Engineering bacterial cells for hydrogen production

Abstract

The invention relates to an electromicrobial system for synthesis of hydrogen, comprising: a gram-negative bacterial cell modified for synthesis of hydrogen, wherein the bacterial cell comprises: i) inner membrane-bound rhodopsin for proton-pumping, ii) membrane-bound hydrogenase, and iii) optionally an electron mediator located in the periplasm and / or on the outer membrane of the bacterial cell; and an electron source for donation of electrons to the bacterial cell.

Description

[0001] Engineering bacterial cells for hydrogen production

[0002] The present invention relates to an electromicrobial system for hydrogen production, for example by photoelectrosynthesis.

[0003] In response to the global energy crisis and environmental challenges, there is a growing emphasis on seeking and utilising clean energy sources (1). Hydrogen emerges as the quintessential clean energy solution, characterised by its high energy density and emission-free combustion (2). Green hydrogen, generated through electrocatalytic water splitting powered by renewable energy, is regarded as the most promising energy carrier for a low-carbon economy (3). The development of cheap, stable, and efficient catalysts for cathodic H2 evolution in an electrochemical system has attracted wide interest (4). Metal materials may be used as the cathode to catalyse water electrolysis, however, substantially increasing the cost (5). Biocatalysts, such as microbial cells and enzymes, are a cost-effective alternative for catalysing H2 synthesis under mild conditions (6).

[0004] The metal-reducing bacterium, Shewanella oneidensis MR-1 is a model electroactive platform, which is usually used for microbial fuel cell (7, 8) and microbial electrosynthesis (9, 10) because of its bidirectional extracellular electron transfer pathway. S. oneidensis MR-1 can be used as a biocatalyst in the electrochemical system for cathodic H2 production by combining extracellular electron transfer and hydrogenase-based metabolism (11). S. oneidensis MR-1 has two hydrogenases, namely, [Ni-Fe] and [Fe-Fe] hydrogenases, located in the periplasmic space with the capacity to catalyse the interconversion between protons and hydrogen (12). The electrode as an external electron donor delivers electrons across the outer membrane into hydrogenases reducing protons to molecular hydrogen. Despite theoretical feasibility, the current wild-type S. oneidensis-bas d. H2 electrosynthesis system exhibits unsatisfactorily low hydrogen yield that largely limits the practical applications (H, 13).

[0005] There is thus a need to develop an alternative or improved methods and materials for the microbial synthesis of H2.

[0006] According to a first aspect of the invention there is provided an electromicrobial system for synthesis of hydrogen, comprising: A) a gram-negative bacterial cell modified for synthesis of hydrogen, wherein the bacterial cell comprises: i) inner membrane-bound rhodopsin for proton-pumping, ii) membrane-bound hydrogenase, and iii) optionally an electron mediator located in the periplasm and / or on the outer membrane of the bacterial cell; and

[0007] B) an electron source for donation of electrons to the bacterial cell.

[0008] The present invention advantageously provides a light-powered electromicrobial system for H2 synthesis. The invention shows that a closed redox loop can be constructed by integrating rhodopsin with an electron donor. If the electron donor can be supplied by an electrode powered by solar panel, a rhodopsin-based photo-electrosynthetic system could drive H2 synthesis with light as the only energy input. This bioenergetic system can be used with only three inputs: light, protons, and electrons. For example, the protons can be supplied by installing Gloeobacter violaceus rhodopsin (GR), augmented by an external photocell that serves as the electron donor (Fig. 1). A hydrogenase closes the electrosynthetic loop by chemically reducing two protons, and catalysing the formation of a H-H bond between them. This engineered bacterium therefore uses only light to convert protons and electrons into H2 molecules, which may be harvested to be used as fuel.

[0009] The present invention proposes that improving microbial electrosynthesis of H2 requires efficient electron transport and favourable periplasmic conditions. S. oneidensis MR-1 is an example of a Gram-negative bacterium that is a well-established bio-nano-factory for synthesising and assembling nanoparticles due to its reducing and detoxification capacities. H2 synthesis of S. oneidensis MR-1 taking place in the narrow periplasmic space yields an opportunity to rationally design the periplasm as a nanoreactor by optimising synergy between nanoparticles and membrane proteins. A favourable reaction space is important for the reductive H2 synthesis half-reaction. Increasing hydrogenase concentration in the periplasm is expected to facilitate the reaction rate. A high proton concentration could also facilitate the catalytic reaction. This system has realised a more than 10-fold increase in hydrogen production rate compared to the wildtype strain, indicating the potential of whole-cell catalysts for efficient hydrogen production. The bacterial cell, such as S. oneidensis MR-1, can be employed to synthesise ferric sulfide (FeS) nanoparticles which are self-assembled and located in the periplasm to mediate the electron transfer. Whilst an electron mediator, such as ferric sulfide (FeS) nanoparticles, is not an essential requirement for the reaction, it can significantly increase efficiency for hydrogen production. Therefore, the provision of an electron mediator is a particularly advantageous embodiment. In addition, the bacterial cell, such as S. oneidensis MR-1 can be engineered to heterologously express GR and its antenna canthaxanthin, which is able to capture light energy to efficiently pump protons from cytoplasm to periplasm. In the periplasm-based nano-bioreactor, electrons are transferred from the cathode to the cells mediated by the extracellular electron transfer pathway and then passed onto the hydrogenases. FeS nanoparticles can play the role of conductive line to facilitate the electron transfer from the outer-membrane proteins to hydrogenase. GR pumping protons into the periplasm increases the periplasmic proton concentration, thereby accelerating H2 synthesis. The hydrogenases use protons and electrons to form H2 molecules. This rationally designed system achieves efficient photoelectrochemical hydrogen production.

[0010] The rhodopsin

[0011] The rhodopsin functions as a light-driven H+ion transporter, which pumps protons from the intracellular (cytoplasmic) side across the cell membrane into the periplasmic space. The transferred protons may be used by the hydrogenase to generate H2.

[0012] Rhodopsin (which may also be known as microbial rhodopsin) is a simple light-driven proton pump found broadly distributed in nature and it can also be easily engineered into different bacterial hosts.

[0013] The bacterial cell may be engineered to express the rhodopsin. The engineering to express the rhodopsin may comprise the transformation and expression of a sequence encoding the rhodopsin. Alternatively, the expression of an endogenous rhodopsin in the bacterial cell may be enhanced, for example by the engineering of a promoter for enhanced expression of the gene encoding an endogenous rhodopsin. Alternatively, a non-functioning or poorly-expressed endogenous rhodopsin may be restored and / or overexpressed by mutation. The rhodopsin may comprise a recombinant rhodopsin. In one embodiment, the rhodopsin is a bacteriorhodopsin. The rhodopsin may comprise a proteorhodopsin (PR) or Gloeobacter rhodopsin (GR). The rhodopsin may comprise Gloeobacter spp. rhodopsin. In a preferred embodiment, the recombinant proton pump is the Gloeobacter violaceus rhodopsin. In another embodiment, the rhodopsin may comprise Gloeobacter violaceus PCC7421 rhodopsin.

[0014] Advantageously, Gloeobacter violaceus rhodopsin has a two-fold faster turnover rate than Proteorhodopsin (PR), and is able to bind carotenoids with a 4-keto group, e.g. salinixanthin and echinenone, to increase the absorption cross-section of the pump. Additionally, Gloeobacter violaceus rhodopsin has a high tolerance to fluctuation in pH.

[0015] Gloeobacter violaceus rhodopsin is originally from thylakoid-less cyanobacterium Gloeobacter violaceus PCC7421 (38) and advantageously has a high efficiency of proton pumping and a rapid photocycle. Furthermore, Gloeobacter violaceus rhodopsin has been shown to combine with retinal analogues to absorb near-infrared light (850- 950nm) (42), which can significantly extend the light-harvesting spectrum and maximise energy harvesting per surface area (43). Gloeobacter violaceus rhodopsin has a p ia=~4.8 (34), compared to proteorhodopsin which has a p Ta=~7.5 (35). Gloeobacter violaceus rhodopsin is functional at a lower pH, a situation that may arise when a bacterial cell expresses the protein ectopically, or overexpresses its endogenous protein.

[0016] The rhodopsin may comprise the amino acid sequence of MGLMTVFSSAPELALLGSTFAQVDPSNLSVSDSLTYGQFNLVYNAFSFAIAAMFASALFF FSAQALVGQRYRLALLVSAIVVSIAGYHYFRIFNSWDAAYVLENGVYSLTSEKFNDAYRY VDWLLTVPLLLVETVAVLTLPAKEARPLLIKLTVASVLMIATGYPGEISDDITTRIIWGTVS TIPFAYILYVLWVELSRSLVRQPAAVQTLVRNMRWLLLLSWGVYPIAYLLPMLGVSGTSA AVGVQVGYTIADVLAKPVFGLLVFAIALVKTKADQESSEPHAAIGAAANKSGGSLIS (Gloeobacter violaceus rhodopsin) (SEQ ID NO: 1).

[0017] The rhodopsin may comprise a modified sequence that affords it greater quantum efficiency than that of Gloeobacter violaceus rhodopsin. This modified sequence may be a naturally-occurring sequence, or may alternatively be a non-naturally-occurring sequence. For function, rhodopsin requires post-translational modification with a covalently conjugated chromophore molecule, such as retinal. Therefore, the bacterial cell may further comprise a chromophore such as retinal.

[0018] Retinal

[0019] The bacterial cell may synthesise retinal (e.g. naturally) and / or the bacterial cell may be engineered such that it is capable of retinal biosynthesis for functional rhodopsin. Additionally or alternatively, the bacterial cell may be provided with retinal, for example by supplementation and / or co-culture with a retinal -producing organism. In one embodiment, the bacterial cell is supplemented with exogenous trans-retinal.

[0020] The bacterial cell may be engineered such that it is capable of P-carotene biosynthesis. The engineered bacterial cell may be further provided with the expression of P-carotene- 15, 15 ’-dioxygenase for conversion of P-carotene into retinal.

[0021] The bacterial cell may be engineered by the transformation with one or more, or all, of the genes selected from dxs, dxr, ispH, ispA, crtE, crtB, crtl, crtY, blh; or alternative genes encoding equivalent functioning enzymes. The genes may be provided with a promoter, such as their native promoter.

[0022] The bacterial cell may be engineered to express one or more, or all, of the enzymes selected from l-deoxy-D-xylulose-5 -phosphate synthase (e.g. encoded by dxs), 1- deoxy-D-xylulose-5-phosphate reductoisomerase (e.g. encoded by dxr), 4-hydroxy-3- methylbut-2-enyl diphosphate reductase (e.g. encoded by ispH), farnesyl diphosphate synthase (e.g. encoded by ispA), geranylgeranyl diphosphate synthase (e.g. encoded by crtE), phytoene synthase (e.g. encoded by crtB), phytoene desaturase (e.g. encoded by crtl), lycopene cyclase (e.g. encoded by crtY), and P-carotene-15, 15’-dioxygenase (e.g. encoded by blh).

[0023] The bacterial cell may be engineered to express one or more, or all, of the enzymes selected from l-deoxy-D-xylulose-5 -phosphate synthase (e.g. encoded by dxs), 1- deoxy-D-xylulose-5-phosphate reductoisomerase (e.g. encoded by dxr), 4-hydroxy-3- methylbut-2-enyl diphosphate reductase (e.g. encoded by ispH), farnesyl diphosphate synthase (e.g. encoded by ispA), geranylgeranyl diphosphate synthase (e.g. encoded by crtE), phytoene synthase (e.g. encoded by crtB), phytoene desaturase (e.g. encoded by crtl), lycopene cyclase (e.g. encoded by crtY), and further engineered to express P- carotene- 15, 15 ’-dioxygenase (e.g. encoded by blh) and a rhodopsin, such as Gloeobacter violaceus rhodopsin.

[0024] The bacterial cell may be engineered to express one or more, or all, of the enzymes selected from l-deoxy-D-xylulose-5-phostaphate synthase (e.g. encoded by dxs), 1- deoxy-D-xylulose-5-phosphate reductoisomerase (e.g. encoded by dxr), 4-hydroxy-3- methylbut-2-enyl diphosphate reductase (e.g. encoded by ispH), farnesyl diphosphate synthase (e.g. encoded by ispA), geranylgeranyl diphosphate synthase (e.g. encoded by crtE), phytoene synthase (e.g. encoded by crtB), phytoene desaturase (e.g. encoded by crtl), lycopene cyclase (e.g. encoded by crtY), and P-carotene-15, 15’-dioxygenase (e.g. encoded by blh ,' and further engineered to express a rhodopsin, such as Gloeobacter violaceus rhodopsin. The expression could be a combination of native expression and recombinant expression, or it may be entirely recombinant.

[0025] The bacterial cell may be engineered by the transformation with a four gene carotenoid biosynthetic pathway comprising crtl, crtY, crtE and crtB and optionally a gene encoding -carotene- 15, 15 ’-dioxygenase (such as blh). The bacterial cell may be engineered by the transformation with crtl and crtY, and the function of endogenous crtE and crtB genes may be restored, for example by insertion of a promoter and / or recombinant crtE and crtB genes. One or more of the crtl, crtY, crtE and crtB genes may be from Erwinia herbicola (Pantoea agglomerans) or E. coli.

[0026] Provision of a carotenoid, such as canthaxanthin

[0027] The bacterial cell may be further provided with one or more carotenoids such as canthaxanthin, salinixanthin or echinenone. Preferably the carotenoid comprises a rhodopsin-binding carotenoid. The carotenoids may be selected from canthaxanthin, astaxanthin, salinixanthin or echinenone; or combinations thereof. Preferably, the bacterial cell is further provided with canthaxanthin and / or echinenone. The GR and carotenoid, such as canthaxanthin, may form a complex, such as a GR-canthaxanthin complex, in the bacterial cell. Advantageously, carotenoids, such as canthaxanthin, act as an antenna of GR to improve the capture of light energy. The GR-canthaxanthin complex provides a 5-fold more proton pumping capacity compared to sole GR.

[0028] The carotenoid, such as canthaxanthin, may be provided as a supplement, such as in the culture media, or the bacterial cell may comprise the enzyme(s) necessary for biosynthesis. The bacterial cell may express, or may be engineered to express, enzyme(s) for carotenoid biosynthesis, such as canthaxanthin biosynthesis.

[0029] Canthaxanthin biosynthesis proceeds from beta-carotene via the action of beta-carotene ketolase e.g. via the action of a protein comprising any of SEQ ID NO: 21-23. In a favoured embodiment, the bacterial cell may express, or may be engineered to express, beta-carotene ketolase.

[0030] The bacterial cell may synthesise canthaxanthin (e.g. naturally) and / or may be engineered such that it is capable of canthaxanthin (also known as P,P-carotene-4,4'- dione) biosynthesis. Additionally or alternatively, the bacterial cell may be provided with canthaxanthin, for example by supplementation and / or co-culture with a canthaxanthin-producing organism.

[0031] The bacterial cell may be engineered such that it is provided with the expression of a P- carotene ketolase (also known as P-C-4-oxygenase) for conversion of P-carotene into canthaxanthin. In an embodiment wherein the bacterial cell comprises S. oneidensis MR-1 that has been engineered such that it is capable of P-carotene biosynthesis, the engineered S. oneidensis MR-1 may be further provided with the expression of a P- carotene ketolase for conversion of P-carotene into canthaxanthin.

[0032] The bacterial cell, such as S. oneidensis MR-1, may be engineered by the transformation with one or more, or all, of the genes selected from Bkt, crtW, crtO, crtZ, Psyl, crtl, crtB, crtE, bhy, crtY, hbfdl, hbfd2, cbfdl, cbfd2,' or alternative genes encoding equivalent functioning enzymes. The genes may be provided with a promoter, such as their native promoter. In one embodiment, the bacterial cell, such as S. oneidensis MR- 1, may be engineered by the transformation with one or more, or all, of the genes selected from crtO, crtl, crtB, crtE, and crtY. In one embodiment, the bacterial cell, such as S. oneidensis MR-1, may be engineered by the transformation with one or more, or all, of the genes selected from crtE, idi, crtl, crtY, and crtB genes, for example of Erwinia herbicola (Pantoea agglomerans) EholO, and a crtO for example of Haematococcus pluvialis. Such genes may be encoded on a plasmid and optionally fused to a promoter, such as Trc promoter. In one embodiment, the bacterial cell may be transformed with a plasmid suitable for providing canthaxanthin synthesis, such as pAC-CANTHipi (SEQ ID NO: 32) (Cunningham FX Jr, Gantt E. Photosynth Res. 2007 May;92(2):245-59. Epub 2007 Jul 17. 10.1007 / sl 1120-007-9210-0 PubMed 17634749 (www .addgene . o rg / 53301 / ) . which is incorporated herein by reference).

[0033] The bacterial cell, such as S. oneidensis MR-1, may be engineered by the transformation with a four gene carotenoid biosynthetic pathway comprising crtl, crtY, crtE and crtB and a gene encoding p-carotene ketolase (such as Bkt, crt , or crtO). One or more of the crtl, crtY, crtE and crtB genes may be from Erwinia herbicola (Pantoea agglomerans) or E. coli. The Bkt gene may be from Chlamydomonas reinhardtii . The crtW gene may be from Paracoccus sp. The crtO gene may be from Gloeobacter violaceus.

[0034] The skilled person will recognise that expression of one or more pathway components may be a combination of native expression and recombinant expression, or it may be entirely recombinant.

[0035] In one embodiment, the bacterial cell may be engineered such that it is capable of both retinal biosynthesis and canthaxanthin biosynthesis. Additionally or alternatively, the bacterial cell may be provided with both retinal and canthaxanthin, for example by supplementation and / or co-culture with a retinal-producing organism and / or a canthaxanthin-producing organism.

[0036] Equivalent genes may be provided from other strains or species. In particular, the bacterial cell may be engineered to provide the carotenoid biosynthetic pathway using any suitable genes for the expression of one or more of l-deoxy-D-xylulose-5 -phosphate reductoisomerase (e.g. encoded by dxr),- geranylgeranyl diphosphate synthase (e.g. encoded by crtE),- zeaxanthin glucosyltransferase (e.g. encoded by crtX); phytoene synthase (e.g. encoded by crtB),- phytoene desaturase (e.g. encoded by crtP),- lycopene cyclase (e.g. encoded by crtY). The phytoene desaturase may comprise the amino acid sequence of MKPTTVIGAGFGGLALAIRLQAAGIPVLLLEQRDKPGGRAYVYEDQGFTFDAGPTVITDP SAIEELFALAGKQLKEYVELLPVTPFYRLCWESGKVFNYDNDQTRLEAQIQQFNPRDVEG YRQFLDYSRAVFKEGYLKLGTVPFLSFRDMLRAAPQLAKLQAWRSVYSKVASYIEDEHL RQAFSFHSLLVGGNPFATSSIYTLIHALEREWGVWFPRGGTGALVQGMIKLFQDLGGEVV LNARVSHMETTGNKIEAVHLEDGRRFLTQAVASNADVVHTYRDLLSQHPAAVKQSNKL QTKRMSNSLFVLYFGLNHHHDQLAHHTVCFGPRYRELIDEIFNHDGLAEDFSLYLHAPCV TDSSLAPEGCGSYYVLAPVPHLGTANLDWTVEGPKLRDRIFAYLEQHYMPGLRSQLVTH RMFTPFDFRDQLNAYHGSAFSVEPVLTQSAWFRPHNRDKTITNLYLVGAGTHPGAGIPGV IGSAKATAGLMLEDLI (Crtl) (SEQ ID NO: 2).

[0037] The lycopene cyclase may comprise the amino acid sequence of MQPHYDLILVGAGLANGLIALRLQQQQPDMRILLIDAAPQAGGNHTWSFHHDDLTESQH RWIAPLVVHHWPDYQVRFPTRRRKLNSGYFCITSQRFAEVLQRQFGPHLWMDTAVAEVN AESVRLKKGQVIGARAVIDGRGYAANSALSVGFQAFIGQEWRLSHPHGLSSPIIMDATVD QQNGYRFVYSLPLSPTRLLIEDTHYIDNATLDPECARQNICDYAAQQGWQLQTLLREEQG ALPITLSGNADAFWQQRPLACSGLRAGLFHPTTGYSLPLAVAVADRLSALDVFTSASIHH AITHFARERWQQQGFFRMLNRMLFLAGPADSRWRVMQRFYGLPEDLIARFYAGKLTLTD RLRILSGKPPVPVLAALQAIMTTHR (CrtY) (SEQ ID NO: 3).

[0038] The geranylgeranyl diphosphate synthase may comprise the amino acid sequence of MTVCAKKHVHLTRDAAEQLLADIDRRLDQLLPVEGERDVVGAAMREGALAPGKRIRPM LLLLTARDLGCAVSHDGLLDLACAVEMVHAASLILDDMPCMDDAKLRRGRPTIHSHYGE HVAILAAVALLSKAFGVIADADGLTPLAKNRAVSELSNAIGMQGLVQGQFKDLSEGDKP RSAEAILMTNHFKTSTLFCASMQMASIVANASSEARDCLHRFSLDLGQAFQLLDDLTDG MTDTGKDSNQDAGKSTLVNLLGPRAVEERLRQHLQLASEHLSAACQHGHATQHFIQAW FDKKLAAVS (CrtE) (SEQ ID NO: 4).

[0039] The phytoene synthase may comprise the amino acid sequence of MNNPSLLNHAVETMAVGSKSFATASKLFDAKTRRSVLMLYAWCRHCDDVIDDQTLGFQ ARQPALQTPEQRLMQLEMKTRQAYAGSQMHEPAFAAFQEVAMAHDIAPAYAFDHLEGF AMDVREAQYSQLDDTLRYCYHVAGVVGLMMAQIMGVRDNATLDRACDLGLAFQLTNI ARDIVDDAHAGRCYLPASWLEHEGLNKENYAAPENRQALSRIARRLVQEAEPYYLSATA GLAGLPLRSAWAIATAKQVYRKIGVKVEQAGQQAWDQRQSTTTPEKLTLLLAASGQALT SRMRAHPPRPAHLWQRPL (CrtB) (SEQ ID NO: 5). The l-deoxy-D-xylulose-5-phostaphate synthase (e.g. encoded by dxs) may comprise the amino acid sequence of

[0040] MHRITILGATGSIGESTLDVVRRHADRYVVHALTAHRQVRKLADQCVEFRPARAVVGTA EAALELETLLRDAGVKTEVSHGEAALESVAADAQTDSVMAAIVGAAGLRPTLAAARAG KRVLLANKE AL VMS GRIFMDAVREHGATLLPIDSEHNAIFQCLP ADDPRY GRGVARVLLT ASGGPFRTRDPATLHDISPDQACAHPNWVMGRKISVDSATMMNKGLEVIEAHWLFGAPA ERIEVLIHPQSIVHSMVAYTDGSVLAQLGNPDMRTPIAYGLAYPERIDAGVTPLDLTVAG GLHFEKPDLVRFPCLGLAFDALRAGGVAPAALNAANEVAVEAFLGGTVRFTDIAGIVRQ VLEATPQGPADTLEAVLSADALAREAAREGVAALAAKR (DXS) (SEQ ID NO: 6).

[0041] The 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (e.g. encoded by ispH may comprise the amino acid sequence of

[0042] MAQPRGFCAGVDRAIEIVERALERFGAPIYVRHEIVHNAYVVAGLRRKGAVFVRELDEVP AGATVIFSAHGVSREVRADAAARGLHVFDATCPLVTKVHVEVSKMRAEGCEIVMIGHRG HPEVEGTMGQASSGMLLVESVADVATLQVTDPSRLAYVTQTTLSVDETREIVAALKARF PQIREPKKQDICYATQNRQDAVKFMAPQVEVVIVVGSPNSSNSNRLRELAERLGVPAYM VDAPEQVRPEWIAGKRRIGLTAGASAPEALAQSIVERLRELGASQVRPLDGIEENMAFPLP RGLLPASAAA (IspH) (SEQ ID NO: 7).

[0043] The farnesyl diphosphate synthase (e.g. encoded by ispA) may comprise the amino acid sequence of

[0044] MSDFAQWMQAQGARTEAALQAALPAAETVPHTLHEAMRYAALSGGKRVRPLLVHAAG EVSGAAPAACDAAACAVEMIHAYSLVHDDMPCMDDDDLRRGRPTVHKAYDEATALLV GDALQTQAFIVLAGAGAIAPAARLQLVAELALASGSTGMAGGQAIDLQNVGRAMTREAL EAMHRMKTGALLRASVRMGALCGEIDAEGLAALDRYAAAVGLAFQVVDDILDVTADTA TLGKTAGKDAAHDKPTYVSLMGLDPARALAGTLRADAHEALAGFGERADRLRDLADLI VLRTH (IspA) (SEQ ID NO: 8).

[0045] The bacterial cell, such as S. oneidensis MR-1, may be transformed with the Erwinia uredovora (Pantoea ananatis) crtEXYIB operon, preferably with its promoter.

[0046] The crtEXYIB operon and promoter may comprise the nucleotide sequence of GTGCAACGTTATGGATTGATGGCGCTTTTGcTCGTTTCCTGCTGGGCCAGCGCGCATA ACATCGTCATCGGGCAGCCCcTTCCGTCGGTTTTTATTGCGGATAAAGGTGAAATGCG GCTGGATGGCGGCAAGGTTAACTATCAAAAAtgGAACAGCCTGTCTCTTCCGGgTCGG ACACGTTTAGTTATTCATGTTGCAGGACGATTGTCGGCCAAAGAGCAGTCCGCCCCGC TTATTGCGGCCCTGCAGCGCGCCAACCTGCCACAAGACCGGTTCCAGACCACAACCAT CGTGAATACAGATGATGCTTTGCCTGGCAGCAGTCTGTTTGTGATTAACAGTATCCGC

[0047] TCCAGTAAAAAAGCCTCACCATGGCAACAATTTATTATCGACAGTAGCGGCGTGGCA

[0048] CAACATCGCTGGCAGCTTAAGCCAGAAGGTGCCGCTGTCATCGTGCTGGACCCTGATG

[0049] GTCAGGTAAAGTTTGCGAAAGACACGGCGCTCAGTGCGGATGATGTTTCTCAGGTCAT

[0050] TGCAACATTGCGTGCGCTGGCAGGCTGATCCTGGCAACCCGGTAAAGGTACCGCACG

[0051] GTCTGCCAATCCGACGGAGGTTTATGAATTTTCCACCTTTTCCACAAGCTCAACTAGT

[0052] ATTAACGATGTGGATTTAGCAAAAAAAACCTGTAACCCTAAATGTAAAATAACGGGT

[0053] AAGCCTGCCAACCATGTTATGGCAGATTAAGCGTCTTTTTGAAGGGCACCGCATCTTT

[0054] CGCGTTGCCGTAAATGTATCCGTTTATAAGGACAGCCCGAATGACGGTCTGCGCAAA

[0055] AAAACACGTTCATCTCACTCGCGATGCTGCGGAGCAGTTACTGGCTGATATTGATCGA

[0056] CGCCTTGATCAGTTATTGCCCGTGGAGGGAGAACGGGATGTTGTGGGTGCCGCGATG

[0057] CGTGAAGGTGCGCTGGCACCGGGAAAACGTATTCGCCCCATGTTGCTGTTGCTGACCG

[0058] CCCGCGATCTGGGTTGCGCTGTCAGCCATGACGGATTACTGGATTTGGCCTGTGCGGT

[0059] GGAAATGGTCCACGCGGCTTCGCTGATCCTTGACGATATGCCCTGCATGGACGATGCG

[0060] AAGCTGCGGCGCGGACGCCCTACCATTCATTCTCATTACGGAGAGCATGTGGCAATAC

[0061] TGGCGGCGGTTGCCTTGCTGAGTAAAGCCTTTGGCGTAATTGCCGATGCAGATGGCCT

[0062] CACGCCGCTGGCAAAAAATCGGGCGGTTTCTGAACTGTCAAACGCCATCGGCATGCA

[0063] AGGATTGGTTCAGGGTCAGTTCAAGGATCTGTCTGAAGGGGATAAGCCGCGCAGCGC

[0064] TGAAGCTATTTTGATGACGAATCACTTTAAAACCAGCACGCTGTTTTGTGCCTCCATG

[0065] CAGATGGCCTCGATTGTTGCGAATGCCTCCAGCGAAGCGCGTGATTGCCTGCATCGTT

[0066] TTTCACTTGATCTTGGTCAGGCATTTCAACTGCTGGACGATTTGACCGATGGCATGAC

[0067] CGACACCGGTAAGGATAGCAATCAGGACGCCGGTAAAATCGACGCTGGTCAATCTGT

[0068] TAGGCCCGAGGGCGGTTGAAGAACGTCTGAGACAACATCTTCAGCTTGCCAGTGAGC

[0069] ATCTCTCTGCGGCCTGCCAACACGGGCACGCCACTCAACATTTTATTCAGGCCTGGTT

[0070] TGACAAAAAACTCGCTGCCGTCAGTTAAGGATGCTGCATGAGCCATTTCGCGGCGATC

[0071] GCACCGCCTTTTTACAGCCATGTTCGCGCATTACAGAATCTCGCTCAGGAACTGGTCG

[0072] CGCGCGGTCATCGGGTGACCTTTATTCAGCAATACGATATTAAACACTTGATCGATAG

[0073] CGAAACCATTGGATTTCATTCCGTCGGGACAGACAGCCATCCCCCCGGCGCGTTAACG

[0074] CGCGTGCTACACCTGGCGGCTCATCCTCTGGGGCCGTCAATGCTGAAGCTCATCAATG

[0075] AAATGGCGCGCACCACCGATATGCTGTGCCGCGAACTCCCCCAGGCATTTAACGATCT

[0076] GGCCGTCGATGGCGTCATTGTTGATCAAATGGAACCGGCAGGCGCGCTCGTTGCTGA

[0077] AGCACTGGGACTGCCGTTTATCTCTGTCGCCTGCGCGCTGCCTCTCAATCGTGAACCG

[0078] GATATGCCCCTGGCGGTTATGCCTTTCGAATACGGGACCAGCGACGCGGCTCGCGAA

[0079] CGTTATGCCGCCAGTGAAAAAATTTATGACTGGCTAATGCGTCGTCATGACCGTGTCA

[0080] TTGCCGAACACAGCCACAGAATGGGCTTAGCCCCCCCGGCAAAAGCTTCACCAGTGT

[0081] TTTTCGCCACTGGCGCAAATCAGCCAGCTTGTTCCTGAACTGGATTTTCCCCGCAAAG

[0082] CGTTACCGGCTTGTTTTCATGCCGTCGGGCCTCTGCGCGAAACGCACGCACCGTCAAC

[0083] GTCTTCATCCCGTTATTTTACATCCTCAGAAAAACCCCGGATTTTCGCCTCGCTGGGCA

[0084] CGCTTCAGGGACACCGTTATGGGCTGTTTAAAACGATAGTGAAAGCCTGTGAAGAAA TTGACGGTCAGCTCCTGTTAGCCCACTGTGGTCGTCTTACGGACTCTCAGTGTGAAGA

[0085] GCTGGCGCGAAGCCGTCATACACAGGTGGTGGATTTTGCCGATCAGTCAGCCGCGCT

[0086] GTCTCAGGCGCAGCTGGCGATCACCCACGGCGGCATGAATACGGTACTGGACGCGAT

[0087] TAATTACCGGACGCCCCTTTTAGCGCTTCCGCTGGCCTTTGATCAGCCCGGCGTCGCG

[0088] TCACGCATCGTTTATCACGGCATCGGCAAGCGTGCTTCCCGCTTTACCACCAGCCATG

[0089] CTTTGGCTCGTCAGATGCGTTCATTGCTGACCAACGTCGACTTTCAGCAGCGCATGGC

[0090] GAAAATCCAGACAGCCCTTCGTTTGGCAGGGGGCACCATGGCCGCTGCCGATATCATT

[0091] GAGCAGGTTATGTGCACCGGTCAGCCTGTCTTAAGTGGGAGCGGCTATGCAACCGCA

[0092] TTATGATCTGATTCTCGTGGGGGCTGGACTCGCGAATGGCCTTATCGCCCTGCGTCTT

[0093] CAGCAGCAGCAACCTGATATGCGTATTTTGCTTATCGACGCCGCACCCCAGGCGGGCG

[0094] GGAATCATACGTGGTCATTTCACCACGATGATTTGACTGAGAGCCAACATCGTTGGAT

[0095] AGCTCCGCTGGTGGTTCATCACTGGCCCGACTATCAGGTACGCTTTCCCACACGCCGT

[0096] CGTAAGCTGAACAGCGGCTACTTTTGTATTACTTCTCAGCGTTTCGCTGAGGTTTTACA

[0097] GCGACAGTTTGGCCCGCACTTGTGGATGGATACCGCGGTCGCAGAGGTTAATGCGGA

[0098] ATCTGTTCGGTTGAAAAAGGGTCAGGTTATCGGTGCCCGCGCGGTGATTGACGGGCG

[0099] GGGTTATGCGGCAAATTCAGCACTGAGCGTGGGCTTCCAGGCGTTTATTGGCCAGGA

[0100] ATGGCGATTGAGCCACCCGCATGGTTTATCGTCTCCCATTATCATGGATGCCACGGTC

[0101] GATCAGCAAAATGGTTATCGCTTCGTGTACAGCCTGCCGCTCTCGCCGACCAGATTGT

[0102] TAATTGAAGACACGCACTATATTGATAATGCGACATTAGATCCTGAATGCGCGCGGC

[0103] AAAATATTTGCGACTATGCCGCGCAACAGGGTTGGCAGCTTCAGACACTGCTGCGAG

[0104] AAGAACAGGGCGCCTTACCCATTACTCTGTCGGGCAATGCCGACGCATTCTGGCAGC

[0105] AGCGCCCCCTGGCCTGTAGTGGATTACGTGCCGGTCTGTTCCATCCTACCACCGGCTA

[0106] TTCACTGCCGCTGGCGGTTGCCGTGGCCGACCGCCTGAGTGCACTTGATGTCTTTACG

[0107] TCGGCCTCAATTCACCATGCCATTACGCATTTTGCCCGCGAGCGCTGGCAGCAGCAGG

[0108] GCTTTTTCCGCATGCTGAATCGCATGCTGTTTTTAGCCGGACCCGCCGATTCACGCTG

[0109] GCGGGTTATGCAGCGTTTTTATGGTTTACCTGAAGATTTAATTGCCCGTTTTTATGCGG

[0110] GAAAACTCACGCTGACCGATCGGCTACGTATTCTGAGCGGCAAGCCGCCTGTTCCGGT

[0111] ATTAGCAGCATTGCAAGCCATTATGACGACTCATCGTTAAAGAGCGACTACATGAAA

[0112] CCAACTACGGTAATTGGTGCAGGCTTCGNTGGCCTGGCACTGGCAATTCGTCTACAAG

[0113] CTGCGGGGATCCCCGTCTTACTGCTTGAACAACGTGATAAACCCGGCGGTCGGGCTTA

[0114] TGTCTACGAGGATCAGGGGTTTACCTTTGATGCAGGCCCGACGGTTATCACCGATCCC

[0115] AGTGCCATTGAAGAACTGTTTGCACTGGCAGGAAAACAGTTAAAAGAGTATGTCGAA

[0116] CTGCTGCCGGTTACGCCGTTTTACCGCCTGTGTTGGGAGTCAGGGAAGGTCTTTAATT

[0117] ACGATAACGATCAAACCCGGCTCGAAGCGCAGATTCAGCAGTTTAATCCCCGCGATG

[0118] TCGAAGGTTATCGTCAGTTTCTGGACTATTCACGCGCGGTGTTTAAAGAAGGCTATCT

[0119] AAAGCTCGGTACTGTCCCTTTTTTATCGTTCAGAGACATGCTTCGCGCCGCACCTCAA

[0120] CTGGCGAAACTGCAGGCATGGAGAAGCGTTTACAGTAAGGTTGCCAGTTACATCGAA

[0121] GATGAACATCTGCGCCAGGCGTTTTCTTTCCACTCGCTGTTGGTGGGCGGCAATCCCT

[0122] TCGCCACCTCATCCATTTATACGTTGATACACGCGCTGGAGCGTGAGTGGGGCGTCTG GTTTCCGCGTGGCGGCACCGGCGCATTAGTTCAGGGGATGATAAAGCTGTTTCAGGAT

[0123] CTGGGTGGCGAAGTCGTGTTAAACGCCAGAGTCAGCCATATGGAAACGACAGGAAAC

[0124] AAGATTGAAGCCGTGCATTTAGAGGACGGTCGCAGGTTCCTGACGCAAGCCGTCGCG

[0125] TCAAATGCAGATGTGGTTCATACCTATCGCGACCTGTTAAGCCAGCACCCTGCCGCGG

[0126] TTAAGCAGTCCAACAAACTGCAGACTAAGCGCATGAGTAACTCTCTGTTTGTGCTCTA

[0127] TTTTGGTTTGAATCACCATCATGATCAGCTCGCGCATCACACGGTTTGTTTCGGCCCGC

[0128] GTTACCGCGAGCTGATTGACGAAATTTTTAATCATGATGGCCTCGCAGAGGACTTCTC

[0129] ACTTTATCTGCACGCGCCCTGTGTCACGGATTCGTCACTGGCGCCTGAAGGTTGCGGC

[0130] AGTTACTATGTGTTGGCGCCGGTGCCGCATTTAGGCACCGCGAACCTCGACTGGACGG

[0131] TTGAGGGGCCAAAACTACGCGACCGTATTTTTGCGTACCTTGAGCAGCATTACATGCC

[0132] TGGCTTACGGAGTCAGCTGGTCACGCACCGGATGTTTACGCCGTTTGATTTTCGCGAC

[0133] CAGCTTAATGCCTATCATGGCTCAGCCTTTTCTGTGGAGCCCGTTCTTACCCAGAGCG

[0134] CCTGGTTTCGGCCGCATAACCGCGATAAAACCATTACTAATCTCTACCTGGTCGGCGC

[0135] AGGCACGCATCCCGGCGCAGGCATTCCTGGCGTCATCGGCTCGGCAAAAGCGACAGC

[0136] AGGTTTGATGCTGGAGGATCTGATATGAATAATCCGTCGTTACTCAATCATGCGGTCG

[0137] AAACGATGGCAGTTGGCTCGAAAAGTTTTGCGACAGCCTCAAAGTTATTTGATGCAA

[0138] AAACCCGGCGCAGCGTACTGATGCTCTACGCCTGGTGCCGCCATTGTGACGATGTTAT

[0139] TGACGATCAGACGCTGGGCTTTCAGGCCCGGCAGCCTGCCTTACAAACGCCCGAACA

[0140] ACGTCTGATGCAACTTGAGATGAAAACGCGCCAGGCCTATGCAGGATCGCAGATGCA

[0141] CGAACCGGCGTTTGCGGCTTTTCAGGAAGTGGCTATGGCTCATGATATCGCCCCGGCT

[0142] TACGCGTTTGATCATCTGGAAGGCTTCGCCATGGATGTACGCGAAGCGCAATACAGCC

[0143] AACTGGATGATACGCTGCGCTATTGCTATCACGTTGCAGGCGTTGTCGGCTTGATGAT

[0144] GGCGCAAATCATGGGCGTGCGGGATAACGCCACGCTGGACCGCGCCTGTGACCTTGG

[0145] GCTGGCATTTCAGTTGACCAATATTGCTCGCGATATTGTGGACGATGCGCATGCGGGC

[0146] CGCTGTTATCTGCCGGCAAGCTGGCTGGAGCATGAAGGTCTGAACAAAGAGAATTAT

[0147] GCGGCACCTGAAAACCGTCAGGCGCTGAGCCGTATCGCCCGTCGTTTGGTGCAGGAA

[0148] GCAGAACCTTACTATTTGTCTGCCACAGCCGGCCTGGCAGGGTTGCCCCTGCGTTCCG

[0149] CCTGGGCAATCGCTACGGCGAAGCAGGTTTACCGGAAAATAGGTGTCAAAGTTGAAC AGGCCGGTCAGCAAGCCTAGGATCAGCGGCAGTCAACGACCACGCCCGAAAAATTAA

[0150] CGCTGCTGCTGGCCGCCTCTGGTCAGGCCCTTACTCCCGGATGCGGGCTCATCCTCCC CGCCCTGCGCATCTCTGGCAGCGCCCGCTCTAGCGCCaTGTCTTTCCCGGAGCGTC (crtEXYIB operon) (SEQ ID NO: 9).

[0151] In one embodiment, the crtX gene may not be provided.

[0152] Additionally, -carotene- 15, 15 ’-dioxygenase may be provided, which may be encoded by the blh gene. The blh gene may be from the uncultured marine bacterium 66A03. The P-carotene-15, 15’-dioxygenase (encoded by the blh gene) may comprise the amino acid sequence of

[0153] MGGLMLIDWCALALVVFIGLPHGALDAAISFSMISSAKRIARLAGILLIYLLLATAFFLIWY QLPAFSLLIFLLISIIHFGMADFNASPSKLKWPHIIAHGGVVTVWLPLIQKNEVTKLFSILTN GPTPILWDILLIFFLCWSIGVCLHTYETLRSKHYNIAFELIGLIFLAWYAPPLVTFATYFCFI HSRRHFSFVWKQLQHMSSKKMMIGSAIILSCTSWLIGGGIYFFLNSKMIASEAALQTVFIG L AALTVPHMILIDFIFRPH S SRIKIKNKGELEGKPIPNPLLGLD STRTG ( -carotene -15, 15’- dioxygenase) (SEQ ID NO: 10).

[0154] The bacterial cell, such as S. oneidensis MR-1, may be further engineered for expression of l-deoxy-D-xylulose-5-phosphate reductoisomerase, which may be by overexpression of the endogenous dxr gene or transformation with a recombinant dxr gene. Preferably, the bacterial cell, such as S. oneidensis MR-1, is transformed with a dxr gene for overexpression. Preferably, a promoter, such as PBAD, is provided for overexpression.

[0155] The l-deoxy-D-xylulose-5-phosphate reductoisomerase may comprise the amino acid sequence of

[0156] MHRITILGATGSIGESTLDVVRRHADRYVVHALTAHRQVRKLADQCVEFRPARAVVGTA EAALELETLLRDAGVKTEVSHGEAALESVAADAQTDSVMAAIVGAAGLRPTLAAARAG KRVLLANKE AL VMS GRIFMDAVREHGATLLPIDSEHNAIFQCLP ADDPRY GRGVARVLLT ASGGPFRTRDPATLHDISPDQACAHPNWVMGRKISVDSATMMNKGLEVIEAHWLFGAPA ERIEVLIHPQSIVHSMVAYTDGSVLAQLGNPDMRTPIAYGLAYPERIDAGVTPLDLTVAG GLHFEKPDLVRFPCLGLAFDALRAGGVAPAALNAANEVAVEAFLGGTVRFTDIAGIVRQ VLEATPQGPADTLEAVLSADALAREAAREGVAALAAKR (l-deoxy-D-xylulose-5- phosphate reductoisomerase) (SEQ ID NO: 11).

[0157] The crtEXYIB operon genes may be promoted by the crtE endogenous promoter (PCUE), or an alternative promoter such as PBAD . Other genes, such as one or more of the dxr, blh and rhodopsin genes (such as Gloeobacter violaceus rhodopsin (GR)) may be under the control of the arabinose inducible PBAD promoter. The skilled person will recognise that any suitable promoter may be used.

[0158] The crtE endogenous promoter (PCUE) may comprise the sequence of GTGCAACGTTATGGATTGATGGCGCTTTTGcTCGTTTCCTGCTGGGCCAGCGCGCATA ACATCGTCATCGGGCAGCCCcTTCCGTCGGTTTTTATTGCGGATAAAGGTGAAATGCG GCTGGATGGCGGCAAGGTTAACTATCAAAAAtgGAACAGCCTGTCTCTTCCGGgTCGG ACACGTTTAGTTATTCATGTTGCAGGACGATTGTCGGCCAAAGAGCAGTCCGCCCCGC TTATTGCGGCCCTGCAGCGCGCCAACCTGCCACAAGACCGGTTCCAGACCACAACCAT CGTGAATACAGATGATGCTTTGCCTGGCAGCAGTCTGTTTGTGATTAACAGTATCCGC TCCAGTAAAAAAGCCTCACCATGGCAACAATTTATTATCGACAGTAGCGGCGTGGCA CAACATCGCTGGCAGCTTAAGCCAGAAGGTGCCGCTGTCATCGTGCTGGACCCTGATG GTCAGGTAAAGTTTGCGAAAGACACGGCGCTCAGTGCGGATGATGTTTCTCAGGTCAT TGCAACATTGCGTGCGCTGGCAGGCTGATCCTGGCAACCCGGTAAAGGTACCGCACG GTCTGCCAATCCGACGGAGGTTTATGAATTTTCCACCTTTTCCACAAGCTCAACTAGT ATTAACGATGTGGATTTAGCAAAAAAAACCTGTAACCCTAAATGTAAAATAACGGGT AAGCCTGCCAACCATGTTATGGCAGATTAAGCGTCTTTTTGAAGGGCACCGCATCTTT CGCGTTGCCGTAAATGTATCCGTTTATAAGGACAGCCCGA (SEQ ID NO: 12).

[0159] The PBAD promoter may comprise the sequence of AAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCT CTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGG ACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTC CACATTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAG ATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCAT (SEQ ID NO: 13).

[0160] The P-carotene hydroxylase may comprise the amino acid sequence of MCQESVIVMQATQPLQTVSQAVPKEFLQADGGFNPNVAMFGIAILLMLANVFGYWQWG LPHWLCFSCSVLALHLSGTVIHDASHNAAHRNTIINAVLGHGSALMLGFAFPVFTRVHLQ HHANVNDPENDPDHFVSTGGPLFLIAARFFYHEIFFFKRRLWRKYELLEWFLSRLVLFTIV FLGIHYGFIGFVMNYWFVPALIVGIALGLFFDYLPHRPFQERNRWKNARVYPSPILNWLIF GQNYHLIHHLWPSIPWYQYQNTYHITKPILDEKGCDQSLGLLEGKNFWSFLYDVFLGIRF HGHNNSQSSDKP (Bhy) (SEQ ID NO: 14), or alternatively the amino acid sequence of MLWIWNALIVFVTVIGMEVIAALAHKYIMHGWGWGWHLSHHEPRKGAFEVNDLYAVV FAALSILLIYLGSTGMWPLQWIGAGMTAYGLLYFMVHDGLVHQRWPFRYIPRKGYLKRL YMAHRMHHAVRGKEGCVSFGFLYAPPLSKLQATLRERHGARAGAARDAQGGEDEPASG K (CrtZ) (SEQ ID NO: 15).

[0161] The phytoene synthase 1 may comprise the amino acid sequence of MAIILVRAASPGLSAADSISHQGTLQCSTLLKTKRPAARRWMPCSLLGLHPWEAGRPSPA VYSSLAVNPAGEAVVSSEQKVYDVVLKQAALLKRQLRTPVLDARPQDMDMPRNGLKEA YDRCGEICEEYAKTFYLGTMLMTEERRRAIWAIYVWCRRTDELVDGPNANYITPTALDR WEKRLEDLFTGRPYDMLDAALSDTISRFPIDIQPFRDMIEGMRSDLRKTRYNNFDELYMY CYYVAGTVGLMSVPVMGIATESKATTESVYSAALALGIANQLTNILRDVGEDARRGRIYL PQDELAQAGLSDEDIFKGVVTNRWRNFMKRQIKRARMFFEEAERGVTELSQASRWPVW ASLLLYRQILDEIEANDYNNFTKRAYVGKGKKLLALPVAYGKSLLLPCSLRNGQT (Psyl) (SEQ ID NO: 16).

[0162] The carotenoid 4-hydroxy-P-ring 4-dehydrogenase may comprise the amino acid sequence of

[0163] TLHGACLPWIETNSLHWKLVKETTVGNTLVSPLNKTQNSRVLVLGGTGKVGGSTAFALS KFSPDLRLVIGGRNREKGDAVVSKLGENSEFVEVNVDSMRSLESAFKDVDLVVHAAGPF QQAEKCTVLEAAISTRTAYVDVCDNTSYSMQAKSFHDKAVAANVPAITTAGIFPGVSNVI AAELVRSARDENTEPQRLRFSYFTAGSGGAGPTSLVTSFLLLGEEVVAYSEGEKVELKPY TGKLNIDFGKGVGKRDVYLWNLPEVRSGHEILGVPTVSARFGTAPFFWNWAMVAMTSL LPPGILRDRNIIEKLANFVYPSVQVFDGIAGECLAMRVDLECANGRNTSAILSHERLSELV GTSTAVFALAILEGSTQAGVWFPEEPEGIAVGDRELLLKRASQGAINFIMKQ (HBFD 1) (SEQ ID NO: 17), or alternatively the amino acid sequence of MAPVLLGLKPTLSTGSVVKETNVGSTLASPLNKTQNSRVLVLGGTGKVGGSTALALSKFS PDLRLVIGGRNREKGDAVVSKLGENSEFVEVNVDSVRSLESALEDVDLVVHAAGPFQQA EKCTVLEAAISTRTAYVDVCDNTSYSMQAKSFHDKAVAANVPAITTAGIFPGVSNVIAAE LVRSARDENTEPQRLRFSYFTAGSGGAGPTSLVTSFLLLGEEVVAYSEGEKVELKPYTGK LNIDFGKGVGKRDVYLWNLPEVRSGHEILGVPTVSARFGTAPFFWNWAMVAMTTLLPPG ILRDRNKIGMLANFVYPSVQIFDGIAGECLAMRVDLECANGRNTFGILSHERLSVLVGTST AVFAMAILEGSTQPGVWFPEEPGGIAISDRELLLQRASQGAINFIMKQ (HBFD2) (SEQ ID NO: 18).

[0164] The carotenoid P-ring 4-dehydrogenase may comprise the amino acid sequence of AISVFSTSYSFHKNLLLHSKQDILNRPCLLFSPVVVESPMRKKKTHRAACICSVAERTRNL DIPQIEEEEENEEELIEQTDSGIIHIKKTLGGKQSRRSTGSIVAPVSCLGILSMIGPAVYFKFS RLMECGDIPVAEMGITFAAFVAAAIGTEFLSGWVHKELWHDSLWYIHKSHHRSRKGRFE FNDVFAIINALPAIALINYGFSNEGLLPGACFGTGLGTTVCGMAYIFLHNGLSHRRFPVGLI AN VP YFHKL A A AHQIHH S GKFQG VPFGLFL GPQELEE VRGGTEELER VI SRT AKRTQ S ST (CBFD 1) (SEQ ID NO: 19), or alternatively the amino acid sequence of AISVFSSGYSFYKNLLLDSKPNILKPPCLLFSPVVIMSPMRKKKKHGDPCICSVAGRTRNL DIPQIEEEEENVEELIEQTDSDIVHIKKTLGGKQSKRPTGSIVAPVSCLGILSMIGPAVYFKF SRLMEGGDIPVAEMGITFATFVAAAVGTEFLSAWVHKELWHESLWYIHKSHHRSRKGRF EFNDVFAIINALPAIALINYGFSNEGLLPGACFGVGLGTTVCGMAYIFLHNGLSHRRFPVW LIANVP YFHKL AAAHQIHHSGKFQGVPFGLFLGPKELEEVRGGTEELERVISRTTKRTQPS T (CBFD2) (SEQ ID NO: 20). The P-carotene ketolase may comprise the amino acid sequence of MGPGIQPTSARPCSRTKHSRFALLAAALTARRVKQFTKQFRSRRMAEDILKLWQRQYHLP REDSDKRTLRERVHLYRPPRSDLGGIAVAVTVIALWATLFVYGLWFVKLPWALKVGETA TSWATIAAVFFSLEFLYTGLFITTHDAMHGTIALRNRRLNDFLGQLAISLYAWFDYSVLHR KHWEHHNHTGEPRVDPDFHRGNPNLAVWFAQFMVSYMTLSQFLKIAVWSNLLLLAGAP LANQLLFMTAAPILSAFRLFYYGTYVPHHPEKGHTGAMPWQVSRTSSASRLQSFLTCYHF DLHWEHHRWPYAPWWELPKCRQIARGAALAPGPLPVPAAAAATAATAAAAAAATGSP APASRAGSASSASAAASGFGSGHSGSVAAQPLSSLPLLSEGVKGLVEGAMELVAGGSSSG GGGEGGKPGAGEHGLLQRQRQLAPVGVMA (Bkt) (SEQ ID NO: 21), or alternatively the amino acid sequence of

[0165] MSAHALPKADLTATSLIVSGGIIAAWLALHVHALWFLDAAAHPILAIANFLGLTWLSVGL FIIAHDAMHGSVVPGRPRANAAMGQLVLWLYAGFSWRKMIVKHMAHHRHAGTDDDPD FDHGGPVRWYARFIGTYFGWREGLLLPVIVTVYALILGDRWMYVVFWPLPSILASIQLFV FGTWLPHRPGHDAFPDRHNARSSRISDPVSLLTCFHFGGYHHEHHLHPTVPWWRLPSTRT KGDTA (CrtW) (SEQ ID NO: 22), or alternatively the amino acid sequence of MMRGSAVKERTSKRLAEGVITHKNDSSGLWWALVIIGLWIFSFAAALRLPIGELSLQAVI GVVILRTFLHTGLFITAHDAMHRTVFPANHRINDWLGTAAVGLYAFMPYRELLIKHQLH HRFPATGKDPDYHDGEHSGFFQWYLKFMKDYMESRNTPFLIAGMAVVFGVCTWLMGVP LVNLALFWLLPLVLSSLQLFYFGTYLPHRQPDGGYRNRHRATSNRLSSFWSFVSCYHFGY HWEHHEYPLVPWHRLPEARR (CrtO) (SEQ ID NO: 23).

[0166] In an embodiment wherein the bacterial cell encodes one or more of the required genes for the retinal and / or canthaxanthin biosynthetic pathways, the skilled person will recognise that there are several options for restoring the relevant pathway. For example, defective or missing genes may be replaced by transformation with a recombinant gene from the same or different strain or species. The gene may be provided with or without a promoter, such as a strong promoter. Alternatively, the expression of an endogenous gene may be restored or increased by the insertion of a promoter for such a gene. Furthermore, a defective gene may be restored by one or more restorative mutations, such as substitutions, deletions or additions.

[0167] If the bacterial cell already expresses one or more functional enzymes of the retinal and / or canthaxanthin biosynthesis pathways, the bacterial cell may only be engineered to express the enzymes that are missing from the pathway. Additionally or alternatively, one or more intermediate molecules in the relevant pathway or pathways may be supplied in the media by supplement or a co-cultured cell. Preferably the transformed genes are chromosomally (i.e. stably) integrated. Preferably, one or more, or all, of the transformed genes are codon optimised for expression in the bacterial cell, such as S. oneidensis MR-1.

[0168] The hydrogenase

[0169] The bacterial cell may be engineered to express the hydrogenase. The engineering to express the hydrogenase may comprise the transformation and expression of a sequence encoding the hydrogenase. Alternatively, the expression of an endogenous hydrogenase in the bacterial cell may be enhanced, for example by the engineering of a promoter for enhanced expression of the gene encoding an endogenous hydrogenase. Alternatively, a non-functioning endogenous hydrogenase may be restored by mutation.

[0170] The hydrogensase may comprise membrane bound Fe-Fe hydrogenase. The hydrogenase may comprise Shewanella spp. hydrogenase. In one embodiment, the hydrogenase may comprise S. oneidensis MR-1 [Fe-Fe] hydrogenase. In a preferred embodiment, the hydrogenase comprises both subunits (A and B) of S. oneidensis MR-1 [Fe-Fe] hydrogenase. The hydrogenase may be encoded by hydA and hydB, which may be overexpressed in the bacterial cell. Overexpression may be provided by providing a promoter to enhance the expression of endogenous hydA and hydB, and / or transforming the bacterial cell with hydA and hydB.

[0171] Advantageously, S. oneidensis MR-1 [Fe-Fe] hydrogenase has excellent catalytic efficiency.

[0172] The hydrogenase subunit A may comprise the sequence of MTTTTYQPGEIQGLIKINASKCKGCDACKQFCPTHAINGASGAVHSIDEDKCLSCGQCLIN CPFSAIEETHSALETVIKKLADKNTTVVGIIAPAVRVAIGEEFGLGTGELVTGKLYGAMNQ AGFKIFDCNFAADLTIMEEGSEFIHRLHANVKGEANAGPLPQFTSCCPGWVRYLETRYPA LLPNLSTAKSPQQMAGTVAKTYGAKVYQMQPENIFTVSVMPCTSKKLEASRPEFNSAWQ YHQEHGANSPSYQDIDAVLTTREMAQLLKLLDIDLANTAEYQGDSLFSEYTGAGTIFGTT GGVMEAALRTAHKVLTGTEMAKLEFEPVRGLKGVKSASVSLFDTELNQDVTVNVAVVH DMGNNIEPVLRDVMAGTSPYHFIEVMNCAGGCVNGGGQPIEGKGSSWLGNI (S. oneidensis MR-1 hydrogenase subunit A) (SEQ ID NO: 24), or a variant thereof. The hydrogenase subunit B may comprise the sequence of MNKKKHLFAEDSFFLSRRKFMAVGAAFVAALAIPIGWFTSKLERRNEYIKARSQGLYKD DSLAKTRVSHANPAVEKYYKEFGGEPLGHMSHELLHTHFVDRTKLSS (S. oneidensis MR-1 hydrogenase subunit B) (SEQ ID NO: 25), or a variant thereof.

[0173] The bacterial cell

[0174] The bacterial cell may comprise any bacterial cell that has, or is engineered to provide, the rhodopsin and the hydrogenase.

[0175] The bacterial cell may comprise Shewanella spp. such as Shewanella oneidensis MR-1. Alternatively, the bacterial cell may comprise E. coli, Pseudomonas spp. such as Pseudomonas putida, Synechocystis spp. such as Synechocystis sp. PCC6803, or Ralstonia spp. such as R. eutropha.

[0176] Advantageously, a Gram-negative bacterial cell can improve the system by concentrating protons and electrons in its periplasmic space, which is favourable for the operation of the hydrogenase. Abundant hydrogenase can increase the H2 synthesis rate.

[0177] Advantageously, S. oneidensis MR-1 has a native hydrogenase, which is encoded by the hydAB gene cluster. Further advantageously, this hydrogenase is capable of catalysing H2 synthesis from protons under physiological conditions.

[0178] The bacterial cell may comprise S. oneidensis MR-1 that is engineered to provide a rhodopsin, and a canthaxanthin (and / or retinal) biosynthesis pathway.

[0179] The retinal biosynthesis pathway of S. oneidensis MR-1 may be provided by transforming .S'. oneidensis MR-1 with the genes of dxr, crtl, crtY, crtE and crtB and a gene for expressing P-carotene-15, 15’-dioxygenase, such as blh. The canthaxanthin biosynthesis pathway of S. oneidensis MR-1 may be provided by further transforming S. oneidensis MR-1 with a gene to for expressing P-carotene ketolase, such as Bkt, crtO, and crt . Preferably one or more promoters may be provided for expression of such genes. In another embodiment, the bacterial cell may be a simple cell that has, or has been engineered to provide the components of the electromicrobial system for synthesis of hydrogen described herein. For example, the simple cell may be Gram-negative and engineered to provide i) an inner membrane-bound rhodopsin for proton-pumping, ii) a membrane-bound hydrogenase, and iii) an electron mediator located in the periplasm.

[0180] The simple cell (which may also be known as a “chromosome-free bacterial cell”) may be in accordance with patent application publication WO2021079145A1, which is herein incorporated by reference. The skilled person will recognise that chromosome free bacterial cells are safe and programmable platforms for synthetic biology, for example as described by Fan et al. (Proc. Natl Acad. Sci. 117, 6752-6761. 2020), which is herein incorporated by reference. The simple cell may be derived from any one of the Gram -negative bacterial cell types described herein, such as S. oneidensis MR-1.

[0181] The skilled person will recognise that a given bacterial cell or simple cell may already have some of the required components for the electromicrobial system for synthesis of hydrogen according to the invention. Therefore, the engineering of such a cell may comprise only the transformation or modification of the necessary genetic information to provide, or activate, the expression of the components that are not already encoded and / or expressed in the cell.

[0182] A population of the bacterial cells may be provided. The population of the bacterial cells may be in a concentration or total number that is viable for hydrogen production, such as viable for maintenance or growth of the bacterial cells. In one embodiment, the population of the bacterial cells that is provided corresponds to up to about 2000 pg biomass protein per cm2of electrode. In one embodiment, the population of the bacterial cells that is provided corresponds to at least about 1 pg biomass protein per cm2of electrode. In one embodiment, the population of the bacterial cells that is provided corresponds to between about 1 pg to about 2000 pg biomass protein per cm2of electrode. The person skilled in the art will recognise that the biomass protein measurement is directly proportional to, and thus an approximate measure of, the number of bacterial cells in the population. The biomass protein may be measured in by appropriate means. In a preferred embodiment, the population of the bacterial cells that is provided corresponds to between about 50 pg and about 500 pg biomass protein per cm2of electrode. The population of bacterial cells may be provided as a biofilm that is adherent to the surface of the electrode, for example the electrode may be a microbially reduced electrode. Alternatively or additionally, the population of bacterial cells, or part of the population of bacterial cells, may initially be provided separately to the electrode, e.g. as a colony for inoculation.

[0183] The electron source

[0184] The electron source may be any system that is capable of donating electrons into the bacterial cell directly or through intermediate molecules such as an electron mediator.

[0185] The electron source may comprise an electrode linked to a source of electricity. The source of electricity may be a solar panel, wind turbine, hydroelectric turbine, nuclear fission or fusion, thermal, such as geothermal, bioenergy, such as bio-fermentation, gravitational potential energy, wave energy, and / or chemical. In embodiments where the source of electricity is chemical, the source may be hydrogen, optionally produced by the electromicrobial system of the invention. In another embodiment, the source of electricity may be a battery. Preferably the source of electricity is from a renewable energy source (i.e. not via the burning of fossil fuels). The electron source may comprise a potentiostat to control the cathode potential and / or a voltage regulator. A voltage regulator can help stabilise the cathode potential to avoid cathode-potential fluctuations that could harm cells.

[0186] In a particularly preferred embodiment, the source of electricity is a solar panel. The solar panel may otherwise be described as an “external photocell”. The solar panel may be electrically connected to an electrode, which can donate electrons to the electron transport chain.

[0187] Advantageously, the use of solar panels for the electron source complements the need for the bacterial cell to utilise a light source for the rhodopsin (proton pump). Therefore, light energy can provide energy for both functions of driving the rhodopsin proton pump and feeding electrons into the cell. In particular, the system may operate from just light energy, protons and electrons as energy sources.

[0188] The electron source may be an electrode connected to a source of electricity. The electrode may be part of an electrolytic cell or galvanic cell. The electrode may comprise a graphene oxide-coated electrode. In one embodiment, the electrode comprises a reduced graphene oxide-coated electrode. Preferably the reduced graphene oxide-coated electrode is microbially reduced. Alternatively, other reduction methods are available to the skilled person, such as chemical reduction, for example using sodium borohydride.

[0189] Microbial reduction of the electrode is advantageously sustainable and avoids the production of undesirable byproducts.

[0190] The electrode may be a cathode linked to a power source and wherein the cathode is in fluid contact with the bacterial cell. The cathode may be in the same media and / or chamber as the bacterial cell. The anode and cathode may be separated by a barrier, which may comprise a proton exchange membrane. In a preferred embodiment, a dual chamber electrode system is provided, which has a membrane only allowing proton transfer between the two chambers.

[0191] A dual chamber system can advantageously avoid the reactive oxygen species generated by the electrode, which could limit or kill the bacterial cells.

[0192] In a preferred embodiment, an electron mediator is provided to supply electrons to the cell and / or facilitate electron transport in the periplasm. The electron mediator may be an endogenous redox mediator to enhance the extracellular electron transfer rate. The electron mediator may comprise or consist of riboflavin, methyl viologen, neutral red, anthraquinone-2,6-disulfonate, potassium ferricyanide, OmcA, iron(II) sulfide (FeS) nanoparticles, and CymA, or combinations thereof. In a preferred embodiment, the electron mediator comprises iron sulphide (FeS) nanoparticles. The iron sulphide (FeS) nanoparticles may be located in the periplasm of the bacterial cell. In one embodiment, the electron mediator comprises or consists of a combination of OmcA, FeS nanoparticles, and CymA (tetraheme cytochrome). In one embodiment, the electron mediator comprises or consists of a combination of OmcA, MtrC, MtrA, MtrB, FeS nanoparticles, and CymA (tetraheme cytochrome). The electron mediator, such as the FeS nanoparticles, may be produced by the bacterial cell, or supplied externally, for example by supplementation of the media. The bacterial cell may be engineered to produce the electron mediator, such as OmcA, FeS nanoparticles, or CymA. In a preferred embodiment, the bacterial cell may natively, or may be engineered to, express membrane proteins that enable the bacterial cell to synthesise FeS nanoparticles de novo by enzyme-catalysed precipitation. The bacterial cell may be incubated with thiosulfate and FeSO4 to form FeS nanoparticles in the periplasm and on the outer membrane.

[0193] The skilled person will recognise that an important step for formation of FeS nanoparticles is the generation of S2". In .S'. oneidensis, phsA is a key gene for reducing S2O to S2-. Therefore, adding Na2S20s to S. oneidensis reduces S2O to S2-, which then forms FeS nanoparticles. The process also happens in other sulfate-reducing bacteria, which are able to reduce SO42to S2-. The bacterial cell may be naturally competent to generate S2-or engineered to generate S2-. The bacterial cell may encode phsA naturally or by engineering.

[0194] In one embodiment, the electron mediator comprises OmcA. For example, in an embodiment wherein the bacterial cell expresses MtrCAB, OmcA acts as an electron mediator, which can react with MtrC to enhance the electron transfer rate. In one embodiment a MtrCBA protein cluster (native or recombinant) and self-assembled iron sulfide (FeS) nanoparticles may act in tandem to facilitate electron transfer within the periplasm.

[0195] In another embodiment, the electron source may be from an electron donor molecule, such as an electron donor molecule capable of oxidation and associated enzyme capable of oxidising the electron donor molecule, such as a dehydrogenase. The electron source may be an organic compound, hydrogen, ammonia or an electrode, or combinations thereof. In one embodiment, the electron donor molecule may be an organic molecule. The electron donor molecule may comprise one or more of the electron donor molecules selected from formate, hydrogen, pyruvate, 2-oxogluterate, sulfur, sulfide, sulphite, thiosulphate, lactate, ethanol, glycerol, malate, succinate, gluconate, x-amines, NADH, and humics, or combinations thereof. The bacterial cell may comprise, or may be engineered to comprise, an enzyme capable of oxidising an electron donor molecule, such as a dehydrogenase or oxidase. The bacterial cell may comprise, or may be engineered to comprise, one or more of formate dehydrogenase; hydrogenase; pyruvate dehydrogenase; 2-oxoglutarate-dehydrogenase; rhodanese; sulfide dehydrogenase; sulfite oxidase; thiosulphate dehydrogenase; lactate oxidase; ethanol dehydrogenase; glycerol dehydrogenase; malate dehydrogenase; succinate dehydrogenase; gluconate dehydrogenase; amine dehydrogenase; and NADH dehydrogenase.

[0196] In one embodiment, the electron source may be a combination of two or more electron sources. For example, the electron source may be a combination of an electrode linked to a source of electricity and an electron donor molecule. The electron source may be a combination of two or more electron donor molecules. The electron source may be one or more electrodes linked to two or more sources of electricity.

[0197] The medium

[0198] The bacterial cell may be provided in a medium for growth and / or maintenance (i.e. culture media). The medium may be any suitable medium that is capable of growth and / or maintenance of the bacterial cell. The medium may be an aqueous NaCl solution e.g. approximately 33-37 g / L NaCl / FUO. The medium may be untreated sea water, or treated sea water.

[0199] The medium may comprise, or be supplemented with, essential nutrients for growth or maintenance of the bacterial cell. In one embodiment, the medium comprises, or is supplemented with, the electron mediator, such as FeS. Additionally or alternatively, the electron mediator, such as FeS, may be produced by the bacterial cell or by a cocultured cell. In one embodiment, FeSC and thiosulfate (e.g. Na2S20s) may be supplemented to the medium of the bacterial cell, to enable the generation of FeS nanoparticles by the bacterial cell itself or by a co-cultured cell.

[0200] The medium may comprise, or be supplemented with, one or more inert gases to achieve anaerobic conditions. The medium may comprise or be supplemented with gaseous N2. Supplementation of the medium with one or more inert gases may be continuous or, alternatively, the supplementation may be implemented in a pulsatile fashion.

[0201] In an embodiment wherein the electrons are provided by an electrode, the electrode, such as the cathode, may be submerged in the medium with the bacterial cell. Additionally or alternatively, the medium may comprise, or be supplemented with, an electron donor molecule. Additionally or alternatively, the medium may comprise, or be supplemented with, one or more carotenoids such as canthaxanthin, salinixanthin and echinenone, and / or one of more carotenoid precursors such as retinal. Preferably, the media comprises, or is supplemented with, canthaxanthin and / or retinal.

[0202] Other cellular components

[0203] The skilled person will recognise that in addition to a rhodopsin, and a hydrogenase, the electromicrobial system for H2 photoelectrosynthesis in accordance with the present invention may additionally require: a quinone pool; and an electron transport chain.

[0204] The skilled person will recognise that such components may be naturally provided in the bacterial cell. One or all of these components may be engineered in the bacterial cell if necessary.

[0205] The quinone pool

[0206] The quinone pool may be an endogenous quinone pool in the membrane of the bacterial cell. The quinone may be menaquinone-7.

[0207] The electron transport chain

[0208] In a preferred embodiment, the bacterial cell has a native functional electron transport chain. The electron transport chain may be an endogenous electron transport chain in the membrane of the bacterial cell. In another embodiment, the bacterial cell may be engineered to provide a functional electron transport chain, for example by the transformation with one or more, or all, of the genes required for a functional electron transport chain.

[0209] In one embodiment, the bacterial cell expresses, or is engineered to express, MtrCAB. MtrCAB is a multi-heme protein complex linking the intracellular electron transport chain with extracellular substrates. Advantageously, the electron transport proteins encoded in MtrCAB genes enables cells to take electrons directly from an electrode.

[0210] Advantageously, the energy transfer efficiency can be increased from 20% to 45% by expression of MtrCAB and carbonic anhydrase (can), and addition of carotenoids, such as canthaxanthin.

[0211] The MtrCAB may be recombinant and / or heterologous. The MtrCAB may be recombinantly expressed.

[0212] In one embodiment, the bacterial cell is transformed with nucleic acid, such as plasmid DNA, encoding the MtrCAB gene cluster. The plasmid may comprise a selection marker. The MtrCAB may be expressed under the control of a suitable promoter, such as the PBAD promoter. The promoter may be inducible or repressible. The MtrCAB gene cluster may be maintained extrachromosomally or stably integrated into the chromosomal DNA.

[0213] MtrA may comprise or consist of the sequence of SEQ ID NO: 26 (MKNCLKMKNLLPALTITMAMSAVMALVVTPNAYASKWDEKMTPEQVEATLDKKFAE GNYSPKGADSCLMCHKKSEKVMDLFKGVHGAIDSSKSPMAGLQCEACHGPLGQHNKGG NEPMITFGKQSTLSADKQNSVCMSCHQDDKRMSWNGGHHDNADVACASCHQVHVAKD PVLSKNTEMEVCTSCHTKQKADMNKRSSHPLKWAQMTCSDCHNPHGSMTDSDLNKPSV NDTCYSCHAEKRGPKLWEHAPVTENCVTCHNPHGSVNDGMLKTRAPQLCQQCHASDGH ASNAYLGNTGLGSNVGDNAFTGGRSCLNCHSQVHGSNHPSGKLLQR).

[0214] MtrB may comprise or consist of the sequence of SEQ ID NO: 27 (MKFKLNLITLALLANTGLAVAADGYGLANANTEKVKLSAWSCKGCVVETGTSGTVGV GVGYNSEEDIRSANAFGTSNEVAGKFDADLNFKGEKGYRASVDAYQLGMDGGRLDVNA GKQGQYNVNVNYRQIATYDSNSALSPYAGIGGNNLTLPDNWITAGSSNQMPLLMDSLNA LELSLKRERTGLGFEYQGESLWSTYVNYMREEKTGLKQASGSFFNQSMMLAEPVDYTTD TIEAGVKLKGDRWFTALSYNGSIFKNEYNQLDFENAFNPTFGAQTQGTMALDPDNQSHT VSLMGQYNDGSNALSGRILTGQMSQDQALVTDNYRYANQLNTDAVDAKVDLLGMNLK VVSKVSNDLRLTGSYDYYDRDNNTQVEEWTQISINNVNGKVAYNTPYDNRTQRFKVAA DYRITRDIKLDGGYDFKRDQRDYQDRETTDENTVWARLRVNSFDTWDMWVKGSYGNR DGSQYQASEWTSSETNSLLRKYNLADRDRTQVEARITHSPLESLTIDVGARYALDDYTDT VIGLTESKDTSYDANISYMITADLLATAFYNYQTIESEQAGSSNYSTPTWTGFIEDQVDVV GAGISYNNLLENKLRLGLDYTYSNSDSNTQVRQGITGDYGDYFAKVHNINLYAQYQATE

[0215] KLALRFDYKIENYKDNDAANDIAVDGIWNVVGFGSNSHDYTAQMLMLSMSYKL).

[0216] MtrC may comprise or consist of the sequence of SEQ ID NO: 28 (MMNAQKSKIALLLAASAVTMALTGCGGSDGNNGNDGSDGGEPAGSIQTLNLDITKVSY ENGAPMVTVFATNEADMPVIGLANLEIKKALQLIPEGATGPGNSANWQGLGSSKSYVDN KNGSYTFKFDAFDSNKVFNAQLTQRFNVVSAAGKLADGTTVPVAEMVEDFDGQGNAPQ YTKNIVSHEVCASCHVEGEKIYHQATEVETCISCHTQEFADGRGKPHVAFSHLIHNVHNA NKAWGKDNKIPTVAQNIVQDNCQVCHVESDMLTEAKNWSRIPTMEVCSSCHVDIDFAA GKGHSQQLDNSNCIACHNSDWTAELHTAKTTATKNLINQYGIETTSTINTETKAATISVQ VVDANGTAVDLKTILPKVQRLEIITNVGPNNATLGYSGKDSIFAIKNGALDPKATINDAGK LVYTTTKDLKLGQNGADSDTAFSFVGWSMCSSEGKFVDCADPAFDGVDVTKYTGMKAD LAFATLSGKAPSTRHVDSVNMTACANCHTAEFEIHKGKQHAGFVMTEQLSHTQDANGK AIVGLDACVTCHTPDGTYSFANRGALELKLHKKHVEDAYGLIGGNCASCHSDFNLESFK KKGALNTAAAADKTGLYSTPITATCTTCHTVGSQYMVHTKETLESFGAVVDGTKDDATS AAQSETCFYCHTPTVADHTKVKM).

[0217] The MtrCAB gene cluster may comprise the sequence of SEQ ID NO: 29 (ATGATGAACGCACAAAAATCAAAAATCGCACTGCTGCTCGCAGCAAGTGCCGTCAC AATGGCCTTAACCGGCTGTGGTGGAAGCGATGGTAATAACGGCAATGATGGTAGTGA TGGTGGTGAGCCAGCAGGTAGCATCCAGACGTTAAACCTAGATATCACTAAAGTAAG CTATGAAAATGGTGCACCTATGGTCACTGTTTTCGCCACTAACGAAGCCGACATGCCA GTGATTGGTCTCGCAAATTTAGAAATCAAAAAAGCACTGCAATTAATACCGGAAGGG GCGACAGGCCCAGGTAATAGCGCTAACTGGCAAGGCTTAGGCTCATCAAAGAGCTAT GTCGATAATAAAAACGGTAGCTATACCTTTAAATTCGACGCCTTCGATAGTAATAAGG TCTTTAATGCTCAATTAACGCAACGCTTTAACGTTGTTTCTGCTGCGGGTAAATTAGC AGACGGAACGACCGTTCCCGTTGCCGAAATGGTTGAAGATTTCGACGGCCAAGGTAA TGCGCCGCAATATACAAAAAATATCGTTAGCCACGAAGTATGTGCTTCTTGCCACGTA GAAGGTGAAAAGATTTATCACCAAGCTACTGAAGTCGAAACTTGTATTTCTTGCCACA CTCAAGAGTTTGCGGATGGTCGCGGCAAACCCCATGTCGCCTTTAGTCACTTAATTCA CAATGTGCATAATGCCAACAAAGCTTGGGGCAAAGACAATAAAATCCCTACAGTTGC ACAAAATATTGTCCAAGATAATTGCCAAGTTTGTCACGTTGAATCCGACATGCTCACC GAGGCAAAAAACTGGTCACGTATTCCAACAATGGAAGTCTGTTCTAGCTGTCACGTA GACATCGATTTTGCTGCGGGTAAAGGCCACTCTCAACAACTCGATAACTCCAACTGTA TCGCCTGCCATAACAGCGACTGGACTGCTGAGTTACACACAGCCAAAACCACCGCAA CTAAGAACTTGATTAATCAATACGGTATCGAGACTACCTCGACAATTAATACCGAAAC TAAAGCAGCCACAATTAGTGTTCAAGTTGTAGATGCGAACGGTACTGCTGTTGATCTC AAGACCATCCTGCCTAAAGTGCAACGCTTAGAGATCATCACCAACGTTGGTCCTAATA ATGCAACCTTAGGTTATAGTGGCAAAGATTCAATATTTGCAATCAAAAATGGAGCTCT

[0218] TGATCCAAAAGCTACTATCAATGATGCTGGCAAACTGGTTTATACCACTACTAAAGAC

[0219] CTCAAACTTGGCCAAAACGGCGCAGACAGCGACACAGCATTTAGCTTTGTAGGTTGG

[0220] TCAATGTGTTCTAGCGAAGGTAAGTTTGTAGACTGTGCAGACCCTGCATTTGATGGTG

[0221] TTGATGTAACTAAGTATACCGGCATGAAAGCGGATTTAGCCTTTGCTACTTTGTCAGG

[0222] TAAAGCACCAAGTACTCGCCACGTTGATTCTGTTAACATGACAGCCTGTGCCAATTGC

[0223] CACACTGCTGAGTTCGAAATTCACAAAGGCAAACAACATGCAGGCTTTGTGATGACA

[0224] GAGCAACTATCACACACCCAAGATGCTAACGGTAAAGCGATTGTAGGCCTTGACGCA

[0225] TGTGTGACTTGTCATACTCCTGATGGCACCTATAGCTTTGCCAACCGTGGTGCGCTAG

[0226] AGCTAAAACTACACAAAAAACACGTTGAAGATGCCTACGGCCTCATTGGTGGCAATT

[0227] GTGCCTCTTGTCACTCAGACTTCAACCTTGAGTCTTTCAAGAAGAAAGGCGCATTGAA

[0228] TACTGCCGCTGCAGCAGATAAAACAGGTCTATATTCTACGCCGATCACTGCAACTTGT

[0229] ACTACCTGTCACACAGTTGGCAGCCAGTACATGGTCCATACGAAAGAAACCCTGGAG

[0230] TCTTTCGGTGCAGTTGTTGATGGCACAAAAGATGATGCTACCAGTGCGGCACAGTCAG

[0231] AAACCTGTTTCTACTGCCATACCCCAACAGTTGCAGATCACACTAAAGTGAAAATGTA

[0232] ATTTGCCCAAGCAGGGGGAGCTCGCTCCCCCTTTCTTGAATTTTGTTGGGACAAATTG

[0233] GGAAGCCTATTATGAAGAACTGCCTAAAAATGAAAAACCTACTGCCGGCACTTACCA

[0234] TCACAATGGCAATGTCTGCAGTTATGGCATTAGTCGTCACACCAAACGCTTATGCGTC

[0235] GAAGTGGGATGAGAAAATGACGCCAGAGCAAGTCGAAGCCACCTTAGATAAGAAGTT

[0236] TGCCGAAGGCAACTACTCCCCTAAAGGCGCCGATTCTTGCTTGATGTGCCATAAGAAA

[0237] TCCGAAAAAGTCATGGACCTTTTCAAAGGTGTCCACGGTGCGATTGACTCCTCTAAGA

[0238] GTCCAATGGCTGGCCTGCAATGTGAGGCATGCCACGGCCCACTGGGTCAGCACAACA

[0239] AAGGCGGCAACGAGCCGATGATCACTTTTGGTAAGCAATCAACCTTAAGTGCCGACA

[0240] AGCAAAACAGCGTATGTATGAGCTGTCACCAAGACGATAAGCGTATGTCTTGGAATG

[0241] GCGGTCACCATGACAATGCCGATGTTGCTTGTGCTTCTTGTCACCAAGTACACGTCGC

[0242] AAAAGATCCTGTGTTATCTAAAAACACGGAAATGGAAGTCTGTACTAGCTGCCATAC

[0243] AAAGCAAAAAGCGGATATGAATAAACGCTCAAGTCACCCACTCAAATGGGCACAAAT

[0244] GACCTGTAGCGACTGTCACAATCCCCATGGGAGCATGACAGATTCCGATCTTAACAA

[0245] GCCTAGCGTGAATGATACCTGTTATTCCTGTCACGCCGAAAAACGCGGCCCAAAACTT

[0246] TGGGAGCATGCACCCGTCACTGAGAATTGTGTCACTTGCCACAATCCTCACGGTAGTG

[0247] TGAATGACGGTATGCTGAAAACCCGTGCGCCACAGCTATGTCAGCAATGTCACGCCA

[0248] GCGATGGCCACGCCAGCAACGCCTACTTAGGTAACACTGGATTAGGTTCAAATGTCG

[0249] GTGACAATGCCTTTACTGGTGGAAGAAGCTGCTTAAATTGCCATAGTCAGGTTCATGG

[0250] TTCTAACCATCCATCTGGCAAGCTATTACAGCGCTAAGGAGACGAGAAAATGAAATT

[0251] TAAACTCAATTTGATCACTCTAGCGTTATTAGCCAACACAGGCTTGGCCGTCGCTGCT

[0252] GATGGTTATGGTCTAGCGAATGCCAATACTGAAAAAGTGAAATTATCCGCATGGAGC

[0253] TGTAAAGGCTGCGTCGTTGAAACGGGCACATCAGGCACTGTGGGTGTCGGTGTCGGTT

[0254] ATAACAGCGAAGAGGATATTCGCTCTGCCAATGCCTTTGGTACATCCAATGAAGTGGC

[0255] GGGTAAATTTGATGCCGATTTAAACTTTAAAGGTGAAAAGGGTTATCGTGCCAGTGTT GATGCTTATCAACTCGGTATGGATGGCGGTCGCTTAGATGTCAATGCGGGCAAACAA

[0256] GGCCAGTACAACGTCAATGTGAACTATCGCCAAATTGCTACCTACGACAGCAATAGC

[0257] GCCCTATCGCCCTACGCGGGTATTGGTGGCAATAACCTCACGTTACCGGATAACTGGA

[0258] TAACAGCAGGTTCAAGCAACCAAATGCCACTCTTGATGGACAGCCTCAATGCCCTCG

[0259] AACTCTCACTTAAACGTGAGCGCACGGGGTTGGGATTTGAATATCAAGGTGAATCCCT

[0260] GTGGAGCACCTATGTTAACTACATGCGTGAAGAGAAAACCGGCTTAAAACAAGCCTC

[0261] TGGTAGCTTCTTCAACCAATCGATGATGTTAGCAGAGCCGGTGGATTACACCACTGAC

[0262] ACCATTGAAGCGGGTGTCAAACTCAAGGGTGATCGTTGGTTTACCGCACTCAGTTACA

[0263] ATGGGTCAATATTCAAAAACGAATACAACCAATTGGACTTTGAAAATGCTTTTAACCC

[0264] CACCTTTGGTGCTCAAACCCAAGGTACGATGGCACTCGATCCGGATAACCAGTCACAC

[0265] ACCGTGTCGCTGATGGGACAGTACAACGATGGCAGCAACGCACTGTCGGGTCGTATT

[0266] CTGACCGGACAAATGAGCCAAGATCAGGCGTTAGTGACGGATAACTACCGTTATGCT

[0267] AATCAGCTCAATACCGATGCCGTCGATGCCAAAGTCGATCTACTGGGTATGAACCTGA

[0268] AAGTCGTTAGCAAAGTGAGCAATGATCTTCGCTTAACAGGTAGTTACGATTATTACGA

[0269] CCGTGACAATAATACCCAAGTAGAAGAATGGACTCAGATCAGCATCAACAATGTCAA

[0270] CGGTAAGGTGGCTTATAACACCCCTTACGATAATCGTACGCAACGCTTTAAAGTTGCC

[0271] GCAGATTATCGCATTACCCGCGATATCAAACTCGATGGTGGTTATGACTTCAAACGTG

[0272] ACCAACGTGATTATCAAGACCGTGAAACCACGGATGAAAATACCGTTTGGGCCCGTT

[0273] TACGTGTAAACAGCTTCGATACTTGGGACATGTGGGTAAAAGGCAGTTACGGTAACC

[0274] GTGACGGCTCACAATACCAAGCGTCTGAATGGACCTCTTCTGAAACCAACAGCCTGTT

[0275] ACGTAAGTACAATCTGGCTGACCGTGACAGAACTCAAGTCGAAGCACGGATCACCCA

[0276] TTCGCCATTAGAAAGCCTGACTATCGATGTTGGTGCCCGTTACGCGTTAGATGATTAT

[0277] ACCGATACTGTGATTGGATTAACTGAGTCAAAAGACACCAGTTATGATGCCAACATC

[0278] AGTTATATGATCACCGCTGACTTACTGGCAACCGCCTTCTACAATTACCAAACCATTG

[0279] AGTCTGAACAGGCGGGTAGCAGCAATTACAGCACCCCAACGTGGACAGGCTTTATAG

[0280] AAGATCAGGTAGATGTGGTCGGTGCAGGTATCAGCTACAACAATCTGCTGGAGAACA

[0281] AGTTACGCCTAGGACTGGACTACACCTATTCCAACTCCGACAGTAACACTCAAGTCAG

[0282] ACAAGGTATCACTGGCGACTATGGTGATTATTTTGCCAAAGTGCATAACATTAACTTA

[0283] TACGCTCAATATCAAGCCACCGAGAAACTCGCGCTGCGCTTCGATTACAAAATTGAG

[0284] AACTATAAGGACAATGACGCCGCAAATGATATCGCCGTTGATGGCATTTGGAACGTC

[0285] GTAGGTTTTGGTAGTAACAGCCATGACTACACCGCACAAATGCTGATGCTGAGCATG

[0286] AGTTACAAACTCTAA)

[0287] OmcA may comprise the amino acid sequence of

[0288] MMKRFNFNTATKAMLGAGLLSLLLTGCGGSDGKDGEDGKPGVVGVNINSTSTLKAKFT

[0289] NATVDAGKVTVNFTLENANGVAVLGLTKDHDLRFGIAQLTPVKEKVGETEADRGYQWQ

[0290] AYINAKKEPGTVPSGVDNLNPSTQFQANVESANKCDTCLVDHGDGSYSYTYQVNVANV

[0291] TEPVKVTYSADATQRATMELELPQLAANAHFDWQPSTGKTEGIQTRNVVSIQACYTCHQ PESLALHGGRRIDIENCASCHTATSGDPESGNSIEFTYMIHAIHKGGERHTFDATGAQVPA PYKIIGYGGKVIDYGKVHYPQKPAADCAACHVEGAGAPANADLFKADLSNQACIGCHTE KPSAHHSSTDCMACHNATKPYGGTGSAAKRHGDVMKAYNDSLGYKAKFSNIGIKNNAL TFDVQILDNKDQPIGKEFISDPSAYTKSSIYFSWGIDKDYPAYTAGSRYSDRGFALSNSKV STYNEATKTFTIDSTNSNLKLPADLTGMNVELYAGVATCFNKGGYGVEDVVATPCSTDT RYAYIQDQPFRFKWNGTDTNSAAEKRRAIIDTAKCSGCHNKEIVHYDNGVNCQACHTPD KGLKTDNTYPGTKVPTSFAWKAHESEGHYLKYAGVQSGTVLKTDCATCHTADKSNVVT GIALGRSPERAWLYGDIKNNGAVIWVSSDAGACLSCHQKYLSDAAKSHIETNGGILNGTS AADVQTRASESCATCHTPSQLMEAHGN (SEQ ID NO: 30).

[0292] CymA may comprise the amino acid sequence of MNWRALFKPSAKYSILALLVVGIVIGVVGYFATQQTLHATSTDAFCMSCHSNHSLKNEV LASAHGGGKAGVTVQCQDCHLPHGPVDYLIKKIIVSKDLYGFLTIDGFNTQAWLDENRK EQADKALAYFRGNDSANCQHCHTRIYENQPETMKPMAVRMHTNNFKKDPETRKTCVDC HKGVAHPYPKG (SEQ ID NO: 31).

[0293] Other functions

[0294] The bacterial cell may be engineered for any suitable function requiring biosynthesis, conversion or degradation of products. The bacterial cell may be engineered for the provision of energy, which may for example drive any biosynthesis or biodegradation pathways in cells other than the bacterial cell itself. For example, in a co-culture of engineered Shewanella oneidensis MR-1 and Ralstonia eutropha, the S. oneidensis MR- 1 can synthesise H2, and the R. eutropha may use the H2 for metabolic oxidation. In one embodiment, an .S'. oneidensis MR-1 microbial cell engineered according to the invention synthesises and provides H2 to an engineered R. eutropha bacterial cell that is capable of CO2 fixation.

[0295] The light source

[0296] In a preferred embodiment, the light source is natural light, such as sunlight. Additionally or alternatively, artificial light may be used.

[0297] Other Aspects According to another aspect of the invention there is provided a Gram-negative bacterial cell that is recombinantly engineered for synthesis of hydrogen, the bacterial cell comprising: an inner membrane-bound rhodopsin; a rhodopsin-binding carotenoid; and a membrane-bound hydrogenase.

[0298] The bacterial cell may further comprise an electron mediator in the periplasm. The bacterial cell may further comprise MtrCAB.

[0299] The bacterial cell may comprise or encode the components necessary to provide the electromicrobial system described herein when provided with an electron source.

[0300] The bacterial cell may be engineered for synthesis of hydrogen in the presence of an electron source for donation of electrons into the bacterial cell. The electron source may be as described herein.

[0301] According to another aspect of the invention there is provided a composition comprising the bacterial cell according to the invention.

[0302] The composition may comprise a population of the bacterial cells. The composition may comprise cell culture media. The cell culture media may be suitable for maintenance, viability and / or growth of the bacterial cell.

[0303] According to another aspect of the invention there is provided the use of the recombinantly engineered bacterial cell according to the invention, or the electromicrobial system of the invention, for hydrogen generation.

[0304] According to another aspect of the invention there is provided a method of H2 generation and / or biosynthesis, the method comprising:

[0305] - providing the electromicrobial system of the invention, and

[0306] - culturing the bacterial cells in the presence of light and water.

[0307] Preferably the light is sunlight. The method may further comprise sub-culturing the bacterial cells for maintenance and / or growth of the culture.

[0308] The method may further comprise the harvesting of the H2, for example by purification or isolation from the culture media and / or cells.

[0309] According to another aspect of the invention there is provided a nucleic acid encoding one or more, or all, of the Bkt, crtO and crtW genes, and further encoding: one or more, or all, of the crtEXYIB operon genes, and , and rhodopsin genes (such as Gloeobacter violaceus rhodopsin (GR)).

[0310] The nucleic acid may further encode the dxr gene. The nucleic acid may further encode blh for retinal biosynthesis.

[0311] The nucleic acid may be DNA. The nucleic acid may be a plasmid.

[0312] According to another aspect of the invention there is provided a nucleic acid encoding a rhodopsin, such as Gloeobacter violaceus rhodopsin (GR); and a hydrogenase, such as HydA and HydB.

[0313] The nucleic acid may comprise the sequence of SEQ ID NO: 33 (pLOl la-HydAB-GR plasmid). The nucleic acid may be pLOl la-HydAB-GR described herein.

[0314] The nucleic acid may further encode genes necessary for canthaxanthin synthesis.

[0315] According to another aspect of the invention there is provided a bacterial cell comprising the nucleic acid of the invention.

[0316] According to another aspect of the invention there is provided a bacterial cell comprising nucleic acid encoding genes necessary for canthaxanthin synthesis and nucleic acid encoding a rhodopsin, such as Gloeobacter violaceus rhodopsin (GR).

[0317] The bacterial cell may further comprise nucleic acid encoding a hydrogenase, such as HydA and HydB. Additionally or alternatively, the bacterial cell may further comprise nucleic acid encoding MtrCAB. Additionally or alternatively, the bacterial cell may further comprise nucleic acid encoding OmcA. Preferably the bacterial cell comprises nucleic acid encoding a hydrogenase, such as HydA and HydB,' MtrCAB; OmcA; a rhodopsin, such as Gloeobacter violaceus rhodopsin (GR); and genes necessary for canthaxanthin synthesis.

[0318] The bacterial cell may further comprise nucleic acid encoding genes necessary for retinal biosynthesis. Additionally or alternatively, the bacterial cell may be naturally competent to generate S2-or engineered to generate S2-. The bacterial cell may encode phsA naturally or by engineering.

[0319] The nucleic acid encoding genes necessary for canthaxanthin synthesis may be a plasmid. The nucleic acid encoding rhodopsin, such as Gloeobacter violaceus rhodopsin (GR), may be a plasmid. The nucleic acid encoding HydA and HydB may be a plasmid. A single plasmid may encode all the components for expression, or two or more plasmids may encode the components for expression (i.e. they may be encoded on separate plasmids or together).

[0320] Each of the above-mentioned components for expression in the bacterial cell may be recombinant. Alternatively, the above-mentioned components for expression in the bacterial cell may be a mixture of endogenous or heterologous components to the bacterial cell. In particular, a bacterial cell may already express some of the components, and may need transformation with only some heterologous genes to be capable of hydrogen synthesis in accordance with the invention.

[0321] The nucleic acid of the invention may be stably integrated on the chromosome, or may be extrachromosomal.

[0322] According to another aspect of the invention there is provided a method of modifying a bacterial cell to generate H2, the method comprising the step of transforming the bacterial cell with the nucleic acid according to the invention.

[0323] The bacterial cell may further be provided with an electron source, e.g. as described herein. The bacterial cell may be further transformed with nucleic acid, such as plamids, described herein. For example, for the expression of the MtrCAB cluster and / or OmcA and / or CymA. One or more, or all of the genes may be provided for transformation on the same plasmid. The bacterial cell may be engineered to express or overexpress a hydrogenase, such as the hydrogenase described herein.

[0324] According to another aspect of the invention there is provided a Gram-negative bacterial cell, such as Shewanella oneidensis MR-1, engineered to express a rhodopsin, such as Gloeobacter rhodopsin (GR), and a rhodopsin-binding carotenoid, such as canthaxanthin; and optionally further engineered to express a hydrogenase.

[0325] According to another aspect of the invention there is provided H2 gas produced from the electromicrobial system or method of the invention herein.

[0326] Where reference is made to a polypeptide or nucleotide sequence, such as a variant polypeptide or nucleotide sequence, the skilled person will understand that one or more amino acid residue or nucleotide substitutions, deletions or additions, may be tolerated, optionally two substitutions may be tolerated in the sequence, such that it maintains its function. The skilled person will appreciate that 1, 2, 3, 4, 5 or more amino acid residues or nucleotides may be substituted, added or removed without affecting function. References to sequence identity may be determined by BLAST sequence alignment (www.ncbi.nlm.nih.gov / BLAST / ) using standard / default parameters. For example, the sequence may have 99% identity and still function according to the invention. In other embodiments, the sequence may have 98% identity and still function according to the invention. In another embodiment, the sequence may have 95% identity and still function according to the invention. In another embodiment, the sequence may have 90%, 85%, or 80% identity and still function according to the invention. In one embodiment, the variation and sequence identity may be according the full length sequence. In other embodiments, the variation may be limited to non-conserved sequences and / or sequences outside of active sites, such as binding domains. Therefore, an active site or binding site of a protein may be 100% identical, whereas the flanking sequences may comprise the stated variations in identity. Such variants may be termed “conserved active site variants”.

[0327] Amino acid substitutions may be conservative substitutions. For example, a modified residue may comprise substantially similar properties as the wild-type substituted residue. For example, a substituted residue may comprise substantially similar or equal charge or hydrophobicity as the wild-type substituted residue. For example, a substituted residue may comprise substantially similar molecular weight or steric bulk as the wild-type substituted residue. With reference to “variant” nucleic acid sequences, the skilled person will appreciate that 1, 2, 3, 4, 5 or more codons may be substituted, added or removed without affecting function. For example, conservative substitutions may be considered.

[0328] The skilled person will appreciate that reference to a bacterial cell may refer to one or more cells, such as a plurality of cells, or a culture of cells.

[0329] The skilled person will appreciate that preferred features of any one embodiment and / or aspect of the invention may be applied to all other embodiments and / or aspects of the invention.

[0330] Examples embodying an aspect of the invention will now be described with reference to the following figures:

[0331] Fig. 1. (A) Schematic of the sustainable bioprocess for H2 bioproduction. Shewanella oneidensis MR-1 uses hydrogenase to catalyse H2 synthesis from protons and electrons, powered by light and electricity. Reduced graphene oxide (rGO) and FeS nanoparticles are introduced to enhance the electron transfer. The cells are engineered to express Gloeobacter rhodopsin (GR) and its antenna canthaxanthin, which by harvests photons and pumps protons. (B) The same schematic but without FeS nanoparticles.

[0332] Fig. 2. (A) TEM images of S. oneidensis MR-1 after 2-day incubation with thiosulfate and FeSCE to form FeS nanoparticles in the periplasm and on the outer membrane. OM: outer membrane; IM: inner membrane. (B) A typical Raman spectrum of FeS nanoparticle synthesised by S. oneidensis . (C) Single cell Raman spectra of cells of S. oneidensis with and without FeS nanoparticles. (D) Fumarate reduction test. The biocathode was poised at -0.5 V (vs Ag / AgCl), and 25 mM fumarate was added at ~50 min.

[0333] Fig. 3. (A) Raman spectrum of graphene oxide (GO) and the reduced GO (rGO) due to the reduction reaction by S. oneidensis . (B) The biomass protein on the electrodes based on carbon paper (CP), CP with rGO and CP with rGO and self- assembled FeS nanoparticles. (C) Average electron uptake rate per cell and (D) H2 production over 72 hours using the bioelectrode based on carbon paper (CP), CP with rGO and CP with rGO and FeS nanoparticles at the cathodic potential of -0.75 vs. SHE. Statistics were performed with Student’s t-test (Data are means ± SD, n = 3).

[0334] Fig. 4. (A) The pictures of cells pellet with (GR+) and without (GR-) Gloeobacter rhodopsin expression. (B) A typical Raman spectrum of a cell with GR complexes identified by a band at -1530 cm-1. (C) The pictures of cells pellet with (CAN+) and without (CAN-) canthaxanthin expression. (D) A typical Raman spectrum of a cell with canthaxanthin (CAN) identified by a band at -1005, 1155 and 1517 cm-1. (E) Extracellular proton concentration changes in response to illumination with white light (-200 pmol / m2 / s).

[0335] Fig. 5. (A) The response of the engineered S. oneidensis cells to light and dark (B) H2 production by the engineered S. oneidensis with GR-CAN and the overexpression of hydrogenases at the potential of -0.75 vs. SHE. Statistics were performed with Student’s t-test (Data are means ± SD, n = 4).

[0336] Fig. 6. TEM image of (A) .S'. oneidensis MR-1 with FeS nanoparticles and (B) the control group without nanoparticles.

[0337] Fig. 7. Size distributions of FeS nanoparticles bio-synthesized by S. oneidensis MR-1 (n = 55).

[0338] Fig. 8. (A) Linear scanning voltammetry (LSV) analysis for the cathode of S. oneidensis . (B) Hydrogen production by carbon paper (CP) with and without S. oneidensis after 48 hours.

[0339] Fig. 9. Percentage of the cells expressing GR and canthaxanthin (CAN) in the population.

[0340] Fig. 10. Absorbance scan of the arabinose-induced S. oneidensis-G and noninduced cell extracts. Fig. 11. Thermodynamic analysis of redox potential and Gibbs free energy of converting protons to hydrogen.

[0341] Fig. 12. Plasmid map of the pLOl la-HydAB-GR.

[0342] Fig. 13. Picture of the practical set-up of the water-splitting electrochemical system using .S'. oneidensis-based cathode.

[0343] Examples

[0344] Engineering nano-bioreactor in bacterial cells for highly efficient hydrogen production by splitting water

[0345] Summary

[0346] Electrocatalytic water splitting driven by renewable energy source is a pivotal strategy for sustainable and clean hydrogen production. Green hydrogen production by splitting water is regarded as a promising energy solution for sustainable development. Microbial cells have been used as catalysts to facilitate the cathodic hydrogen evolution in the electrochemical system. However, the low hydrogen yield limits the application of biocatalysts although they could be a cost-effective alternative to the traditional metal catalysts. In this study, we present a novel approach by synergising nano-material engineering and synthetic biology to create a nano-bioreactor system for highly efficient hydrogen production. We engineered the periplasmic space (20-30 nm) of an electroactive bacterium, Shewanella oneidensis MR-1, to serve as the nano-bioreactor to enhance the interaction between electrons and protons, catalysed by membrane bond Fe-Fe hydrogenase, for hydrogen generation. To optimise electron transfer, we used the microbially reduced graphene oxide-coated electrode, which facilitated efficient electron transfer from the electrode to the cells. Inside the cells, native MtrCBA protein cluster and self-assembled iron sulfide (FeS) nanoparticles acted in tandem to facilitate electron transfer within the periplasm. To improve proton transfer, we engineered Shewanella oneidensis MR-1 to express Gloeohacter rhodopsin (GR) and light harvesting antenna canthaxanthin (CAN), which achieved high proton pumping efficiency illuminated by the light. In the light, hydrogen production rate increased by 35.6% because of the light-powered GR-CAN and overexpression of hydrogenases further improve hydrogen production rate over 50%. The engineered nano-bioreactor of .S'. oneidensis MR-1 has achieved a hydrogen yield of 80 pmol / mg-protein / day, over 10 times that of the control group with wild-type MR-1 cells alone. This innovative integration of biotic-abiotic systems provides new insights into designing efficient microbial electrosynthesis.

[0347] Results

[0348] Biogenic FeS nanoparticles assembled into S. oneidensis for enhanced inward electron transfer

[0349] S. oneidensis cells, as bio-nano-factories, have been reported to reduce a variety of multifunctional nanomaterials such as silver (16) and cadmium nanoparticles (13, 28). In this study, S. oneidensis MR-1 was used to synthesise iron sulfide (FeS) nanoparticles in the membrane because of its highly reductive and unique electrical properties along with easily accessible raw materials (29). Transmission electron microscopy (TEM) image of the cell with synthesised FeS shows that the formed nano-sized particles (15.5± 5.8 nm) were mostly located in the periplasmic space with a small number of the nanoparticles attached to the outer membrane of cells (Fig. 2A, Fig. 6 and Fig. 7). Raman micro-spectroscopy was used to verify the biosynthesised FeS nanoparticles at the single cell level (Fig. 2B). Two characteristic Raman bands at 219 and 282 cm1have been identified in the spectrum of pure FeS (Sigma-Aldrich Co., UK), and these bands can be attributed to the characteristic Fe-S vibrations (30). Single-cell Raman analysis revealed that Raman spectra of the cells assembled with FeS nanoparticles also displayed the bands associated with the Fe-S vibrations (Fig. 2C), which is in line with the TEM result of a self-assembling bio-nano hybrid cells of S. oneidensis MR-1.

[0350] From the TEN image, we can see a large number of FeS nanoparticles located in the periplasm, which could facilitate the periplasmic electron transfer. Electrochemical tests were performed to evaluate the effect of FeS nanoparticles on the inward extracellular electron transfer of S. oneidensis . We introduced fumarate as an electron acceptor into the S. oneidensis-bas A electrochemical system. In the anaerobic cathodic condition at a potential of -0.5 V (vs. Ag / AgCl), 25 mM fumarate was injected into the cathode chamber with .S'. oneidensis and S. oneidensis- . The addition of fumarate is expected to significantly enhance electron-transfer processes because S. oneidensis contains a periplasmic fumarate reductase catalysing the reduction of fumarate to succinate (15). We measured the current consumption and observed a prompt decrease in the cathodic current for both strains with and without FeS nanoparticles (Fig. 2D). Notably, the S. oneidensis-FeS strain demonstrated over twofold increase in inward current density (-37 pA / cm2) compared to S. oneidensis without FeS nanoparticles (-17 pA / cm2). This indicated that the self-assembled FeS nanoparticles in the S. oneidensis- FeS strain enhanced the inward electron transfer rate.

[0351] Constructing a biologically reduced graphene oxide-bacteria-FeS nanoparticles hybrid system for enhanced F production

[0352] S. oneidensis can be employed as a biocatalyst to transform protons into H2 by hydrogenases (11). Linear scanning voltammetry (LSV) analysis was used to measure hydrogen evolution reaction activity for the carbon paper cathode pre-grown with .S'. oneidensis at neutral pH. The LSV results show a significant cathodic current with an onset potential of around -0.56 V vs. SHE for the S. oneidensis MR-1 biocathode, in contrast to weak reduction currents observed in the control group of the carbon paperbased electrode (Fig. 8A). These results suggest that S. oneidensis MR-l-based biocathode should catalyse proton reduction with an overpotential of around 0.15 V, compared to the theoretical hydrogen evolution potential of -0.41 V vs. SHE. When we set the cathodic potential of -0.7 V vs. SHE, we observed a substantial production of hydrogen production at the biocathode, whereas almost no hydrogen was detected in the control cathode without S. oneidensis MR-1 (Fig. 8B).

[0353] The microbial electrochemical system can be combined with material engineering to enhance H2 production. Graphene has been widely applied to enhance the electrochemical properties of the biocathode, due to its high conductivity and large surface area (16, 17). To enhance the electrochemical performance, we used microbially reduced graphene oxide to coat the electrode. The outer membrane cytochromes of S. oneidensis MR-1 have been reported to catalyse graphene oxide (GO) to reduced graphene oxide (rGO) (31). We mixed S. oneidensis MR-1 cells with GO and incubated the mixture for 24 hours at room temperature. Raman spectroscopy was used to compare the spectra of the S. oneidensis MR-1-G0 samples before and after the incubation (Fig. 3A). The initial GO exhibits two sharp peaks, in which the D peak (around 1350 cm ’ ) is associated with the K-point phonons of Aigsymmetry, and the G peak (around 1580— 1600 cm ' ) is attributed to the zone centre phonons of E2gsymmetry (32). We observed an obvious red shift of the G band of the samples, transitioning from 1594 to 1581 cm"1after microbial reduction (Fig. 3A). This shift suggests the formation of rGO from GO, mediated by S. oneidensis MR-1. The microbially-reduced graphene oxide was dropped onto the carbon paper to form a thin layer, resulting in an rGO-coated cathode for subsequent microbial H2 electrosynthesis. We found an increase in biomass content on the rGO-coated electrode compared to the control carbon paper (Fig. 3B), which could be due to the large surface area of graphene. We further compare their electron uptake rates, demonstrating that the current consumption per milligram of cell protein was enhanced from 0.00464 pmol electrons / pg / hour to 0.00636 pmol electrons / pg / hour (Fig. 3C). This indicates that the introduction of graphene oxide has increased conductivity, accelerating the electron-transfer processes between the cells of S. oneidensis MR-1 and rGO sheets. The H2 production yield has increased from 7.82 pmol / mg / day to 25.69 pmol / mg / day (Fig. 3D). Then we combined rGO-biocathode with FeS nanoparticles. In the presence of FeS nanoparticles, electron uptake rate and hydrogen yield were further enhanced to 0.00817 pmol electrons / pg / hour and 43.11 pmol / mg / day (Fig. 3D), respectively, suggesting that nanomaterial engineering significantly enhanced the microbial H2 electrosynthesis.

[0354] Increasing proton pumping into the periplasm by synthetic biology approaches Outward proton-pumping rhodopsin is the most abundant microbial rhodopsin in nature, which has been widely used as a synthetic biology tool (33, 34). The bacteria with the rhodopsin are able to harvest light energy to pump out protons into periplasm, generating a proton gradient. Herein, we choose Gloeobacter rhodopsin (GR), from Gloeobacter violaceus, which has a high turnover rate and a fast photocycling rate (35). The plasmid pLOl la-GR was transferred into S. oneidensis MR-1, and the expression of GR was induced by 1 mM arabinose. The cell pellet of the induced group showed a pink colour compared to the uninduced control (Fig. 4A). Single-cell Raman spectroscopy showed a typical peak for GR at around 1530 cm-1, which is consistent with other studies (Fig. 4B). At the single-cell level, around 95% of the cell population were counted containing GR (Number of measured cell n=198, Fig. 9A), indicating the gene expression of GR in most S. oneidensis MR-l-GR. We used a plate reader to scan the absorbance of the cell extracts and observed the obvious absorbance at -540 nm compared to the control group, further confirming a strong light absorption of GR in .S'. oneidensis MR-l-GR (Fig. 10). Previous studies showed GR can be combined with carotenoids such as canthaxanthin and echinenone to enhance the proton-pumping capacity (36). We engineered S. oneidensis to synthesise canthaxanthin. The expression of canthaxanthin turned the colour of the cell pellet of S. oneidensis MR-l-GR-CAN (Table S I), shift from red to a little orange red (Fig. 4C). Single-cell Raman spectroscopy confirmed the characteristic peaks for canthaxanthin at around 1005, 1155, 1517 cm-1(Fig. 4D). Single cell Raman analysis confirms that over 99% population of .S'. oneidensis MR-l-GR-CAN expressed canthaxanthin (Number of measured cell n=198, Fig. 9B). To investigate the proton pumping capacities of cells with GR and GR- CAN, we monitor the pH dynamic change of the cell suspension in an unbuffered solution. Light is turned on for 50 seconds and then off when pH remains stable. The extracellular protons of S. oneidensis MR-l-GR-CAN showed a higher level than that of the S. oneidensis MR-l-GR, showing a ~50% increase in pH variation (Fig. 4E). This indicates that the introduction of canthaxanthin antenna can effectively enhance the proton pumping efficiency of the rhodopsin-expressing cells.

[0355] Synergising proton-pumping rhodopsin with overexpressed hydrogenases for enhanced H2 production

[0356] To investigate the electron uptake capacity of the engineered cells with .S'. oneidensis MR-l-GR-CAN in the light, strains were pre-grown and inoculated into bioelectrochemical systems with rGO and FeS nanoparticles (Fig. 1). The working electrode was poised at a cathodic potential of -0.75 vs. SHE. The cathode chamber was equipped with white LED lights to activate proton pumping in the GR-CAN complex of S. oneidensis MR-l-GR-CAN. The response of the cathodic current to the light was tested by switching on and off the LED lights. In the light conditions, the current consumption was significantly enhanced compared to those in the dark conditions. The current density was boosted from -58 pA / cm2to -64 pA / cm2within 40 minutes, indicating the proton motive force generated by GR enhanced the electron uptake by the engineered cells (Fig. 5A). Then we compared the hydrogen production between the nano-electrochemical system with and without expression of GR-CAN in .S'. oneidensis MR-l-GR-CAN. At a potential of -0.75 V vs. SHE, the hydrogen yield of the microbial electrochemical system was increased by 35.6% (Fig. 5B). This could be attributed to two major advantages of the GR-CAN. One is that the proton gradient generated by the GR-CAN complex promoted the electron transfer rate (Fig. 5A). Secondly, enhanced pumping of protons into the periplasmic nanoscale space created a more favourable environment by elevating the concentrations of electrons and protons and facilitating the hydrogenase-catalysed interaction for H2 synthesis. Based on thermodynamic analysis of the hydrogen synthesis (Fig. 11), the increase of proton and electron concentrations leads to a decrease of Gibbs free energy, lowering the energy barrier for the conversion of protons to hydrogen. Therefore, the periplasmic bio- nanoreactor significantly promoted the generation of H2.

[0357] In addition, a high enzyme concentration created in the periplasmic environment could also accelerate the reaction. Hydrogenases of S. oneidensis were efficient enzymes for catalytic H2 turnover (37). Here, the [Fe-Fe] hydrogenase encoded by hydA and hydB were overexpressed to further improve hydrogen production. The result shows that the hydrogen yield of hydrogenase-overexpressing cells achieved 80 pmol / mg-protein / d, which is 10 times that of wildtype S. oneidensis without modification. This hydrogen production rate is significantly higher than that of traditional systems based on wholecell catalysts (Table 1). This study demonstrates that the combination of nanomaterial engineering with synthetic biology unlocks the potential of microbial H2 electrosynthesis.

[0358] Discussion

[0359] This study aims to establish a sustainable and efficient system for green hydrogen production. A periplasmic nano-photo-electrochemical system has been constructed by synergizing the abiotic nanomaterials and biotic components. The system could be extended for the synthesis of other valuable chemicals such as formate (10) and succinate (38). The key materials used in this work, including the microbially reduced GO and FeS nanoparticles, are synthesised by biological methods. Compared to the traditional borohydride reduction methods, these protocols are eco-friendlier.

[0360] In this study, biogenic FeS nanoparticles play an important role in enhancing electrontransfer processes. S. oneidensis synthesises and assembles FeS nanoparticles, mirroring the natural processes observed in sulfate-reducing bacteria (SRB). In nature, 97% of sulfides on earth are produced by SRB (39). Most SRB can synthesise FeS nanoparticles by using ferrous compounds and their produced free sulfide (40). SRB assembled with FeS nanoparticles on the membrane and periplasm become electroactive because FeS nanoparticles are electrically conductive and can interact with biological components (41). The reductive capability of FeS nanoparticles also makes it a suitable mediator in the microbial electrosynthesis system. In fact, molecular ensembles of iron and sulfide are common in biological systems. For example, iron-sulfur (Fe-S) clusters are involved in important biochemical pathways, such as photosynthesis and respiration (42). Interestingly, the Fe-S clusters are important structures of hydrogenase which act as a connective line to deliver electrons to the central active site (37). Inspired by the naturally occurring FeS and the chains of Fe-S in hydrogenases, we introduced the FeS nanoparticles to the bio-nano-electrochemical system which showed a significant enhancement in electron transfer and H2 production.

[0361] Sustainable synthetic biology is regarded as an important tool to improve microbial synthesis performance. Microbial rhodopsins are one of the major photosystems in nature, which convert light energy to biochemical energy (43). Among them, outward proton-pumping microbial rhodopsin is widely used as an optogenetic tool to regulate the metabolism of engineered microorganisms (26, 33, 44). The proton motive force generated by the proton-pumping microbial rhodopsin has been demonstrated to increase intracellular ATP levels (45), survival rate (46), biomass growth (27) and bioproduction yield (26). In this study, the light-activated GR combined with canthaxanthin not only enhanced the electron uptake rate of cells but also provided a favourable condition in periplasm with elevated proton content. Although the introduction of rhodopsin boosts the H2 synthesis, the mechanism remains largely unexplored. For example, the dynamics of proton flux across the membranes are unclear. In addition, a practical application of the microbial photo-electrochemical system could require a more efficient rhodopsin, we expect to leverage artificial intelligence to engineer rhodopsin with superior properties, such as high tolerance to pH, high proton pumping capacity and broad spectrum of light absorbance.

[0362] Materials and methods

[0363] Bacterial strains, culture conditions and plasmid construction

[0364] All bacterial strains and plasmids used in this study are shown in Table S I . A. coli strains were grown in Lysogeny Broth (LB) at 37 °C under aeration by shaking at 200 rpm. .S'. oneidensis strains were grown in LB broth at 30 °C under aeration by shaking at 150 rpm. If required, 25 pg / ml chloramphenicol and 12.5 pg / ml tetracycline were added in the culture medium. All plasmid constructions were performed in E. coli DH5a. The plasmid pLOl la-GR contains the Gloeobacter rhodopsin gene. The plasmid pLOl la-GR-HydAB contains the GR gene and the HydAB genes from .S'. oneidensis MR-1 (Fig. 12). The plasmid pAC-BETAipi (Addgene: Plasmid #53301) contains the gene cluster for canthaxanthin synthesis (47). Induction of GR expression was accompanied by the addition of exogenous trans-retinal (Sigma-Aldrich) to achieve a final concentration of 5 pg / ml. Biosynthesis of iron sulfide nanoparticles

[0365] .S'. oneidensis strains were precultured in LB at 150 rpm for 24 hours. The cultured cells were collected by centrifugation at 3000 g for 3 min and washed twice times with phosphate buffer saline. Then the cells were incubated in 100 mL anaerobic mineral medium with 0.5 mM Na2S20s and 20 mM DL-lactate, with an initial ODeoo of 0.5, at 30 °C, stirring at 150 rpm. After 24 h incubation, 1 mM FeSCL was added, and cultured for another 24 h to form FeS nanoparticles. The anaerobic mineral medium contains 5.85 g / 1 NaCl, 0.3 g / 1 NaOH, 1.5 g / 1 NH4C1, 0.1 g / 1 KC1, 11.91 g / 1 (4-(2-hydroxyethyl)- 1 -piperazineethanesulfonic acid) HEPES, and 0.6 g / 1 NaELPCL FhO. The trace mineral minerals solution and amino acids solution were also added, and the compositions can be found in the previous study (48). The FeS nanoparticles formation is operated under the anaerobic condition by pumping in pure N2.

[0366] Microbial reduction of graphene oxide to modify the electrode

[0367] For the reduction of graphene oxide, 5 ml of graphene oxide (3 mg / ml) solution is mixed with 10 ml of 100 mL of 20-h culture of S. oneidensis MR-1 strains. The mixture is shaken at 200 rpm, 25 °C. The microbially reduced GO is collected after 24 hours. The rGO composite is washed three times by centrifugation and sonication with distilled water. The carbon paper (CP) is cut into pieces with an aera surface of 4 cm2(2 cm x 2 cm). 2 ml of the microbially-reduced GO solution is dropped on carbon paper and air dried to form a GO-coated electrode. In the subsequent experiments, the biomass on the electrode is quantified by the bicinchoninic acid (BCA) assay kit (49).

[0368] Transmission electron microscope (TEM) characterisation

[0369] S. oneidensis MR-1 cells with and without FeS nanoparticles were collected by centrifugation at 3000 g for 3 min, then fixed in 4% formaldehyde and 2.5% glutaraldehyde in 0.1 M PIPES buffer pH 7.2 for 1 hour at room temperature ahead of storage at 4°C. The following day samples were then washed with buffer (0.1 M PIPES buffer pH 7.2), centrifuged, and the cell pellets enrobed in 2.5% low melting point agarose. Dissected cubes (~lmm3) of enrobed cells were treated with 50 mM glycine in buffer, then washed again in buffer ahead of secondary fixation with 1% (w / v) osmium tetroxide and 1.5% potassium ferrocyanide in buffer. Samples were washed extensively with Milli-Q water and stained with 0.5% uranyl acetate (aq.) then washed again with Milli-Q water. The samples were then dehydrated through an ethanol series and infiltrated with and embedded in TAAB low-viscosity epoxy resin. The samples were then polymerised at 60°C for 24 hours. Sections of 90 nm were cut from the resin blocks using a Leica UC7 Ultramicrotome and collected onto 3 mm copper grids. The sections were then post-stained with lead citrate and imaged using a JEOL Flash 120kV TEM equipped with a Gatan Rio camera.

[0370] Raman spectroscopy characterisation

[0371] Single S. oneidensis cells and FeS nanoparticles were characterised by Raman microspectroscopy. Before the measurements, samples were washed three times with distilled water to remove traces of culture medium and extracellular metabolites. 1.5-pl suspension is dropped onto an aluminium-coated slide and air dried for subsequent measurements. The cells were observed under a microscope after washing. Raman spectroscopic acquisition was performed using a LabRAM HR Evolution confocal Raman microscope using a 100x / 0.75 air objective (HORIBA, UK). Single-cell Raman spectra were obtained using a 532-nm neodymium-yttrium aluminium garnet laser with a 300 grooves mm-1diffraction grating and were acquired in the range of 100-3200 cm" \ The laser power was set at ~80 mW which was attenuated by neutral density filters before focusing onto the samples. Spectra were recorded with LABSPEC 6 software (HORIBA, UK). All raw spectra were pre-processed within LABSPEC 6 software by cosmic ray correction, polyline baseline fitting and subtraction and vector normalization of the entire spectral region.

[0372] Linear sweep voltammetry (LSV) analysis

[0373] LSV tests were conducted on the carbon paper-based electrode and biocathode with .S'. oneidensis cells. The polarization curves were obtained within a potential range between -0.85 V and -0.25 V vs. SHE with a scanning rate of 1 mV / s. The LSV was controlled by the potentiostat (PalmSens, Netherlands). The current was recorded and the current density was calculated based on the current per square centimetre of the cathode projected area.

[0374] Electrochemical test for fumarate reduction

[0375] The H-shaped dual-chamber reactor was constructed by connecting two chambers with a proton exchange membrane (PEM) Nafion membrane (Fig. 13). The reference electrode (RE-5B, BASi, USA) was Ag / AgCl in a 3M KC1 solution (+0.197 V vs. Standard Hydrogen Electrode, SHE). The working electrode is carbon paper, and the counter electrode is platinum wire. Precultured S. oneidensis suspensions were transferred to 200 ml minimal medium with 20 mM DL-lactate in 500 ml flasks. After 12-h growth at 30 °C with 150 rpm, the cell suspension concentration was adjusted to an ODeoo of ~0.5 and injected into the working chamber with a working volume of 50 mL. In contrast, the counter chamber was filled with mineral medium without lactate and bacteria. The working chamber was purged with pure N2 gas to remove oxygen. The biocathode was imposed at -0.5 V (vs. Ag / AgCl) by the potentiostat (PalmSens, Netherlands). Once the cathodic current stabilized, 25 mM of fumarate was introduced to monitor the current change by the potentiostat (PalmSens, Netherlands).

[0376] Tests of the proton pumping capacity of the engineered cells in the light

[0377] The precultured S. oneidensis strains were harvested and washed with an unbuffed solution containing 10 mM NaCl, 10 mM MgSCL ^lLO. and 0.1 mM CaCh. Then cells were resuspended in the unbuffered solution to obtain an ODeoo of -1.5. The cell suspension was placed in the dark until the pH was stabilized, which was measured by a pH meter (Hanna edge). Following stabilization, the suspension was exposed to illumination with an intensity of -200 pmol / m / s for 50 seconds. Real-time pH values were recorded and the ApH was calculated.

[0378] Tests of the response of the electrochemical systems with the engineered cells to light

[0379] A photo-electrochemical system was constructed by surrounding the working chamber of the electrochemical system with 5 meters of white LED light strips. The strains with and without induction of GR-CAN complex were precultured as above and formed a biofilm on the electrode. The light was switched on for 40 minutes and then switched off. The currents were measured by the potentiostat (PalmSens, Netherlands).

[0380] Cathodic Hydrogen production

[0381] The photo-electrochemical system was constructed as described above. The S. oneidensis cells were incubated in the working chamber for 48 hours to form a biofilm on the electrode. Then planktonic cells and mineral medium in the working chamber were removed and replaced with the medium containing 50 mM HEPES and 50 mM NaCl. Then working electrode was poised at -0.75 vs. SHE for 72 hours. Hydrogen was detected in the headspace using gas chromatography equipped with the Carboxen-1010 column. For the light-driven hydrogen synthesis, LED light strips were switched on, providing illumination with a light intensity of approximately 200 pmol / m / s. During the hydrogen production, the working chamber was maintained at 25 °C without agitation.

[0382] References

[0383] All references are herein incorporated by reference.

[0384] 1. D. E. H. J. Gernaat et al. , Climate change impacts on renewable energy supply. Nature Climate Change 11, 1 19-125 (2021).

[0385] 2. M. Yue et al. , Hydrogen energy systems: A critical review of technologies, applications, trends and challenges. Renewable and Sustainable Energy Reviews 146, 1 1 1 180 (2021).

[0386] 3. J. A. Turner, Sustainable Hydrogen Production. Science 305, 972-974 (2004).

[0387] 4. M. Wang, L. Chen, L. Sun, Recent progress in electrochemical hydrogen production with earth-abundant metal complexes as catalysts. Energy & Environmental Science 5, 6763-6778 (2012).

[0388] 5. R. Bashyam, P. Zelenay, A class of non-precious metal composite catalysts for fuel cells. Nature 443, 63-66 (2006).

[0389] 6. L. Leone et al. , Enzymatic and Bioinspired Systems for Hydrogen Production. International Journal of Molecular Sciences 24, 8605 (2023).

[0390] 7. C. Yang et al. , Carbon dots-fed Shewanella oneidensis MR-1 for bioelectricity enhancement. Nature Communications 11, 1379 (2020).

[0391] 8. F. Li et al. , Modular engineering to increase intracellular NAD(H / +) promotes rate of extracellular electron transfer of Shewanella oneidensis. Nature Communications

[0392] 9. 3637 (2018).

[0393] 9. K. C. Ford, M. A. TerAvest, The electron transport chain of Shewanella oneidensis MR-1 can operate bidirectionally to enable microbial electrosynthesis. Applied and Environmental Microbiology 0, e01387-01323.

[0394] 10. H. Chen et al. , A feasible strategy for microbial electrocatalytic CO2 reduction via whole-cell-packed and exogenous-mediator-free rGO / Shewanella biohydrogel. Chemical Engineering Journal 460, 141863 (2023).

[0395] 1 1. Y. Fu et al. , Extracellular electron transfer of Shewanella oneidensis MR-1 for cathodic hydrogen evolution reaction. Electrochimica Acta 305, 528-533 (2019). 12. G. Meshulam-Simon, S. Behrens, A. D. Choo, A. M. Spormann, Hydrogen Metabolism in Shewanella oneidensis MR-1. Applied and Environmental Microbiology 73, 1 153-1 165 (2007).

[0396] 13. H.-X. Han et al. , Reversing Electron Transfer Chain for Light-Driven Hydrogen Production in Biotic-Abiotic Hybrid Systems. Journal of the American Chemical Society 144, 6434-6441 (2022).

[0397] 14. X. Jiang et al. , Nanoparticle Facilitated Extracellular Electron Transfer in Microbial Fuel Cells. Nano Letters 14, 6737-6742 (2014).

[0398] 15. Y. Yang et al. , Enhancing Bidirectional Electron Transfer of Shewanella oneidensis by a Synthetic Flavin Pathway. ACS Synthetic Biology 4, 815-823 (2015).

[0399] 16. B. Cao et al. , Silver nanoparticles boost charge -extraction efficiency in Shewanella microbial fuel cells. Science 373, 1336-1340 (2021).

[0400] 17. T. Lin et al. , Engineered Shewanella oneidensis-reduced graphene oxide biohybrid with enhanced biosynthesis and transport of flavins enabled a highest bioelectricity output in microbial fuel cells. Nano Energy 50, 639-648 (2018).

[0401] 18. A. Okamoto, K. Hashimoto, K. H. Nealson, R. Nakamura, Rate enhancement of bacterial extracellular electron transport involves bound flavin semiquinones. Proceedings of the National Academy of Sciences 110, 7856-7861 (2013).

[0402] 19. K. Abuyen, M. Y. El-Naggar, Soluble Iron Enhances Extracellular Electron Uptake by Shewanella oneidensis MR-1. ChemElectroChem 10, e202200965 (2023).

[0403] 20. X. Wu et al. , A Role for Microbial Palladium Nanoparticles in Extracellular Electron Transfer. Angewandte Chemie International Edition 50, 427-430 (201 1).

[0404] 21. L.-J. Tian et al. , Directed Biofabrication of Nanoparticles through Regulating Extracellular Electron Transfer. Journal of the American Chemical Society 139, 12149- 12152 (2017).

[0405] 22. B. Luo et al. , A Periplasmic Photosensitized Biohybrid System for Solar Hydrogen Production. Advanced Energy Materials 11, 2100256 (2021).

[0406] 23. Y.-S. Son et al. , Improved bio-hydrogen production by overexpression of glucose-6-phosphate dehydrogenase and FeFe hydrogenase in Clostridium acetobutylicum. International Journal of Hydrogen Energy 46, 36687-36695 (2021).

[0407] 24. J. Y. H. Kim, B. Jo, Y. Jo, H. Cha, Improved production of biohydrogen in light- powered Escherichia coli by co-expression of proteorhodopsin and heterologous hydrogenase. Microbial Cell Factories 11, 2 (2012).

[0408] 25. O. Beja et al. , Bacterial Rhodopsin: Evidence for a New Type of Phototrophy in the Sea. Science 289, 1902-1906 (2000). 26. Y. Toya et al. , Optogenetic reprogramming of carbon metabolism using lightpowering microbial proton pump systems. Metabolic Engineering 'll, 227-236 (2022).

[0409] 27. P. A. Davison et al. , Engineering a Rhodopsin-Based Photo-Electrosynthetic System in Bacteria for CO2 Fixation. ACS Synthetic Biology 11, 3805-3816 (2022).

[0410] 28. E. H. Edwards et al. , Shewanella oneidensis MR-1 respires CdSe quantum dots for photocatalytic hydrogen evolution. Proceedings of the National Academy of Sciences 120, e2206975120 (2023).

[0411] 29. Z. Dang et al. , Regulating the synthesis rate and yield of bio-assembled FeS nanoparticles for efficient cancer therapy. Nanoscale 13, 18977-18986 (2021).

[0412] 30. J. Ding et al. , Single-Particle Analysis for Structure and Iron Chemistry of Atmospheric Particulate Matter. Analytical Chemistry 92, 975-982 (2020).

[0413] 31. G. Wang, F. Qian, C. W. Saltikov, Y. Jiao, Y. Li, Microbial reduction of graphene oxide by Shewanella. Nano Research 4, 563-570 (201 1).

[0414] 32. A. C. Ferrari, J. Robertson, Interpretation of Raman spectra of disordered and amorphous carbon. Physical Review B 61, 14095-14107 (2000).

[0415] 33. W. Tu, J. Xu, I. P. Thompson, W. E. Huang, Engineering artificial photosynthesis based on rhodopsin for CO2 fixation. Nature Communications 14, 8012 (2023).

[0416] 34. E. T. Johnson et al. , Enhancement of Survival and Electricity Production in an Engineered Bacterium by Light-Driven Proton Pumping. Applied and Environmental Microbiology 76, 4123-4129 (2010).

[0417] 35. M. R. Miranda et al. , The photocycle and proton translocation pathway in a cyanobacterial ion-pumping rhodopsin. Biophys J 96, 1471-1481 (2009).

[0418] 36. K. Chuon, J.-g. Shim, S.-G. Cho, M. Song, K.-H. Jung, Natural selection of carotenoid binding in Gloeobacter rhodopsin. Algal Research 74, 103232 (2023).

[0419] 37. W. Lubitz, H. Ogata, O. Rudiger, E. Reijerse, Hydrogenases. Chemical Reviews 114, 4081-4148 (2014).

[0420] 38. G. Quek, R. J. Vazquez, S. R. McCuskey, F. Lopez-Garcia, G. C. Bazan, An n- Type Conjugated Oligoelectrolyte Mimics Transmembrane Electron Transport Proteins for Enhanced Microbial Electrosynthesis. Angewandte Chemie International Edition 62, e202305189 (2023).

[0421] 39. D. Rickard, Sulfidic sediments and sedimentary rocks (Newnes, 2012).

[0422] 40. A. Picard, A. Gartman, P. R. Girguis, What Do We Really Know about the Role of Microorganisms in Iron Sulfide Mineral Formation? Frontiers in Earth Science 4 (2016). 41. X. Deng, N. Dohmae, A. H. Kaksonen, A. Okamoto, Biogenic Iron Sulfide Nanoparticles to Enable Extracellular Electron Uptake in Sulfate-Reducing Bacteria. Angewandte Chemie International Edition 59, 5995-5999 (2020).

[0423] 42. C. Ayala-Castro, A. Saini, F. W. Outten, Fe-S Cluster Assembly Pathways in Bacteria. Microbiology and Molecular Biology Reviews 'll, 1 10-125 (2008).

[0424] 43. L. Gomez-Consarnau et al. , Microbial rhodopsins are major contributors to the solar energy captured in the sea. Science Advances 5, eaaw8855 (2019).

[0425] 44. M. Sano et al. , Conversion of Mevalonate to Isoprenol Using Light Energy in Escherichia coli without Consuming Sugars for ATP Supply. ACS Synthetic Biology 11, 3966-3972 (2022).

[0426] 45. J. M. Walter, D. Greenfield, C. Bustamante, J. Liphardt, Light-powering Escherichia coli with proteorhodopsin. Proceedings of the National Academy of Sciences 104, 2408-2412 (2007).

[0427] 46. Y. Song et al. , Proteorhodopsin Overproduction Enhances the Long-Term Viability of Escherichia coli. Applied and Environmental Microbiology 86, e02087- 02019 (2019).

[0428] 47. F. X. Cunningham, E. Gantt, A portfolio of plasmids for identification and analysis of carotenoid pathway enzymes: Adonis aestivalis as a case study. Photosynthesis Research 92, 245-259 (2007).

[0429] 48. C. K. Ng et al. , Genetic engineering biofilms in situ using ultrasound-mediated DNA delivery. Microbial Biotechnology 14, 1580-1593 (2021).

[0430] 49. D. Coursolle, D. B. Baron, D. R. Bond, J. A. Gralnick, The Mtr Respiratory Pathway Is Essential for Reducing Flavins and Electrodes in Shewanella oneidensis. J Bacteriol 192, 467-474 (2010).

[0431] 50. Y. Honda, R. Yuki, R. Hamakawa, H. Fujii, Photo-Electro-Biochemical H2 Production Using the Carbon Material-Based Cathode Combined with Genetically Engineered Escherichia coli Whole-Cell Biocatalysis. ChemSusChem 17, e202300958 (2024). Table 1. Comparison to other microbial electrochemical systems for H2 production

[0432] Cathode

[0433] Synthetic Electrode Abiotic

[0434] Strains potential (vs. H2 yield Reference biology materials materials

[0435] SHE)

[0436] 462.7

[0437] Carbon pmol / d

[0438] E. coli Hydrogenase Methylviologen — 1.3 (50) paper (-7.34 pmol / mg / d)

[0439] .S'. Carbon 24.5

[0440] / / -0.758 (11) oneidensis felt mL / m3 / d

[0441] .S'. Carbon -4.6

[0442] / / -0.76 (13) oneidensis felt pmol / mg / d

[0443] .S'. Carbon 7.82 This

[0444] / / -0.75 oneidensis paper pmol / mg / d study

[0445] Carbon

[0446] S. GR-CAN, FeS 80.37 This paper with -0.75 oneidensis hydrogenase nanoparticles pmol / mg / d study rGO

[0447] Table SI. Strains and plasmids used in this study

[0448] Strains Comments Reference

[0449] E. coli DH5a Commercially obtained (NEW England, UK) Lab collection

[0450] Shewanella oneidensis Wild-type strain Lab

[0451] MR-1 collection

[0452] Shewanella oneidensis Shewanella oneidensis MR- 1-GR harbouring

[0453] This study

[0454] MR- 1-GR pLOl la-GR

[0455] Shewanella oneidensis Shewanella oneidensis MR- 1-GR harbouring

[0456] This study

[0457] MR-l-GR-CAN pLOl la-GR and pAC-CANTHipi

[0458] Shewanella oneidensis Shewanella oneidensis MR- 1-GR harbouring

[0459] MR-l-GR-CAN- pLOl la-GR-HydAB pAC-CANTHipi This study

[0460] HydAB

[0461] Plasmids Comments Reference pLOl la (Tcr, RK2 ori, Expression vector with PBAD promoter and

[0462] Mob+) downstream cloning sites (gift from Oliver Lenz, (1)

[0463] Technische Universitat Berlin, Germany). pLOl la-GR pLOl la containing the gene for GR rhodopsin from Gloeobacter violaceus PCC7421 pAC-CANTHipi pAC-BETAipi with the gene cluster of the canthaxanthin synthetic pathway pLOl la-HydAB-GR pLOl la containing GR gene rhodopsin from

[0464] Gloeobacter violaceus PCC7421, HydA and 7 / vt / This study genes from Shewanella oneidensis MR-1

[0465] Reference

[0466] (1) Davison, P. A., Tu, W., Xu, J., Della Valle, S., Thompson, I. P., Hunter, C. N., & Huang, W. E. (2022). Engineering a rhodopsin-based photo-electrosynthetic system in bacteria for CO2 fixation. ACS Synthetic Biology, 11(11), 3805-3816.

[0467] (2) Cunningham, F. X., & Gantt, E. (2007). A portfolio of plasmids for identification and analysis of carotenoid pathway enzymes: Adonis aestivalis as a case study. Photosynthesis Research, 92, 245-259.

[0468] SEQUENCES

[0469] > pAC-CANTHipi sequence 11140 bp (SEQ ID NO: 32) gaattccggatgagcattcatcaggcgggcaagaatgtgaataaaggccggataaaacttgtgcttattt ttctttacggtctttaaaaaggccgtaatatccagctgaacggtctggttataggtacattgagcaactg actgaaatgcctcaaaatgttctttacgatgccattgggatatatcaacggtggtatatccagtgatttt tttctccattttagcttccttagctcctgaaaatctcgataactcaaaaaatacgcccggtagtgatctt atttcattatggtgaaagttggaacctcttacgtgccgatcaacgtctcattttcgccaaaagttggccc agggcttcccggtatcaacagggacaccaggatttatttattctgcgaagtgatcttccgtcacaggtat ttattcggcgcaaagtgcgtcgggtgatgctgccaacttactgatttagtgtatgatggtgtttttgagg tgctccagtggcttctgtttctatcagctgtccctcctgttcagctactgacggggtggtgcgtaacggc aaaagcaccgccggacatcagcgctagcggagtgtatactggcttactatgttggcactgatgagggtgt cagtgaagtgcttcatgtggcaggagaaaaaaggctgcaccggtgcgtcagcagaatatgtgatacagga tatattccgcttcctcgctcactgactcgctacgctcggtcgttcgactgcggcgagcggaaatggctta cgaacggggcggagatttcctggaagatgccaggaagatacttaacagggaagtgagagggccgcggcaa agccgtttttccataggctccgcccccctgacaagcatcacgaaatctgacgctcaaatcagtggtggcg aaacccgacaggactataaagataccaggcgtttccccctggcggctccctcgtgcgctctcctgttcct gcctttcggtttaccggtgtcattccgctgttatggccgcgtttgtctcattccacgcctgacactcagt tccgggtaggcagttcgctccaagctggactgtatgcacgaaccccccgttcagtccgaccgctgcgcct tatccggtaactatcgtcttgagtccaacccggaaagacatgcaaaagcaccactggcagcagccactgg taattgatttagaggagttagtcttgaagtcatgcgccggttaaggctaaactgaaaggacaagttttgg tgactgcgctcctccaagccagttacctcggttcaaagagttggtagctcagagaaccttcgaaaaaccg ccctgcaaggcggttttttcgttttcagagcaagagattacgcgcagaccaaaacgatctcaagaagatc atcttattaatcagataaaatatttctagatttcagtgcaatttatctcttcaaatgtagcacctgaagt cagccccatacgatataagttgtaattctcatgtttgacagcttatcatcgataagctATCTCGGTAGTG GGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTCAACCACCAT CAAACAGGATTTTCGCC

[0470] TGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAGGC

[0471] GGTGAAGGGCAATCAGCT

[0472] GTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAA

[0473] ACCGCCTCTCCCCGCGCG

[0474] TTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGG

[0475] GCAGTGAGCGCAACGCA

[0476] ATTAATGTGAGTTAGCGCGAATTGATCTGGTTTGACAGCTTATCATCGACTGC ACGGTGCACCAATGCTT

[0477] CTGGCGTCAGGCAGCCATCGGAAGCTGTGGTATGGCTGTGCAGGTCGTAAAT CACTGCATAATTCGTGTC

[0478] GCTCAAGGCGCACTCCCGTTCTGGATAATGTTTTTTGCGCCGACATCATAACG GTTCTGGCAAATATTCT

[0479] GAAATGAGCTGTTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTG

[0480] AGCGGATAACAATTTCA

[0481] CACAGGAAACAGCGCCGCTGAGAAAAAGCGAAGCGGCACTGCTCTTTAACAA TTTATCAGACAATCTGTG TGGGCACTCGACCGGAATTATCGATTAACTTTATTATTAAAAATTAAAGAGGT ATATATTAATGTATCGA

[0482] TTAAATAAGGAGGAATAAACCATGGGGGGTTCTCATCATCATCATCATCATGG TATGGCTAGCATGACTG

[0483] GTGGACAGCAAATGGGTCGGGATCTGTACGACGATGACGATAAGGATCGATG GGGATCCGAGCTCaagcg tttgtgcgcctcgacgtggccagtctgcactgccttgaacccgcgagtctcccgccgcactgactgccat agcacagctagacgaatgcagctagcagcgacagtaatgttggagcagcttaccggaagcgctgaggcac tcaaggagaaggagaaggaggttgcaggcagctctgacgtgttgcgtacatgggcgacccagtactcgct tccgtcagaagagtcagacgcggcccgcccgggactgaagaatgcctacaagccaccaccttccgacaca aagggcatcacaatggcgctaGCtgtcatcggctcctgggccgcagtgttcctccacgccatttttcaaa tcaagcttccgacctccttggaccagctgcactggctgcccgtgtcagatgccacagctcagctggttag cggcaGCagcagcctgctGCacatcgtcgtagtattctttgtcctggagttcctgtacacaggccttttt atcaccacgcatgatgctatgcatggcaccatcgccatgagaaacaggcagcttaatgacttcttgggca gagtatgcatctccttgtacgcctggtttgattacaacatgctgcaccgcaagcattgggagcaccacaa ccacactggcgaggtgggcaaggaccctgacttccacaggggaaaccctggcattgtgccctggtttgcc agcttcatgtccagctacatgtcgatgtggcagtttgcgcgcctcgcatggtggacggtggtcatgcagc tgctgggtgcgccaatggcgaacctgctggtgttcatggcggccgcgcccatcctgtccgccttccgctt gttctactttggcacgtacatgccccacaagcctgagcctggcgccgcgtcaggctcttcaccagccgtc atgaactggtggaagtcgcgcactagccaggcgtccgacctggtcagctttctgacctgctaccacttcg acctgcactgggagcaccaccgctggcccttcgccccctggtgggagctgcccaactgccgccgcctgtc tggccgaggtctggttcctgcctagctggacacacAGCTTtaatgcggtagtttatcacagttaaattgc taacgcagtcaggcaccgtgtatgaaatctaacaatgcgctcatcgtcatcctcggcaccgtcaccctgg atgctgtaggcataggcttggttatgccggtactgccgggcctcttgcgggatatcgtccattccgacag catcgccagtcactatggcgtgctgctagcgctatatgcgttgatgcaatttctatgcgcacccgttctc ggagcactgtccgaccgctttggccgccgcccagtcctgctcgcttcgctacttggagccactatcgact acgcgatcatggcgaccacacccgtcctgtggatctaaaggcacagcgtctcatgcttcgcacaatgtaa aactgcttcagaacctggcgagagctatccgcgcggtctacggttaactgatactaaaagacaattcagc gggtaaccttgcaatggtgagtggcagtaaagcgggcgtttcgcctcatcgcgaaatagaagtaatgaga caatccattgacgatcacctggctggcctgttacctgaaaccgacagccaggatatcgtcagccttgcga tgcgtgaaggcgtcatggcacccggtaaacggatccgtccgctgctgatgctgctggccgcccgcgacct ccgctaccagggcagtatgcctacgctgctcgatctcgcctgcgccgttgaactgacccataccgcgtcg ctgatgctcgacgacatgccctgcatggacaacgccgagctgcgccgcggtcagcccactacccacaaaa aatttggtgagagcgtggcgatccttgcctccgttgggctgctctctaaagcctttggtctgatcgccgc caccggcgatctgccgggggagaggcgtgcccaggcggtcaacgagctctctaccgccgtgggcgtgcag ggcctggtactggggcagtttcgcgatcttaacgatgccgccctcgaccgtacccctgacgctatcctca gcaccaaccacctcaagaccggcattctgttcagcgcgatgctgcagatcgtcgccattgcttccgcctc gtcgccgagcacgcgagagacgctgcacgccttcgccctcgacttcggccaggcgtttcaactgctggac gatctgcgtgacgatcacccggaaaccggtaaagatcgcaataaggacgcgggaaaatcgacgctggtca accggctgggcgcagacgcggcccggcaaaagctgcgcgagcatattgattccgccgacaaacacctcac ttttgcctgtccgcagggcggcgccatccgacagtttatgcatctgtggtttggccatcaccttgccgac tggtcaccggtcatgaaaatcgcctgataccgcccttttgggttcaagcagtacataacgatggaaccac attac agg agtagtg atg aatg aagg acg ag eg c cttgttc ag cgtaag aacg atcatctgg atategtt ctcgacccccgtcgcgccgtaactcaggctagcgcaggttttgagcgctggcgctttacccactgcgccc tgccagagctgaattttagcgacatcacgctggaaaccaccttcctgaatcggcagctacaggctccgct gctgatcagctccatgaccggcggcgttgagcgctcgcgccatatcaaccgccacctcgccgaggcggcg caggtgctaaaaattgcgatgggggtgggctcccagcgcgtcgccattgagagcgacgcgggcttagggc tggataaaaccctgcggcagctggctccggacgtgccgctgctggcgaacctcggcgcggcgcagctgac cggcagaaaaggtattgattacgcccgacgggccgtggagatgatcgaggcggatgcgctgattgtgcac ctaaacccgctgcaggaggcgctacagcccggcggcgatcgcgactggcgcggacggctggcggctattg aaactctggtccgcgagctgcccgttccgctggtggtgaaagaggtgggagccggtatctcccgaaccgt ggccgggcagctgatcgatgccggcgttaccgtgattgacgtcgcgggcgcgggcggcaccagctgggcc gccgttgaaggcgagcgggcggccaccgagcagcagcgcagcgtggccaacgtctttgccgactggggga tccccaccgctgaggcgctggttgacattgccgaggcctggccgcagatgccccttattgcctcgggcgg gattaaaaacggcgtcgacgcggcgaaagcgctgcggctcggcgcgtgcatggtagggcaggccgccgcc gtgctcggcagcgcaggcgtctccacggagaaggtgatcgatcacttcaacgtgattattgagcagctgc gggtggcctgcttctgcaccggcagccgcagcctgagcgatctaaagcaggctgatatccgctatgtgcg ggatacgccatgagccattttgccattgtggcaccgccgctctacagtcatgcggtggcgctgcatgccc tggcgctggagatggcccaacgcggccactcgaccagccgcgctacgttgccgaggctaatctggtgatc acccacggcggtctcaataccgtactggatgcgctggctgccgcgacgccggtgctggcggtgccactct ctttcgaccagcccgccgtggctgcccggctggtctataacgggctgggtcgccgggtatcgcgctttgc cagacagcagacgctggcggatgagattgcccaactgctgggggatgagacgctgcatcagcgtctggcg acggcccgccagcagcttaacgacgccgggggcacgccccgtgcggcgaccctgattgaacaggccatag cagggagtgagagcgtatcgtgagggatctgattttagtcggcggcggcctggccaacgggctgatcgcc tggcgtctgcgccagcgctacccgcagcttaacctgctgctgatcgaggccggggagcagcccggcggga accatacctggtcattccatgaagacgatctgactcccgggcagcacgcctggctggccccgctggtggc ccacgcctggccgggctatgaggtgcagtttcccgatcttcgccgtcgcctcgcgcgcggctactactcc attacctcagagcgctttgccgaggccctgcatcaggcgctgggggagaacatctggctaaactgttcgg tgagcgaggtgttacccaatagcgtgcgccttgccaacggtgaggcgctgcttgccggagcggtgattga cggacgcggcgtgaccgccagttcggcgatgcaaaccggctatcagctctttcttggtcagcagtggcgg ctgacacagccccacggcctgaccgtaccgatcctgatggatgccacggtggcgcagcagcagggctatc gctttgtctacacgctgccgctctccgccgacacgctgctgatcgaggatacgcgctacgccaatgtccc gcagcgtgatgataatgccctacgccagacggttaccgactatgctcacagcaaagggtggcagctggcc cagcttgaacgcgaggagaccggctgtctgccgattaccctggcgggtgacatccaggctctgtgggccg atgcgccgggcgtgccgcgctcgggaatgcgggctgggctatttcaccctaccactggctattcgctgcc gctggcggtggcccttgccgacgcgattgccgacagcccgcggctgggcagcgttccgctctatcagctc acccggcagtttgccgaacgccactggcgcaggcagggattcttccgcctgctgaaccggatgcttttcc tggccgggcgcgaggagaaccgctggcgggtgatgcagcgcttttatgggctgccggagcccaccgtaga gcgcttttacgccggtcggctctctctctttgataaggcccgcattttgacgggcaagccaccggttccg ctgggcgaagcctggcgggcggcgctgaaccattttcctgacagacgagataaaggatgaaaaaaaccgt tgtgattggcgcaggctttggtggcctggcgctggcgattcgcctgcaggcggcagggatcccaaccgta ctgctggagcagcgggacaagcccggcggtcgggcctacgtctggcatgaccagggctttacctttgacg ccgggccgacggtgatcaccgatcctaccgcgcttgaggcgctgttcaccctggccggcaggcgcatgga ggattacgtcaggctgctgccggtaaaacccttctaccgactctgctgggagtccgggaagaccctcgac tatgctaacgacagcgccgagcttgaggcgcagattacccagttcaacccccgcgacgtcgagggctacc ggcgctttctggcttactcccaggcggtattccaggagggatatttgcgcctcggcagcgtgccgttcct ctcttttcgcgacatgctgcgcgccgggccgcagctgcttaagctccaggcgtggcagagcgtctaccag tcggtttcgcgctttattgaggatgagcatctgcggcaggccttctcgttccactccctgctggtaggcg gcaaccccttcaccacctcgtccatctacaccctgatccacgcccttgagcgggagtggggggtctggtt ccctgagggcggcaccggggcgctggtgaacggcatggtgaagctgtttaccgatctgggcggggagatc gaactcaacgcccgggtcgaagagctggtggtggccgataaccgcgtaagccaggtccggctggcggatg gtcggatctttgacaccgacgccgtagcctcgaacgctgacgtggtgaacacctataaaaagctgctcgg ccaccatccggtggggcagaagcgggcggcagcgctggagcgcaagagcatgagcaactcgctgtttgtg ctctacttcggcctgaaccagcctcattcccagctggcgcaccataccatctgttttggtccccgctacc gggagctgatcgacgagatctttaccggcagcgcgctggcggatgacttctcgctctacctgcactcgcc ctgcgtgaccgatccctcgctcgcgcctcccggctgcgccagcttctacgtgctggccccggtgccgcat cttggcaacgcgccgctggactgggcgcaggaggggccgaagctgcgcgaccgcatctttgactaccttg aagagcgctatatgcccggcctgcgtagccagctggtgacccagcggatctttaccccggcagacttcca cgacacgctggatgcgcatctgggatcggccttctccatcgagccgctgctgacccaaagcgcctggttc cgcccgcacaaccgcgacagcgacattgccaacctctacctggtgggcgcaggtactcaccctggggcgg gcattcctggcgtagtggcctcggcgaaagccaccgccagcctgatgattgaggatctgcaatgagccaa ccgccgctgcttgaccacgccacgcagaccatggccaacggctcgaaaagttttgccaccgctgcgaagc tgttcgacccggccacccgccgtagcgtgctgatgctctacacctggtgccgccactgcgatgacgtcat tgacgaccagacccacggcttcgccagcgaggccgcggcggaggaggaggccacccagcgcctggcccgg ctgcgcacgctgaccctggcggcgtttgaaggggccgagatgcaggatccggccttcgctgcctttcagg aggtggcgctgacccacggtattacgccccgcatggcgctcgatcacctcgacggctttgcgatggacgt ggctcagacccgctatgtcacctttgaggatacgctgcgctactgctatcacgtggcgggcgtggtgggt ctgatgatggccagggtgatgggcgtgcgggatgagcgggtgctggatcgcgcctgcgatctggggctgg ccttccagctgacgaatatcgcccgggatattattgacgatgcggctattgaccgctgctatctgcccgc cgagtggctgcaggatgccgggctgaccccggagaactatgccgcgcgggagaatcgggccgcgctggcg cgggtggcggagcggcttattgatgccgcagagccgtactacatctcctcccaggccgggctacacgatc tgccgccgcgctgcgcctgggcgatcgccaccgcccgcagcgtctaccgggagatcggtattaaggtaaa agcggcgggaggcagcgcctgggatcgccgccagcacaccagcaaaggtgaaaaaattgccatgctgatg gcggcaccggggcaggttattcgggcgaagacgacgagggtgacgccgcgtccggccggtctttggcagc gtcccgtttaggcgggcggccatgacgttcacgcaggatcgcctgtaggtcggcaggcttgcgggcgtaa ataaaaccgaaggagacgcagccctcccggccgcgcaccgcgtggtgcaggcggtgggcgacgtagagcc gcttcaggtagccccggcgcgggatcccgcctcgccgtgctgtccggtctcaacctgatccgccagaatc gagccaacgggatcggccagcacgaattcggtatgcggagagtgtttggcaaaccatgcctgcaagccac ccagcgtgccgccggagccaacgcctactaccacggcatcaaccctgcctgccagctggtcgaacagctc cggtgcggtggtggtggcgtgcgccagcgggttggccgggttagagaactgatcaatatagtaagcaccc ggcgtctcctctgccaggcggcgggcgtagtcctgatagtactccgggtggccctttgtcacgtcggagc gggtcaggcgcacatcaacacccagcgcacgcaggtggtagatcctctacgccggacgcatcgtggccgg catcaccggcgccacaggtgcggttgctggcgcctatatcgccgacatcaccgatggggaagatcgggct cgccacttcgggctcatgagcgcttgtttcggcgtgggtatggtggcaggccccgtggccgggggactgt tgggcgccatctccttggggtcgaatttgctttcgaatttctgccattcatccgcttattatcacttatt caggcgtagcaccaggcgtttaagggcacccattaagcattctgccgacatggaagccatcacagacggc atgatgaacctgaatcgccagcggcatcagcaccttgtcgccttgcgtataatatttgcccatggtgaaa acgggggcgaagaagttgtccatattggccacgtttaaatcaaaactggtgaaactcacccagggattgg ctgagacgaaaaacatattctcaataaaccctttagggaaataggccaggttttcaccgtaacacgccac atcttgcgaatatatgtgtagaaactgccggaaatcgtcgtggtattcactccagagcgatgaaaacgtt tcagtttgctcatggaaaacggtgtaacaagggtgaacactatcccatatcaccagctcaccgtctttca ttgccatacg

[0484] > pLOl la-HydAB-GR sequence 12512 bps (SEQ ID NO: 33)

[0485] ACATGGTACTCCGTCAAGCCGTCAATTGTCTGATTCGTTACCAATTATGACAA CTTGACGGCTACATCATTCACTTTTTCTTCACAACCGGCACGGAACTCGCTCG GGCTGGCCCCGGTGCATTTTTTAAATACCCGCGAGAAATAGAGTTGATCGTCA AAACCAACATTGCGACCGACGGTGGCGATAGGCATCCGGGTGGTGCTCAAAA GCAGCTTCGCCTGGCTGATACGTTGGTCCTCGCGCCAGCTTAAGACGCTAATC CCTAACTGCTGGCGGAAAAGATGTGACAGACGCGACGGCGACAAGCAAACAT GCTGTGCGACGCTGGCGATATCAAAATTGCTGTCTGCCAGGTGATCGCTGATG TACTGACAAGCCTCGCGTACCCGATTATCCATCGGTGGATGGAGCGACTCGTT AATCGCTTCCATGCGCCGCAGTAACAATTGCTCAAGCAGATTTATCGCCAGCA GCTCCGAATAGCGCCCTTCCCCTTGCCCGGCGTTAATGATTTGCCCAAACAGG TCGCTGAAATGCGGCTGGTGCGCTTCATCCGGGCGAAAGAACCCCGTATTGGC AAATATTGACGGCCAGTTAAGCCATTCATGCCAGTAGGCGCGCGGACGAAAG TAAACCCACTGGTGATACCATTCGCGAGCCTCCGGATGACGACCGTAGTGATG AATCTCTCCTGGCGGGAACAGCAAAATATCACCCGGTCGGCAAACAAATTCT CGTCCCTGATTTTTCACCACCCCCTGACCGCGAATGGTGAGATTGAGAATATA ACCTTTCATTCCCAGCGGTCGGTCGATAAAAAAATCGAGATAACCGTTGGCCT CAATCGGCGTTAAACCCGCCACCAGATGGGCATTAAACGAGTATCCCGGCAG CAGGGGATCATTTTGCGCTTCAGCCATACTTTTCATACTCCCGCCATTCAGAG AAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTAC TGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTA ACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAAT CACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTGCTATGC CATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCA ACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGGCTAGCTAAGGAGGAGACCC CATGGGAGAGCTCATGACAACGACAACTTATCAACCAGGAGAAATCCAAGGG

[0486] CTGATCAAGATTAATGCATCCAAATGCAAAGGATGTGATGCCTGTAAACAATT

[0487] CTGCCCAACCCATGCCATTAATGGCGCTTCGGGTGCAGTACACTCTATCGATG

[0488] AAGATAAATGCTTAAGCTGCGGACAGTGTTTAATTAACTGTCCATTTAGCGCT

[0489] ATTGAGGAAACCCACAGCGCACTTGAAACCGTGATTAAAAAGCTCGCTGATA

[0490] AAAATACCACCGTGGTCGGGATTATCGCGCCTGCGGTACGGGTGGCGATTGG

[0491] TGAAGAATTTGGCTTAGGTACAGGTGAGCTAGTAACAGGCAAACTCTACGGT

[0492] GCCATGAATCAAGCTGGCTTTAAAATTTTCGACTGTAACTTCGCCGCCGATTT

[0493] GACCATTATGGAAGAAGGCAGTGAGTTTATTCATCGCCTGCACGCCAATGTAA

[0494] AAGGTGAAGCTAACGCAGGCCCATTGCCGCAATTTACCTCCTGCTGCCCAGGC

[0495] TGGGTACGCTACCTCGAAACCCGCTACCCTGCACTTTTACCTAACCTATCGAC

[0496] CGCCAAATCACCTCAGCAAATGGCAGGGACTGTCGCCAAAACCTACGGCGCC

[0497] AAGGTATATCAAATGCAGCCAGAGAATATTTTCACTGTCTCTGTAATGCCTTG

[0498] CACCTCGAAAAAGCTCGAAGCCTCCCGTCCCGAATTTAACTCGGCTTGGCAAT

[0499] ATCATCAGGAACACGGCGCAAACTCGCCCTCCTACCAAGATATTGATGCCGTG

[0500] CTCACCACAAGGGAAATGGCTCAGTTACTCAAACTGCTCGATATCGATCTCGC

[0501] GAATACCGCGGAATATCAAGGCGATAGTTTGTTCTCTGAATACACTGGCGCGG

[0502] GCACAATTTTTGGAACAACCGGCGGGGTGATGGAAGCGGCGCTGCGTACCGC

[0503] CCATAAAGTACTGACTGGAACTGAAATGGCTAAGCTGGAATTTGAACCCGTA

[0504] CGCGGGCTAAAAGGCGTGAAATCAGCCTCTGTCAGCCTGTTTGATACAGAGCT

[0505] TAACCAAGATGTGACCGTCAATGTCGCCGTAGTGCACGACATGGGCAACAAC

[0506] ATTGAGCCCGTACTGCGCGATGTGATGGCTGGCACCTCTCCTTATCACTTTATT

[0507] GAGGTGATGAACTGCGCTGGCGGTTGCGTCAACGGCGGAGGCCAACCTATTG

[0508] AAGGTAAAGGCTCTTCATGGCTGGGTAACATTTAACGGGAAACAGAAATGAA

[0509] CAAGAAAAAACACCTATTTGCCGAGGACAGTTTCTTTCTGTCACGCCGTAAAT

[0510] TTATGGCTGTCGGTGCCGCGTTTGTGGCCGCACTCGCGATCCCCATCGGCTGG

[0511] TTTACCAGCAAGCTTGAACGCCGTAATGAGTACATTAAGGCCAGAAGCCAAG

[0512] GGCTATACAAGGACGACAGCCTAGCAAAAACCCGCGTCAGCCATGCTAACCC

[0513] CGCGGTAGAAAAGTACTACAAAGAATTCGGTGGCGAGCCATTAGGACATATG

[0514] TCCCACGAGCTACTGCACACCCACTTTGTCGATCGCACCAAATTAAGCTCTTG

[0515] AAGATCTGCTTGGAGCCACCCGCAGTTCGAAAAATAATAAGCTTGACCTGTG

[0516] AAGTGAAAAATGGCGCACATTGTGCGACATTTTTTTTGTCTGCCGTTTACCGC

[0517] TACTGCGTCACGGATCTCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGG

[0518] GTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCC

[0519] GCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTC

[0520] AAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCAC

[0521] CTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCC

[0522] CTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGG

[0523] ACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGA

[0524] TTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTT

[0525] AACAAAAATTTAACGCGAATTTTAACAAAATCTCGAATTCAAGAAACCAATT

[0526] GTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGC

[0527] TAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGAC

[0528] CAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAA

[0529] GTCCACATTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTA

[0530] TCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTT

[0531] TCTCCATACCCGTTTTTTTGGGCTAGCTAAGGAGGAGACCCCATGGGTTTGAT

[0532] GACCGTATTTTctTcTGCACCTGAACTTGCCCtTcTCGGATCAACCTTTGCCCAGG

[0533] TCGATCCTTCAAACTTATCGGTCTCAGATTCGcTGACCTATGGTCAGTTCAATC

[0534] TGGTTTACAACGCTTTCTCGTTTGCCATCGCGGCAATGTTCGCATCTGCCCTCT

[0535] TCTTCTTCAGCGCTCAGGCACTCGTCGGTCAACGATACCGGTTGGCCTTGCTT

[0536] GTTTCAGCAATTGTTGTGAGTATCGCTGGGTACCACTACTTTCGGATCTTCAAT

[0537] AGTTGGGATGCTGCCTACGTTCTGGAGAATGGCGTGTATTCCCTGACTAGCGA AAAATTCAACGACGCCTACCGCTATGTGGATTGGCTGTTGACCGTGCCTCTGT TGCTGGTGGAGACAGTGGCAGTGCTGACGTTGCCTGCAAAGGAGGCAAGACC CTTGCTGATCAAACTGACGGTGGCTTCAGTTCTGATGATTGCCACGGGCTACC

[0538] CCGGCGAGATTTCTGACGACATTACGACTCGCATCATCTGGGGTACGGTCAGC ACGATTCCCTTCGCCTACATCCTCTATGTGTTGTGGGTCGAACTGTCCAGGTCC CTTGTCCGCCAGCCCGCTGCTGTACAAACCCTGGTCCGCAACATGCGGTGGCT GCTGTTGCTCTCCTGGGGTGTTTACCCGATCGCATACCTTCTACCCATGCTTGG AGTATCCGGTACGTCCGCGGCTGTCGGCGTTCAGGTTGGCTATACGATCGCAG ACGTGCTGGCGAAGCCTGTATTTGGTCTTCTAGTCTTCGCGATTGCACTCGTG AAAACAAAAGCAGATCAAGAAAGCAGTGAACCACATGCCGCAATAGGTGCTG CTGCAAATAAATCGGGAGGCAGTCTTATCTCCTAGCGGCCGCAGATCTGCTTG GAGCCACCCGCAGTTCGAAAAATAATAAGCTTGACCTGTGAAGTGAAAAATG

[0539] GCGCACATTGTGCGACATTTTTTTTGTCTGCCGTTTACCGCTACTGCGTCACAG ATCTGAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGC GTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAA TAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAAT GGCGAATGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTC ACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAA GCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTG CTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTG TCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTG

[0540] ATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGCACCCT TTCTCGGTCCTTCAACGTTCCTGACAACGAGCCTCCTTTTCGCCAATCCATCGA CAATCACCGCGAGTCCCTGCTCGAACGCTGCGTCCGGACCGGCTTCGTCGAAG GCGTCTATCGCGGCCCGCAACAGCGGCGAGAGCGGAGCCTGTTCAACGGTGC

[0541] CGCCGCGCTCGCCGGCATCGCTGTCGCCGGCCTGCTCCTCAAGCACGGCCCCA ACAGTGAAGTAGCTGATTGTCATCAGCGCATTGACGGCGTCCCCGGCCGAAA AACCCGCCTCGCAGAGGAAGCGAAGCTGCGCGTCGGCCGTTTCCATCTGCGG

[0542] TGCGCCCGGTCGCGTGCCGGCATGGATGCGCGCGCCATCGCGGTAGGCGAGC AGCGCCTGCCTGAAGCTGCGGGCATTCCCGATCAGAAATGAGCGCCAGTCGT

[0543] CGTCGGCTCTCGGCACCGAATGCGTATGATTCTCCGCCAGCATGGCTTCGGCC AGTGCGTCGAGCAGCGCCCGCTTGTTCCTGAAGTGCCAGTAAAGCGCCGGCT

[0544] GCTGAACCCCCAACCGTTCCGCCAGTTTGCGTGTCGTCAGACCGTCTACGCCG ACCTCGTTCAACAGGTCCAGGGCGGCACGGATCACTGTATTCGGCTGCAACTT TGTCATGATTGACACTTTATCACTGATAAACATAATATGTCCACCAACTTATC

[0545] AGTGATAAAGAATCCGCGCGTTCAATCGGACCAGCGGAGGCTGGTCCGGAGG CCAGACGTGAAACCCAACATACCCCTGATCGTAATTCTGAGCACTGTCGCGCT CGACGCTGTCGGCATCGGCCTGATTATGCCGGTGCTGCCGGGCCTCCTGCGCG ATCTGGTTCACTCGAACGACGTCACCGCCCACTATGGCATTCTGCTGGCGCTG TATGCGTTGGTGCAATTTGCCTGCGCACCTGTGCTGGGCGCGCTGTCGGATCG

[0546] TTTCGGGCGGCGGCCAATCTTGCTCGTCTCGCTGGCCGGCGCCACTGTCGACT ACGCCATCATGGCGACAGCGCCTTTCCTTTGGGTTCTCTATATCGGGCGGATC GTGGCCGGCATCACCGGGGCGACTGGGGCGGTAGCCGGCGCTTATATTGCCG

[0547] ATATCACTGATGGCGATGAGCGCGCGCGGCACTTCGGCTTCATGAGCGCCTGT TTCGGGTTCGGGATGGTCGCGGGACCTGTGCTCGGTGGGCTGATGGGCGGTTT CTCCCCCCACGCTCCGTTCTTCGCCGCGGCAGCCTTGAACGGCCTCAATTTCCT GACGGGCTGTTTCCTTTTGCCGGAGTCGCACAAAGGCGAACGCCGGCCGTTAC GCCGGGAGGCTCTCAACCCGCTCGCTTCGTTCCGGTGGGCCCGGGGCATGACC GTCGTCGCCGCCCTGATGGCGGTCTTCTTCATCATGCAACTTGTCGGACAGGT GCCGGCCGCGCTTTGGGTCATTTTCGGCGAGGATCGCTTTCACTGGGACGCGA CCACGATCGGCATTTCGCTTGCCGCATTTGGCATTCTGCATTCACTCGCCCAG GCAATGATCACCGGCCCTGTAGCCGCCCGGCTCGGCGAAAGGCGGGCACTCA

[0548] TGCTCGGAATGATTGCCGACGGCACAGGCTACATCCTGCTTGCCTTCGCGACA CGGGGATGGATGGCGTTCCCGATCATGGTCCTGCTTGCTTCGGGTGGCATCGG

[0549] AATGCCGGCGCTGCAAGCAATGTTGTCCAGGCAGGTGGATGAGGAACGTCAG

[0550] GGGCAGCTGCAAGGCTCACTGGCGGCGCTCACCAGCCTGACCTCGATCGTCG

[0551] GACCCCTCCTCTTCACGGCGATCTATGCGGCTTCTATAACAACGTGGAACGGG

[0552] TGGGCATGGATTGCAGGCGCTGCCCTCTACTTGCTCTGCCTGCCGGCGCTGCG

[0553] TCGCGGGCTTTGGAGCGGCGCAGGGCAACGAGCCGATCGCTGATCGTGGAAA

[0554] CGATAGGCCTATGCCATGCGGGTCAAGGCGACTTCCGGCAAGCTATACGCGC

[0555] CCTAGAATTGTCAATTTTAATCCTCTGTTTATCGGCAGTTCGTAGAGCGCGCC

[0556] GTGCGTCCCGAGCGATACTGAGCGAAGCAAGTGCGTCGAGCAGTGCCCGCTT

[0557] GTTCCTGAAATGCCAGTAAAGCGCTGGCTGCTGAACCCCCAGCCGGAACTGA

[0558] CCCCACAAGGCCCTAGCGTTTGCAATGCACCAGGTCATCATTGACCCAGGCGT

[0559] GTTCCACCAGGCCGCTGCCTCGCAACTCTTCGCAGGCTTCGCCGACCTGCTCG

[0560] CGCCACTTCTTCACGCGGGTGGAATCCGATCCGCACATGAGGCGGAAGGTTTC

[0561] CAGCTTGAGCGGGTACGGCTCCCGGTGCGAGCTGAAATAGTCGAACATCCGT

[0562] CGGGCCGTCGGCGACAGCTTGCGGTACTTCTCCCATATGAATTTCGTGTAGTG

[0563] GTCGCCAGCAAACAGCACGACGATTTCCTCGTCGATCAGGACCTGGCAACGG

[0564] GACGTTTTCTTGCCACGGTCCAGGACGCGGAAGCGGTGCAGCAGCGACACCG

[0565] ATTCCAGGTGCCCAACGCGGTCGGACGTGAAGCCCATCGCCGTCGCCTGTAG

[0566] GCGCGACAGGCATTCCTCGGCCTTCGTGTAATACCGGCCATTGATCGACCAGC

[0567] CCAGGTCCTGGCAAAGCTCGTAGAACGTGAAGGTGATCGGCTCGCCGATAGG

[0568] GGTGCGCTTCGCGTACTCCAACACCTGCTGCCACACCAGTTCGTCATCGTCGG

[0569] CCCGCAGCTCGACGCCGGTGTAGGTGATCTTCACGTCCTTGTTGACGTGGAAA

[0570] ATGACCTTGTTTTGCAGCGCCTCGCGCGGGATTTTCTTGTTGCGCGTGGTGAA

[0571] CAGGGCAGAGCGGGCCGTGTCGTTTGGCATCGCTCGCATCGTGTCCGGCCACG

[0572] GCGCAATATCGAACAAGGAAAGCTGCATTTCCTTGATCTGCTGCTTCGTGTGT

[0573] TTCAGCAACGCGGCCTGCTTGGCCTCGCTGACCTGTTTTGCCAGGTCCTCGCC

[0574] GGCGGTTTTTCGCTTCTTGGTCGTCATAGTTCCTCGCGTGTCGATGGTCATCGA

[0575] CTTCGCCAAACCTGCCGCCTCCTGTTCGAGACGACGCGAACGCTCCACGGCGG

[0576] CCGATGGCGCGGGCAGGGCAGGGGGAGCCAGTTGCACGCTGTCGCGCTCGAT

[0577] CTTGGCCGTAGCTTGCTGGACCATCGAGCCGACGGACTGGAAGGTTTCGCGG

[0578] GGCGCACGCATGACGGTGCGGCTTGCGATGGTTTCGGCATCCTCGGCGGAAA

[0579] ACCCCGCGTCGATCAGTTCTTGCCTGTATGCCTTCCGGTCAAACGTCCGATTC

[0580] ATTCACCCTCCTTGCGGGATTGCCCCGACTCACGCCGGGGCAATGTGCCCTTA

[0581] TTCCTGATTTGACCCGCCTGGTGCCTTGGTGTCCAGATAATCCACCTTATCGGC

[0582] AATGAAGTCGGTCCCGTAGACCGTCTGGCCGTCCTTCTCGTACTTGGTATTCC

[0583] GAATCTTGCCCTGCACGAATACCAGCTCCGCGAAGTCGCTCTTCTTGATGGAG

[0584] CGCATGGGGACGTGCTTGGCAATCACGCGCACCCCCCGGCCGTTTTAGCGGCT

[0585] AAAAAAGTCATGGCTCTGCCCTCGGGCGGACCACGCCCATCATGACCTTGCCA

[0586] AGCTCGTCCTGCTTCTCTTCGATCTTCGCCAGCAGGGCGAGGATCGTGGCATC

[0587] ACCGAACCGCGCCGTGCGCGGGTCGTCGGTGAGCCAGAGTTTCAGCAGGCCG

[0588] CCCAGGCGGCCCAGGTCGCCATTGATGCGGGCCAGCTCGCGGACGTGCTCAT

[0589] AGTCCACGACGCCCGTGATTTTGTAGCCCTGGCCGACGGCCAGCAGGTAGGC

[0590] CTACAGGCTCATGCCGGCCGCCGCCGCCTTTTCCTCAATCGCTCTTCGTTCGTC

[0591] TGGAAGGCAGTACACCTTGATAGGTGGGCTGCCCTTCCTGGTTGGCTTGGTTT

[0592] CATCAGCCATCCGCTTGCCCTCATCTGTTACGCCGGCGGTAGCCGGCCAGCCT

[0593] CGCAGAGCAGGATTCCCGTTGAGCACCGCCAGGTGCGAATAAGGGACAGTGA

[0594] AGAAGGAACACCCGCTCGCGGGTGGGCCTACTTCACCTATCCTGCCCGGCTGA

[0595] CGCCGTTGGATACACCAAGGAAAGTCTACACGAACCCTTTGGCAAAATCCTGT

[0596] ATATCGTGCGAAAAAGGATGGATATACCGAAAAAATCGCTATAATGACCCCG

[0597] AAGCAGGGTTATGCAGCGGAAAAGATCCGTCGACCCTTTCCGACGCTCACCG

[0598] GGCTGGTTGCCCTCGCCGCTGGGCTGGCGGCCGTCTATGGCCCTGCAAACGCG

[0599] CCAGAAACGCCGTCGAAGCCGTGTGCGAGACACCGCGGCCGCCGGCGTTGTG

[0600] GATACCTCGCGGAAAACTTGGCCCTCACTGACAGATGAGGGGCGGACGTTGA CACTTGAGGGGCCGACTCACCCGGCGCGGCGTTGACAGATGAGGGGCAGGCT

[0601] CGATTTCGGCCGGCGACGTGGAGCTGGCCAGCCTCGCAAATCGGCGAAAACG

[0602] CCTGATTTTACGCGAGTTTCCCACAGATGATGTGGACAAGCCTGGGGATAAGT

[0603] GCCCTGCGGTATTGACACTTGAGGGGCGCGACTACTGACAGATGAGGGGCGC

[0604] GATCCTTGACACTTGAGGGGCAGAGTGCTGACAGATGAGGGGCGCACCTATT

[0605] GACATTTGAGGGGCTGTCCACAGGCAGAAAATCCAGCATTTGCAAGGGTTTC

[0606] CGCCCGTTTTTCGGCCACCGCTAACCTGTCTTTTAACCTGCTTTTAAACCAATA

[0607] TTTATAAACCTTGTTTTTAACCAGGGCTGCGCCCTGTGCGCGTGACCGCGCAC

[0608] GCCGAAGGGGGGTGCCCCCCCTTCTCGAACCCTCCCGGCCCGCTAACGCGGG

[0609] CCTCCCATCCCCCCAGGGGCTGCGCCCCTCGGCCGCGAACGGCCTCACCCCAA

[0610] AAATGGCAGCCAAGCTGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTT

[0611] TATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAG

[0612] CACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGG

[0613] AGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCT

[0614] CACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAG

[0615] ATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTT

[0616] GATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTC

[0617] AGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCG

[0618] TAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTG

[0619] CCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGC

[0620] GCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCA

[0621] AGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTG

[0622] GCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATA

[0623] GTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAG

[0624] CCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGC

[0625] TATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGT

[0626] AAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAA

[0627] ACG TTC TC TT TG GG TT GA AT TC GT CT TT CA GTA TCG AT GC GCT GG GT GC GG CG GG GT AT GT CC CG TC AC TA GC GC AT AC ATG AA AC CT GT CG CA AG GC CG AT AC CG

[0628] GCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTC

Claims

CLAIMS1. An electromicrobial system for synthesis of hydrogen, comprising:A) a gram-negative bacterial cell modified for synthesis of hydrogen, wherein the bacterial cell comprises: i) inner membrane-bound rhodopsin for proton-pumping, ii) membrane-bound hydrogenase, and iii) optionally an electron mediator located in the periplasm and / or on the outer membrane of the bacterial cell; andB) an electron source for donation of electrons to the bacterial cell.

2. The electromicrobial system according to claim 1, wherein the rhodopsin comprises Gloeobacter rhodopsin (GR).

3. The electromicrobial system according to claim 1 or claim 2, wherein the bacterial cell synthesises retinal naturally and / or the bacterial cell is engineered to synthesise retinal; and / or the bacterial cell is provided with retinal.

4. The electromicrobial system according to any preceding claim, wherein the bacterial cell is further provided with one or more rhodopsin-binding carotenoids.

5. The electromicrobial system according to claim 4, wherein the one or more rhodopsin-binding carotenoids comprise canthaxanthin or echinenone.

6. The electromicrobial system according to claim 4 or 5, wherein the bacterial cell is engineered to express one or more enzyme(s) for biosynthesis of the carotenoid.

7. The electromicrobial system according to any preceding claim, wherein the bacterial cell is engineered to express the hydrogenase.

8. The electromicrobial system according to any preceding claim, wherein the hydrogensase comprises membrane-bound Fe-Fe hydrogenase.

9. The electromicrobial system according to any preceding claim, wherein the hydrogenase is encoded by hydA and hydB.

10. The electromicrobial system according to any preceding claim, wherein the bacterial cell comprises Shewanella spp., E. coli, Pseudomonas spp., Synechocystis spp., or Ralstonia spp.

11. The electromicrobial system according to any preceding claim, wherein the bacterial cell comprises S. oneidensis MR-1.

12. The electromicrobial system according to any preceding claim, wherein the electron source comprises an electrode linked to a source of electricity.

13. The electromicrobial system according to claim 12, wherein the source of electricity is a solar panel, wind turbine, hydroelectric turbine, nuclear fission or fusion, thermal, such as geothermal, bioenergy, such as bio-fermentation, gravitational potential energy, wave energy, and / or chemical.

14. The electromicrobial system according to claim 12 or 13, wherein the electrode comprises a reduced graphene oxide-coated electrode.

15. The electromicrobial system according to any preceding claim, further comprising a dual chamber electrode system.

16. The electromicrobial system according to any preceding claim, wherein the electron mediator comprises iron(II) sulfide (FeS) nanoparticles in the periplasm of the bacterial cell and / or OmcA.

17. The electromicrobial system according to any preceding claim, wherein the electron mediator comprises or consists of a combination of OmcA, MtrC, MtrA, MtrB, FeS nanoparticles, and CymA.

18. The electromicrobial system according to any preceding claim, wherein the bacterial cell expresses, or is engineered to express, MtrCAB.

19. A Gram -negative bacterial cell that is recombinantly engineered for synthesis of hydrogen, the bacterial cell comprising:an inner membrane-bound rhodopsin; a rhodopsin-binding carotenoid; and a membrane-bound hydrogenase.

20. The Gram -negative bacterial cell according to claim 19, wherein the bacterial cell further comprises an electron mediator in the periplasm.

21. A composition comprising the bacterial cell or a population of bacterial cells according to claim 20.

22. Use of the electromicrobial system according to any of claims 1-18, or the Gramnegative bacterial cell according to claim 19 or 20, for hydrogen generation.

23. A method of H2 generation, the method comprising:- providing the electromicrobial system according to any of claims 1-18, and- culturing the bacterial cells in the presence of light and water.

24. A nucleic acid encoding one or more, or all, of the Bkt, crtO and crtW genes, and further encoding: one or more, or all, of the crtEXYIB operon genes, and a rhodopsin.

25. A nucleic acid encoding an inner membrane-binding rhodopsin for proton-pumping; and a membrane-binding hydrogenase and / or enzymes for canthaxanthin synthesis.

26. A bacterial cell comprising the nucleic acid according to claims 24 or 25.

27. The bacterial cell according to claim 26, further comprising nucleic acid encoding MtrCAB and / or OmcA.

28. A method of modifying a bacterial cell to generate H2, the method comprising the step of transforming the bacterial cell with the nucleic acid according to claims 24 or 25.

29. Shewanella oneidensis MR-1 engineered to express Gloeobacter rhodopsin (GR), and a rhodopsin-binding carotenoid; and optionally further engineered to express a hydrogenase.

30. H2 gas produced from the electromicrobial system according to any of claims 1-18 or method of claim 23.

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