016), as well as complex multistep pathways like an optimized methylerythritol phosphate pathway for isoprene production (Gao et al. 2016) and a de novo engineered 2,3-butanediol biosynthetic pathway (Oliver et al. 2013). However, some redox enzymes require dedicated reductases to supply reducing power for catalysis, which may complicate engineering because of a need to adjust both enzyme and reductase activity (Paddon and Keasling 2014). These enzymes may instead be coupled directly to the electron carrier proteins used by the photosynthetic apparatus, and this is the main topic of the following discussion.
Examples of biotechnological applications coupling enzymes to photosynthetic reducing power via ferredoxin or flavodoxin
Gnanasekaran et al. (2015)
Jensen et al. (2012)
Bacillus megaterium ATCC 13368
Goñi et al. (2009)
Berepiki et al. (2016)
Iron–sulfur cluster enzymes
Reviewed by Dubini and Ghirardi (2014)
Fixen et al. (2016)
Hydroxymethylbutenyl-diphosphate synthase (IspG)
Gao et al. (2016)
Non-heme diiron enzymes
Synechococcus elongatus PCC7942
Zhang et al. (2013)
P450s may, however, catalyze many other reactions, such as N- or S-hydroxylations, epoxidations, dealkylations, and many more (Sono et al. 1996). Catalysis takes place by a complex cycle, wherein reductive activation of O2 produces a highly reactive ferryl-oxo radical that attacks the substrate R–H bond (Rittle and Green 2010; Munro et al. 2013a, b). The required electrons are usually provided by dedicated P450 reductases although some P450s obtain the necessary reducing power from H2O2 or by oxidizing NADH directly (Munro et al. 2007b). Eukaryotic P450 reductases are diflavin enzymes that constitute fused FAD/NADPH-binding (FNR-like) and FMN-binding (flavodoxin-like) domains, joined by a short linker peptide and anchored to the ER membrane by an N-terminal transmembrane domain (Porter and Kasper 1986; Porter 1991; Wang et al. 1997). Electron flow is reversed relative to that of NADP+ photoreduction in photosynthesis: first a 2-electron reduction of FAD by NAPDH, followed by a sequential 1-electron transfer via FMN onto the P450 heme (Vermilion et al. 1981; Murataliev et al. 2004). The midpoint potentials of the FMN semiquinone/hydroquinone couple range from −250 to −130 mV (Das and Sligar 2009), consistent with a reversed direction of electron flow compared to flavodoxins and ferredoxins involved in photosynthesis. Consequently, both ferredoxin and flavodoxin should be thermodynamically adequate to drive P450 catalysis.
Ferredoxin was already suggested 20 years ago to enable coupling of cytochrome P450 activity to photosynthesis (Lacour and Ohkawa 1999), but the first demonstrations of this idea were only recently realized in vitro (Jensen et al. 2011) and in vivo (Nielsen et al. 2013; Gnanasekaran et al. 2015, 2016; Wlodarczyk et al. 2016). Although work on redirecting photosynthetic reducing equivalents remains mostly proof of concept, light-driven P450 activity has yielded reasonable titers. Expression of the biosynthetic pathway consisting of two P450s (CYP79A1 and CYP71E1) and a glucosyltransferase (UGT85B1) yielded 1–2 mg g DW−1 and 3–5 mg L−1, respectively, of the cyanogenic glucoside dhurrin in N. tabacum chloroplast (Gnanasekaran et al. 2016) or in Synechocystis PCC 6803 (Wlodarczyk et al. 2016). In another example, 40 µg g DW−1 of the antimicrobial diterpenoid isopimaric acid resulted from transiently expressing CYP720B4 from Sitka spruce in tobacco (Gnanasekaran et al. 2015). While the physiological consequences of coupling cytochrome P450 enzymes to photosynthetic reducing power are not yet fully explored, introduction of the mammalian CYP1A1 into Synechococcus elongatus PCC 7002 caused a 31% increase in photosynthetic electron transfer rates (Berepiki et al. 2016), which suggests that the photosynthetic machinery has the capacity to tolerate increased sink activity.
It may be possible to redirect reducing equivalents from the photosynthetic electron transport chain at other points than photosystem I/ferredoxin. However, since most photosynthetic electron transfer steps occur within large multiprotein complexes embedded in the thylakoid membrane, only a few mobile electron carriers are accessible. Besides ferredoxin, these consist of plastocyanin or cytochrome c6 in the thylakoid lumen and the PQ/PQH2 pool in the thylakoid membrane. The lumenal localization of plastocyanin and cytochrome c6 insulates them from most of the biosynthetic machinery in the stroma of chloroplasts or the cytoplasm of cyanobacteria, and their positive midpoint potentials of +340 to +390 mV (Antal et al. 2013) render them mostly unusable for metabolic engineering. However, a mutant of photosystem II containing a single Lys-to-Glu mutation near the Q A site on the luminal surface of the PSII complex was recently shown to pass electrons directly to a (positively charged) mitochondrial cytochrome c (Larom et al. 2010, 2015). While cytochrome c is an unsuitable carrier to support many biosynthetic reactions, the fact that this novel tapping point harbors a potential of −30 to −80 mV (Antal et al. 2013) and allows electron transfer to positively charged carrier proteins makes it possible to assemble entirely novel redox chains from this point. The consequences of introducing sinks so early in the photosynthetic electron transfer chain are still unclear, but uncoupling reducing power supply from the proton gradient might be advantageous for highly reductive but low ATP-dependent pathways (Oliver and Atsumi 2014).
Plastoquinone is lipid soluble and resides within the thylakoid membrane leaflet. Although this localization makes it relatively inaccessible, several different enzymatic processes use it as either a reductant or an oxidant, and the PQ/PQH2 ratio also transmits the redox status of the photosynthetic apparatus (Rintamaki et al. 2000; Puthiyaveetil et al. 2008). As the redox state of the plastoquinol pool affects overall photosynthetic electron transport rates, several enzymes are thought to regulate it. For example, the cytochrome bd oxidase of cyanobacteria and the plant plastid terminal oxidase oxidize plastoquinol to dissipate excess reducing power (Carol and Kuntz 2001; Berry et al. 2002), while PGR5/PGRL1 and NAD(P)H dehydrogenase reduce it as part of cyclic electron transport to increase ATP production (Iwai et al. 2010; Kramer and Evans 2011). The pool also participates in the biosynthesis of carotenoids and chlorophyll. Plastoquinone is reduced during turnover of the enzymes phytoene desaturase and ζ-carotene desaturase, and plastoquinol oxidized in the conversion of Mg-protoporphyrin IX monomethyl ester to protochlorophyllide (Berthold and Stenmark 2003; Steccanella et al. 2015). While few plastoquinol-dependent enzymes of interest to the biotechnological community are known, both aerobic cyclase and plastid terminal oxidase belong to the non-heme diiron oxygenase family (Berthold and Stenmark 2003). Another member of this family is the aldehyde-deformylating oxygenase of cyanobacteria, which can be reduced by charged derivatives of hydrophobic phenazine. Since these compounds possess midpoint potentials (+63 to +155 mV) (Ksenzhek et al. 1977) similar to those of the plastoquinone/plastoquinol couple (+80 to +110 mV) (Antal et al. 2013), it raises the possibility that these enzymes could also be reduced by plastoquinol.
Native competition for photosynthetic reducing power
When attempting to redirect reducing power from ferredoxin towards heterologous enzymes, making these enzymes competitive against the many native processes that compete for electrons from reduced ferredoxin (Fig. 3) constitutes a major challenge. While the primary electron sinks—such as NADP+ photoreduction—are relatively well understood, recent proteomic studies on ferredoxin interaction partners of Synechocystis and Chlamydomonas ferredoxins have identified ~200 possible ferredoxin-dependent proteins (Hanke et al. 2011; Peden et al. 2013; Cassier-Chauvat and Chauvat 2014). This highlights the incompleteness of our knowledge of ferredoxin-dependent processes and underlines the importance of competitiveness for heterologous enzymes. Consistent with the pivotal importance of NADPH as a central reducing currency, FNR consumes the most electrons from ferredoxin (Fig. 3a) to provide continuous NADPH supply for CO2 fixation, which accounts for up to 80% of photosynthetic reducing power (Holfgrefe et al. 1997; Backhausen et al. 2000). Reductive activation and inactivation of chloroplast enzymes of, e.g., the Calvin cycle, starch biosynthesis, and nitrogen assimilation pathways by the chloroplast thioredoxin system in response to the redox status of the photosynthetic apparatus also constitute a major electron sink (Fig. 3b) (Lemaire et al. 2007; Meyer et al. 2009). Thioredoxins are small disulfide reductases that reduce protein disulfides by a thiol–disulfide exchange mechanism. Chloroplast thioredoxins are kept in a reduced state by the enzyme ferredoxin:thioredoxin reductase (Dai 2000). While the exact proportion of reducing power being consumed by thioredoxin redox regulation is not well established, it is probably substantial; protein thiols must be maintained in a reduced state to ensure continuous assimilatory activity (Scheibe and Dietz 2012).
Nitrogen assimilation in chloroplasts and cyanobacteria (Fig. 3c) involves two ferredoxin-dependent enzymes—nitrite reductase and glutamine oxoglutarate aminotransferase (Knaff and Hirasawa 1991)—and consumes up to 20% of photosynthetic reducing equivalents (Champigny 1995; Holfgrefe et al. 1997). Reduction of sulfite to sulfide relies on a ferredoxin-dependent sulfite reductase to supply sulfur for cysteine, methionine, and iron–sulfur cluster synthesis (Fig. 3d) (Yonekura-Sakakibara et al. 2000). Biosynthesis and breakdown of chlorophyll and carotenoid pigments at the chloroplast inner envelope leaflet (Fig. 3e) are also known or thought to involve ferredoxin-dependent enzymes such as chlorophyll a oxygenase and pheophorbide a oxygenase (Reinbothe et al. 2006), and two bacterial-type carotenoid hydroxylating cytochromes P450s LUT1 and LUT5 (Tian et al. 2004; Kim and DellaPenna 2006; Kim et al. 2010). Likewise, biosynthesis of phycobilisome bilin pigments in cyanobacteria requires ferredoxin-dependent heme oxygenase and bilin reductase enzymes (Dammeyer and Frankenberg-Dinkel 2008). Such high abundance of enzymes that compete for reduced ferredoxin seriously limits the amount of reducing power that can be redirected (Yacoby et al. 2011; Nielsen et al. 2013; Mellor et al. 2016). Therefore, optimization of light-driven biosynthesis requires improving the competitiveness of heterologous enzymes, strategies towards which will be the topic of the final section.
How can we improve coupling to photosynthetic electron transport?
The development of strategies that enable heterologous enzymes to compete with natural processes for reduced ferredoxin benefits from understanding how partitioning of electrons occurs between these natural acceptors in photosynthetic organisms. Different acceptor enzymes are arranged hierarchically (Backhausen et al. 2000). While we do not know all the details of this hierarchy, NADP+ photoreduction (and CO2 fixation) sits at its top, followed by fixation of nitrogen and sulfur, and redox regulation (Fig. 3). Affinity towards ferredoxin appears to be an important positional determinant in this hierarchy. Evidence for this includes differing affinities of photosystem I, FNR, and sulfite reductase (listed in order of increasing K M) towards the major leaf ferredoxin in Arabidopsis (Hanke et al. 2004), and also between ferredoxin and FNR isoforms found in the roots and leaves of maize (Matsumura et al. 1999; Onda et al. 2000). FNR probably sequesters most reducing power, in part by being the most abundant partner—its concentration is estimated to be 90–170 µM in cyanobacteria (Moal and Lagoutte 2012), and because it relies on an abundant and readily available substrate. In contrast to NADP+, which is constantly recycled during metabolic reactions, the availability of nitrate/nitrite is more variable. This requires the nitrogen assimilation machinery to compete with constitutive sinks and be poised for nitrogen assimilation upon availability. Consistent with this, the addition of high concentrations of nitrite to isolated chloroplasts reduces CO2 fixation, and ferredoxin-dependent nitrite reductase has a 100-fold lower K M for ferredoxin than FNR, which probably increases its competitiveness for reducing equivalents (Holfgrefe et al. 1997; Backhausen et al. 2000; Hanke et al. 2004; Hirasawa et al. 2009). Therefore, ferredoxins engineered towards increased affinity towards heterologous enzymes should increase the flow of reducing power into such pathways. Ferredoxin variants must first and foremost retain high affinity towards photosystem I, which complicates any manipulation of affinity. Directed evolution—the generation of random or semi-random variants of an enzyme combined with screening of large variant libraries for desirable parameters (Dalby 2014)—might be well suited to tackle this task. However, this approach is contingent on the development of high-throughput methods to screen ferredoxin-dependent enzyme activity, approaches to which were recently proposed (Atkinson et al. 2016).
Another approach to direct more reducing power to heterologous 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 mutants with reduced activity, which had 140-fold and 10- to 15-fold increased hydrogen productivities, respectively (Baebprasert et al. 2011; Pinto et al. 2013). Similarly, knocking out the genes coding for the proteins PGR5 and PGRL1, which mediate the back-donation of electrons from ferredoxin into the PQ pool during cyclic electron flow (Fig. 3f), caused a 10-fold increase in hydrogen production in Chlamydomonas (Steinbeck et al. 2015). While protection of the hydrogenase due to higher oxygen consumption partly explained this increase, removal of a pathway that dissipates excess reducing power may also have forced the cell to channel excess reducing power to the hydrogenase as an alternative sink. Likewise, metabolic network analyses have suggested that the removal of electron-dissipating and NADPH-consuming pathways from cyanobacteria could increase productivity for ethanol and other biofuels (Erdrich et al. 2014; Shabestary and Hudson 2016).
Improved competitiveness for reducing power may also result from fusing enzymes with ferredoxin. This should reduce the likelihood that ferredoxin will interact with other proteins transferring electrons to the fusion partner and possibly accelerate overall association rates. The bacterial cytochrome P450 BM3 (Fig. 4a) is a well-studied natural P450-reductase fusion, which contains both P450 and diflavin reductase moieties within a single polypeptide. It achieves the highest catalytic rates measured for a cytochrome P450 because of very fast electron transfer between the closely co-localized reductase and P450 domains (Munro et al. 2002; Lewis and Arnold 2009). Consequently, it has inspired many efforts to mimic its unique domain organization (Munro et al. 2007a; Girhard et al. 2015). Fusion with ferredoxin as a method of coupling enzymes to photosynthesis was first demonstrated in vitro on the Chlamydomonas hydrogenase HydA (Fig. 4b). This yielded 2.5-fold higher hydrogen productivities in the presence of competing FNR and NADP+ in vitro (Yacoby et al. 2011) and also showed 4.5-fold increased hydrogenase activity in vivo (Eilenberg et al. 2016). In a similar manner, fusing ferredoxin with the first cytochrome P450 of the dhurrin biosynthetic pathway, CYP79A1 (Fig. 4b), via a flexible linker resulted in an enzyme less susceptible to competition by the presence of FNR, evident from a 1.5-fold improved light-driven activity in vivo (Mellor et al. 2016). This P450 was also fused directly to the M-subunit of photosystem I in the cyanobacterium Synechococcus sp. PCC 7002 to place it close to the source of reduced ferredoxin. This approach yielded active CYP79A1, but the effectiveness was not compared with that of non-fused enzyme (Lassen et al. 2014a). While results from fusion with ferredoxin in photosynthetic systems are still somewhat limited, examples from non-photosynthetic systems suggest that the approach could become viable in photosynthetic organisms. Particularly, noteworthy examples are a 20-fold increase in in vivo activity of the soybean P450 isoflavone synthase fused with Catharanthus roseus P450 reductase (Leonard and Koffas 2007) and a >100-fold increase in activity of the bacterial P450cam anchored to a reductase system, consisting of a ferredoxin and ferredoxin reductase via a trimeric protein scaffold (Hirakawa and Nagamune 2010).
Photosynthetic organisms express different ferredoxin isoforms involved in distinct physiological processes. These include a root isoform, which supports NADP+ reduction less efficiently partly because it possesses a redox potential close to the NADP+/NADPH couple (Matsumura et al. 1997; Hanke et al. 2004; Terauchi et al. 2009). Because ferredoxins involved in photosynthesis support the reduction of NADP+ to NADPH, they have redox potentials much more negative than those required for the reduction of, e.g., cytochrome P450s, whose reductases possess redox potentials of −200 to −270 mV (Das and Sligar 2009; Simtchouk et al. 2013). This suggests another avenue of optimization, namely control of electron partitioning by introducing ferredoxins with more positive redox potentials, which would then be less able to support NADP+ photoreduction. Redox potentials of both ferredoxin and flavodoxin have been modified—in some cases, by as much as +150 mV—through site-directed mutagenesis (Hoover et al. 1999; Hurley et al. 2006). Electrons passed onto such an engineered ferredoxin would essentially be unavailable for NADP+ photoreduction, thereby increasing the likelihood of electron transfer to heterologous enzymes. In this context, it is also important to note that a highly negative redox potential is not always desirable. Electron transfer reactions between donors (e.g., ferredoxin) and acceptors, which differ greatly in redox potentials, risk falling within the Marcus inverted region (Marcus 1992), resulting in slower electron transfer rates than intuitively expected. Thus, the potential of ferredoxins notwithstanding, it may be advisable to select the optimal redox partner for a given redox enzyme from a panel of carriers with suitable redox potentials, and taking into consideration redox potentials of potential off-target enzymes.
Photosynthetic organisms have many attractive features for metabolic engineering, particularly in relation to pathways involving cytochrome P450s and other redox enzymes, because they supply both the reducing power and the O2 required for turnover. The importance of electron carriers in biotechnological applications of photosynthetic organisms is apparent from several recent examples where successful redox tuning increased the productivity of novel metabolic routes. Thus, there is much unexplored potential for redirecting photosynthetic reducing power, and engineering of photosynthetic electron transfer chains to accommodate heterologous enzymes could be an important aspect of successful metabolic engineering of photosynthetic organisms.
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