Genetic Engineering, Synthetic Biology and the LightReactions of Photosynthesis1[OPEN]
Dario Leister2
Plant Molecular Biology, Faculty of Biology, Ludwig-Maximilians-University Munich, D-82152Planegg-Martinsried, Germany
ORCID ID: 0000-0003-1897-8421 (D.L.).
Oxygenic photosynthesis is imperfect, and the evo-lutionarily conditioned patchwork nature of the lightreactions in plants provides ample scope for their im-provement (Leister, 2012; Blankenship and Chen, 2013).In fact, only around 40% of the incident solar energy isused for photosynthesis. Two obvious ways of reduc-ing energy loss are to expand the spectral band used forphotosynthesis and to shift saturation of the process tohigher light intensities. Indeed, even minor enhance-ments to the efficiency or stress resistance of the lightreactions of photosynthesis should have a positive im-pact on biomass production and yield (Leister, 2012;Blankenship and Chen, 2013; Long et al., 2015).
However, modifications to the essential structure ofthe light reactions of plant photosynthesis are currentlylimited by two main factors. One is the high degree ofconservation of their structural components, whichlimits the efficiency gains attainable by conventionalbreeding approaches (Dann and Leister, 2017). Thesecond is that the organization of these structuralcomponents into multiprotein complexes requires thesimultaneous tailoring of several proteins, some ofthem encoded by different genetic systems in differentsubcellular compartments (nucleus and chloroplasts) inland plants. Therefore, the successful modification ofthe light reactions of plant photosynthesis has beenlimited to a few cases. Similarly, modification of theactivity of auxiliary proteins involved in the regulationof the light reactions to enhance plant growth and yieldonly recently resulted in successful outcomes and islimited to a small subset of regulatory proteins (Pribilet al., 2010; Kromdijk et al., 2016; Głowacka et al., 2018).
In addition to enhancing the light reactions for im-proved biomass production and yield, concepts havebeen developed for the direct coupling of photosyn-thesis to other important pathways that are connectedindirectly to photosynthesis in natural systems, for in-stance, because they reside in different subcellularcompartments (Lassen et al., 2014b). Direct couplingwould be expected to boost the production of rare
compounds in cells and contribute to the biotechno-logical production of high-value compounds in vivo.
In vitro, it is possible to functionally link componentsof photosynthesis with entirely unrelated biotic orabiotic catalysts or with abiotic electrode materials. Infact, photosystem I (PSI) is naturally adapted for highlyefficient light harvesting and charge separation (Kargulet al., 2012; Nguyen and Bruce, 2014; Martin andFrymier, 2017) and has been described as the most ef-ficient natural nano-photochemical machine (Nelson,2009). Upon light excitation, PSI produces the mostpowerful naturally occurring reducing agent, P700*,which, together with an exceptionally long-livedcharge-separated state, provides sufficient driving
AADVANCES
• Individual photosynthetic proteins can be
exchanged between species, but the
replacement of proteins embedded in
photosynthetic multiprotein complexes is
complicated due to their “frozen metabolic
state.”
• Entire multiprotein (sub)complexes can be
exchanged via “synthetic photosynthetic
modules” that contain a sufficient number of
proteins to overcome the “frozen metabolic
state,” as well as all genetic elements and
auxiliary factors for their efficient expression,
biogenesis, and function.
• Overexpression of photosynthetic proteins that
are not organized in complexes can enhance
photosynthetic efficiency and growth.
• Photosynthesis can be coupled in vivo to
previously unrelated enzymes and pathways to
enable light-driven production of valuable
compounds.
• Photosystem I is a highly efficient and robust
nano-photochemical machine that can be
technically applied in vitro for the production of
hydrogen or electricity.
1This work was funded by the Deutsche Forschungsgemeinschaft(TRR 175 and GRK 2062).
2Author for contact: [email protected].[OPEN]Articles can be viewed without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.18.00360
778 Plant Physiology�, March 2019, Vol. 179, pp. 778–793, www.plantphysiol.org � 2019 American Society of Plant Biologists. All Rights Reserved. www.plantphysiol.orgon June 29, 2020 - Published by Downloaded from
force to reduce protons to H2 at neutral pH. PSI oper-ates with a quantum yield close to 1, and currently, nosynthetic system has approached its remarkable effi-ciency. Moreover, PSI preparations generally are ro-bust, especially those obtained from extremophilicmicroalgae (Kubota et al., 2010; Haniewicz et al., 2018).The superior qualities of PSI have stimulated strategiesdesigned to generate in vitro hybrids of PSI and varioustypes of redox-active catalysts or other materials.In sum, the light reactions of photosynthesis are a
prime target for genetic engineering and synthetic bi-ology approaches for three major reasons. (1) En-hancement of the process in vivo to increase theefficiency of light use promises to increase biomass andcrop yields. (2) Coupling of the light reactions of pho-tosynthesis to previously unconnected pathways willenable us to utilize the reducing power of the light re-actions directly to produce large amounts of high-valuecompounds in vivo. (3) The high efficiency and ro-bustness of PSI should allow it to be used in hybridswith biotic or abiotic components to generate hydro-gen, simple carbon-based solar fuels, or electricityin vitro. In this review, the background to and recentdevelopments in these three strategies are discussed.
NATURAL BUILDING BLOCKSOF PHOTOSYNTHESIS
During photosynthesis, carbon dioxide (CO2) isconverted into organic compounds, principally sugars,using sunlight as energy. In plants, algae, and cyano-bacteria, photosynthesis uses water as the electron do-nor for the chemical reduction of CO2 and releasesoxygen. Algae and plants derive from a lineage thatarose from an endosymbiotic relationship between aprotist and a cyanobacterium. Chloroplasts, the pho-tosynthetic organelles in modern plants, are in fact thedescendants of this ancient symbiotic cyanobacterium
and possess an internal membrane system that resem-bles the thylakoid membranes of modern-day cyano-bacteria. Indeed, during the evolutionary transitionfrom cyanobacteria to chloroplasts, the overall organi-zation and mode of action of the photosynthetic ma-chinery was retained (Box 1). However, significantchanges have occurred in the subunit composition ofphotosystems (Fig. 1), their posttranslational modifi-cation (Pesaresi et al., 2011), the harvesting of light en-ergy, pigment composition, and the regulation ofphotosynthesis (Box 2; Holt et al., 2004; Mullineauxand Emlyn-Jones, 2005; Rochaix, 2007; de Bianchiet al., 2010).Its evolutionary history makes photosynthesis well
suited for synthetic biology strategies. The evolution-ary diversification of the photosynthetic machineryhas provided a set of building blocks, ranging fromsingle proteins such as the soluble electron transporters(plastocyanin, cytochrome c6, flavodoxin, and ferre-doxin) to multiprotein complexes like photosystemsand antenna complexes (phycobiliosomes and light-harvesting complexes [LHCs]). In principle, the build-ing blocks should be interchangeable betweencyanobacteria, algae, and plants. Instances of theswapping of homologous photosynthetic proteins be-tween species by means of genetic engineering arediscussed in the next section to highlight the compli-cations associated with apparently straightforwardapproaches. The focus then shifts to genuinely syntheticapproaches, in which specific photosynthetic buildingblocks have been introduced into photosynthetic spe-cies that lack them. In the subsequent two sections, thecombination of nonphotosynthetic (bio-bio hybrids,using photosynthesis as an electron source for unre-lated biological processes) and nonbiological (bio-nanohybrids, using photosynthesis as an electron source fornonbiological processes) building blocks with compo-nents of the photosynthetic machinery is described. Anoverview of these approaches is provided in Figure 2.
Figure 1. Subunit composition of cya-nobacterial (Synechocystis) and plant(Arabidopsis) thylakoid multiproteincomplexes. Subunits specific to eitherthe cyanobacterium Synechocystis orthe flowering plant Arabidopsis are in-dicated by black shading; subunits withdomains specific to one group of orga-nisms are shown in dark gray, and con-served subunits are shown in light gray.c6, Cytochrome c6; Cyt b6f, cytochromeb6f complex; Fd, ferredoxin; FLV, fla-vodiiron protein; FNR, ferredoxin-NADP reductase; Fv, flavodoxin; LHCI(II), light-harvesting complex I (II). Forreasons of clarity, the ATP synthase andNAD(P)H dehydrogenase complex arenot shown.
Theoretically, it should be relatively easy to exchangeindividual conserved photosynthetic proteins betweendifferent species, even in such distantly related orga-nisms as cyanobacteria and plants. However, in prac-tice, these simple genetic engineering exercises can beproblematic (Table 1). Several individual proteins fromcyanobacterial PSII (D1, CP43, CP47, and PsbH) andPSI (PsaA) have indeed been genetically replaced bytheir plant counterparts with varying success. Syn-echocystis PCC6803 (Synechocystis) strains expressingthe D1 protein from Poa annua or PsbH frommaize (Zeamays) could still perform photosynthesis, albeit with
less efficiency than the wild-type strain (Nixon et al.,1991; Chiaramonte et al., 1999), but the replacement ofSynechocystis CP43 or CP47 by their homologs fromspinach (Spinacia oleracea) was incompatible with pho-toautotrophy (Carpenter et al., 1993; Vermaas et al.,1996). Similarly, in Synechocystis strains equippedwith Arabidopsis (Arabidopsis thaliana) PsaA, PSI func-tion was severely disrupted (Viola et al., 2014).
Such complications resulting from the replacement ofcore subunits of photosystems, despite their high sim-ilarity (78%–86% identity between the cyanobacterialand plant proteins; Table 1), reflect the so-called "frozenmetabolic state" of the photosynthetic multiproteincomplexes. This termwas coined byGimpel et al. (2016)but was introduced originally as "frozen metabolic
accident" by Shi et al. (2005). The frozen metabolic ac-cident concept refers to the observation that selectionhas not significantly altered biophysically and physio-logically inefficient photosynthetic proteins (includingthe D1 protein of PSII or Rubisco of the Calvin-Bensoncycle) over billions of years of evolution. In fact, bio-informatic analysis of photosynthetic cyanobacterialgenes suggests that the evolution rate of proteins at thecore of the photosynthetic apparatus is highly con-strained by protein-protein, protein-lipid, and protein-cofactor interactions (Shi et al., 2005). This provides aninternal selection pressure, conserving the sequence ofproteins in photosynthetic multiprotein complexes and,in the case of prokaryotes, also the genomic organiza-tion of their genes (Shi et al., 2005). The term frozenmetabolic accident was generalized to frozen metabolicstate by Gimpel et al. (2016) and implies that, in eachphotosynthetic species, slightly distinct modules of thecores of photosynthetic multiprotein complexes haveevolved that are optimized with respect to their in-trinsic interactions and that rarely tolerate the alterationof single proteins by exchange or mutation. In conse-quence, the simultaneous exchange of entire sets of coreproteins (or modules) with all their intrinsic interac-tions should be more feasible than substituting indi-vidual proteins that may disrupt the frozen metabolic
state. Such an experiment has been performed in thegreen alga Chlamydomonas reinhardtii, where the six PSIIcore proteins D1, CP47, CP43, D2, cytochrome b559-a,and cytochrome b559-b were swapped for their homo-logs from two other green algal species (Gimpel et al.,2016). Photoautotrophy was not affected by the ex-change of this synthetic biology module, although thefully altered strains performed suboptimally comparedwith strains in which only one to five genes were ex-changed. However, in control experiments where thesynthetic C. reinhardtii six-gene module was reintro-duced to the C. reinhardtii deletion strain that lackedall six genes, only about 86% of wild-type PSII func-tionality was rescued. Tentative explanations for thisdecreased efficiency include the following: (1) off-target effects of the PSII gene deletions on the operoncomponents and tRNA genes associated with thesePSII loci; (2) misregulation of the transferred PSII genesbecause their expression cassettes lacked unidenti-fied cis-acting DNA elements; and (3) perturbation ofpolycistron-dependent posttranscriptional regulationof the transformed genes, since theywere no longer partof an operon (Gimpel et al., 2016).Taken together, the outcomes of these replacement
experiments indicate that exchanging conserved pho-tosynthetic proteins from multiprotein complexes can
BBOX 2. Where Photosynthesis Varies Most: Antennas, Pigments, and Regulation
The photosynthetic machineries of cyanobacteria
and plants differ markedly in their light-harvesting
pigment-protein complexes and their regulation.
Cyanobacteria harvest light with phycobilisomes,
giant soluble complexes associated with the inner
membrane surface that contain phycobiliproteins.
Plants employ intramembranous antennae, the
light-harvesting complexes I (LHCI) and II (LHCII).
Additional Lhc proteins (CP24, CP26, CP29 and
PsbS) are present in the PSII of plants, but not in
cyanobacteria (see Fig. 1).
In addition, the pigment composition-- particularly
that of the light-harvesting systems--differs
between cyanobacteria and plants. In both
cyanobacteria and plants, PSI and PSII (without
CP24, CP26 and CP29) bind chlorophyll (Chl) a and
β-carotene molecules, but phycobilisomes contain
linear tetrapyrroles (bilins) as pigments. LHCI
contains Chl a and b and the carotenoids
violaxanthin, lutein, and β-carotene. In LHCII and
the inner antenna proteins CP24, CP26, and CP29,
neoxanthin substitute for β-carotene. Both
cyanobacteria and plants utilize Chl a and β-
carotene, but plants use Chl b and the carotenoids
lutein, violaxanthin, and neoxanthin, although
cyanobacteria can synthesize their precursors
(zeaxanthin and lycopene).
Photosynthetic electron flow is regulated at
various levels, of which three are highlighted here.
(1) Adjustment of the ratio of linear to cyclic
electron flow (CEF) controls the output of
photosynthesis in terms of the ATP:NADPH ratio.
One major CEF route is antimycin A-sensitive and
involves the PGRL1/PGR5 complex in plants;
cyanobacteria lack this complex (Leister and
Shikanai, 2013; see Fig. Box 1 and Fig. 1). (2) In
plants, excess energy absorbed during exposure to
high light levels can be dissipated by LHCII via a
mechanism known as the energy-dependent
component (qE) of non-photochemical quenching
(NPQ), which involves the xanthophyll cycle (with
enzymes like violaxanthin de-epoxidase and
zeaxanthin epoxidase) and the PSII subunit PsbS,
and is activated by a decrease in pH in the
thylakoid lumen (Holt et al., 2004; de Bianchi et al.,
2010). (3) Reversible relocation of phycobilisomes
(Mullineaux and Emlyn-Jones, 2005) or LHCII
(Rochaix, 2007) from PSII to PSI depending on light
conditions is used to balance the distribution of
excitation energy between the photosystems and
is referred to as “state transitions.” In plants, but
not in cyanobacteria, the process depends on
reversible protein phosphorylation (Pesaresi et al.,
be problematic, given the highly integrated nature ofthe photosynthetic machinery. Therefore, approachesdesigned to enhance photosynthesis, such as introduc-ing the high light-resistant D1 protein from a greenalga that lives under extreme conditions (Treves et al.,2016) into crop plants, do not appear promising. In-stead, entire (sub)complexes with their internal net-work of evolutionarily optimized interactions shouldbe transferable between species.
EXCHANGE OF NONCONSERVEDPHOTOSYNTHETIC MODULES
Inspection of the repertoire of subunits of the pho-tosynthetic machinery in the three model species Syn-echocystis PCC6803, C. reinhardtii, and Arabidopsis,which represent different stages in the evolution ofoxygen-generating photosynthesis, shows that themost dramatic changes in the photosynthetic proteomeoccurred during the transition from the cyanobacterialendosymbiont (for which Synechocystis serves as
proxy) to the chloroplast of unicellular algae (withC. reinhardtii as proxy; Fig. 3; Supplemental Table S1). Inparticular, phycobilisomes, flavodoxin, and severalphotosystem subunits were lost, while LHCs, somenovel photosystem subunits, and several proteins in-volved in alternative electron pathways or photo-protection evolved. During the transition from algalchloroplasts to those of flowering plants, relatively fewproteins were lost (e.g. flavodiiron proteins and thecanonical cytochrome c6) or acquired (Lhcb6/CP24;Fig. 3; Supplemental Table S1). The NAD(P)H dehy-drogenase (NDH) complex involved in antimycinA-insensitive cyclic electron flow (Box 1) is a specialcase, since the chloroplast NDH from flowering plantstraces back to the cyanobacterial complex, but thecomplex was lost during evolution in C. reinhardtii(Fig. 3; Supplemental Table S1).
Several attempts have been made to introduce pho-tosynthetic proteins into species that lack the corre-sponding homolog (Table 2). These heterologousexpression approaches have been successful for thesoluble electron transporters flavodoxin, cytochrome
Figure 2. Overview of genetic engi-neering and synthetic biology approachesrelated to the light reactions of photo-synthesis. Different shading is used toindicate cyanobacterial (blue) and plant(green) proteins (complexes) and pro-teins (complexes) that are typically notassociated directly with photosynthesis(red). Yellow shading indicates nonbio-logical materials. For designations ofproteins, see Figure Box 1.
c6, and flavodiiron proteins. Indeed, cyanobacterialflavodoxin can at least partially replace plant ferredoxinand confer enhanced stress tolerancewhen expressed inaddition to ferredoxin (Tognetti et al., 2006, 2007;Blanco et al., 2011). Similarly, red algal cytochrome c6enhances the growth and photosynthesis of Arabi-dopsis plants (Chida et al., 2007). Since Arabidopsislacks a functional cytochrome c6 that can transfer elec-trons from the cytochrome b6f complex to PSI (Molina-Heredia et al., 2003; Weigel et al., 2003), it is plausiblethat the more oxidized plastoquinone pool in thetransgenic plants is a direct consequence of additionalPSI reduction mediated by the algal protein (Chidaet al., 2007). Interestingly, it seems to make no differ-ence if an endogenous soluble electron transport pro-tein (Box 3) or its heterologous equivalent from a distantspecies is overexpressed. For instance, overexpressionof the endogenous soluble proteins plastocyanin andferredoxin also can enhance growth in plants (Pesaresiet al., 2009; Lin et al., 2013; Chang et al., 2017; Zhouet al., 2018). Interestingly and rather unexpectedly,this concept also can work for certain proteins that arepart of multiprotein complexes (Simkin et al., 2017; seeBox 3). This indicates that the quantity of such proteinsis relevant for growth enhancement and not their evo-lutionary origin.More recently, the two Physcomitrella patens fla-
vodiiron protein (FLV) genes FlvA and FlvB wereintroduced into Arabidopsis, which, like other angio-sperms, has lost FLVs during evolution (Yamamotoet al., 2016). FLVs are the main mediators of pseudo-cyclic electron flow in photosynthetic organisms, butheterologous expression of FLVs in Arabidopsis had noeffect on steady-state photosynthesis and growth of thetransgenic plants (Yamamoto et al., 2016). However, theArabidopsis FLV lines displayed higher photosyntheticyields just after the onset of actinic light following a
long dark adaptation, suggesting that the FLVs medi-ated a large electron sink during the induction of pho-tosynthesis. In fluctuating light experiments, theArabidopsis FLV lines had much less PSI acceptor sidelimitation, implying that the large FLV-mediated elec-tron sink makes photosynthesis more resistant to thefluctuating light. Consequently, this protective effect ofFLVs is more pronounced in Arabidopsis lines thatare more sensitive to fluctuating light, like the pgr5mutant defective in antimycin A-sensitive cyclic elec-tron flow (Box 1; Leister and Shikanai, 2013; Yamamotoet al., 2016). Similarly, the expression of FLVs in a rice(Oryza sativa) line with markedly reduced cyclic elec-tron flow restored CO2 assimilation and growth rate towild-type levels (Wada et al., 2018). Like FLVs, LHcx/LHCSR proteins play a role in photoprotection in greenalgae and mosses, but they have been lost in angio-sperms during evolution. The heterologous expressionof P. patens LHCSR1 in Nicotiana benthamiana and Ni-cotiana tabacum yielded an active protein that hasproperties similar if not identical to those of mossLHCSR1 (Pinnola et al., 2015).Efforts to create LHCII complexes like those in plants
by heterologously expressing the membrane-spanninglight-harvesting chlorophyll a/b-binding protein Lhcbfrom Pisum spp. (pea) in Synechocystis were unsuc-cessful. Although the pea Lhcb protein was synthesizedin Synechocystis and integrated into the membrane, itdid not accumulate to steady-state levels detectable byimmunoblot analysis (He et al., 1999). Possible expla-nations are that Lhcb is degraded rapidly, either be-cause its unfamiliar structure makes it a good substratefor the cyanobacterial proteolytic system or it cannotfold/assemble properly due to the lack of plant-specificpigments or assembly factors. Interestingly, chloro-phyll b production (Box 2) after the introduction ofplant chlorophyll a oxygenase is boosted when Lhcb is
Table 1. Replacement of conserved individual or multiple photosynthetic proteins
Gene/Protein Description Protein Identity Reference
psbA/D1 Synechocystis D1 was replaced by its homolog from Poa annua,which resulted in slower growth. This is likely to be due to
increased charge recombination between the donor and acceptorsides of the reaction center.
86% Nixon et al. (1991)
psbB/CP47 Synechocystis CP47 was replaced by its homolog from spinachand photoautotrophy was lost.
80% Vermaas et al. (1996)
psbC/CP43 Synechocystis CP43 was replaced by its homolog from spinachand photoautotrophy was lost.
85% Carpenter et al.(1993)
psbH/PsbH Synechocystis PsbH was replaced by its counterpart from maize.The resulting strain displayed increased light sensitivity and lower
chlorophyll content.
78% Chiaramonte et al.(1999)
psaA/PsaA Synechocystis PsaA was replaced by its equivalent fromArabidopsis. This resulted in reduced photoautotrophic growth
and a drastically reduced chlorophyll-phycocyanin ratio.
All six core subunits of C. reinhardtii were replaced by theircounterparts from two different green algae (Volvox carteri and
Scenedesmus obliquus). The resulting strains werephotoautotrophic but showed reduced photosynthetic efficiency,and the heterologous proteins reached only between 10% and
expressed, even though LHCII does not accumulate indetectable amounts (Xu et al., 2001).
Even soluble multiprotein complexes can beexpressed heterologously, as has been demonstratedimpressively from the carbon-fixation end of photo-synthesis. For example, by coexpression of five auxil-iary factors, a functional plant Rubisco complex wasassembled successfully in Escherichia coli (Aigner et al.,2017). This result is in line with the frozen metabolicstate concept, showing that photosynthetic proteinsembedded in a network of interactions with otherproteins and auxiliary factors can be introduced intodistantly related species, but only with their own in-teraction networks. While Gimpel et al. (2016) showed
that efficient gene expression needs to be accountedfor when designing synthetic modules for the transferof photosystem subunits from one species to another,Aigner et al. (2017) demonstrated that auxiliary factorsrequired for the biogenesis of photosynthetic (sub)complexes need to be considered in such experiments,adding additional facets to the frozen metabolic stateconcept.
Auxiliary factors required for the accumulation ofphotosynthetic multiprotein complexes include thefollowing: (1) cofactors like iron-sulfur clusters andpigments (Box 2); (2) chaperones that are required for theinsertion of cofactors (e.g. pigments into LHCs; Schmid,2008); and (3) assembly factors. Assembly factors are
Figure 3. Evolutionary plasticity of the photo-synthetic proteome. The changes in the compo-sition of the inventory of photosynthetic proteins(top) during evolution from the cyanobacterialendosymbiont to the chloroplast in floweringplants (bottom) are shown. As proxies for theoriginal endosymbiont and the unicellularchloroplast-containing protist that gave rise toflowering plants, the model cyanobacteriumSynechocystis PCC6803 (left), the model greenalga C. reinhardtii (middle), and the model flow-ering plant Arabidopsis (right) are used. At topleft, the entire inventory of photosynthetic pro-teins in Synechocystis PCC6803 is listed andassigned to the five different classes PSII, PSI,other electron transport components (other ET),antenna, and photoprotection, whereby thetransition from other ET to photoprotection isfluid. The proteins that have been acquired or lostduring evolution inC. reinhardtii and Arabidopsisare listed in the middle and right sections, re-spectively, of the top part. Note that, for reasons ofsimplicity, we have not considered the ATP syn-thase complex here. The NDH complex (or Nda2in the case of C. reinhardtii) appears only as awhole (without its individual subunits) here.NDH listed in parentheses indicates that theNDH complex is specifically lost only inC. reinhardtii and, therefore, that the plant NDHcomplex is not a reacquisition. Accordingly,Nda2 replaces the NDH complex in C. rein-hardtii. A detailed catalog of the indicated pro-teins with their full names, functions, and furtherliterature links is available in Supplemental TableS1. The bottom part provides a sketch of the ev-olution of flowering plants that traces back to theendosymbiosis between a unicellular eukaryoteand a cyanobacterium, resulting (besides the redalgal and glaucophyte lineages that are notshown) in chloroplast-containing protists thatevolved further to plants.
required to support the stepwise assembly and func-tionality of the multiprotein/pigment complexes, andmany of these are conserved between photosyntheticspecies (Nixon et al., 2010; Nickelsen and Rengstl, 2013;Jensen and Leister, 2014). Therefore, the exchange ofentire multiprotein complexes between distantly re-lated species will not be simple due to the repertoire ofauxiliary factors that has changed markedly duringevolution. Since the species that Gimpel et al. (2016)
studied were closely related, this aspect could beneglected. In consequence, the impact of these auxiliaryfactors on the formation of multiprotein/pigmentcomplexes will have to be fully elucidated to under-stand which factors are sufficient to assemble a photo-synthetic multiprotein complex; previous (genetic)approaches only identified factors required for thisprocess. Hence, the transfer of entire photosynthetic(sub)complexes between distantly related species by a
Table 2. Introduction of heterologous photosynthetic proteins (protein complexes)
Gene/Protein Description Reference
PetJ/cytochrome c6 Growth and photosynthesis of Arabidopsis plants was enhanced by theexpression of a red algal (Porphyra yezoensis) cytochrome c6 gene.
Chida et al. (2007)
Fld/flavodoxin Tobacco lines expressing a plastid-targeted cyanobacterial flavodoxin inaddition to endogenous ferredoxin display increased tolerance to
environmental stress. Flavodoxin can at least partially replace ferredoxin intobacco.
Tognetti et al. (2006,2007; Blanco et al.
(2011
FlvA+B/flavodiiron protein (FLV) FlvA and FlvB from the moss Physcomitrella patens mediate pseudocyclicelectron flow in Arabidopsis.
Yamamoto et al. (2016)
Lhcb/LHCII Pea Lhcb protein is synthesized in Synechocystis and integrated into themembrane, but it is then degraded, such that it cannot be detected by
immunoblot analysis.
He et al. (1999)
BBOX 3. Overexpression of Photosynthetic Proteins Can Enhance Photosynthesis
Boosting the efficiency of the photosynthetic light
reactions by synthetic biology is a long-term
project, whereas optimizing the process under
agriculturally relevant conditions is already
feasible by much simpler means. A prominent
example of this is represented by the parallel
overexpression of the three photoprotective
proteins PsbS, violaxanthin de-epoxidase (VDE),
and zeaxanthin epoxidase (ZE) in tobacco
(Kromdijk et al., 2016). This PsbS-VDE-ZE
overexpressor line displayed an accelerated
photoprotective response to natural shading
events, resulting in increased plant dry matter
productivity under field conditions. Moreover,
tobacco plants overexpressing PsbS alone showed
less stomatal opening in response to light,
decreasing water loss under field conditions
(Glowacka et al., 2018).
Also, overexpression of soluble electron
transporters can enhance photosynthesis. These
proteins can be easily overexpressed because their
actions are not dependent on strict
stoichiometries. For instance, overexpression of
both the endogenous thylakoid lumen protein
plastocyanin and the heterologous red algal
cytochrome c6 protein can enhance growth in
plants (Chida et al., 2007; Pesaresi et al., 2009; Zhou
et al., 2018), and a similar effect is seen for
cyanobacterial flavodoxin and endogenous plant
ferredoxins (Tognetti et al., 2006; Tognetti et al.,
2007; Blanco et al., 2011; Lin et al., 2013; Chang et
al., 2017).
A third instance for enhancing photosynthesis is
provided by overexpression of the tobacco Rieske
protein (PETC) in Arabidopsis (Simkin et al., 2017),
resulting in enhanced levels of other Cyt b6f
complex subunits, together with enhanced plant
growth and dry weight production. This implies
that PETC may be rate-limiting for the
accumulation of the Cyt b6f complex and that the
additional tobacco PETC copies can escape the
limitations of the “frozen metabolic state” due to
the high similarity with their Arabidopsis
counterparts.
These instances of enhancing photosynthesis by
“simple” overexpression of (endogenous)
photosynthetic proteins raise the question of why
evolution has not already produced such plants in
nature by positively selecting mutations in
regulatory regions that increase the expression of
such genes. The most plausible explanation is that,
under natural conditions, overexpression of these
genes involves a trade-off. This hypothetical trade-
off should be related to plant fitness in terms of
synthetic photosynthetic module must include entiresets of strongly interacting proteins (to address the fro-zen metabolic state), as well as all genetic elements andauxiliary factors sufficient for the efficient expression,biogenesis, and function of the proteins in the module.
BIO-BIO HYBRIDS: USING PHOTOSYNTHESIS ASAN ELECTRON SOURCE FOR UNRELATEDBIOLOGICAL PROCESSES
The idea of using the reducing power of photosyn-thesis to drive unrelatedmetabolic reactions has inspiredseveral biotechnological concepts. The photosynthesis-driven formation of secondary metabolites has beendemonstrated in vivo, whereas the light-driven genera-tion of H2 by bio-bio hybrids so far works only in vitroand is closely related to bio-nano approaches. Therefore,the coupling of photosynthesis to previously unrelatedpathways is discussed here first, followed by bio-biosystems for H2 generation.
The redirection of PSI-reducing equivalents to drivereactions catalyzed by cytochrome P450 enzymes hasbeen achieved in vivo by genetic modifications ofplants and cyanobacteria (Lassen et al., 2014b; Nielsenet al., 2016; Mellor et al., 2017). Cytochrome P450sconstitute the largest family of plant enzymes that acton various endogenous and xenobiotic molecules(Rasool and Mohamed, 2016). Their extreme versa-tility and irreversibility of catalyzed reactions makethese enzymes very attractive for use in biotechnol-ogy, medicine, and phytoremediation. P450s aremonooxygenases that insert an oxygen atom intohydrophobic molecules, which enhances their reac-tivity and hydrophilicity. Most eukaryotic P450s re-quire NADPH:cytochrome P450 reductase as theelectron donor.
The endoplasmic reticulum (ER) membrane gener-ally is accepted to be the primary subcellular repositoryof eukaryotic P450s and their NADPH:cytochromeP450 reductase. By relocating cytochrome P450s to thechloroplasts, the reducing power of photosynthesis can
Table 3. Combination of PSI with nonphotosynthetic proteins or complexes: bio-bio hybrids
Gene/Protein Description Reference
-----------------------------------------------------------------------------PSI-P450 hybrids----------------------------------------------------------------------Fusion enzyme of rat CYP1A1and yeast NADPH-P450reductase (in vitro)
Spinach chloroplasts were combined with microsomes from yeast expressingthe rat CYP1A1/CPR fusion enzyme. In this system, NADP+ was
photosynthetically reduced to NADPH to supply the electrons for P450, thusenabling the light-driven conversion of 7-ethoxycoumarin to 7-
hydroxycoumarin.
Kim et al. (1996)
CP79A1 from sorghum(in vitro and in vivo)
The ER-derived P450 CYP79A1 was capable of converting Tyr tohydroxyphenyl-acetaldoxime, employing ferredoxin reduced by barley PSI(without the need for NADPH and CPR). In the next step, CYP79A1 wasfused to cyanobacterial PsaM or Arabidopsis ferredoxin. The engineered
fusions exhibited light-driven activity both in vivo and in vitro.
Jensen et al. (2011);Lassen et al. (2014a);Mellor et al. (2016)
CYP79A1, CYP71E1, andUGT85B1 from sorghum (invivo)
The two P450s CYP79A1 and CYP71E1 and the UDP-glucosyltransferaseUGT85B1 were targeted to N. benthamiana or Synechocystis thylakoid
membranes, where they converted Tyr to dhurrin employingphotosynthetically reduced ferredoxin.
Nielsen et al. (2013);Wlodarczyk et al. (2016)
---------------------------------------------------------------------PSI-hydrogenase hybrids---------------------------------------------------------------------Proteobacterial [NiFe]hydrogenase genetically fusedto cyanobacterial PSI (in vitro)
A fusion of cyanobacterial PsaE to an [NiFe] hydrogenase from theb-proteobacterium Ralstonia eutropha was reconstituted into a PsaE-
deficient PSI from Synechocystis by self-assembly. In a modified version ofthis system, cytochrome c3 from Desulfovibrio vulgaris was cross-linked tothe docking site of ferredoxin in PsaE, targeting electrons directly from PSI
via cytochrome c3 to the hydrogenase. This gave the hydrogenase acompetitive advantage over the natural acceptors of electrons from PSI. In athird variant of this system, the R. eutropha hydrogenase was genetically
fused to Synechocystis PsaE and reconstituted into a PsaE-deficient PSI fromSynechocystis. His tagging of PsaF enabled the assembly of the PSI-
hydrogenase hybrid onto a gold electrode.
Ihara et al. (2006a,2006b; Krassen et al.
(2009
Green algal [FeFe]hydrogenase genetically fusedto ferredoxin (in vitro)
The C. reinhardtii hydrogenase was fused to ferredoxin. This switched thebias of electron transfer from FNR to hydrogenase and resulted in an
increased rate of hydrogen photoproduction in vitro.
Yacoby et al. (2011)
Bacterial [FeFe] hydrogenasewired to cyanobacterial PSI(in vitro)
To enable the transfer of electrons from the terminal Fe4S4 cluster in PSI ofSynechococcus sp. PCC 7002 to the distal Fe4S4 cluster in the hydrogenasefrom Clostridium acetobutylicum, the two components were covalently
coupled to each other via a molecular wire, the thiolated organic moleculeoctanedithiol. This allowed electrons to tunnel through the wire from PSI tothe hydrogenase. Self-assembly of the PSI-wire-hydrogenase complex was
be targeted directly to the reactions catalyzed by theseenzymes, thus providing the basis for the large-scaleproduction of valuable products. Initial attempts tocouple P450s with photosynthesis were conductedin vitro (Table 3; Fig. 4A). Spinach chloroplasts werecombined with microsomes from yeast cells that hadbeen stably transformed with a fusion gene expressingthe rat CYP1A1-NADPH:cytochrome P450 reductasefusion enzyme (Kim et al., 1996). These experimentsconfirmed that it is feasible to drive P450-mediated re-actions using electrons derived from photosyntheticallygenerated NADPH. Intriguingly, the NADPH:cyto-chrome P450 reductase is not always essential, asdemonstrated by coincubating CYP79A1 from sor-ghum (Sorghum bicolor) with barley (Hordeum vulgare)PSI and employing ferredoxin to deliver electrons di-rectly from PSI to the P450 (Jensen et al., 2011). Thisapproach also was shown to be practicable in vivo.CYP79A1, either alone or in combination withCYP71E1 and the UDP-glucosyltransferase UGT85B1,was first targeted in vivo to cyanobacterial or plantthylakoid membranes, where they catalyzed the same
reactions as in their original cellular compartment (theER), utilizing photosynthetically reduced ferredoxin asthe electron donor (Nielsen et al., 2013; Lassen et al.,2014a; Gnanasekaran et al., 2016; Wlodarczyk et al.,2016). Genetic fusions also have been employed forthe coupling of P450s to PSI. CYP79A1 has been fusedto either cyanobacterial PsaM or Arabidopsis ferre-doxin, and the engineered enzyme showed light-drivenactivity both in vivo and in vitro (Lassen et al., 2014a;Mellor et al., 2016). In the latter case, the efficiency of thesystem was enhanced because the fusion could com-pete better with endogenous electron sinks coupled tometabolic pathways (Mellor et al., 2016).In contrast to PSI-P450 hybrids, PSI-hydrogenase
hybrids currently function only in vitro. Unlike fossilfuels, H2 is environmentally benign, as it produces onlywater when combusted. Therefore, in principle, har-nessing of the reducing power of photosynthesis for thedirect production of H2 (i.e. ultimately using sunlightand water) would yield a fully sustainable system ofenergy generation. PSI, but not PSII, provides a stan-dard midpoint potential that is sufficiently negative to
Figure 4. Design of bio-bio hybrids. A, Har-nessing the reducing power of photosynthesisfor cytochrome P450 enzymes (red) has beendemonstrated in vitro and in vivo. In vitroapproaches were based either on a CPR-CYP1A1 fusion protein that could use NADPHproduced by photosynthesis or on CYP79A1that can utilize photoreduced ferredoxin di-rectly. The latter approach also was realizedin vivo by fusing CYP79A1 to ferredoxin (notshown) or the PSI subunit PsaM. The mostelaborate approach reported utilizes three en-zymes (CYP79A1, CYP71E1, and UGT85B1) tocouple dhurrin synthesis with photosynthesis.B, PSI-hydrogenase complexes are based eitheron genetic fusions of the hydrogenase (Hyd) toPsaE or ferredoxin or on covalent linking of thehydrogenase and PSI via a molecular wire.Currently, these bio-bio hybrids function onlyin vitro.
power the reduction of protons to H2 (Utschig et al.,2015). Hence, approaches have been developed to en-gineer PSI to produce H2 either as a replacement or inaddition to its natural product NADPH (see Figure Box1) by redirecting PSI electrons to a catalytic component.This catalytic component can be abiotic or biotic (hy-drogenases). The feasibility of coupling H2 generationto photosynthesis was demonstrated 45 years ago bymixing chloroplast preparations with ferredoxin andhydrogenase (Benemann et al., 1973). Twenty-five yearslater, hydrogen evolution by direct electron transfer(without ferredoxin) from PSI to hydrogenases wasaccomplished for the first time (McTavish, 1998).
Hydrogenases catalyze the reversible interconver-sion of H2 into protons and electrons and are wide-spread in nature; they occur in bacteria and archaea butalso in some eukarya. In vivo, hydrogenases can me-diate photosynthetic H2 production, albeit mostly in-directly or under anaerobic conditions due to theiroxygen sensitivity (Ghirardi, 2015; Oey et al., 2016).Hydrogenases can be classified according to theirmetal-ion composition (e.g. [NiFe] and [FeFe] hydro-genases; Lubitz et al., 2014; Martin and Frymier, 2017).[FeFe] hydrogenases preferentially catalyze proton re-duction and can evolve H2 at high rates but are ex-tremely sensitive to oxygen and are the only type ofhydrogenases found in eukaryotic microorganisms.[NiFe] hydrogenases are less sensitive to oxygen butpreferentially oxidize H2 under physiological condi-tions (Lubitz et al., 2014). In vitro, both types of hy-drogenases have been linked directly to PSI (Table 3;Fig. 4B). PSI-[NiFe] hydrogenase complexes have beengenerated by fusing the hydrogenase genetically to thePsaE protein (Ihara et al., 2006b). PSI-[FeFe] hydro-genase complexes were obtained by genetically fusingthe hydrogenase to ferredoxin (Yacoby et al., 2011) orcovalently linking the FeS clusters present in the hy-drogenase to PSI via a molecular wire (Lubner et al.,2010, 2011).
Two parameters characterize the efficiency of thesein vitro systems: their H2 production rate and longevity.While low rates of H2 production (in the range from 0.1to 10 mmol H2 mg21 chlorophyll h21) were describedfor the early chloroplast extract experiments and ge-netic fusions of PSI to [NiFe] or [FeFe] hydrogenases(McTavish, 1998; Ihara et al., 2006b; Yacoby et al., 2011),higher rates of between 2,000 and 3,000 mmol H2 mg21
chlorophyll h21 have been achieved with wired PSI-[FeFe] hydrogenase complexes and genetically fusedPSI-[NiFe] hydrogenase complexes assembled on agold electrode (Krassen et al., 2009; Lubner et al., 2011).Few data are available with respect to the longevityof the PSI-hydrogenase systems; however, a mini-mum lifetime of 64 d was reported for a wired [FeFe]hydrogenase-PSI complex at room temperature andunder ambient illumination (Lubner et al., 2010).
The future use of hydrogenases in photosynthesis-driven H2 production will depend strongly onwhether it is possible to overcome the oxygen sensi-tivity of many hydrogenases, for instance by employing
oxygen-tolerant [NiFe] hydrogenases (Burgdorf et al.,2005; Schiffels et al., 2013). If this is not possible, theirefficient use in vivo in thylakoids, which inevitablygenerate oxygen during linear electron flow, will beimpossible. However, as demonstrated with the goldsurface system (Krassen et al., 2009), the design of novelmatrices into which the hybrid system can be incorpo-rated may enhance the efficiency markedly.
BIO-NANO HYBRIDS: USE OF PHOTOSYNTHESIS ASA SOURCE OF ELECTRONS FORNONBIOLOGICAL PROCESSES
From bio-bio hybrids, it is only a small step to de-veloping bio-nano hybrids, as demonstrated by PSI
Figure 5. Design of selected bio-nano hybrids. A, PSI-platinum hybridsare generated by combining platinum nanoparticles (dots) or nano-clusters (star) with PSI. Platinum nanoparticles also can be linked to PSIvia nanowires. B, PSI-based photocurrent-generating system. A varietyof such systems have been developed, which consist of PSI moleculesimmobilized on electrodes and implement electron transfer bymeans ofdiffusible redox mediators or nanowires. Moreover, all-solid-state PSI-based solar cells and systems in which cytochrome c was employed tointerface PSI with electrode materials have been generated (Gordiichuket al., 2014; Gizzie et al., 2015; Ciornii et al., 2017; Janna Olmos et al.,2017). The cross-containing circle indicates a current-using device, andthe yellow rectangles symbolize the electrodes.
hybrids that employ abiotic catalysts for photosynthetichydrogen production instead of hydrogenase. In fact,abiotic catalysts have the advantage of bypassingthe lability of hydrogenases in the presence of oxygen.PSI-platinum hybrids have been produced by combin-ing PSI with platinum nanoparticles (either directlyor via tethering by nanowires) or platinum nano-clusters (Kargul et al., 2012; Fukuzumi, 2015; Utschiget al., 2015; Fig. 5A; Table 4). Current PSI-platinumhybrids are less efficient than the most advancedPSI-hydrogenase systems but are extremely robust(Utschig et al., 2015). However, future widespreadusage of the PSI-platinum systemwill be limited by thehigh cost of platinum. A more economical alternativeto precious metals are earth-abundant molecular cat-alysts. However, hybrids consisting of PSI and earth-abundant molecular catalysts have a much shorterworking life than platinum-based configurations(Utschig et al., 2015), likely due to the instability of themolecular catalyst.PSI-based photocurrent-producing devices consti-
tute another class of photosynthesis-derived nano-bio systems (Table 4; Fig. 5). In such devices, PSI isimmobilized onto electrodes. Many variants of thisconcept have been tested, such as varying the electrodematerials, immobilization/orientation strategies, and/or artificial redox mediators (Nguyen and Bruce,2014; Janna Olmos and Kargul, 2015; Plumeré andNowaczyk, 2016; Kargul et al., 2018). PSI must beimmobilized on the electrode surface in such a way thatelectron transfers between the electrodes and the oxi-dizing (P700) and reducing (FB, the terminal [4Fe-4S]cluster, or an intermediate electron transporter) sidesof PSI can proceed with the required efficiency. Elec-trons are transferred between the electrodes and theoxidizing or reducing sides of PSI either by a diffusibleredox mediator or molecular wires. Examples of elec-trode materials and redox mediators are provided inTable 4. After P700 photoexcitation, electrons aretransferred from P700 via several factors (P700→ A0 →
A1→ FX→ FA→ FB) to the iron-sulfur cluster FB (Box 1).As in the case of PSI-hydrogenase and PSI-platinumnanoparticles, PSI can be wired to its electrodes. Thishas been achieved by wiring the A1 cofactor (phyllo-quinone) to the substrate surface, such that electronsare transferred directly from A1 to the electrode, thusbypassing the downstream FeS clusters (FX, FA, and FB)in the stromal domain of PSI (Terasaki et al., 2007, 2009;Miyachi et al., 2009, 2010).Modifications of PSI have been utilized to enhance
the efficiency of the photocurrent-generating system(Das et al., 2004; Frolov et al., 2005; Carmeli et al., 2010).As mentioned previously, fusions to the stroma-facedPSI subunit PsaE can be used to link new components toPSI; however, in this case, PsaD fusions were used. Tothis end, recombinant His-tagged PsaD was immobi-lized on the functionalized electrode surface, whichthen was exposed to native PSI complexes, resulting inimmobilized PSI with P700 facing away from the elec-trode (Das et al., 2004). Another way to control theorientation of PSI during immobilization involves in-troducing Cys mutations. To allow for direct thiolcoupling to an gold surface, various residues on thelumen-exposed face of PSI were replaced by Cys andtested (Frolov et al., 2005). PSI attachment was achievedwith all single mutants, even those placed farther fromthe P700 site, suggesting that a specific location is notrequired as long as the Cys is exposed at the luminalsurface of PSI. The concept of targeted attachment viaintroduced Cys residues was exploited in subsequentstudies to link PSI to maleimide-functionalized galliumarsenide (Frolov et al., 2008), to immobilize PSI betweenthe substrate and a metallized scanning near-field op-tical microscopy tip (Gerster et al., 2012), and to bindPSI to carbon nanotubes (Kaniber et al., 2010). Anothermodification of PSI is represented by the attachment ofplasmonic metal nanoparticles, which resulted in en-hanced light absorption (Carmeli et al., 2010) even inthe green part of the solar spectrum that is not absorbednormally (Szalkowski et al., 2017). This suggests that it
Table 4. Combination of PSI with nonbiological materials: bio-nano hybrids
PSI-based photocurrent-generating devices contain PSI from either plants orcyanobacteria immobilized on electrode materials such as gold, graphene,indium tin oxide, fluorine-doped tin oxide, TiO2, glass, ZnO, or alumina.The most commonly utilized immobilization strategies involve the usage of
organothiol-based self-assembled monolayers. Electron donors to PSIinclude sodium ascorbate, 2,6-dichlorophenolindophenol, reduced
ferricyanide, osmium complexes, ruthenium hexamine trichloride, or theimmobilization wire (NTA-Ni-His6-PSI). Acceptors of PSI electrons includenaphthoquinone-derivative molecular wire, methyl viologen, oxidized
ferricyanide, composite Bis-aniline nanoparticle-ferredoxin, and MethyleneBlue. Also, all-solid-state PSI-based solar cells that do not employ anyexogenous redox mediators or buffer solutions have been generated.
Reviewed in Gordiichuket al. (2014); Nguyen andBruce (2014); Gizzieet al. (2015); JannaOlmos et al. (2017)
is possible to enhance light absorption by PSI in vitrothrough the attachment of abiotic components that actas optical antennae to extend the spectrum of photonsavailable for P700 activation.
Taken together, these efforts demonstrate that PSI-based photocurrent-generating systems are still in anexploratory phase, with many variants under devel-opment. In the next phase, viable building blocks andreference systems should be established that can serveas starting points for the systematic engineering of su-perior systems. This will require the modification of PSIfor optimal effect in artificial systems and undoubtedlywill differ substantially from the original environmentin which PSI was molded by biological evolution.
CONCLUSION AND OUTLOOK
Enhancing photosynthesis and growth can be ach-ieved by a simple overexpression of certain photosyn-thetic proteins, and PSI can be coupled to previouslyunconnected biotic or abiotic components to generatevaluable compounds, hydrogen, or electricity. More-over, entire photosynthetic complexes can be expressedfunctionally in distantly related species (as demon-strated for Rubisco; Aigner et al., 2017). Thus, what arethe next goals and which challenges need to be over-come? Two challenges for the in vivo systems are ob-vious: (1) enhancing the efficiency of in vivo bio-biohybrids; and (2) upscaling the size of synthetic photo-synthetic modules to cover entire photosystems orcomplex antenna systems.
With respect to the enhancement of in vivo cyano-bacterial and algal systems harboring hybrid configu-rations in which photosynthesis directly drivespreviously unconnected pathways, the use of labora-tory evolution offers a unique opportunity to tailorthese systems for their intended purpose. Laboratoryevolution utilizes the high rate of evolution typical inmicrobial systems (in particular, if suitable selectionconditions can be designed) to fine-tune and optimizeprocesses through the selection of advantageous ge-netic variation. While this strategy has been employedin E. coli and yeast with impressive success, now pho-tosynthetic microbial systems are emerging as attrac-tive targets for this approach (Leister, 2018).
The exchange of entire multiprotein complexes be-tween distantly related species will not be a simpleexercise, because the frozen metabolic state of the corephotosynthetic complexes will necessitate the exchangeof major parts of photosystems rather than individualsubunits. The Rubisco case study by Aigner et al. (2017)represents a promising proof of principle, but one needsto consider that the synthetic Rubisco module com-prised only the two different subunits present inthe mature complex and five auxiliary factors for itsassembly. Entire photosystems will require muchlarger synthetic photosynthetic modules, and manyof the auxiliary factors have not been identifiedyet. Therefore, when designing these complex synthetic
photosynthetic modules, we will identify the set ofcomponents that are sufficient (and not only necessary)to drive photosynthesis, providing an unprecedent-edly deep understanding of this fundamental andcomplex process.
Given that the problems described above can besolved, what will be the next steps in the synthetic bi-ology of the light reactions of photosynthesis (seeOutstanding Questions)? The combination of syntheticphotosynthetic modules from diverse species could al-low the design of novel variants of photosynthesis.Numerous instances of such recombined photosyn-thetic variants can be imagined, including plants thatemploy cyanobacteria-derived phycobilisomes forhighly sufficient photosynthesis under low-light con-ditions, cyanobacteria that employ plant-derived LHCsas antennae to shift the light saturation of photosyn-thesis to higher intensities, or the integration of cya-nobacterial chlorophyll d and chlorophyll f (whichcan absorb far-red and near infrared light) into algalor plant photosynthesis to expand the spectral regionavailable to drive photosynthesis (Loughlin et al.,2013; Ho et al., 2016). Moreover, such variants ofrecombined photosynthesis could be optimized furtherby laboratory evolution within a suitable microbialchassis. However, such recombined photosyntheticvariants cannot be considered a truly novel type ofphotosynthesis because they would only bring togetherpreexisting pieces that evolution has separated. Nev-ertheless, they could be an important step toward theambitious goal to design truly novel synthetic photo-synthetic modules that contain more efficient substi-tutes of the frozenmetabolic accidents discussed above.
Ample proof of functionality for in vivo hybrids(between photosynthesis and previously unconnectedmetabolic pathways) and in vitro hybrids (between PSIand biotic or abiotic catalysts) has been obtained, butsuch systems will only be commercially successful ifthey can compete with established nonbiological sys-tems. Tailoring of PSI in cyanobacteria, where tech-niques like gene replacement and gene modificationare routine, may contribute to further improving the
OOUTSTANDING QUESTIONS
• Can photosynthesis be enhanced by combining
“synthetic photosynthetic modules” from
different species?
• Can the “frozen metabolic accidents” be
substituted by more efficient proteins via re-
designing “synthetic photosynthetic modules”?
• Can a fundamentally different type of
photosynthesis be realized?
• Can bio-bio and bio-nano hybrids be generated
efficiently and provide valuable substances and energy safely and economically?
efficiency and robustness of in vitro and in vivo hy-brids. One promising route could be to identify thoseparts of the original PSI (which evolved under con-straints imposed by the cyanobacterial cell) that arenecessary for hybrid devices (a minimal PSI). Such bio-nano systems can be optimized to include novel non-biological components that replace or complementnatural pigments and/or the protein-based backboneof photosystems, going far beyond the limited toolboxof nature’s chemistry. Such novel systems, inspired bynatural photosynthesis, could be used to engage biol-ogists in the design of fundamentally different types ofphotosynthesis in living organisms.
Supplemental Data
The following supplemental materials are available.
Supplemental Table S1. Absence/presence of photosynthetic proteins inthe three model species Synechocystis PCC6803, C. reinhardtii, andArabidopsis.
ACKNOWLEDGMENTS
I thank Paul Hardy for critical reading of the article.
Received March 22, 2018; accepted June 14, 2018; published July 10, 2018.
LITERATURE CITED
Aigner H, Wilson RH, Bracher A, Calisse L, Bhat JY, Hartl FU, Hayer-Hartl M (2017) Plant RuBisCo assembly in E. coli with five chloroplastchaperones including BSD2. Science 358: 1272–1278
Benemann JR, Berenson JA, Kaplan NO, Kamen MD (1973) Hydrogenevolution by a chloroplast-ferredoxin-hydrogenase system. Proc NatlAcad Sci USA 70: 2317–2320
Blanco NE, Ceccoli RD, Segretin ME, Poli HO, Voss I, Melzer M, Bravo-Almonacid FF, Scheibe R, Hajirezaei MR, Carrillo N (2011) Cyano-bacterial flavodoxin complements ferredoxin deficiency in knocked-downtransgenic tobacco plants. Plant J 65: 922–935
Blankenship RE, Chen M (2013) Spectral expansion and antenna reductioncan enhance photosynthesis for energy production. Curr Opin ChemBiol 17: 457–461
Burgdorf T, Lenz O, Buhrke T, van der Linden E, Jones AK, Albracht SP,Friedrich B (2005) [NiFe]-hydrogenases of Ralstonia eutropha H16:modular enzymes for oxygen-tolerant biological hydrogen oxidation.J Mol Microbiol Biotechnol 10: 181–196
Carmeli I, Lieberman I, Kraversky L, Fan Z, Govorov AO, Markovich G,Richter S (2010) Broad band enhancement of light absorption in pho-tosystem I by metal nanoparticle antennas. Nano Lett 10: 2069–2074
Carpenter SD, Ohad I, Vermaas WF (1993) Analysis of chimeric spinach/cyanobacterial CP43 mutants of Synechocystis sp. PCC 6803: thechlorophyll-protein CP43 affects the water-splitting system of photo-system II. Biochim Biophys Acta 1144: 204–212
Chang H, Huang HE, Cheng CF, Ho MH, Ger MJ (2017) Constitutive ex-pression of a plant ferredoxin-like protein (pflp) enhances capacity ofphotosynthetic carbon assimilation in rice (Oryza sativa). Transgenic Res26: 279–289
Chiaramonte S, Giacometti GM, Bergantino E (1999) Construction andcharacterization of a functional mutant of Synechocystis 6803 harbour-ing a eukaryotic PSII-H subunit. Eur J Biochem 260: 833–843
Chida H, Nakazawa A, Akazaki H, Hirano T, Suruga K, Ogawa M, SatohT, Kadokura K, Yamada S, Hakamata W, et al (2007) Expression of thealgal cytochrome c6 gene in Arabidopsis enhances photosynthesis andgrowth. Plant Cell Physiol 48: 948–957
Ciornii D, Riedel M, Stieger KR, Feifel SC, Hejazi M, Lokstein H, ZouniA, Lisdat F (2017) Bioelectronic circuit on a 3D electrode architecture:
enzymatic catalysis interconnected with photosystem I. J Am Chem Soc139: 16478–16481
Dann M, Leister D (2017) Enhancing (crop) plant photosynthesis by in-troducing novel genetic diversity. Philos Trans R Soc Lond B Biol Sci372: 20160380
Das R, Kiley PJ, Segal M, Norville J, Yu AA, Wang L, Trammell SA,Reddick LE, Kumar R, Stellacci F, et al (2004) Integration of photo-synthetic protein molecular complexes in solid-state electronic devices.Nano Lett 4: 1079–1083
de Bianchi S, Ballottari M, Dall’osto L, Bassi R (2010) Regulation of plantlight harvesting by thermal dissipation of excess energy. Biochem SocTrans 38: 651–660
Frolov L, Rosenwaks Y, Carmeli C, Carmeli I (2005) Fabrication of aphotoelectronic device by direct chemical binding of the photosyntheticreaction center protein to metal surfaces. Adv Mater 17: 2434–2437
Frolov L, Rosenwaks Y, Richter S, Carmeli C, Carmeli I (2008) Photoe-lectric junctions between GaAs and photosynthetic reaction center pro-tein. J Phys Chem C 112: 13426–13430
Fukuzumi S (2015) Artificial photosynthetic systems for production ofhydrogen. Curr Opin Chem Biol 25: 18–26
Gerster D, Reichert J, Bi H, Barth JV, Kaniber SM, Holleitner AW,Visoly-Fisher I, Sergani S, Carmeli I (2012) Photocurrent of a singlephotosynthetic protein. Nat Nanotechnol 7: 673–676
Ghirardi ML (2015) Implementation of photobiological H2 production: theO2 sensitivity of hydrogenases. Photosynth Res 125: 383–393
Gimpel JA, Nour-Eldin HH, Scranton MA, Li D, Mayfield SP (2016) Re-factoring the six-gene photosystem II core in the chloroplast of the greenalgae Chlamydomonas reinhardtii. ACS Synth Biol 5: 589–596
Gizzie EA, Niezgoda JS, Robinson MT, Harris AG, Jennings GK,Rosenthal SJ, Cliffel DE (2015) Photosystem I-polyaniline/TiO2 solid-state solar cells: simple devices for biohybrid solar energy conversion.Energy Environ Sci 8: 3572–3576
Głowacka K, Kromdijk J, Kucera K, Xie J, Cavanagh AP, Leonelli L,Leakey ADB, Ort DR, Niyogi KK, Long SP (2018) Photosystem IIsubunit S overexpression increases the efficiency of water use in a field-grown crop. Nat Commun 9: 868
Gnanasekaran T, Karcher D, Nielsen AZ, Martens HJ, Ruf S, Kroop X,Olsen CE, Motawie MS, Pribil M, Møller BL, et al (2016) Transfer ofthe cytochrome P450-dependent dhurrin pathway from Sorghum bicolorinto Nicotiana tabacum chloroplasts for light-driven synthesis. J Exp Bot67: 2495–2506
Gordiichuk PI, Wetzelaer GJ, Rimmerman D, Gruszka A, de Vries JW,Saller M, Gautier DA, Catarci S, Pesce D, Richter S, et al (2014) Solid-state biophotovoltaic cells containing photosystem I. Adv Mater 26:4863–4869
Haniewicz P, Abram M, Nosek L, Kirkpatrick J, El-Mohsnawy E, OlmosJDJ, Kou�ril R, Kargul JM (2018) Molecular mechanisms of photo-adaptation of photosystem I supercomplex from an evolutionary cya-nobacterial/algal intermediate. Plant Physiol 176: 1433–1451
He Q, Schlich T, Paulsen H, Vermaas W (1999) Expression of a higherplant light-harvesting chlorophyll a/b-binding protein in Synechocystissp. PCC 6803. Eur J Biochem 263: 561–570
Ho MY, Shen G, Canniffe DP, Zhao C, Bryant DA (2016) Light-dependentchlorophyll f synthase is a highly divergent paralog of PsbA of photo-system II. Science 353: aaf9178
Holt NE, Fleming GR, Niyogi KK (2004) Toward an understanding of themechanism of nonphotochemical quenching in green plants. Biochem-istry 43: 8281–8289
Ihara M, Nakamoto H, Kamachi T, Okura I, Maeda M (2006a) Photoin-duced hydrogen production by direct electron transfer from photosys-tem I cross-linked with cytochrome c3 to [NiFe]-hydrogenase.Photochem Photobiol 82: 1677–1685
Ihara M, Nishihara H, Yoon KS, Lenz O, Friedrich B, Nakamoto H,Kojima K, Honma D, Kamachi T, Okura I (2006b) Light-driven hy-drogen production by a hybrid complex of a [NiFe]-hydrogenase andthe cyanobacterial photosystem I. Photochem Photobiol 82: 676–682
Janna Olmos JD, Kargul J (2015) A quest for the artificial leaf. Int J BiochemCell Biol 66: 37–44
Janna Olmos JD, Becquet P, Gront D, Sar J, Dąbrowski A, Gawlik G,Teodorczyk M, Pawlak D, Kargul J (2017) Biofunctionalisation ofp-doped silicon with cytochrome c553 minimises charge recombinationand enhances photovoltaic performance of the all-solid-state photosys-tem I-based biophotoelectrode. RSC Advances 7: 47854–47866
Kaniber SM, Brandstetter M, Simmel FC, Carmeli I, Holleitner AW (2010)On-chip functionalization of carbon nanotubes with photosystem I.J Am Chem Soc 132: 2872–2873
Kargul J, Janna Olmos JD, Krupnik T (2012) Structure and function ofphotosystem I and its application in biomimetic solar-to-fuel systems.J Plant Physiol 169: 1639–1653
Kargul J, Bubak G, Andryianau G (2018) Biophotovoltaic systems basedon photosynthetic complexes. In K Wandelt, ed, Encyclopedia of Inter-facial Chemistry: Surface Science and Electrochemistry, Vol Vol 7.Elsevier, Amsterdam, Netherlands, pp 43–63
Kim YS, Hara M, Ikebukuro K, Miyake J, Ohkawa H, Karube I (1996)Photo-induced activation of cytochrome P450/reductase fusion enzymecoupled with spinach chloroplasts. Biotechnol Tech 10: 717–720
Krassen H, Schwarze A, Friedrich B, Ataka K, Lenz O, Heberle J (2009)Photosynthetic hydrogen production by a hybrid complex of photo-system I and [NiFe]-hydrogenase. ACS Nano 3: 4055–4061
Kromdijk J, Głowacka K, Leonelli L, Gabilly ST, Iwai M, Niyogi KK,Long SP (2016) Improving photosynthesis and crop productivity byaccelerating recovery from photoprotection. Science 354: 857–861
Kubota H, Sakurai I, Katayama K, Mizusawa N, Ohashi S, Kobayashi M,Zhang P, Aro EM, Wada H (2010) Purification and characterization ofphotosystem I complex from Synechocystis sp. PCC 6803 by expressinghistidine-tagged subunits. Biochim Biophys Acta 1797: 98–105
Lassen LM, Nielsen AZ, Olsen CE, Bialek W, Jensen K, Møller BL, JensenPE (2014a) Anchoring a plant cytochrome P450 via PsaM to the thyla-koids in Synechococcus sp. PCC 7002: evidence for light-driven biosyn-thesis. PLoS ONE 9: e102184
Lassen LM, Nielsen AZ, Ziersen B, Gnanasekaran T, Møller BL, JensenPE (2014b) Redirecting photosynthetic electron flow into light-drivensynthesis of alternative products including high-value bioactive natu-ral compounds. ACS Synth Biol 3: 1–12
Leister D (2012) How can the light reactions of photosynthesis be improvedin plants? Front Plant Sci 3: 199
Leister D (2018) Experimental evolution in photoautotrophic microorgan-isms as a means of enhancing chloroplast functions. Essays Biochem 62:77–84
Leister D, Shikanai T (2013) Complexities and protein complexes in theantimycin A-sensitive pathway of cyclic electron flow in plants. FrontPlant Sci 4: 161
Lin YH, Pan KY, Hung CH, Huang HE, Chen CL, Feng TY, Huang LF(2013) Overexpression of ferredoxin, PETF, enhances tolerance to heatstress in Chlamydomonas reinhardtii. Int J Mol Sci 14: 20913–20929
Long SP, Marshall-Colon A, Zhu XG (2015) Meeting the global food de-mand of the future by engineering crop photosynthesis and yield po-tential. Cell 161: 56–66
Loughlin P, Lin Y, Chen M (2013) Chlorophyll d and Acaryochloris marina:current status. Photosynth Res 116: 277–293
Lubitz W, Ogata H, Rüdiger O, Reijerse E (2014) Hydrogenases. ChemRev 114: 4081–4148
Lubner CE, Knörzer P, Silva PJ, Vincent KA, Happe T, Bryant DA,Golbeck JH (2010) Wiring an [FeFe]-hydrogenase with photosystem Ifor light-induced hydrogen production. Biochemistry 49: 10264–10266
Lubner CE, Applegate AM, Knörzer P, Ganago A, Bryant DA, Happe T,Golbeck JH (2011) Solar hydrogen-producing bionanodevice outper-forms natural photosynthesis. Proc Natl Acad Sci USA 108: 20988–20991
Martin BA, Frymier PD (2017) A review of hydrogen production by pho-tosynthetic organisms using whole-cell and cell-free systems. Appl Bi-ochem Biotechnol 183: 503–519
McTavish H (1998) Hydrogen evolution by direct electron transfer fromphotosystem I to hydrogenases. J Biochem 123: 644–649
Mellor SB, Nielsen AZ, Burow M, Motawia MS, Jakubauskas D, MøllerBL, Jensen PE (2016) Fusion of ferredoxin and cytochrome P450 enablesdirect light-driven biosynthesis. ACS Chem Biol 11: 1862–1869
Mellor SB, Vavitsas K, Nielsen AZ, Jensen PE (2017) Photosynthetic fuelfor heterologous enzymes: the role of electron carrier proteins. Photo-synth Res 134: 329–342
Miyachi M, Yamanoi Y, Yonezawa T, Nishihara H, Iwai M, Konno M,Iwai M, Inoue Y (2009) Surface immobilization of PSI using vitamin K1-
like molecular wires for fabrication of a bio-photoelectrode. J NanosciNanotechnol 9: 1722–1726
Miyachi M, Yamanoi Y, Shibata Y, Matsumoto H, Nakazato K, Konno M,Ito K, Inoue Y, Nishihara H (2010) A photosensing system composed ofphotosystem I, molecular wire, gold nanoparticle, and double surfac-tants in water. Chem Commun (Camb) 46: 2557–2559
Molina-Heredia FP, Wastl J, Navarro JA, Bendall DS, Hervás M, HoweCJ, De La Rosa MA (2003) Photosynthesis: a new function for an oldcytochrome? Nature 424: 33–34
Mullineaux CW, Emlyn-Jones D (2005) State transitions: an example ofacclimation to low-light stress. J Exp Bot 56: 389–393
Nelson N (2009) Plant photosystem I: the most efficient nano-photochemical machine. J Nanosci Nanotechnol 9: 1709–1713
Nguyen K, Bruce BD (2014) Growing green electricity: progress andstrategies for use of photosystem I for sustainable photovoltaic energyconversion. Biochim Biophys Acta 1837: 1553–1566
Nickelsen J, Rengstl B (2013) Photosystem II assembly: from cyanobacteriato plants. Annu Rev Plant Biol 64: 609–635
Nielsen AZ, Ziersen B, Jensen K, Lassen LM, Olsen CE, Møller BL,Jensen PE (2013) Redirecting photosynthetic reducing power towardbioactive natural product synthesis. ACS Synth Biol 2: 308–315
Nielsen AZ, Mellor SB, Vavitsas K, Wlodarczyk AJ, Gnanasekaran T,Perestrello Ramos H de Jesus M, King BC, Bakowski K, Jensen PE(2016) Extending the biosynthetic repertoires of cyanobacteria andchloroplasts. Plant J 87: 87–102
Nixon PJ, Rögner M, Diner BA (1991) Expression of a higher plant psbAgene in Synechocystis 6803 yields a functional hybrid photosystem IIreaction center complex. Plant Cell 3: 383–395
Nixon PJ, Michoux F, Yu J, Boehm M, Komenda J (2010) Recent advancesin understanding the assembly and repair of photosystem II. Ann Bot106: 1–16
Oey M, Sawyer AL, Ross IL, Hankamer B (2016) Challenges and oppor-tunities for hydrogen production from microalgae. Plant Biotechnol J 14:1487–1499
Pesaresi P, Scharfenberg M, Weigel M, Granlund I, Schröder WP, FinazziG, Rappaport F, Masiero S, Furini A, Jahns P, et al (2009) Mutants,overexpressors, and interactors of Arabidopsis plastocyanin isoforms:revised roles of plastocyanin in photosynthetic electron flow and thy-lakoid redox state. Mol Plant 2: 236–248
Pesaresi P, Pribil M, Wunder T, Leister D (2011) Dynamics of reversibleprotein phosphorylation in thylakoids of flowering plants: the roles ofSTN7, STN8 and TAP38. Biochim Biophys Acta 1807: 887–896
Pinnola A, Ghin L, Gecchele E, Merlin M, Alboresi A, Avesani L, PezzottiM, Capaldi S, Cazzaniga S, Bassi R (2015) Heterologous expression ofmoss light-harvesting complex stress-related 1 (LHCSR1), the chloro-phyll a-xanthophyll pigment-protein complex catalyzing non-photochemical quenching, in Nicotiana sp. J Biol Chem 290: 24340–24354
Plumeré N, Nowaczyk MM (2016) Biophotoelectrochemistry of photo-synthetic proteins. Adv Biochem Eng Biotechnol 158: 111–136
Pribil M, Pesaresi P, Hertle A, Barbato R, Leister D (2010) Role of plastidprotein phosphatase TAP38 in LHCII dephosphorylation and thylakoidelectron flow. PLoS Biol 8: e1000288
Rasool S, Mohamed R (2016) Plant cytochrome P450s: nomenclature andinvolvement in natural product biosynthesis. Protoplasma 253:1197–1209
Rochaix JD (2007) Role of thylakoid protein kinases in photosynthetic ac-climation. FEBS Lett 581: 2768–2775
Schiffels J, Pinkenburg O, Schelden M, Aboulnaga HA, Baumann ME,Selmer T (2013) An innovative cloning platform enables large-scaleproduction and maturation of an oxygen-tolerant [NiFe]-hydrogenasefrom Cupriavidus necator in Escherichia coli. PLoS ONE 8: e68812
Shi T, Bibby TS, Jiang L, Irwin AJ, Falkowski PG (2005) Protein inter-actions limit the rate of evolution of photosynthetic genes in cyano-bacteria. Mol Biol Evol 22: 2179–2189
Simkin AJ, McAusland L, Lawson T, Raines CA (2017) Overexpression ofthe RieskeFeS protein increases electron transport rates and biomassyield. Plant Physiol 175: 134–145
Szalkowski M, Janna Olmos JD, Buczy�nska D, Mackowski S, KowalskaD, Kargul J (2017) Plasmon-induced absorption of blind chlorophylls inphotosynthetic proteins assembled on silver nanowires. Nanoscale 9:10475–10486
Terasaki N, Yamamoto N, Tamada K, Hattori M, Hiraga T, Tohri A, SatoI, Iwai M, Iwai M, Taguchi S, et al (2007) Bio-photosensor: cyano-bacterial photosystem I coupled with transistor via molecular wire. Bi-ochim Biophys Acta 1767: 653–659
Terasaki N, Yamamoto N, Hiraga T, Yamanoi Y, Yonezawa T, NishiharaH, Ohmori T, Sakai M, Fujii M, Tohri A, et al (2009) Plugging a mo-lecular wire into photosystem I: reconstitution of the photoelectricconversion system on a gold electrode. Angew Chem Int Ed Engl 48:1585–1587
Tognetti VB, Palatnik JF, Fillat MF, Melzer M, Hajirezaei MR, Valle EM,Carrillo N (2006) Functional replacement of ferredoxin by a cyano-bacterial flavodoxin in tobacco confers broad-range stress tolerance.Plant Cell 18: 2035–2050
Tognetti VB, Zurbriggen MD, Morandi EN, Fillat MF, Valle EM,Hajirezaei MR, Carrillo N (2007) Enhanced plant tolerance to ironstarvation by functional substitution of chloroplast ferredoxin with abacterial flavodoxin. Proc Natl Acad Sci USA 104: 11495–11500
Treves H, Raanan H, Kedem I, Murik O, Keren N, Zer H, Berkowicz SM,Giordano M, Norici A, Shotland Y, et al (2016) The mechanismswhereby the green alga Chlorella ohadii, isolated from desert soil crust,exhibits unparalleled photodamage resistance. New Phytol 210:1229–1243
Vermaas WF, Shen G, Ohad I (1996) Chimaeric CP47 mutants of the cy-anobacterium Synechocystis sp. PCC 6803 carrying spinach sequences:construction and function. Photosynth Res 48: 147–162
Viola S, Rühle T, Leister D (2014) A single vector-based strategy formarker-less gene replacement in Synechocystis sp. PCC 6803. Microb CellFact 13: 4
Wada S, Yamamoto H, Suzuki Y, Yamori W, Shikanai T, Makino A (2018)Flavodiiron protein substitutes for cyclic electron flow without com-peting CO2 assimilation in rice. Plant Physiol 176: 1509–1518
Weigel M, Varotto C, Pesaresi P, Finazzi G, Rappaport F, Salamini F,Leister D (2003) Plastocyanin is indispensable for photosynthetic elec-tron flow in Arabidopsis thaliana. J Biol Chem 278: 31286–31289
Wlodarczyk A, Gnanasekaran T, Nielsen AZ, Zulu NN, Mellor SB,Luckner M, Thøfner JFB, Olsen CE, Mottawie MS, Burow M, et al(2016) Metabolic engineering of light-driven cytochrome P450 depen-dent pathways into Synechocystis sp. PCC 6803. Metab Eng 33: 1–11
Xu H, Vavilin D, Vermaas W (2001) Chlorophyll b can serve as the majorpigment in functional photosystem II complexes of cyanobacteria. ProcNatl Acad Sci USA 98: 14168–14173
Yacoby I, Pochekailov S, Toporik H, Ghirardi ML, King PW, Zhang S(2011) Photosynthetic electron partitioning between [FeFe]-hydrogenaseand ferredoxin:NADP+-oxidoreductase (FNR) enzymes in vitro. ProcNatl Acad Sci USA 108: 9396–9401
Yamamoto H, Takahashi S, Badger MR, Shikanai T (2016) Artificial re-modelling of alternative electron flow by flavodiiron proteins in Ara-bidopsis. Nat Plants 2: 16012
Zhou XT, Wang F, Ma YP, Jia LJ, Liu N, Wang HY, Zhao P, Xia GX, ZhongNQ (2018) Ectopic expression of SsPETE2, a plastocyanin from Suaedasalsa, improves plant tolerance to oxidative stress. Plant Sci 268: 1–10
