WO2010036880A2 - Selection system for di-iron [fefe] hydrogenase properties - Google Patents

Selection system for di-iron [fefe] hydrogenase properties Download PDF

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WO2010036880A2
WO2010036880A2 PCT/US2009/058361 US2009058361W WO2010036880A2 WO 2010036880 A2 WO2010036880 A2 WO 2010036880A2 US 2009058361 W US2009058361 W US 2009058361W WO 2010036880 A2 WO2010036880 A2 WO 2010036880A2
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hydrogenase
gene
exogenous
coli
enzyme
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WO2010036880A3 (en
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Edwin H. Wintermute
Christina Agapakis
Patrick Boyle
Pamela A. Silver
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President And Fellows Of Harvard College
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0067—Oxidoreductases (1.) acting on hydrogen as donor (1.12)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

Definitions

  • [FeFe] hydrogenases in applications such as biofuel production is limited by the oxygen sensitivity of the enzymes, as well as other sub-optimal properties including catalytic rate.
  • the invention provides compositions and methods for in vivo selection of hydrogenase enzymes for desired biological properties. Methods of the invention are used on any member of the FeFe hydrogenase family of enzymes. Moreover, methods of the invention enable the derivation of enzymes optimized for a wide range of properties, including, but not limited to, oxygen tolerance, kinetic rate, and thermostability.
  • the invention features an oxygen-tolerant FeFe hydrogenase enzyme and a microorganism, e.g., E. coli, comprising such an oxygen-tolerant FeFe hydrogenase enzyme.
  • a microorganism e.g., E. coli
  • the viability of the microorganism is dependent upon function of the hydrogenase enzyme.
  • E. coli growth is supported by wild type hydrogenase, cells are only viable in atmospheres with reduced levels of oxygen, e.g., in atmospheres of 8-10% oxygen. Normal air is 21% oxygen.
  • the methods of the invention have yielded a microorganism that is viable in an atmosphere comprising greater than 10% oxygen due to an oxygen-tolerant hydrogenase.
  • Such a microorganism is viable in an atmosphere comprising greater than 12%, 14%, 16%, 18%, 20% or more of oxygen.
  • Such an enzyme and microorganisms harboring a gene encoding and expressing such an enzyme are advantageous over existing enzymes and microbes used in biofuel production.
  • a microorganism was engineered to screen variants, e.g., mutants, of an hydrogenase enzyme to identify those with certain desirable characteristics.
  • the microorganism comprises a mutation in an NAD(P)H-dependent or ferrodoxin-dependent enzyme gene rendering the gene product absent, inactive, or reduced in level of expression.
  • the microorganism also includes a first nucleic acid encoding a ferrodoxin-dependent enzyme and a second nucleic acid encoding a heterologous FeFe hydrogenase.
  • the microorganism further comprises a heterologous ferrodoxin-encoding sequence and may also comprise one or more nucleic acids encoding a heterologous hydrogenase maturation factor.
  • the microorganism is an enteric bacteria such as Escherichia coli (E. coli) or Salmonella typhimurium.
  • the microorganism is engineered to lack a bacterial sulfite reductase and instead expresses plant-derived sulfite reductase.
  • An exemplary microorganism is an Escherichia coli (E. coli) containing a mutation in an endogenous sulfite reductase gene, an exogenous sulfite reductase gene, an exogenous ferredoxin gene, an exogenous hydrogenase gene, and at least one exogenous hydrogenase maturation cofactor gene.
  • the exogenous hydrogenase gene encodes a FeFe hydrogenase.
  • the mutation in an endogenous sulfite reductase gene is a deletion of the cysl open reading frame.
  • the mutation is any insertion, deletion, inversion, translocation, transposition, excision, truncation, frameshift, or point mutation that causes the resulting gene product to be non-functional.
  • the mutation is also any RNA or DNA mediated silencing technology that prevents transcription of the gene or translation of the mRNA into a polypeptide.
  • the mutation occurs in any ferredoxin-dependent enzyme, including, but not limited to, nitrite reductase and glutamate synthase. Moreover, the mutation is any insertion, deletion, inversion, translocation, transposition, excision, truncation, frameshift, or point mutation that causes the resulting gene product to be non-functional.
  • the mutation is also any RNA or DNA mediated silencing technology that prevents transcription of the gene or translation of the mRNA into a polypeptide.
  • the exogenous sulfite reductase gene is isolated from Zea mays.
  • the exogenous ferredoxin gene is isolated from Spinacia oleracea.
  • the said exogenous hydrogenase gene comprises a catalytic hydrogenase subunit isolated from Clostridium acetobutylicum.
  • the exogenous hydrogenase maturation cofactor gene is isolated from Chlamydomonas reinhardtii.
  • Exogenous hydrogenase genes of the invention contain nucleotide sequences isolated from one species. Alternatively, or in addition, exogenous hydrogenase genes contain nucleotide sequences isolated from more than one species. In a preferred embodiment, intact hydrogenase gene sequences from multiple species are randomly spliced, intermixed, and reassembled. The resulting multi-species hydrogenase gene chimeras are introduced into E. coli containing a mutation in gene encoding for a ferredoxin-dependent enzyme, an exogenous gene encoding for said ferredoxin-dependent enzyme or a homolog thereof, an exogenous ferredoxin, and at least one hydrogenase gene maturation factor. The resulting E. coli is grown in media lacking the ferredoxin-dependent gene product and under selective pressure.
  • the invention provides a method of selecting for a biological property of a hydrogenase enzyme, including the steps of (a) providing an engineered E. coli comprising a mutation in a gene encoding a ferredoxin-dependent enzyme; (b) introducing into the engineered E. coli an exogenous gene encoding the ferredoxin-dependent-enzyme or a homolog thereof, an exogenous ferredoxin gene, an exogenous hydrogenase gene, and at least one exogenous hydrogenase maturation factor; and (c) growing the engineered E. coli in media lacking the product of the ferredoxin-dependent enzyme and under selective pressure; wherein the survival of the engineered E.
  • the hydrogenase enzyme is a FeFe hydrogenase.
  • the ferredoxin-dependent enzyme is sulfite reductase, nitrite reductase, or glutamate synthase.
  • the ferredoxin-dependent enzyme is sulfite reductase and its product is cysteine.
  • the mutation in a gene encoding a ferredoxin-dependent enzyme is a deletion of the cysl open reading frame of sulfite reductase.
  • the mutation is any insertion, deletion, inversion, translocation, transposition, excision, truncation, frameshift, or point mutation that causes the resulting gene product to be non-functional.
  • the mutation is also any RNA or DNA mediated silencing technology that prevents transcription of the gene or translation of the mRNA into a polypeptide.
  • the exogenous gene encoding said ferredoxin- dependent-enzyme or a homolog thereof is the sulfite reductase gene.
  • the sulfite reductase gene is isolated from Zea mays.
  • the exogenous ferredoxin gene is isolated from Spinacia oleracea.
  • the exogenous hydrogenase gene comprises a catalytic hydrogenase subunit isolated from Clostridium acetobutylicum.
  • the exogenous hydrogenase maturation cofactor gene is isolated from Chlamydomonas reinhardtii.
  • the biological property selected is oxygen tolerance, thermostability, or resistance to oxidizing agents or poisons.
  • selective pressure is oxygen, high temperature, low temperature, hydrogen peroxide, nitrous oxide, an oxidizing agent, a heavy metal, cyanide, or any compound known to interfere with hydrogenase activity.
  • the biological property is oxygen tolerance and the selective pressure is presence of oxygen.
  • the selection occurs in vivo.
  • the invention further provides a method of selecting for oxygen tolerance of a [FeFe] hydrogenase enzyme, including the steps of (a) providing an engineered E. coli comprising a mutation in a sulfite reductase gene; (b) introducing into said engineered E.
  • coli an exogenous sulfite reductase gene or a homolog thereof, an exogenous ferredoxin gene, an exogenous hydrogenase gene, and at least one exogenous hydrogenase maturation factor; and (c) growing said engineered E. coli in media lacking cystein and in the presence of oxygen; wherein the survival of said engineered E. coli in the presence of oxygen indicates the presence of a functional hydrogenase enzyme, thereby identifying those hydrogenase enzymes having oxygen tolerance.
  • the invention also provides a method of selecting for increased catalytic rate of a [FeFe] hydrogenase enzyme, including the steps of (a) providing an engineered E. coli containing a mutation in a sulfite reductase gene; (b) introducing into the engineered E. coli an exogenous sulfite reductase gene or a homolog thereof, an exogenous ferredoxin gene, an exogenous hydrogenase gene, and at least one exogenous hydrogenase maturation factor, wherein the expression of the hydrogenase gene is low; and (c) growing the engineered E. coli in media lacking cysteine; wherein the survival of the engineered E.
  • Figure 1 is a schematic representation of the artificial cysteine production pathway introduced into engineered E. coli of the invention.
  • Figure 2 is a schematic representation of pCrHydEF-CrHydG.
  • Figure 3 is a schematic representation of pCrHydAl.
  • Figure 4 is a schematic representation of pCaHydA.
  • Figure 5 is a schematic representation of pSoFd-ZmSIR.
  • Figure 6 is a line graph showing hydrogenase-dependent growth of E. coli.
  • compositions and methods of the invention provide a means for driving the evolution of desirable qualities of enzymes, such as [FeFe] hydrogenases, in vivo by inserting an exogenous synthetic pathway into an engineered microorganism that can be grown under selective pressure. Consequently, the selected [FeFe] hydrogenases demonstrate qualities that are physiologically relevant, e.g. optimally functioning in vivo. This selection method is superior to and in contrast to alternative and established methods of mutating enzymes and testing enzymatic activities as well as binding efficacies in vitro and out of any biological context.
  • the selected [FeFe] hydrogenases of the invention catalyze reactions that produce molecular hydrogen with superior properties with respect to their selection criteria.
  • a [FeFe] hydrogenase selected for function in the presence of oxygen should be less sensitive than known hydrogenases, or insensitive, to atmospheric or molecular oxygen. This oxygen insensitive hydrogenase is then introduced into microorganisms that produce improved quantities of molecular hydrogen when exposed to air.
  • a core aspect of the invention is the construction of an artificial biosynthetic pathway that uses hydrogen uptake or production to allow growth of a microbial cell.
  • the following is a particular example of such an artificial pathway, the basic principles of which can be generalized as described below.
  • Compositions and methods of the invention provide an engineered E. coli strain, in which the endogenous sulfite reductase gene and the protein it encodes, which uses NAD(P)H as a source of reducing equivalents, are deleted and replaced with a sulfite reductase from Zea mays (maize).
  • This maize-derived enzyme normally uses a reduced Fe 2 S 2 'plant-type' ferredoxin that is found in photosynthetic organisms but is not found in E. coli. Therefore, the E. coli is engineered to express a plant-type Fe 2 S 2 ferredoxin, which may be the spinach chloroplast ferredoxin but which can be any protein of this class. Because E. coli does not normally express an Fe 2 S 2 ferredoxin, other proteins within E. coli are, in general, not adapted to interact with this type of ferredoxin. Thus, an E. coli strain expressing only the maize sulfite reductase and an Fe 2 S 2 ferredoxin will not be able to grow in the absence of reduced sulfur. Consequently, the E.
  • FeFe hydrogenase such as the hydrogenase from Clostridium acetobutylicum, Clostridium pasteurianum, or Chlamydomonas reinhardtii.
  • FeFe hydrogenases, and FeFe hydrogenases in general use Fe 2 S 2 ferredoxins as their primary or exclusive direct redox partner, hi addition to expressing an FeFe hydrogenase, the strain is engineered to express maturation factors for this type of hydrogenase, such as HydEF and HydG of Chlamydomonas reinhardtii. These factors are necessary and sufficient for maturation of a wide variety of FeFe hydrogenase in heterologous organisms.
  • the resulting E. coli strain lacks its endogenous sulfite reductase, expresses sulfite reductase from maize, expresses the Fe 2 S 2 ferredoxin from spinach, and expresses the FeFe hydrogenase from, for example, C. acetobutylicum, and the FeFe hydrogenase maturation factors HydEF and HydG from Chlamydomonas reinhardtii.
  • This E. coli strain cannot grow under normal conditions in the absence of a source of reduced sulfur and is effectively a cysteine auxotroph, but is able to grow in the absence of reduced sulfur when grown anaerobically in the presence of hydrogen.
  • This genetic assay for hydrogenase function is used in a variety of ways to select for hydrogenases with various improved properties. For example, oxygen-resistant hydrogenases are selected by growing the strain in the presence of various concentrations of oxygen.
  • heat-resistant hydrogenases are selected by growing the strain at various temperatures, hi another variation, hydrogenases with a higher catalytic rate are selected by reducing the level of hydrogenase expression, so that the unselected hydrogenase activity is not sufficient to allow rapid growth, hi a further variation, hydrogenases that interact with a given type of ferredoxin may be selected if the starting hydrogenase does not interact with a chosen ferredoxin.
  • the invention also provides a number of related alternative strategies for the genetic assay of hydrogenase function.
  • other bacterial strains are used instead of E. coli, such as Salmonella typhimurium or any other enteric bacteria that lack an Fe 2 S 2 ferredoxin or any other bacteria whose genetic characteristics are appropriate.
  • An appropriate genetic background entails (a) well characterized and non-redundant native metabolic pathways for producing the metabolite (i.e. cysteine) under selection; (b) the absence or near-absence of native metabolism for providing reduced ferredoxin. Ideally, the organism becomes dependent on the exogenous pathway. If the organsim natively expresses multiple redundant pathways for reducing sulfur, for example, it would be difficult or impossible to eliminate them all.
  • an organism with few or one endogenous pathway for reducing sulfur or performing the function of the exogeously-introduced ferredoxin-dependent enzyme is preferred.
  • methods of the invention are designed to ensure that the ferredoxin in the exogenous pathway is being reduced only by the hydrogenase. If multiple native or endogenous pathways within the organism produce reduced ferredoxin, the selection would fail. Consequently, an organism with a few or one endogeous ferredoxin is preferred.
  • a variety of metabolic genes may be used instead of ferredoxin-dependent sulfite reductase, such as ferredoxin-dependent nitrate reductase, ferredoxin-dependent glutamate synthase, or any other enzyme that uses an Fe 2 S 2 ferredoxin instead of NADPH or NADH as a source of reducing equivalents.
  • ferredoxin-dependent sulfite reductase such as ferredoxin-dependent nitrate reductase, ferredoxin-dependent glutamate synthase, or any other enzyme that uses an Fe 2 S 2 ferredoxin instead of NADPH or NADH as a source of reducing equivalents.
  • Such enzymes may be found in photosynthetic organisms that use reduced Fe 2 S 2 ferredoxin generated by photosystem I as a common currency of reducing equivalents.
  • a variety OfFe 2 S 2 ferredoxins are also used.
  • a general form of this aspect of the invention is: a bacterium that (a) is engineered to express an Fe 2 S 2 ferredoxin, which normally lacks an Fe 2 S 2 ferredoxin; (b) is engineered to express an Fe 2 S 2 ferredoxin-dependent enzyme, which lacks the corresponding activity of that Fe 2 S 2 ferredoxin-dependent enzyme either as a result of mutation or as a natural property; and (c) is engineered to expresses a hydrogenase that is either capable of interacting with the Fe 2 S 2 ferredoxin or can be selected to do so.
  • Oxidation 2H + + D red ⁇ H 2 + D ⁇ O 0 x
  • [32] [FeFe] -hydrogenases are a group of proteins with a highly homologous catalytic domain and highly variable accessory domains (Ghirardi, M.L. et al. 2007. Annual Review of Plant Biology 58, 71-91). Their active site sits at the edge of the catalytic domain, and in most hydrogenases it is covered on the other side by an N-terminal "ferredoxin-like" domain, which contains several iron-sulfur clusters (Ghirardi, M.L. et al. 2007. Annual Review of Plant Biology 58, 71-91; Peters, J. W. et al. 1998. Science. 282, 1853-1858;).
  • Electrons are shuttled from the protein surface through these clusters to the active site where they are used to make hydrogen.
  • compositions and methods of the invention encompass polynucleotide and polypeptide sequences that encode for hydrogenase enzymes from any species.
  • Exemplary species include, but are not limited to, Clostridium acetobutylicum, Clostridium difficile, Clostridium pasteurianum, Clostridium perfringens, Clostridium thermocellum, Megasphaera elsdenii, Desulfovibrio desulfuricans, Desulfovibrio fructosovoran, Desulfovibrio vulgaris, Thermotoga maritime, Chlamydomonas reinhardtii, Chlorella fusca, Scenedesmus obliquus, Entamoeba histolytica, Nyctotherus ovalis, Spironucleus barkhanus, and Trichomonas vaginalis.
  • Hydrogenase genes are introduced exogenously into E. coli.
  • the invention provides methods of introducing a hydrogenase gene, or any portion thereof, isolated from Clostridium acetobutylicum into an E. coli.
  • the invention provides methods of introducing a hydrogenase maturation cofactor gene, or any portion thereof, isolated from Chlamydomonas reinhardtii into an E. coli.
  • Hydrogenase and hydrogenase maturation factor gene sequences of all species are encompassed by the invention. Hydrogenase and hydrogenase maturation factor genes of photosynthetic plants and bacteria are preferred.
  • Hydrogenase and hydrogenase maturation factor sequences or any portion thereof, having 50%, 60%, 70%, 80%, 90%, or 100% identity to the sequences provided below, or any point in between, are encompassed by the invention.
  • Clostridium acetobutylicum hydrogenase subunit (ferredoxin) is encoded by the following mRNA sequence (NCBI Accession No. NC_003030 REGION: 3378774..3380126 and SEQ K) NO: l)(For all mRNA transcripts incorporated into the present application, the initiator methionine, encoded by the codon "atg,” is bolded and capitalized to delineate the start of the coding region.):
  • Clostridium acetobutylicum hydrogenase expression factor (hybG) is encoded by the following amino acid sequence (NCBI Accession No. NP 347444.1 and SEQ ID NO: 4): MCLAWGEVLKISDCRGV ⁇ VGNLKJIEVFMHLVPEVKIGQYVLIHAGCAIEIIDEKAAKETLEILRKLSDD
  • Clostridium acetobutylicum hydrogenase formation factor (hybE) is encoded by the following mRNA sequence (NCBI Accession No. NC_003030 REGION: 934635..935642 and
  • SEQ ID NO: 5 1 ATGgagaata agatattatt aagccatgga agtggtggta aacaaactag tagtctaata
  • Clostridium acetobutylicum hydrogenase maturation factor (hybF) is encoded by the following mRNA sequence (NCBI Accession No. NC_003030 REGION: 935067..937955 and
  • Clostridium acetobutylicum hydrogenase maturation factor (hybF) is encoded by the following amino acid sequence (NCBI Accession No. NP_347447.1 and SEQ ID NO: 8): MKYVDEFRNGDYAKTLVRLIQKLTKKXINIMEICGSHT]VLAJGFRYG]KDILPSNI ⁇
  • Clostridium acetobutylicum hydrogenase expression-formation factor (hybD) is encoded by the following mRNA sequence (NCBI Accession No. NC_003030 REGION:
  • Clostridium acetobutylicum hydrogenase maturation factor (hybF) is encoded by the following amino acid sequence (NCBI Accession No. NP_347446.1 and SEQ ID NO: 10):
  • Clostridium acetobutylicum hydrogenase expression-formation factor is encoded by the following amino acid sequence (NCBI Accession No. NP_347447.1 and SEQ ID NO: 11): MKYVDEFRNGDYAKTLVRLIQKLTKKKINIMEICGSHTMAmRYGIKDILPSNIRLISGPGCPVCVTSQGYID TALELSLSREVIIATFGDMIRVPGTKTSLMKRKAEGADlXI ⁇ SPMDALTLAENNPLKKVVFLSVGFETTTPI TAITILEAKKRGVKNIFFLTSNKMVPPVMRTLVEDKELNITGFLLPGNVSAIIGKKP YEFLSSEYNVSGWTG FEPLDILKGLKVLroilNNNASVrVNEYKRVVRDEGNVTALRYIKEVFEVTDSTWRGIGNIEKSGYKINTEYE QFDAVKQF ⁇ fYKECDSSSECRCGDILKGKITPIECSLFKKACT
  • Chlamydomonas reinhardtii iron hydrogenase (HYDl) is encoded by the following mRNA sequence (NCBI Accession No. XM_001693324 and SEQ ID NO: 12):
  • Chlamydomonas reinhardtii iron hydrogenase (HYD2) is encoded by the following mRNA sequence (NCBI Accession No. XM_001694451 and SEQ ID NO: 14): i caaatataca agtgctgaca aacaagaccc acaacaattc ccctgaagac tattcgtcgc
  • Chlamydomonas reinhardtii iron hydrogenase (HYD2) is encoded by the following amino acid sequence (NCBI Accession No. XPJ)01694503.1 and SEQ ID NO: 15):
  • Chlamydomonas reinhardtii iron-hydrogenase HydAl (hydAl) is encoded by the following mRNA sequence (NCBI Accession No. AY055755 and SEQ ID NO: 16):
  • Chlamydomonas reinhardtii iron-hydrogenase HydAl (hydAl) is encoded by the following amino acid sequence (NCBI Accession No. AAL23572.1 and SEQ E) NO: 17): MSALVLKPCAAVSIRGSSCRARQVAPRAPLAASTVRVALATLEAP ARRLGNVACAAAAP AAEAPLSHVQQ ALAELAKPKDDPTRKHVCVQVAPAVRVAIAETLGLAPGATTPKQLAEGLRRLGFDEVFDTLFGADLT ⁇ MEE GSELLHRLTEHLEAHPHSDEPLPMFTSCCPGWIAMLEKS YPDLIP YVSSCKSPQMMLAAMVKSYLAEKKGI
  • Chlamydomonas reinhardtii iron-hydrogenase HydA2 (hydA2) is encodedbythe followingmRNA sequence (NCBIAccessionNo. AY055756 and SEQ ID NO: 18): 1 tgacaaacaa gacccacaac aattcccctg aagactattc gtcgcaggca caaccgagcg
  • Chlamydomonas reinhardtii iron-hydrogenase HydA2 (hydA2) is encoded by the following amino acid sequence (NCBI Accession No. AAL23573.1 and SEQ ID NO: 19):
  • Chlamydomonas reinhardtii iron hydrogenase assembly protein (HYDEF) is encoded by the following mRNA sequence (NCBI Accession No. XM_001691413 and SEQ ID NO: 20):
  • Chlamydomonas reinhardtii iron hydrogenase assembly protein (HYDEF) is encoded by the following amino acid sequence (NCBI Accession No. XP_001691465.1 and SEQ ID NO:
  • Chlamydomonas reinhardtii Fe-hydrogenase assembly protein (HydG) is encoded by the following mRNA sequence (NCBI Accession No. AY582740 and SEQ ID NO: 22):
  • Chlamydomonas reinhardtiiFe-hydrogenase assemblyprotein (HydG) is encodedbythe following amino acid sequence (NCBIAccessionNo. AAS92602.1 and SEQ ID NO: 23):
  • Chlamydomonas reinhardtii Fe-hydrogenase assembly protein (HydEF) is encoded by the following mRNA sequence (NCBI Accession No. AY582739 and SEQ ID NO: 24): i ATGgcgcaca gcctcagcgc acacagccgt caggctggtg acagaaagct tggcgcgggc
  • gagaccaccc agctggacct ggagatgcgc gcgcgcgccg cctccaccac cacagctgag
  • Ferredoxins are iron-sulfur proteins that mediate electron transfer in a range of metabolic reactions.
  • Ferredoxins are small proteins containing iron and sulfur atoms organized as iron- sulfur clusters. These proteins act like capacitors because they can accept or discharge electrons. As a result of this ability, the oxidation states of the iron atoms are changed (usually, by +2 or +3). As such, ferredoxin acts as an electron transfer agent in biological redox reactions.
  • Ferredoxins are classified according to the nature of their iron-sulfur clusters and by sequence similarity.
  • chloroplast-type or "plant-type”.
  • the active center is a [Fe 2 S 2 ] cluster, where the iron atoms are tetrahedrally coordinated both by inorganic sulfur atoms and by sulfurs provided by four conserved cysteine (Cys) residues.
  • Fe 2 S 2 ferredoxins function as electron carriers in the photosynthetic electron transport chain and as electron donors to various cellular proteins, such as glutamat ⁇ synthase, nitrate reductase and sulfite reductase.
  • the [Fe 4 S 4 ] ferredoxins are subdivided into low-potential (bacterial-type) and high- potential (HiPIP) ferredoxins.
  • Bacterial-type ferredoxins are further subdivided into groups, based on their sequence properties. Most contain at least one conserved domain, including four cysteine residues that bind to a [Fe 4 S 4 ] cluster, hi Pyrococcus fu ⁇ osus Fe 4 S 4 ferredoxin, one of the conserved Cys residues is substituted with aspartic acid.
  • [66] 3-D structures are known for a number of monocluster and dicluster bacterial-type ferredoxins.
  • the fold belongs to the ⁇ + ⁇ class, with 2-7 ⁇ -helices and four ⁇ -strands forming a barrel-like structure, and an extruded loop containing three "proximal" Cys ligands of the iron- sulfur cluster.
  • HiPIPs High potential iron-sulfur proteins
  • HiPIPs form a unique family OfFe 4 S 4 ferredoxins that function in anaerobic electron transport chains.
  • HiPIPs have so far been characterized structurally, their folds belonging to the ⁇ + ⁇ class.
  • the [Fe 4 S 4 ] cluster adopts a cubane-like conformation and is ligated to the protein via four Cys residues.
  • compositions and methods of the invention encompass polynucleotide and polypeptide sequences that encode for exogenous ferredoxin enzymes.
  • the invention provides methods of introducing a ferredoxin gene, or any portion thereof, isolated from Spinacia oleracea into a cell from E.coli.
  • Ferredoxin gene sequences of all species are encompassed by the invention. Ferredoxin genes of photosynthetic plants and bacteria are preferred. In a particularly preferred embodiment, ferredoxin I of Spinacia olearcea is used.
  • Ferredoxin sequences or any portion thereof, having 50%, 60%, 70%, 80%, 90%, or 100% identity to the sequences provided below, or any point in between, are encompassed by the invention.
  • Ferredoxin I Spinach chloroplast ferredoxin, unprocessed form
  • FNR Spinacia oleracea Ferredoxin NADP+ oxidoreductase precursor
  • Spinacia oleracea Ferredoxin is encoded by the following amino acid sequence (NCBI Accession No. CAA52867.1 and SEQ ID NO: 34):
  • Engineerred E. coli of the invention contain at least one mutation in a sulfite reductase gene. Moreover, engineerred E. coli of the invention contain at least one exogenous sulfite reductase gene. In combination with other exogenous genes and their products, e.g. hydrogenase, hydrogenase maturation factor(s), and ferredoxin, the following reaction is performed:
  • H 2 S The product of this reaction, H 2 S, is used by the E. coli to generate cysteine and methionine, which are essential for cell growth and survival. Thus, if any component of the artificial pathway is non-functional, the reaction will not proceed, H 2 S will not be produced, and therefore, sufficient levels of cyteine will not be generated by the engineered E. coli.
  • compositions and methods of the invention encompass polynucleotide and polypeptide sequences that encode for a sulfite reductase enzyme. Specifically, compositions and methods of the invention encompass polynucleotide and polypeptide sequences that include a mutation.
  • This mutation is any insertion, deletion, inversion, translocation, transposition, excision, truncation, frameshift, or point mutation in a polynucleotide or polypeptide sequence encoding for a sulfite reductase enzyme that causes theresulting geneproductto be non-functional. Furthermore, the mutation is also anyRNA orDNAmediated silencingtechnologythatprevents transcription of the gene ortranslation ofthe mRNA into apolypeptide. Mutations ofthe invention occur at any pointwithinthe sequence encoding forthe alpha subunit, beta component, orbothregions ofthe sulfitereductase enzyme. Exemplarysequences areprovidedbelow.
  • compositions andmethods ofthe invention encompass polynucleotide and polypeptide sequences that encode forexogenous sulfitereductase enzymes.
  • the invention provides methods ofintroducing a sulfitereductase gene, or anyportion thereof, isolated fromZea mays into acell fromE.coli.
  • Contemplated sequences include sulfite reductase genes, or anyportionthereof, from any species ofbacteria orphotosynthetic organism. Specifically, sequenceshaving 50%, 60%, 70%, 80%, 90%, or 100% identityto the sequences providedbelow, oranypoint inbetween, are encompassedbythe invention. [85] E.
  • coli sulfitereductase is encodedbythe followingmRNA sequence (NCBI Accession No. NZ_AAJV02000023, REGION: 73896..75608 and SEQ ID NO: 35) (reverse complement shown):
  • E. coli sulfite reductase, alpha subunit, cysJ is encoded by the following amino acid sequence (NCBI Accession No. ZP_03046139.1 and SEQ ID NO: 36): MTTQVPPSALLPLNPEQLARLQAATTDLTPTQLAWVSGYFWGVLNQQPAALAATPAPAAEMPGITIISASQ TGNAJ ⁇ VAEALRDDLLAAKINVKL ⁇ S ⁇ AGDYKFKQ]A.SEKLLIVYTSTQGEGEPPEEAVALHKFLFSKKAP KLENTAFAVFSLGDSSYEFFCQSGKDFDSKLAELGGERLLDRVD ADVEYQAAASEWRARWD ALKSRAPV AAPSQSVATGAVNEIHSSPYSKDAPLVASLSVNQKITGRNSEKDVRHIEIDLGDSGLRYQPGDALGVWYQN DPALVKELVELLWLKGDEPVTVEGKTLPLNEALQWHFELTVN
  • Zea Mays ferredoxin-sulfite reductaseprecursor(sir) is encodedbythe following mRNA sequence (NCBIAccessionNo. NM_001111832 and SEQ ID NO: 38):
  • Engineerred E. coli of the invention contain at least one mutation in a nitrite reductase gene. Moreover, engineerred E. coli of the invention contain at least one exogenous nitrite reductase gene.
  • compositions and methods of the invention encompass polynucleotide and polypeptide sequences that encode for a sulfite reductase enzyme.
  • compositions and methods of the invention encompass polynucleotide and polypeptide sequences that include a mutation.
  • This mutation is any insertion, deletion, inversion, translocation, transposition, excision, truncation, frameshift, or point mutation in a polynucleotide or polypeptide sequence encoding for a sulfite reductase enzyme that causes the resulting gene product to be non-functional.
  • the mutation is also any RNA or DNA mediated silencing technology that prevents transcription of the gene or translation of the mRNA into a polypeptide. Exemplary sequences are provided below.
  • compositions and methods of the invention encompass polynucleotide and polypeptide sequences that encode for exogenous nitrite reductase enzymes.
  • the invention provides methods of introducing a nitrite reductase gene, or any portion thereof, isolated from a green plant or an algae into a cell from E.coli.
  • Contemplated sequences include nitrite reductase genes, or any portion thereof, from any species of bacteria or photosynthetic organism. Specifically, sequences having 50%, 60%, 70%, 80%, 90%, or 100% identity to the sequences provided below, or any point in between, are encompassed by the invention.
  • E. coli nitrite reductase, nirA is encoded by the following mRNA sequence (NCBI Accession No. NC_000913, Region 1396798..1397550 and SEQ E) NO: 40)(reverse complement shown):
  • E. coli nitrite reductase, nirA is encoded by the following amino acid sequence (NCBI Accession No. NP_415850.1 and SEQ ID NO: 41) MIPEKMIRMQSGGCAIHCQDCSISQLCIPFTLNEHELDQLDNIIERKKPIQKGQTLFKAGDELKSLYAIRSGTI KSYTITEQGDEQITGFHLAGDLVGFD AIGSGHHPSFAQALETSMVCEIPFETLDDLSGKMPNLRQQMMRLM SGEIKGDQDMILLLSKKNAEERLAAFlYNLSRRFAQRGFSPREFRLTMTRGDIGNYLGLTVETISRLLGRFQK SGMLAVKGKYITIENNDALAQLAGHTRNVA [96] E. colinitritereductase, nirB, is encodedbythe followingmRNA sequence (NCBI Accession No. NP_415850.1 and SEQ ID NO: 41) MIPEKMIRMQSGGCAIHCQDCSISQ
  • E. coli nitrite reductase, nirB is encoded by the following amino acid sequence (NCBI Accession No. NP_417824.1 and SEQ ID NO: 43):
  • E. coli nitrite reductase, nirD is encoded by the following mRNA sequence (NCBI Accession No. NC_000913, Region 3494573..3494899 and SEQ ID NO: 44):
  • E. coli nitrite reductase, nirD is encoded by the following amino acid sequence (NCBI Accession No. NP_417825.1 and SEQ ID NO: 45):
  • Engineerred E. coli of the invention contain at least one mutation in a glutamate synthase gene. Moreover, engineerred E. coli of the invention contain at least one exogenous glutamate synthase gene. [101] Compositions and methods of the invention encompass polynucleotide and polypeptide sequences that encode for a sulfite reductase enzyme. Specifically, compositions and methods of the invention encompass polynucleotide and polypeptide sequences that include a mutation.
  • This mutation is any insertion, deletion, inversion, translocation, transposition, excision, truncation, frameshift, or point mutation in a polynucleotide or polypeptide sequence encoding for a sulfite reductase enzyme that causes the resulting gene product to be non-functional.
  • the mutation is also any RNA or DNA mediated silencing technology that prevents transcription of the gene or translation of the mRNA into a polypeptide. Exemplary sequences are provided below.
  • compositions and methods of the invention encompass polynucleotide and polypeptide sequences that encode for exogenous glutamate synthase enzymes.
  • the invention provides methods of introducing a glutamate synthase gene, or any portion thereof, isolated from a green plant or an algae into a cell from E. coli.
  • Contemplated sequences include glutamate synthase genes, or any portion thereof, from any species of bacteria or photosynthetic organism. Specifically, sequences having 50%, 60%, 70%, 80%, 90%, or 100% identity to the sequences provided below, or any point in between, are encompassed by the invention.
  • E. coli glutamate synthase,gltB is encoded by the following mRNA sequence (NCBI Accession No.
  • E. coli glutamate synthase,gltB is encoded by the following amino acid sequence (NCBI Accession No. NP_417679.1 and SEQ ID NO: 47):
  • E. coli glutamate synthase, gltD is encoded by the following mRNA sequence (NCBI Accession No. NC_000913, Region 3357220..3358638 and SEQ ID NO: 48):
  • E. coli glutamate synthase, gltD is encoded by the following amino acid sequence (NCBI Accession No. NP_417680.1 and SEQ ID NO: 49): MSQNWQFroLQRVDPPKKPLKIRKJEFVEire ⁇
  • oxidation describes the loss of electrons by a molecule, atom or ion. Alternatively, “oxidation” describes an increase in oxidation number. Conversely, the term “reduction” as used herein describes the gain of electrons by a molecule, atom or ion. Alternatively, “reduction” describes a decrease in oxidation number.
  • redox as used herein describes a pair, series, or plurality of reduction and oxidation reactions occurring wherein the oxidation state of at least one molecule, atom, or ion changes.
  • Polynucleotides and polypeptides of the invention are isolated from photosvnthetic organisms of the invention.
  • All photosynthetic organisms are encompassed by the present invention.
  • Exemplary organisms include, but are not limited to, green plants and bacteria.
  • the organism is Spinacia olearcea.
  • Other contemplated plants encompassed by the invention include, but are not limited to, Arabidopsis and all species of corn.
  • the organism is an aquatic phototroph.
  • Aquatic phototrophs encompassed by the invention include, but are not limited to, the eukaryotic Chlorophyta division comprising the Chlamydomonas and Chlorella genera, as well as the bacterial Phyla Cyanobacteria, Chloroflexi, Chlorobi, and the Alpha Proteohacteria.
  • the photosynthetic organism is Chlamydomonas reinhardtii.
  • the photosynthetic organism is Clostridium acetobutylicum.
  • the photosynthetic organism is Clostridium saccharobutylicum.
  • the organism is cyanobacteria, also known as blue-green algae, blue-green bacteria, or cyanophyta.
  • Cyanobacteria of the invention are isolated directly from fresh or salt water.
  • cyanobacteria of the invention are isolated from nature or cultured.
  • Exemplary cyanobacteria include, but are not limited to, those of the following genera, Anabaena, Oscillataria, Halospirulina, Planktothricoides, Prochlorococcus, Prochloron, and Prochlorothrix.
  • Polynucleotides and polypeptides of the invention are isolated from non-photosynthetic organisms of the invention. Moreover, endogenous and exogenous polynucleotide and polypeptides of the invention are inserted into cells of non-photosynthetic organisms. [115] Alternatively, or in addition, a non-photosynthetic bacterium is preferred. In this embodiment, Escherichia coli (E. coli) are used for directed evolution of one or more polypeptide species of an engineered hydrogenase.
  • bacteria encompassed by the invention include, but are not limited to, species of the following genera, Clostridium (specifically, C. pasteurianum), Ralstonia (specifically, R. eutrophia), Thermosynechococcus (specifically, T. longates), Thermotoga (specifically, T. neapolitana), and Desulfovibrio (specifically, D. gigas, D.desulfuricans, and D. baculatum).
  • Clostridium specifically, C. pasteurianum
  • Ralstonia specifically, R. eutrophia
  • Thermosynechococcus specifically, T. longates
  • Thermotoga specifically, T. neapolitana
  • Desulfovibrio specifically, D. gigas, D.desulfuricans, and D. baculatum.
  • yeast comprises the use of all strains of Saccharomyces cerevisiae.
  • the term “endogenous gene” is meant to describe a gene that exists in the genome of the host cell. Conversely, the term “exogenous gene” is meant to describe a gene that does not exist within the genome of a host cell.
  • the term, “transgenic” is meant to describe any polynucleotide or polypeptide sequence, gene, or cell that is isolated from one species and introduced into another species.
  • isolated is meant to describe a polynucleotide sequence, polypeptide sequence, or gene, that is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
  • An isolated polynucleotide is, for example, a recombinant DNA or RNA molecule, provided one of the nucleic acid sequences normally found immediately flanking that recombinant RNA molecule in a naturally-occurring molecule is removed or absent.
  • isolated polynucleotides include, without limitation, a recombinant DNA or RNA that exists as a separate molecule (e.g., a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences as well as recombinant DNA or RNA that is incorporated into a vector, an autonomously replicating plasmid, or into the genomic DNA or RNA of a prokaryote or eukaryote.
  • an isolated polynucleotide can include a recombinant DNA or RNA molecule that is part of a hybrid, chimeric, or fusion polynucleotide.
  • Isolated polypeptides of the invention are encoded by isolated polynucleotides. Alternatively, or in addition, isolated polypeptides are segregated away from other polypeptides and/or cellular material by art-recognized methods. Isolated polypeptides of the invention exist as homomeric or heteromeric monomers, dimers, tetramers, oligomers. Isolated polypeptides of the invention are folded, misfolded, or denatured. Furthermore, isolated polypeptides of the invention contain one or more subunits. [121] Isolated cells of the invention contain polynucleotides and polypeptides of the invention. As such, an isolated cell of the invention is engineered, altered, or manipulated. Isolated cells of the invention are derived from any species of organism. Alternatively, or in addition, isolated cells are unicellular organisms.
  • Endogenous and exogenous polypeptides and polynucleotides of the invention are introduced into cells.
  • introduction is meant to describe any process by which a polynucleotide or polypeptide sequence crosses a plasma membrane of a host cell.
  • Exemplary methods of introducing polynucleotide or polypeptide sequences into host cells include, but are not limited to, electroporation, heat shock, magnetofection, gene gun, endocytosis, vesicle fusion, and lipofection.
  • polynucleotides and polypeptides of the invention are bound, coupled, operably linked, fused, or tethered, to compounds that facilitate transportation of these sequences into a cell.
  • a polynucleotide or polypeptide is bound to a cationic polymer, a nanoparticle, or calcium phosphate.
  • biological property is meant to describe a property of a enzyme, for instance, a hydrogenase enzyme, that is affected by the cellular or extracellular environment.
  • a hydrogenase enzyme catalyzes the reversible oxidation of molecular hydrogen (2H + ⁇ H 2 ).
  • the presence of certain environmental factors including, but not limited to, atmospheric molecular oxygen, high temperature, low temperature, oxidizing agents, and heavy metals.
  • biological property is meant to describe operational parameters of the enzyme such as catalytic rate.
  • catalytic rate is equivalent to kinetic rate.
  • catalytic rate is meant to describe the rate, speed, or efficiency (speed with minimal energy or starting material of the reaction, e.g. 2H + or H 2 ) at which a hydrogenase converts 2H + ⁇ H 2 or H 2 — » 2H + .
  • ferredoxin-dependent enzyme is meant to describe any enzyme that requires the presence of a functional ferredoxin polypeptide in order to fulfill any of its functions.
  • exemplary ferredoxin-dependent enzymes of E. coli include, but are not limited, to sulfite reductase (cysl, cysJ), nitrite reductase (nirAB), and glutamate synthetase (gltBD).
  • the term “homolog” is meant to describe a ferredoxin-dependent enzyme from any species or an enzyme with a polypeptide sequence that that is 50%, 60%, 70%, 80%, 90%, or 100% (or any point in between) identical to a ferredoxin-dependent enzyme.
  • the term “homolog” is meant to describe a ferredoxin-dependent enzyme from any species or an enzyme encoded by a polynucleotide sequence that is 50%, 60%, 70%, 80%, 90%, or 100% (or any point in between) identical to the polynucleotide sequence that encodes for a ferredoxin-dependent enzyme.
  • selective pressure is meant to describe an environmental condition in which an engineered E. coli of the invention is grown that challenges the ability of the E. coli to survive.
  • Selective pressure is specific to the type of biological property of a hydrogenase enzyme that is desired. For example, if an oxygen tolerant hydrogenase enzyme is desired, then the engineered E. coli is grown in the presence of atmospheric oxygen, wherein the atmospheric oxygen applies selective pressure upon the survival of the E. coli.
  • the engineered E. coli is grown at higher or lower temperatures than the hydrogenase can normally tolerate and be catalytically active.
  • the range of temperatures include those temperatures, high or low, at which the E. coli would normally be unable to grow.
  • the selective pressure is inceased or decreased temperature.
  • the expression levels of the exogenous hydrogenase gene are decreased, such that each hydrogenase expressed within the engineered E. coli must catalyze reactions at a faster rate for the engineered E. coli to survive.
  • the selective pressure is decreased hydrogenase expression.
  • engineered E. coli containing hydrogenase enzymes having the desired biology property survive whereas engineered E. coli that contain hydrogenase enzymes without the desired biological property die.
  • engineered E. coli containing hydrogenase enzymes having the desired biology property survive for a longer period of time than engineered E. coli that contain hydrogenase enzymes without the desired biological property.
  • engineered E. coli containing hydrogenase enzymes having the desired biology property proliferate at a faster rate, and survive better or thrive, compared to engineered E. coli that contain hydrogenase enzymes without the desired biological property.
  • the term "functional" enzyme is meant to describe an enzyme, e.g. hydrogenase, ferredoxin, sulfite reductase, etc., that catalyzes a reaction.
  • a functional enzyme catalyzes a given reaction at a particular rate.
  • a non-functional enzyme catalyzes a reaction at a rate that is too slow to produce a required product for a cell or to maintain survival.
  • An E. coli strain of the genotype ⁇ cysl [pSIR-Fd (Zea sulfite reductase, Spinacea Ferredoxi ⁇ ); pHydAl (Clostridium acetobutylicum hydrogenase active subunit); pHydEF, HydG (Chlamydomonas reinhardtii hydrogenase maturation factors)/ is mutagenized by a standard procedure with, for example, N-nitrosoguanidine, ethylmethane sulfonate, or ultraviolet light.
  • the mutagenized bacteria are plated on selective agar plates containing M9 minimal medium with added ferrous iron in the form Of(FeSO 4 at 10 ⁇ M, or M63 medium, or another minimal medium.
  • the medium is lacking in cysteine, methionine, and any other form of reduced sulfur but may optionally contain other amino acids, vitamins, and nutrients depending on other features of the genotype of the E. coli strain background.
  • the plates are poured according to standard procedures, but are incubated for at least 4 days at room temperature in anaerobic conditions, for example, in an MBRAUN anaerobic glove box (M. Braun Group).
  • the bacteria are grown for many generations to allow fixation of any mutations, particularly mutations that are linked to a high-copy plasmid and that are recessive.
  • the bacteria are spun down and the pellet is transferred to an anaerobic chamber. In the chamber, the pellet is resuspended in a buffer that has been stored in the chamber for several days in a loose-capped container so that any dissolved oxygen will be removed.
  • the bacteria are resuspended to a final concentration of about 10 7 cells/ml and 0.1 mis is spread on a selective plate.
  • the mutagenized bacteria are titered on selective and non-selective plates.
  • Mutants are then characterized according to the genetic location of the causative mutation or mutations. As an initial step, mutant isolates are first tested as to whether the mutant phenotype co-segregates with one of the plasmids in the strain. Each mutant can be tested in two ways. In the first method, recipient bacterial strains are generated that are identical to the non- mutagenized parent strain except that each recipient is lacking one of the plasmids and, is thus, sensitive to the corresponding antibiotic. Plasmid DNA is prepared from each mutant.
  • the recipient strains are transformed with this DNA and selected for resistant to the relevant antibiotic.
  • the resulting transformed strains are tested for the mutant phenotype.
  • a mutant strain is grown in the absence of antibiotic selection for a long period of time, and a set of segregants are identified that lack each one of the antibiotic resistance markers. Such segregants are identified, for example, by replica-plating or by toothpicking. The rate of formation of such segregants is enhanced by growth in the presence of acridine orange according to standard procedures (Miller, Experiments in Molecular Genetics). If the mutant phenotype appears to co-segregate with a given plasmid, the gene or genes that contribute to the artificial hydrogenase-dependent pathway are sequenced.
  • the mutation causing the mutant phenotype will not be linked to a plasmid but will instead be in the host chromosome and can be mapped.
  • Such methods are well known in the art of bacterial genetics, and include Hfr crosses and Pl transductions.
  • the method of linked transposon mutagenesis is used, in which drug-resistant transposon insertions linked to a mutation of interest are generated, and then the drug-resistance phenotype is mapped.
  • Example 2 Construction of a Bacterial Strain Whose Growth is Conditionally Dependent on the Activity of an Exogenous FeFe Hydrogenase Using Cysteine Auxotrophv as a Phenotype [134] To construct a bacterial strain whose growth depends on the activity of an FeFe hydrogenase, the following plasmids were constructed:
  • Plasmids were produced using the Novagen Duet backbone vectors (Merck KGaA, Darmstadt Germany), an integrated system of compatible vectors used for expressing multiple genes simultaneously in E coli.
  • CaHydA (HydA from Clostridium acetobutylicum) is encodedbythe following polynucleotide sequence and SEQ ID NO: 50 (PCRproduct from aconstructprovidedbyMatt Posewitz): atgggcgcggccgcatctagaatgggcaaaacaataatcttaaatggcaatgaagtgcatacagataagatattac tatccttgagctagcaagagaaaataatgtagatatcccaacactctgctttttaaaggattgtggcaattttggaa aatgcggagtctgtatggtagaggtagaaggcaagggctttagagctgcttgtgtgtgccaaagttgaagatggaatg gtaataaacacagaatccgatgaagtaaaacacaga
  • CrHydA (HydAl from Chlamydomonas reinhardti ⁇ ) is encoded by the following polynucleotide sequence and SEQ ID NO: 52 (Synthetic construct designed to express the C. reinhardii protein while removing the >GC bias that complicates exogenous expression): atgggcgcggccgcatctagagctgcaccagccgcagaagctcctttgtctcatgttcaacaggcccttagccgagct tgcaaaccaaaggatgaccctactagaaaacacgtatgtgtccaagtggccccagctgttagggtagcaattgctg aaacacttggttggcccctggagcaaccactccaaagcagttagctgagggcctaagaaggcttggttttgatgaa gtgtgtg
  • CrHydEF from Chlamydomonas reinhardtii
  • SEQ ID NO: 54 synthetic construct designedto express the C. reinhardiiprotein whileremovingthe >GC bias that complicates exogenous expression: atgggcgcggccgcatctagagctgcacatgcctctgcttcaaagcaactccagatgttcctgtagacgatcttcc acctgcccacgctagagcagccgtcgcgcgcgctaataggagagccagggcaatggcttccgcgcgaagcagctgccg agacattaggtgactttctaggacttggcaagggtggattgagtccaggcgcaaccgctaacttagagaacaa gtgctaggtgttcttgagg
  • CrHydEF (from Chlamydomonas reinhardti ⁇ ) is encodedbythe following amino acid sequence and SEQ ID NO: 55:
  • CrHydG (from Chlamydomonas reinhardtii) is encoded by the following polynucleotide sequence and SEQ ID NO: 56 (Synthetic construct designed to express the C. reinhardii protein while removing the >GC bias that complicates exogenous expression): atggaattcgcggccgcatctagaactgctcatggtaaagcatctgccacaagagaatatgctggagattttttgcc aggcaccactatttcacacgcatggtccgttgagagggaaacacatcagatacaggaatcctgccgagtggataa acgaaagctgcaatccataaggccttagaaaccagtaaagctgacgcacaagatgctggtagagtaaagagagattcta gccaaggcaaaggctggtagagt
  • CrHydG (from Chlamydomonas reinhardtii) is encodedbythe following amino acid sequence and SEQ ID NO: 57: Mefaaasrtahgkasatreyagdflpgttishawsverethhryrnpaewineaaihkaletskadaqdagrvreil akakekafvtehapvnaesksefvqgltleecatlinvdsnnvelmneifdtalaikeriygnrwlfaplyianhc mntctycafrsankgmersiltdddlreevaalqrqghrrilaltgehpkytfdnflhavnviasvktepegsirri nveipplsvsdmrrlkntdsvgtfvlfqetyhrdtfkvmhpsgpksdfdf
  • SoFD fromSpinacea oleracea is encodedbythe followingpolynucleotide sequence and SEQ IDNO: 58 (Synthetic construct, codon optimized): atggctgcatataaagttactttggtaacaccaaccggtaatgtcgaatttcaatgtcctgatgacgtgtacatttt agacgccgctgaggaagagggaatagatctaccatattcttgcagagcaggctcatgttccagttgcgccggtaagc ttaaaactggaagcttgaaccaggatgaccaatctttcttagatgatgaccagatcgatgaaggctgggttctaaca tgtgctgcataccctgtatcagacgtcaccattgaaactcataaggaggaggaggttgggt
  • SEQ ID NO: 59 maaykvtlvtptgnvefqcpddvyildaaeeegidlpyscragscsscagklktgslnqddqsfIdddqidegwvlt caaypvsdvtiethkeeelta
  • zmSIR (from Zea mays) is encoded by the following polynucleotide sequence and SEQ ID NO:
  • zmSIR (from Zea mays) is encoded by the following polynucleotide sequence and SEQ ID NO: 61: makkdasevkrskveiikeksnf lryplneelvseapninesavqlikfhgsyqqtdrdvrgqknysfmlrtknpcg kvpnqlylamdtladefgigtlrlttrqtfqlhgvlkknlktvlstviknmgstlgacgdlnrnvlapaapyvkkdi
  • Example 3 Bacterial Growth Conditions in an Environment Lacking Oxygen and Containing Hydrogen
  • Bacteria were plated on standard petri dishes containing M9 minimal agar media as described above. Petri dishes were enclosed in airtight Vacu-Quik jars (Almore International), and a defined atmosphere was established in the jars using the method of serial evacuation and replacement with customized gas mixtures. Anaerobic hydrogen atmospheres contained 5% hydrogen and the balance nitrogen by volume.
  • Example 4 Mutagenesis of a Bacterial Strain Whose Growth is Conditionally Dependent on the Activity of an Exogenous FeFe Hydro genase and Iisolation of Mutants with Enhanced Oxygen Resistance.
  • An E. coli strain BL21(DE3) with the genotype ⁇ cysl [pCaHydA, pCrHydEF-CrHydG, pSoFD-ZmSIR] was exposed to ultraviolet light at an intensity empirically determined to kill 90% of growing cells (ImJ). Cells were allowed to recover for 4 hours in the dark, in selective media, before being plated out on minimal media and exposed to a customized atmosphere. In the case of selections for oxygen tolerance, the final atmosphere contained, by partial pressure, 20% oxygen, 5% hydrogen and the balance nitrogen. Colonies visible after 4 days were recultured in oxygen-rich conditions in both the presence or absence of hydrogen gas, to assay the hydrogen-dependence of the observed growth phenotype.
  • Example 5 Construction of a Bacterial Strain Whose Growth is Conditionally Dependent on the
  • An E. coli strain of the genotype AnirB [pNIR-Fd (Zea nitrite reductase, Spinacea Ferredoxi ⁇ j; pHydAl (Clostridium acetobutylicum hydrogenase active subunit); pHydEF, HydG (Chlamydomonas reinhardtii hydrogenase matruation factors)/ is mutagenized by a standard procedure with, for example, N-nitrosoguanidine, ethylmethane sulfonate, or ultraviolet light. Miller, Experiments in Molecular Genetics (Cold Spring Harbor Press, Cold Spring Harbor, New York) describes protocols for use of these and other mutagens.
  • the mutagenized bacteria are plated on selective agar plates containing minimal medium with added ferrous iron in the form of (FeSO 4 at 10 ⁇ M, or M63 medium, or another minimal medium.
  • the media contains an oxidized nitrogen source such as nitrate or nitrite, but no reduced nitrogen in the form of ammonia or amino acids. Vitamins and nutrients that without metabolically accessilble reduced nitrogen may be added, depending on other features of the genotype of the E. coli strain background.
  • the plates are poured according to standard procedures, but are incubated for at least 4 days at room temperature in anaerobic conditions, for example in a an MBRAUN anerobic glove box (M. Braun Group). Mutagenesis procedures and the isolation and characterization of desired mutations proceeds otherwise as in the example above.
  • Example 6 Construction of a Bacterial Strain Whose Growth is Conditionally Dependent on the Activity of an Exogenous FeFe Hydro genase Using Reduced Glutamate Auxotrophv as a Phenotype
  • An E. coli strain of the genotype AgdhA AgItB [pGlsF-Fd (Glutamate synthase from Synechocystis sp. PCC 6308, Spinacea Ferredoxi ⁇ ); pHydAl (Clostridium acetobutylicum hydrogenase active subunit); pHydEF, HydG (Chlamydomonas reinhardtii hydrogenase matruation factors)/ is mutagenized by a standard procedure with, for example, N- nitrosoguanidine, ethylmethane sulfonate, or ultraviolet light.
  • the mutagenized bacteria are plated on selective agar plates containing minimal medium with added ferrous iron in the form Of(FeSO 4 at 10 ⁇ M, or M63 medium, or another minimal medium.
  • the media contains no glutamate or glutamine, which must be synthesized by the exogenous pathway to allow cell survival. Vitamins and nutrients that without metabolically accessible glutamate maybe added, depending on other features of the genotype of the E. coli strain background.
  • the plates are poured according to standard procedures, but are incubated for at least 4 days at room temperature in anaerobic conditions, for example in a an MBRAUN anerobic glove box (M. Braun Group). Mutagenesis procedures and the isolation and characterization of desired mutations proceeds otherwise as in the example above.
  • Example 7 Hydro genase-dependent growth
  • An engineered strain of E. coli that required the function of a hydrogenase enzyme for viability under certain conditions was generated and isolated. Such strains and methods to isolate strains with such desirable properties are useful as a tool to perform directed evolution experiments on hydrogenase enzymes.
  • a number of hydrogenase properties are engineered using this system, e.g., oxygen-tolerance. The property of oxygen tolerance greatly increases the commercial utility of the hydrogenase enzyme.
  • the behavior of the system has been characterized and quantified in several ways. The oxygen-tolerance of the hydrogenase in the system has been quantified. When E. coli growth is supported by the hydrogenase, cells are only viable in atmospheres with reduced levels of oxygen.
  • the data shown in Figure 6 indicate that the cells require hydrogenase function to grow, and that hydrogenase function is destroyed by oxygen.

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Abstract

The present invention provides engineered microorganisms such as E.coli and methods for selecting for desired biological properties of hydrogenase enzymes in vivo. Using the methods of the invention, hydrogenase enzymes are selected for properties including, but not limited to, oxygen tolerance, thermostability, and increased catalytic rate.

Description

SELECTION SYSTEM FOR DI-IRON [FeFe] HYDROGENASE PROPERTIES
BACKGROUND OF THE INVENTION
[01] The utility of [FeFe] hydrogenases in applications such as biofuel production is limited by the oxygen sensitivity of the enzymes, as well as other sub-optimal properties including catalytic rate.
SUMMARY OF THE INVENTION
[02] The utility of [FeFe] hydrogenases in applications such as biofuel production is limited by the oxygen sensitivity of the enzymes, as well as other sub-optimal properties including catalytic rate. The invention provides compositions and methods for in vivo selection of hydrogenase enzymes for desired biological properties. Methods of the invention are used on any member of the FeFe hydrogenase family of enzymes. Moreover, methods of the invention enable the derivation of enzymes optimized for a wide range of properties, including, but not limited to, oxygen tolerance, kinetic rate, and thermostability.
[03] Accordingly, the invention features an oxygen-tolerant FeFe hydrogenase enzyme and a microorganism, e.g., E. coli, comprising such an oxygen-tolerant FeFe hydrogenase enzyme. The viability of the microorganism is dependent upon function of the hydrogenase enzyme. When E. coli growth is supported by wild type hydrogenase, cells are only viable in atmospheres with reduced levels of oxygen, e.g., in atmospheres of 8-10% oxygen. Normal air is 21% oxygen. The methods of the invention have yielded a microorganism that is viable in an atmosphere comprising greater than 10% oxygen due to an oxygen-tolerant hydrogenase. Such a microorganism is viable in an atmosphere comprising greater than 12%, 14%, 16%, 18%, 20% or more of oxygen. Such an enzyme and microorganisms harboring a gene encoding and expressing such an enzyme are advantageous over existing enzymes and microbes used in biofuel production.
[04] A microorganism was engineered to screen variants, e.g., mutants, of an hydrogenase enzyme to identify those with certain desirable characteristics. For example, the microorganism comprises a mutation in an NAD(P)H-dependent or ferrodoxin-dependent enzyme gene rendering the gene product absent, inactive, or reduced in level of expression. The microorganism also includes a first nucleic acid encoding a ferrodoxin-dependent enzyme and a second nucleic acid encoding a heterologous FeFe hydrogenase. The microorganism further comprises a heterologous ferrodoxin-encoding sequence and may also comprise one or more nucleic acids encoding a heterologous hydrogenase maturation factor. For example, the microorganism is an enteric bacteria such as Escherichia coli (E. coli) or Salmonella typhimurium. The microorganism is engineered to lack a bacterial sulfite reductase and instead expresses plant-derived sulfite reductase.
[05] An exemplary microorganism is an Escherichia coli (E. coli) containing a mutation in an endogenous sulfite reductase gene, an exogenous sulfite reductase gene, an exogenous ferredoxin gene, an exogenous hydrogenase gene, and at least one exogenous hydrogenase maturation cofactor gene. In a preferred embodiment of the invention, the exogenous hydrogenase gene encodes a FeFe hydrogenase.
[06] hi one aspect of the invention, the mutation in an endogenous sulfite reductase gene is a deletion of the cysl open reading frame. Moreover, the mutation is any insertion, deletion, inversion, translocation, transposition, excision, truncation, frameshift, or point mutation that causes the resulting gene product to be non-functional. Furthermore, the mutation is also any RNA or DNA mediated silencing technology that prevents transcription of the gene or translation of the mRNA into a polypeptide.
[07] hi another aspect of the invention, the mutation occurs in any ferredoxin-dependent enzyme, including, but not limited to, nitrite reductase and glutamate synthase. Moreover, the mutation is any insertion, deletion, inversion, translocation, transposition, excision, truncation, frameshift, or point mutation that causes the resulting gene product to be non-functional.
Furthermore, the mutation is also any RNA or DNA mediated silencing technology that prevents transcription of the gene or translation of the mRNA into a polypeptide. [08] hi alternative embodiments of the invention, the exogenous sulfite reductase gene is isolated from Zea mays. The exogenous ferredoxin gene is isolated from Spinacia oleracea. The said exogenous hydrogenase gene comprises a catalytic hydrogenase subunit isolated from Clostridium acetobutylicum. The exogenous hydrogenase maturation cofactor gene is isolated from Chlamydomonas reinhardtii.
[09] Exogenous hydrogenase genes of the invention contain nucleotide sequences isolated from one species. Alternatively, or in addition, exogenous hydrogenase genes contain nucleotide sequences isolated from more than one species. In a preferred embodiment, intact hydrogenase gene sequences from multiple species are randomly spliced, intermixed, and reassembled. The resulting multi-species hydrogenase gene chimeras are introduced into E. coli containing a mutation in gene encoding for a ferredoxin-dependent enzyme, an exogenous gene encoding for said ferredoxin-dependent enzyme or a homolog thereof, an exogenous ferredoxin, and at least one hydrogenase gene maturation factor. The resulting E. coli is grown in media lacking the ferredoxin-dependent gene product and under selective pressure.
[10] The invention provides a method of selecting for a biological property of a hydrogenase enzyme, including the steps of (a) providing an engineered E. coli comprising a mutation in a gene encoding a ferredoxin-dependent enzyme; (b) introducing into the engineered E. coli an exogenous gene encoding the ferredoxin-dependent-enzyme or a homolog thereof, an exogenous ferredoxin gene, an exogenous hydrogenase gene, and at least one exogenous hydrogenase maturation factor; and (c) growing the engineered E. coli in media lacking the product of the ferredoxin-dependent enzyme and under selective pressure; wherein the survival of the engineered E. coli under selective pressure indicates the presence of a functional hydrogenase enzyme, thereby identifying those hydrogenase enzymes having the desired biological property. In a preferred embodiment of the invention, the hydrogenase enzyme is a FeFe hydrogenase. [11] In one aspect of the invention, the ferredoxin-dependent enzyme is sulfite reductase, nitrite reductase, or glutamate synthase. Preferably, the ferredoxin-dependent enzyme is sulfite reductase and its product is cysteine. In another aspect of the invention, the mutation in a gene encoding a ferredoxin-dependent enzyme is a deletion of the cysl open reading frame of sulfite reductase. Moreover, the mutation is any insertion, deletion, inversion, translocation, transposition, excision, truncation, frameshift, or point mutation that causes the resulting gene product to be non-functional. Furthermore, the mutation is also any RNA or DNA mediated silencing technology that prevents transcription of the gene or translation of the mRNA into a polypeptide. In a further aspect of the invention, the exogenous gene encoding said ferredoxin- dependent-enzyme or a homolog thereof is the sulfite reductase gene.
[12] In certain embodiments of the invention, the sulfite reductase gene is isolated from Zea mays. The exogenous ferredoxin gene is isolated from Spinacia oleracea. The exogenous hydrogenase gene comprises a catalytic hydrogenase subunit isolated from Clostridium acetobutylicum. The exogenous hydrogenase maturation cofactor gene is isolated from Chlamydomonas reinhardtii. [13] In certain embodiments of the invention, the biological property selected is oxygen tolerance, thermostability, or resistance to oxidizing agents or poisons. Accordingly, selective pressure is oxygen, high temperature, low temperature, hydrogen peroxide, nitrous oxide, an oxidizing agent, a heavy metal, cyanide, or any compound known to interfere with hydrogenase activity. Preferably, the biological property is oxygen tolerance and the selective pressure is presence of oxygen. In another preferred embodiment the selection occurs in vivo. [14] The invention further provides a method of selecting for oxygen tolerance of a [FeFe] hydrogenase enzyme, including the steps of (a) providing an engineered E. coli comprising a mutation in a sulfite reductase gene; (b) introducing into said engineered E. coli an exogenous sulfite reductase gene or a homolog thereof, an exogenous ferredoxin gene, an exogenous hydrogenase gene, and at least one exogenous hydrogenase maturation factor; and (c) growing said engineered E. coli in media lacking cystein and in the presence of oxygen; wherein the survival of said engineered E. coli in the presence of oxygen indicates the presence of a functional hydrogenase enzyme, thereby identifying those hydrogenase enzymes having oxygen tolerance.
[15] The invention also provides a method of selecting for increased catalytic rate of a [FeFe] hydrogenase enzyme, including the steps of (a) providing an engineered E. coli containing a mutation in a sulfite reductase gene; (b) introducing into the engineered E. coli an exogenous sulfite reductase gene or a homolog thereof, an exogenous ferredoxin gene, an exogenous hydrogenase gene, and at least one exogenous hydrogenase maturation factor, wherein the expression of the hydrogenase gene is low; and (c) growing the engineered E. coli in media lacking cysteine; wherein the survival of the engineered E. coli indicates the presence of a functional hydrogenase, thereby identifying those hydrogenase enzymes having an increased catalytic rate. [16] Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. All references cited herein are hereby incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[17] Figure 1 is a schematic representation of the artificial cysteine production pathway introduced into engineered E. coli of the invention.
[18] Figure 2 is a schematic representation of pCrHydEF-CrHydG. [19] Figure 3 is a schematic representation of pCrHydAl.
[20] Figure 4 is a schematic representation of pCaHydA.
[21] Figure 5 is a schematic representation of pSoFd-ZmSIR.
[22] Figure 6 is a line graph showing hydrogenase-dependent growth of E. coli.
DETAILED DESCRIPTION
[23] The utility of [FeFe] hydrogenases in applications such as biofuel production has been limited by the oxygen sensitivity of the enzymes, as well as other properties including catalytic rate. Compositions and methods of the invention provide a means for driving the evolution of desirable qualities of enzymes, such as [FeFe] hydrogenases, in vivo by inserting an exogenous synthetic pathway into an engineered microorganism that can be grown under selective pressure. Consequently, the selected [FeFe] hydrogenases demonstrate qualities that are physiologically relevant, e.g. optimally functioning in vivo. This selection method is superior to and in contrast to alternative and established methods of mutating enzymes and testing enzymatic activities as well as binding efficacies in vitro and out of any biological context. The selected [FeFe] hydrogenases of the invention catalyze reactions that produce molecular hydrogen with superior properties with respect to their selection criteria. For instance, a [FeFe] hydrogenase selected for function in the presence of oxygen should be less sensitive than known hydrogenases, or insensitive, to atmospheric or molecular oxygen. This oxygen insensitive hydrogenase is then introduced into microorganisms that produce improved quantities of molecular hydrogen when exposed to air.
[24] A core aspect of the invention is the construction of an artificial biosynthetic pathway that uses hydrogen uptake or production to allow growth of a microbial cell. The following is a particular example of such an artificial pathway, the basic principles of which can be generalized as described below. [25] Compositions and methods of the invention provide an engineered E. coli strain, in which the endogenous sulfite reductase gene and the protein it encodes, which uses NAD(P)H as a source of reducing equivalents, are deleted and replaced with a sulfite reductase from Zea mays (maize). This maize-derived enzyme normally uses a reduced Fe2S2 'plant-type' ferredoxin that is found in photosynthetic organisms but is not found in E. coli. Therefore, the E. coli is engineered to express a plant-type Fe2S2 ferredoxin, which may be the spinach chloroplast ferredoxin but which can be any protein of this class. Because E. coli does not normally express an Fe2S2 ferredoxin, other proteins within E. coli are, in general, not adapted to interact with this type of ferredoxin. Thus, an E. coli strain expressing only the maize sulfite reductase and an Fe2S2 ferredoxin will not be able to grow in the absence of reduced sulfur. Consequently, the E. coli is also engineered to express an FeFe hydrogenase such as the hydrogenase from Clostridium acetobutylicum, Clostridium pasteurianum, or Chlamydomonas reinhardtii. These FeFe hydrogenases, and FeFe hydrogenases in general, use Fe2S2 ferredoxins as their primary or exclusive direct redox partner, hi addition to expressing an FeFe hydrogenase, the strain is engineered to express maturation factors for this type of hydrogenase, such as HydEF and HydG of Chlamydomonas reinhardtii. These factors are necessary and sufficient for maturation of a wide variety of FeFe hydrogenase in heterologous organisms.
[26] hi sum, the resulting E. coli strain lacks its endogenous sulfite reductase, expresses sulfite reductase from maize, expresses the Fe2S2 ferredoxin from spinach, and expresses the FeFe hydrogenase from, for example, C. acetobutylicum, and the FeFe hydrogenase maturation factors HydEF and HydG from Chlamydomonas reinhardtii. This E. coli strain cannot grow under normal conditions in the absence of a source of reduced sulfur and is effectively a cysteine auxotroph, but is able to grow in the absence of reduced sulfur when grown anaerobically in the presence of hydrogen.
[27] This genetic assay for hydrogenase function is used in a variety of ways to select for hydrogenases with various improved properties. For example, oxygen-resistant hydrogenases are selected by growing the strain in the presence of various concentrations of oxygen.
Alternatively or in addition, heat-resistant hydrogenases are selected by growing the strain at various temperatures, hi another variation, hydrogenases with a higher catalytic rate are selected by reducing the level of hydrogenase expression, so that the unselected hydrogenase activity is not sufficient to allow rapid growth, hi a further variation, hydrogenases that interact with a given type of ferredoxin may be selected if the starting hydrogenase does not interact with a chosen ferredoxin.
[28] The invention also provides a number of related alternative strategies for the genetic assay of hydrogenase function. For example, other bacterial strains are used instead of E. coli, such as Salmonella typhimurium or any other enteric bacteria that lack an Fe2S2 ferredoxin or any other bacteria whose genetic characteristics are appropriate. An appropriate genetic background entails (a) well characterized and non-redundant native metabolic pathways for producing the metabolite (i.e. cysteine) under selection; (b) the absence or near-absence of native metabolism for providing reduced ferredoxin. Ideally, the organism becomes dependent on the exogenous pathway. If the organsim natively expresses multiple redundant pathways for reducing sulfur, for example, it would be difficult or impossible to eliminate them all. As such, an organism with few or one endogenous pathway for reducing sulfur or performing the function of the exogeously-introduced ferredoxin-dependent enzyme is preferred. Also, methods of the invention are designed to ensure that the ferredoxin in the exogenous pathway is being reduced only by the hydrogenase. If multiple native or endogenous pathways within the organism produce reduced ferredoxin, the selection would fail. Consequently, an organism with a few or one endogeous ferredoxin is preferred.
[29] A variety of metabolic genes may be used instead of ferredoxin-dependent sulfite reductase, such as ferredoxin-dependent nitrate reductase, ferredoxin-dependent glutamate synthase, or any other enzyme that uses an Fe2S2 ferredoxin instead of NADPH or NADH as a source of reducing equivalents. Such enzymes may be found in photosynthetic organisms that use reduced Fe2S2 ferredoxin generated by photosystem I as a common currency of reducing equivalents. A variety OfFe2S2 ferredoxins are also used. Thus, a general form of this aspect of the invention is: a bacterium that (a) is engineered to express an Fe2S2 ferredoxin, which normally lacks an Fe2S2 ferredoxin; (b) is engineered to express an Fe2S2 ferredoxin-dependent enzyme, which lacks the corresponding activity of that Fe2S2 ferredoxin-dependent enzyme either as a result of mutation or as a natural property; and (c) is engineered to expresses a hydrogenase that is either capable of interacting with the Fe2S2 ferredoxin or can be selected to do so.
[30] The following table describes such systems using E. coli K12.
Table 1
Figure imgf000009_0001
Hydro genases
[31] A hydrogenase is an enzyme that catalyses the reversible oxidation of molecular hydrogen (H2). Hydrogenases play a vital role in anaerobic metabolism. Hydrogen uptake (H2 oxidation) is coupled to the reduction of electron acceptors such as oxygen, nitrate, sulfate, carbon dioxide, and fumarate, whereas proton reduction (H2 evolution) is essential in pyruvate fermentation and in the disposal of excess electrons. Both low-molecular weight compounds and proteins such as ferredoxins, cytochrome c3, and cytochrome c6 can act as physiological electron donors (D) or acceptors (A) for hydrogenases. Reduction = H2 + A0x → 2H+ + Ared
Oxidation = 2H+ + Dred → H2 + D ■O0x
[FeFe] -hydro genases
[32] [FeFe] -hydrogenases are a group of proteins with a highly homologous catalytic domain and highly variable accessory domains (Ghirardi, M.L. et al. 2007. Annual Review of Plant Biology 58, 71-91). Their active site sits at the edge of the catalytic domain, and in most hydrogenases it is covered on the other side by an N-terminal "ferredoxin-like" domain, which contains several iron-sulfur clusters (Ghirardi, M.L. et al. 2007. Annual Review of Plant Biology 58, 71-91; Peters, J. W. et al. 1998. Science. 282, 1853-1858;). Electrons are shuttled from the protein surface through these clusters to the active site where they are used to make hydrogen. The naturally-occurring [FeFe]-hydrogenases from algae, such as Chlamydomonas reinhardtii, do not contain this N-terminal region. Instead, ferredoxin is able to dock to the protein and donate electrons from its own iron-sulfur cluster directly to the FeFe active site (Chang, CH. et al. 2007. Biophysical Journal, 93, 3034-3035). [33] The lack of this domain also contributes to the C. reinhardtii hydrogenases sensitivity to oxygen, which is 400 times greater than that of hydrogenases that do possess this domain (Brenner, M.P. Editor. 2006. JASON, Engineering Microorganisms for Energy Production, The MITRE Corporation). [34] Compositions and methods of the invention encompass polynucleotide and polypeptide sequences that encode for hydrogenase enzymes from any species. Exemplary species include, but are not limited to, Clostridium acetobutylicum, Clostridium difficile, Clostridium pasteurianum, Clostridium perfringens, Clostridium thermocellum, Megasphaera elsdenii, Desulfovibrio desulfuricans, Desulfovibrio fructosovoran, Desulfovibrio vulgaris, Thermotoga maritime, Chlamydomonas reinhardtii, Chlorella fusca, Scenedesmus obliquus, Entamoeba histolytica, Nyctotherus ovalis, Spironucleus barkhanus, and Trichomonas vaginalis. Hydrogenase genes are introduced exogenously into E. coli. For example, the invention provides methods of introducing a hydrogenase gene, or any portion thereof, isolated from Clostridium acetobutylicum into an E. coli. Moreover, the invention provides methods of introducing a hydrogenase maturation cofactor gene, or any portion thereof, isolated from Chlamydomonas reinhardtii into an E. coli. [35] Hydrogenase and hydrogenase maturation factor gene sequences of all species are encompassed by the invention. Hydrogenase and hydrogenase maturation factor genes of photosynthetic plants and bacteria are preferred.
[36] Hydrogenase and hydrogenase maturation factor sequences, or any portion thereof, having 50%, 60%, 70%, 80%, 90%, or 100% identity to the sequences provided below, or any point in between, are encompassed by the invention.
[37] Clostridium acetobutylicum hydrogenase subunit (ferredoxin) is encoded by the following mRNA sequence (NCBI Accession No. NC_003030 REGION: 3378774..3380126 and SEQ K) NO: l)(For all mRNA transcripts incorporated into the present application, the initiator methionine, encoded by the codon "atg," is bolded and capitalized to delineate the start of the coding region.):
1 ATGaataaca agtatataga acttttcaaa tcactcgtag attcctatta caacgatact
61 tttgattctt ttgtatatca catcctttca gatgaagagg tagataaaaa agagctctca
121 aaagtaatat catccttatg tggcgttagt gttgaattta aagatactga aacttacata
181 agtgaattaa aaaaagcaat ctcaaattat aaatgtactg ataacatagt agaaaaaatc 241 aaagaatgcg attcttcttg tcacagcaac gaaggagaaa ctccctgcca aaaatcatgt 301 ccatttgatg ctatactggt agataaaaac actaagactt cacacataca gaaagattta 361 tgcacagatt gcggcaactg tataacttca tgtccttctg gatcaatatt ggataaaatt 421 gaatttatgc ctctattaaa tctatttaaa aataatgaga cagtaatagc tgctgtagca 481 cctgccatag ctggtcagtt tggagaaaat gtttcacttg aaatgcttag aactgctttt 541 aaaaaggttg gatttgcaga tatggtagaa gtggcttttt ttgcagatat gcttactata 601 aaggaagctt ttgaatttaa tgaactagta aattcaaagg atgatttaat gataacctct 661 tgttgttgcc caatgtgggt ttccatgata cgaaaaatat acaaggattt agctagacac 721 gtttcacctt cggtctctcc tatgattgcc tctggaagag ttataaaaaa actaaatcca 781 aattgcaagg tggtttttat aggtccttgt attgctaaaa aagccgaatc tagaagtcaa 841 gatataagtg atgcaataga tttcgtgctt accttcgaag agttaaaagg tatatttgat
901 gtactagata tagatccaga aaagctgcca gaaacacata caaagagtta tgcctcaaga
961 gagggcagac tttatggtcg cacaggtgga gtatctacct ctgtagatga agctgtaaaa
1021 agaattttcc ctaacaaaca tcatttattt aaatccacaa aagtagatgg tgttaaggat
1081 tgtaaggata tacttaataa aacgcaggct ggtaatatag gtgcaaattt tttagaaggt 1141 atgggctgtg ttggtggctg cgttggtggt ccaaaagcta tagttcacaa agatcaggga 1201 cgtgaaagtg taaataaaac agcagaaagt tctgaaatta aaatatctgt tgatagtgaa 1261 cgaatgaagg atattttaag tcgaatagga attaactcaa tagaagattt cggagataaa 1321 tctaaagtag atatttttga aagaagattt taa [38] Clostridium acetobutylicum hydrogenase subunit (ferredoxin) is encoded by the following amino acid sequence (NCBI Accession No. NP 349825.1 and SEQ ID NO: 2):
MNNKYIELFKSLVDSYYNDTFDSFΛRΠILLSDEEVDKKELSKVISSLCGVSVEFKDTETYISELKKAISNYKCT DNIVEKIKECDSSCHSNEGETPCQKSCPFDAILVDKNTKTSHIQKDLCTDCGNCITSCPSGSILDKIEFMPLLN LFKNNETVIAAVAPAIAGQFGENVSLEMLRTAFKKVGFADMVEVAFFADMLTIKEAFEFNELVNSKDDLMI TSCCCPMWVSMIRKIYKDLARHVSPSVSPMIASGRVIKJΠ.NPNCKVVFIGPCIAKKAESRSQDISDAΓDFVLT FEELKGIFDVLDROPEKLPETHTKSYASREGRLYGRTGGVSTSVDEAVKMFPNKHHLFKSTKVDGVKDCKD ILNKTQAGMGANFLEGMGCVGGCVGGPKARΉKDQGRESVNKTAESSEIKISVDSEPJVIKDILSRIGINSIED FGDKSKVDIFERRF [39] Clostridium acetobutylicum hydrogenase expression factor (hybG) is encoded by the following mRNA sequence (NCBI Accession No. NC_OO3O3O REGION: 934376..934591 and
SEQ ID NO: 3):
1 ATGtgtttgg ctgttccagg agaagttttg aaaataagtg attgtagggg tgttgttaaa 61 gttggaaatt taaagagaga ggtgtttatg catcttgtcc ctgaggttaa gattggacaa 121 tatgttttaa ttcatgcggg ctgtgcaatt gaaataattg atgaaaaggc agcaaaagaa 181 actcttgaga ttttaaggaa gttatcagac gattaa
[40] Clostridium acetobutylicum hydrogenase expression factor (hybG) is encoded by the following amino acid sequence (NCBI Accession No. NP 347444.1 and SEQ ID NO: 4): MCLAWGEVLKISDCRGV\^VGNLKJIEVFMHLVPEVKIGQYVLIHAGCAIEIIDEKAAKETLEILRKLSDD
[41] Clostridium acetobutylicum hydrogenase formation factor (hybE) is encoded by the following mRNA sequence (NCBI Accession No. NC_003030 REGION: 934635..935642 and
SEQ ID NO: 5): 1 ATGgagaata agatattatt aagccatgga agtggtggta aacaaactag tagtctaata
61 aataatttgt ttgtgaaata ttttgataat aatattataa aagcgatgaa tgattctgca
121 caaataaagt tagttggtac tagaattgca tttacaacag attcgtttgt catttctccg
181 cagtttttta agggagggga tatcggtaaa cttgcagtgt gtggaacagt aaatgattta
241 tcagtaagtg gtgcaagacc atgttatcta acaagtgcat ttattataga agaaggatat 301 tcaatggaaa gccttgagct cattgtaaaa tctatggccg aaactgctaa aaaagcagga 361 gttaaaattg tggctggaga tactaaggtg gttgaaaagg gaaatgtaga tggtgtgtat 421 ataaatacta caggcatagg ggagctgtat aataatataa atattagtgc tagtagggca 481 gaggttggag atttaattat tgtgaatgga aatattggag aacatggtat gagtattatg 541 tgtgaaagaa gtggaataga tatatatgga gaattgaaaa gtgattgtga tcctttaaat 601 gaattagtag gttgtatgct caaggtatgt aaagatattc atgttatcag agatgctact 661 agagggggca ttgcagcagt tttaaatgaa atagcagaag ctagtgatgt atctgttgag 721 cttaattatg agaatatacc tgtaagtgag gaagtaaagg gagcgtgtga attacttggt 781 ttagatccat tatatattgc taatgagggg aagctttgtt gttttgtacc caaagaatat 841 gcactaaatg tgcttaaaga gatgaaaaag aatgtattgg gcaagaatgc tgctattatt 901 ggaaaagtag ttgaagaaac tcattataag gtatatttga aaactgttgt tggaggaaag 961 agaatagtag atatgtcgtc tggtgaacag ttccctagaa tatgttaa [42] Clostridium acetobutylicum hydrogenase formation factor (hybE) is encoded by the following amino acid sequence (NCBI Accession No. NP 347445.1 and SEQ ID NO: 6):
MENKILLSHGSGGKQTSSLINl^FX^YFDNMIKAMNDSAQIKLVGTRIAFTTDSFVISPQFFKGGDIGKLAV CGTVNDLSVSGARPCYLTSAFIIEEGYSMESLELIλ^SMAETAKKAGVKIVAGDTKVVEKGNVDGVYINTT GIGELYNNIMSASRAEVGDLIIVNGNIGEHGMSIMCERSGIDlYGELKSDCDPLNELVGCMLKVCKDIHVIR DATRGGIAAVLNEIAEASDVSVELNYENIPVSEEVKGACELLGLDPLYIANEGKLCCFVPKEYALNVLKEM KKNVLGKNAAIIGKVVEETHYKVYLKTWGGKRIVDMSSGEQFPRIC
[43] Clostridium acetobutylicum hydrogenase maturation factor (hybF) is encoded by the following mRNA sequence (NCBI Accession No. NC_003030 REGION: 935067..937955 and
SEQ IDNO: 7): i ATGtttaaaa ggttattatt aaaaatagaa gggattgtac aaggtgtggg ttttagacct
61 tttgtttata gacaggctag tttgcttggt ttaaagggtt gggtcagcaa taattctgct
121 ggggtatata ttgatgtaga aggtgaaagt tcaaatttat atgaattcat tgataaactg
181 aaatatgata aacctttttt gtgtagaata gaaaatatcg ctattgagga aaaaacagct
241 gtgaattaca ggagttttaa aataaaaaga agtgaggata aatataataa aactactctc
301 atttcccctg atataggtat atgtgaaaaa tgtattgagg atatcacaaa tccatcaagt
361 aaaaggtata agtatccctt tgctagttgt acaaattgtg gacctagatt ttctatatta
421 aaggctattc cgtatgatag aaaaaacacg actatgaaca aatttaagct gtgttcagct
481 tgtgataaag aatacaatga ttataacaat aggagatttt atgctgaaac taattcctgc
541 aaaatttgcg gtcctcatat ttggattgaa aatcataatg gagtaaaaat agaagtcgat
601 gacgaattaa attggacaag aaaaaagctt aaagaaggca aaatttttgc tataaaaggc
661 ttaggcggct ttcaacttgt ttgtgacgca gagaatgaag aagcaataaa gaagcttaga
721 cagaagaaga atagacaaca taaaccattt gctgttatgg ttaaagatat tgaaactgca
781 aaaaagtatt gcagtgtaaa ttataaagaa gagaaattac taaaaagtaa agtaaaacct
841 atagttatac taaatagatc taattataat aaaataccta aggtgatagc tccatatcaa
901 aaaacattgg gagttatgct tccatatact ccattacatt acttgctatt ttcagatgat
961 atagaggttc ttataatgac tagtgcaaat gcccatggac tgcctttgga gtataaaaat
1021 gaaagcgcaa ttgataaact gggaaatatt gcagattatt ttttgcaaca taatagagat
1081 ataaatgtgc caatagatga ttcaattgta agggtagtaa atggacaaga atgtgttata
1141 agacgagcac gaggatatac accaaatcca attaaaagtg ataacgtaag agaaatattg
1201 gcttgtggat ctaatatgaa gaatacattt tgtatagcaa agaataattt tatgttttta
1261 agtcaatata atggcgattt agaaaatgtg gaaaccatag ataggtacat aaataacatt
1321 agacattttg aaaaaatatt taaatttact ccggggtata tagcttgtga tatgcatccc
1381 aattatttat ctattatata cgacaattat tcaaagctac ctaaaataga ggttcagcac
1441 catcatgctc atatagttag ttgtttaaca gaaaataaag ttaaaggaaa agttatagga
1501 attgcctatg atgggacagg gtatggaaca gataaaaaag tatggggagg ggaatttttt
1561 atttgtgata caaaaaagtt tatcaggctt gcacatctta aatatgtgaa aatgcctggc
1621 gctgaaatgg caataagaga aaattggaga atggctgtag cttatattta cagtgttttt
1681 gaatctggta aatataatga aaaacactta ttgaaaacta taatgaaatt atatggtgaa
1741 aaggcaatta agcttataga tataattaaa gcaaatataa attgtccgga aacctctagt
1801 atggggcgct tatttgatgc agctgcaagc ttaattggag ttagagataa tattacgtat
1861 gaaggacagg cagctagtga gcttgaagca gttattgaat taaaatgtaa gcagtattat
1921 gaatatgata taagatttaa agatgataac tatattatag agggtgataa agttataaaa
1981 ggtattatac aagataaaat atcaggggtt accatggggg agatgtcctc taaatttcat
2041 aatactgtag ttgaattttc aaaggatgta tgtaaattgc ttaggaagaa aactgcgatt
2101 aatcaggtag ccttaagcgg tggagtattt caaaattcct atttgttaaa aaatttagtt
2161 gattctttaa aaaaagaggc ttttatagtt tatacaaata aagaaatacc aacaaatgat
2221 ggaggtattg ccttaggaca gatcattgtg gcaaacgaaa ttttaaataa tagagaagga
2281 ataaaataa [44] Clostridium acetobutylicum hydrogenase maturation factor (hybF) is encoded by the following amino acid sequence (NCBI Accession No. NP_347447.1 and SEQ ID NO: 8): MKYVDEFRNGDYAKTLVRLIQKLTKKXINIMEICGSHT]VLAJGFRYG]KDILPSNI^
TALELSLSREVIIATFGDMΠIVPGTKTSLMKRKAEGADIKIVYSPMDALTLAENNPLKKVVFLSVGFETTTPI TAITILEAKKRGVKMFFLTSNKMWPX^MTLVEDKEU
FEPLDILKGLKVLMILNNNASVIΛT^YKJRVVRDEGNVTALRYIKΕVFEVTDSTWRGIGNIEKSGYKINTEYE QFDAΛTCQFNINYKECDSSSECRCGDILKGKITPIECSLFKKACTPDHPVGSCMVSYEGVCAAYYKFLNT
[45] Clostridium acetobutylicum hydrogenase expression-formation factor (hybD) is encoded by the following mRNA sequence (NCBI Accession No. NC_003030 REGION:
937955..939040 and SEQ ID NO: 9):
1 ATGaaatatg ttgatgaatt tagaaatggg gattatgcaa aaactcttgt gagattaata
61 cagaaactta ctaagaaaaa aataaatata atggagatat gcggcagtca tactatggct
121 atatttagat atggaattaa agatatactt ccaagtaata taaggttgat ttctgggccg 181 gggtgtcccg tttgtgttac gtctcaaggt tatattgata ctgctttaga gctttcttta 241 agtagagagg ttatcattgc aacttttgga gatatgataa gggttccggg aacaaaaact 301 tctctaatga aaagaaaagc tgagggagct gatattaaaa tagtttattc accaatggat 361 gcattgactt tggcagaaaa taatccgtta aaaaaagtag tctttctttc tgtaggtttt 421 gaaacaacaa cacctattac agcaattaca atattggagg ctaaaaaaag aggtgttaag 481 aatatatttt ttttgacgtc aaataagatg gttcctccag ttatgaggac acttgtagaa 541 gataaggaac ttaacataac tggtttttta cttcctggaa atgttagtgc tattattgga 601 aaaaaaccat atgaattttt gagcagtgag tataatgtat ctggtgttgt aacaggcttt 661 gagccgttgg atatacttaa gggcttaaag gtacttatag atattataaa taacaatgct 721 tcagtaatag taaatgaata taagagggta gtaagagatg agggtaacgt tacggcatta 781 agatatataa aagaagtatt tgaagttact gatagcacat ggagaggaat agggaacatt
841 gaaaaaagtg gatataagat taatacagaa tatgagcaat ttgatgctgt aaaacagttt
901 aatataaatt ataaagaatg cgatagcagt tctgaatgta gatgtggaga tatacttaaa
961 gggaaaataa ctccaataga atgttcgcta tttaaaaaag catgcacacc tgatcatcct
1021 gtaggttctt gtatggtgtc ttatgaaggt gtatgtgcag cgtattataa attccttaat 1081 acatga
[46] Clostridium acetobutylicum hydrogenase maturation factor (hybF) is encoded by the following amino acid sequence (NCBI Accession No. NP_347446.1 and SEQ ID NO: 10):
MFKIO.LLKIEGIVQGVGFRPFVYRQASLLGLKGWVSNNSAGVYID VEGESSNLYEFIDKLKYDKPFLCRIEN IMEEKTAλTSfYRSFKJKRSEDKYNKTTLISPDIGICEKCIEDITNPSSKRYKYPFASCTNCGPRPSILKAIP YDRK
NTTMNKFKLCSACDKEYNDYNNRRFYAETNSCKICGPHlWffi
GLGGFQLVCDAENEEAlKKLRQKKNRQHOΨAVMVKDIETAKKYCSλ^^
NKIPKVL\PYQKTLGVMLPYTPLHYLLFSDDIEVLMTSANAHGLPLEYKNESAroKI.GN]ADYFLQHNRDI
NVProDSIVRVVNGQECVIRRARGYTPNPrKSDNVREILACGSNMKNTFCIAKNNFMFLSQYNGDLENVETI DRYINMRHFEKIFKFTPGYIACDMHPNYLSirmSrrSKl^^
GYGTDKKVWGGEFFICDTKXFmLAHLKYVE^VIPGAEMAIRENWRMAVAYIYSWESGKYNEKHLLKTIM
KLYGEKAIKLIDIIKANINCPETSSMGRLFD AAASLIGVRDNITYEGQAASELEAVIELKCKQYYEYDIRFKD
DNYIIEGDKXOKGΠQDKISGVTMGEMSSKPHNTVVEFSKDVCKLLRKKTAINQVALSGGVFQNSYLLKNLV
DSLKXEAFRnfTNKEffTNDGGIALGQIWANEILNNREGIK
[47] Clostridium acetobutylicum hydrogenase expression-formation factor (hybD) is encoded by the following amino acid sequence (NCBI Accession No. NP_347447.1 and SEQ ID NO: 11): MKYVDEFRNGDYAKTLVRLIQKLTKKKINIMEICGSHTMAmRYGIKDILPSNIRLISGPGCPVCVTSQGYID TALELSLSREVIIATFGDMIRVPGTKTSLMKRKAEGADlXIλΥSPMDALTLAENNPLKKVVFLSVGFETTTPI TAITILEAKKRGVKNIFFLTSNKMVPPVMRTLVEDKELNITGFLLPGNVSAIIGKKP YEFLSSEYNVSGWTG FEPLDILKGLKVLroilNNNASVrVNEYKRVVRDEGNVTALRYIKEVFEVTDSTWRGIGNIEKSGYKINTEYE QFDAVKQF^^^^fYKECDSSSECRCGDILKGKITPIECSLFKKACTPDHPVGSCMVSYEGVCAAYYKFLNT
[48] Chlamydomonas reinhardtii iron hydrogenase (HYDl) is encoded by the following mRNA sequence (NCBI Accession No. XM_001693324 and SEQ ID NO: 12):
1 atcttacatg aacacacaaa cactctcgca ggcactagcc tcaaaccctc gaaacctttt 61 tccaacagtt tacaccccaa ttcggacgcc gctccaagct cgctccgttg ctccttcatc 121 gcaccaccta ttatttctaa tatcgtagac gcgacaagAT Gtcggcgctc gtgctgaagc 181 cctgcgcggc cgtgtctatt cgcggcagct cctgcagggc gcggcaggtc gccccccgcg 241 ctccgctcgc agccagcacc gtgcgtgtag cccttgcaac acttgaggcg cccgcacgcc 301 gcctaggcaa cgtcgcttgc gcggctgccg cacccgctgc ggaggcgcct ttgagtcatg 361 tccagcaggc gctcgccgag cttgccaagc ccaaggacga ccccacgcgc aagcacgtct 421 gcgtgcaggt ggctccggcc gttcgtgtcg ctattgccga gaccctgggc ctggcgccgg 481 gcgccaccac ccccaagcag ctggccgagg gcctccgccg cctcggcttt gacgaggtgt 541 ttgacacgct gtttggcgcc gacctgacca tcatggagga gggcagcgag ctgctgcacc 601 gcctcaccga gcacctggag gcccacccgc actccgacga gccgctgccc atgttcacca 661 gctgctgccc cggctggatc gctatgctgg agaaatctta cccggacctg atcccctacg 721 tgagcagctg caagagcccc cagatgatgc tggcggccat ggtcaagtcc tacctagcgg 781 aaaagaaggg catcgcgcca aaggacatgg tcatggtgtc catcatgccc tgcacgcgca 841 agcagtcgga ggctgaccgc gactggttct gtgtggacgc cgaccccacc ctgcgccagc 901 tggaccacgt catcaccacc gtggagctgg gcaacatctt caaggagcgc ggcatcaacc 961 tggccgagct gcccgagggc gagtgggaca atccaatggg cgtgggctcg ggcgccggcg 1021 tgctgttcgg caccaccggc ggtgtcatgg aggcggcgct gcgcacggcc tatgagctgt 1081 tcacgggcac gccgctgccg cgcctgagcc tgagcgaggt gcgcggcatg gacggcatca 1141 aggagaccaa catcaccatg gtgcccgcgc ccgggtccaa gtttgaggag ctgctgaagc
1201 accgcgccgc cgcgcgcgcc gaggccgccg cgcacggcac ccccgggccg ctggcctggg 1261 acggcggcgc gggcttcacc agcgaggacg gcaggggcgg catcacactg cgcgtggccg
1321 tggccaacgg gctgggcaac gccaagaagc tgatcaccaa gatgcaggcc ggcgaggcca
1381 agtacgactt tgtggagatc atggcctgcc ccgcgggctg tgtgggcggc ggcggccagc 1441 cccgctccac cgacaaggcc atcacgcaga agcggcaggc ggcgctgtac aacctggacg 1501 agaagtccac gctgcgccgc agccacgaga acccgtccat ccgcgagctg tacgacacgt 1561 acctcggaga gccgctgggc cacaaggcgc acgagctgct gcacacccac tacgtggccg 1621 gcggcgtgga ggagaaggac gagaagaagt gaggagcgcc agaggctctt tgggcggaga 1681 cagcttcaaa gcgagggggc gtattagcag taccgtaaat atgcactgat gggtgatgcg 1741 ggtgtcctcc tttatattga atggggtcaa aataggcggc gggtcaaatg tttccttttt 1801 gagtggtgtc acagcatggg gcacgtgtgc ggaggccagt tgccctccag tgcacgcgct 1861 cccggtgtgt ggccgcactg gccttggata atgcaccggt ggaggattat ggaagagggg 1921 gactcagaag gctcattatt ggacaatgcc tggtctcttc cacattggtg tgagcgcggc 1981 tccgcatagg ctgttcactg cacgctggca ttaggcgtag gtactggcat gagggagcgc 2041 ggcttgctaa ccgaatggcg tatccctcca gggcacgtcg gaatggcgcg tgcccatcaa 2101 cgcaaattct tggccttcat cgcttctgga tattgaagct gcacaaacct gcattctatt 2161 tgcttgttta cacgtgcccc aatcttggtt ggaagctaaa catgtttggg aacaattcat 2221 cttactaaag cgtgtggggg ttgaggatgc gcacgttgtg cgctggtggg tgggcgggaa 2281 cgtgggtagc atttaggcta gctggcatac gacaacgggg cccgtgagga ttgagcactt 2341 gactcgcgaa cttatgaacg tagcgcttta tacccaccgt atgcgattga cgttggtgta 2401 ggcaaccagg cggtaggaag gcggagagat gcattgcaaa cgcctgtaaa agaacggcat 2461 agctactaga cactctgatg tggacccttg gcgcagccac gacaggagag gtgtgcatca 2521 gccgcttgta agcacgcact tctgag [49] Chlamydomonas reinhardtii iron hydrogenase (HYDl) is encoded by the following amino acid sequence (NCBI Accession No. XP_001693376.1 and SEQ ID NO: 13):
MSALVLKPCAAVSIRGSSCRARQVAPRAPLAASTVRVALATLEAPARRLGNVACAAAAPAAEAPLSHVQQ ALAELAKPKDDPTRKHVCVQVAPAVRVAIAETLGLAPGATTPKQLAEGLRRLGFDEVFDTLFGADLTIMEE GSELLHRLTEHLEAHPHSDEPLPMFTSCCPGWIAMLEKS YPDLIPYVSSCKSPQMMLAAMVKSYLAEKKGI
APKDMVMVSiMP CTRKQSEADRDWFCVD ADPTLRQLDHVITTVΈLGNIFKERGINLAELPEGEWDNPMGV
GSGAGVLFGTTGGVMEAALRTAYELFTGTPLPRLSLSEVRGMDGIKETMTMWAPGSKFEELLKHRAAAR AEAAAHGTPGPLAWDGGAGFTSEDGRGGITLRVAVANGLGNAKKLITKMQAGEAKYDFVEIMACPAGCV GGGGQPRSTDKAITQKRQAALYNLDEKSTLRRSHENPSIRELYDTYLGEPLGHKAHELLHTHYVAGGVEE KDEKK
[50] Chlamydomonas reinhardtii iron hydrogenase (HYD2) is encoded by the following mRNA sequence (NCBI Accession No. XM_001694451 and SEQ ID NO: 14): i caaatataca agtgctgaca aacaagaccc acaacaattc ccctgaagac tattcgtcgc
61 aggcacaacc gagcgaaccg cggcaacact gtccctggcc ccgactcagc ctacttgttc
121 tcaattagct agtgtctgcg gatagattag caggATGgcg cttggtcttc ttgccgagct
181 gcgcgcgggt caggccgtgg catgtgctcg ccgcaccaat gcccctgctc accctgcggc
241 agtggtgccc tgcctgccta gccgtgcggg caagttcttc aatctaagtc agaaggtccc
301 ttcgtcgcag tctgcgcgcg gctcgaccat tcgtgtggca gcgaccgcaa ctgatgctgt
361 tccgcactgg aagctcgcgc tcgaggagct tgacaagccc aaggatggcg gccgcaaggt
421 cttgatcgcg caagtggccc cggccgtccg cgttgccatt gctgagtcat tcggcctggc
481 cccgggtgcc gtgtcgccgg gcaaactagc caccggcttg cgtgccctcg gcttcgacca
541 ggtgtttgac acgctgttcg cggccgacct gaccatcatg gaggagggca cggagctgct
601 gcaccgcctc aaggagcacc tggaggccca cccgcactcc gatgagccgc tgcccatgtt
661 caccagctgc tgccccggct gggtcgccat gatggagaag tcctaccctg agctcattcc
721 cttcgtcagc tcctgcaaga gtccccagat gatgatgggc gccatggtga agacctacct
781 gtccgagaag cagggcatcc ccgccaagga catcgtcatg gtctccgtca tgccttgcgt
841 gcgcaagcag ggcgaggctg accgcgagtg gttctgtgtg agcgagccgg gcgtgcgcga
901 cgtggaccac gtaatcacca ccgccgagct gggcaacatc ttcaaggagc gtggcatcaa
961 cctgcccgag ctgcccgaca gtgactggga ccagccgctg ggcctgggct ccggcgccgg
1021 cgtactgttc ggcaccaccg gcggcgtcat ggaggcggcg ctgcgcacgg cctacgagat
1081 agtgaccaag gagcccctgc cgcgcctcaa cctgagcgag gtgcgcggct tggacggcat
1141 caaggaggcg tccgtgacgc tggtccccgc tccgggctcc aagttcgccg agctggtggc
1201 ggagcgcctg gcgcacaagg tcgaggaggc ggccgcggct gaggcggcgg cggcggtgga
1261 gggcgccgtg aagccgccca tcgcgtacga cggcggccag ggtttctcca cggatgacgg
1321 caagggcggc ctgaagctgc gggtggcggt ggcgaacggc ctgggcaacg ccaagaagct
1381 gatcggcaag atggtatctg gcgaggccaa gtacgacttc gtagaaatca tggcctgccc
1441 tgccggctgc gtgggcggcg gcggccagcc ccgctccacc gacaagcaga tcacccagaa
1501 gcggcaggcg gctctgtacg acctggacga gcgcaacacg ctgcgccgca gccacgaaaa
1561 cgaggcggtc aaccagctgt acaaggagtt cctgggcgag cccctgtccc accgcgccca
1621 cgagctgctg cacacccact acgtgccagg cggcgccgag gccgatgctt agggcacagc
1681 tcttgcgtgg tatgtgtaaa cttatgcgtc cgagtgagcc ttggacttgg agctgcggaa
1741 gccgactgcc gaggcgggga gcgcccgtgc ggagcatatg cgatgtatgg tggtagtggg
1801 aggcggctgt ggggatcact atgacacgga cacttcgaat aaggttgcgc agctagcgct
1861 aaggggtggt gcgccctggt tcaatacaca tacatcccca cgcggcaggt aggggcatgt
1921 tataagtgag atcatcatgt tttgctcgga gttcagatac ttaaggactg aaaggttgga
1981 ttattgcttt gaacggaaca ggaggtcggg tggattacaa ttgtcctgga acggtgcgga
2041 tttagcatct tgacgtacac agaatgttac atctagtgga cacgcgcggc actgagcgca
2101 tacatcatgc gcatgtgcgc cttcgcctgc acgcacgtac gtaaccgacg gcggtattcg
2161 tcgcgaagtg tgtgtgtcat taggtttgtg tttgtcgtga tgtggtggtg gcaaaactgg
2221 gactcatgta cggtacggta tgggctgtgc caccgtacat gtggcgatgt gattgtgagt
2281 ctcctgctgt ctcacgcgca gggggaggtg caaccacccg ttgtgcgagc cctctttata
2341 taataagcta agcgtccttt caaagctgag ttttcgaatg gtgtggcgcc aggccgccag 2401 ctgtttttgg tgtgaggtag gtacgggccc atgggctttg acagtgcctg gaagtgctca 2461 cgcgcgtgtg tgaccagttg accaccggca ggcgcaacat ggcgcacaga cagtgtccta 2521 tgacctgtaa cactgccctg cctta
[51] Chlamydomonas reinhardtii iron hydrogenase (HYD2) is encoded by the following amino acid sequence (NCBI Accession No. XPJ)01694503.1 and SEQ ID NO: 15):
MALGLLAELRAGQAVACARRTNAPAHPAAWPCLPSRAGKFFNLSQKVPSSQSARGSTIRVAATATDAVP
HWKXALEELDKPKDGGRKVLIAQVAPAVRVAIAESFGLAPGAVSPGKLATGLRALGFDQVFDTLFAADLTI
MEEGTELLHRLKEHLEAHPHSDEPLPMFTSCCPGWVAMMEKSYPELIPFVSSCKSPQMMMGAMVKTYLSE KQGffAKDIVlVrVSλTSffCVRKQGEADREWFCVSEPGVRDVDHVITTAELGNIFKERGINLPELPDSD WDQPL GLGSGAGVLFGTTGGVMEAALRTAYEIVTKEPLPRLNLSEVRGLDGIKEASVTLVPAPGSKFAELVAERLA HKVEEAAAAEAAAAVEGAVKPPIAYDGGQGFSTDDGKGGLKLRVAVANGLGNAKKLIGKMVSGEAKYD FVEIMACP AGCVGGGGQPRSTDKQITQKRQAALYDLDERNTLRRSHENEAVNQLYKEFLGEPLSHRAHEL LHTHYVPGGAEADA
[52] Chlamydomonas reinhardtii iron-hydrogenase HydAl (hydAl) is encoded by the following mRNA sequence (NCBI Accession No. AY055755 and SEQ ID NO: 16):
1 ctcaaaccct cgaaaccttt ttccaacagt ttacacccca attcggacgc cgctccaagc
61 tcgctccgtt gctccttcat cgcaccacct attatttcta atatcgtaga cgcgacaagA 121 TGtcggcgct cgtgctgaag ccctgcgcgg ccgtgtctat tcgcggcagc tcctgcaggg
181 cgcggcaggt cgccccccgc gctccgctcg cagccagcac cgtgcgtgta gcccttgcaa
241 cacttgaggc gcccgcacgc cgcctaggca acgtcgcttg cgcggctgcc gcacccgctg
301 cggaggcgcc tttgagtcat gtccagcagg cgctcgccga gcttgccaag cccaaggacg
361 accccacgcg caagcacgtc tgcgtgcagg tggctccggc cgttcgtgtc gctattgccg 421 agaccctggg cctggcgccg ggcgccacca cccccaagca gctggccgag ggcctccgcc
481 gcctcggctt tgacgaggtg tttgacacgc tgtttggcgc cgacctgacc atcatggagg
541 agggcagcga gctgctgcac cgcctcaccg agcacctgga ggcccacccg cactccgacg
601 agccgctgcc catgttcacc agctgctgcc ccggctggat cgctatgctg gagaaatctt
661 acccggacct gatcccctac gtgagcagct gcaagagccc ccagatgatg ctggcggcca 721 tggtcaagtc ctacctagcg gaaaagaagg gcatcgcgcc aaaggacatg gtcatggtgt
781 ccatcatgcc ctgcacgcgc aagcagtcgg aggctgaccg cgactggttc tgtgtggacg
841 ccgaccccac cctgcgccag ctggaccacg tcatcaccac cgtggagctg ggcaacatct
901 tcaaggagcg cggcatcaac ctggccgagc tgcccgaggg cgagtgggac aatccaatgg
961 gcgtgggctc gggcgccggc gtgctgttcg gcaccaccgg cggtgtcatg gaggcggcgc 1021 tgcgcacggc ctatgagctg ttcacgggca cgccgctgcc gcgcctgagc ctgagcgagg
1081 tgcgcggcat ggacggcatc aaggagacca acatcaccat ggtgcccgcg cccgggtcca
1141 agtttgagga gctgctgaag caccgcgccg ccgcgcgcgc cgaggccgcc gcgcacggca
1201 cccccgggcc gctggcctgg gacggcggcg cgggcttcac cagcgaggac ggcaggggcg
1261 gcatcacact gcgcgtggcc gtggccaacg ggctgggcaa cgccaagaag ctgatcacca 1321 agatgcaggc cggcgaggcc aagtacgact ttgtggagat catggcctgc cccgcgggct
1381 gtgtgggcgg cggcggccag ccccgctcca ccgacaaggc catcacgcag aagcggcagg 1441 cggcgctgta caacctggac gagaagtcca cgctgcgccg cagccacgag aacccgtcca 1501 tccgcgagct gtacgacacg tacctcggag agccgctggg ccacaaggcg cacgagctgc 1561 tgcacaccca ctacgtggcc ggcggcgtgg aggagaagga cgagaagaag tgaggagcgc 1621 cagaggctct ttgggcggag acagcttcaa acjcgaggggg cgtattagca gtaccgtaaa 1681 tatgcactga tgggtgatgc gggtgtcctc ctttatattg aatggggtca aaataggcgg 1741 cgggtcaaat gtttcctttt tgagtggtgt cacagcatgg ggcacgtgtg cggaggccag 1801 ttgccctcca gtgcacgcgc tcccggtgtg tggccgcact ggccttggat aatgcaccgg 1861 tggaggatta tggaagaggg ggactcagaa ggctcattat tggacaatgc ctggtctctt 1921 ccacattggt gtgagcgcgg ctccgcatag gctgttcact gcacgctggc attaggcgta 1981 ggtactggca tgagggagcg cggcttgcta accgaatggc gtatccctcc agggcacgtc 2041 ggaatggcgc gtgcccatca acgcaaattc ttggccttca tcgcttctgg atattgaagc 2101 tgcacaaacc tgcattctat ttgcttgttt acacgtgccc caatcttggt tggaagctaa 2161 acatgtttgg gaacaattca tcttactaaa gcgtgtgggg gttgaggatg cgcacgttgt
2221 gcgctggtgg gtgggcggga acgtgggtag catttaggct agctggcata cgacaacggg
2281 gcccgtgagg attgagcact tgactcgcga acttatgaac gtagcgcttt atacccaccg
2341 tatgcgattg acgttggtgt aggcaaccag gcggtaggaa ggcggagaga tgcattgcaa 2401 acgcctgtaa aagaacggca tagaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa
[53] Chlamydomonas reinhardtii iron-hydrogenase HydAl (hydAl) is encoded by the following amino acid sequence (NCBI Accession No. AAL23572.1 and SEQ E) NO: 17): MSALVLKPCAAVSIRGSSCRARQVAPRAPLAASTVRVALATLEAP ARRLGNVACAAAAP AAEAPLSHVQQ ALAELAKPKDDPTRKHVCVQVAPAVRVAIAETLGLAPGATTPKQLAEGLRRLGFDEVFDTLFGADLTΓMEE GSELLHRLTEHLEAHPHSDEPLPMFTSCCPGWIAMLEKS YPDLIP YVSSCKSPQMMLAAMVKSYLAEKKGI
APKDMVMVSIMPCTRKQSEADRDWFCVDADPTLRQLDHVITTVELGNIFKERGINLAELPEGEWDNPMGV GSGAGVLFGTTGGVMEAALRTAYELFTGTPLPRLSLSEVRGMDGIKETNITMVPAPGSKFEELLKHRAAAR AEAAAHGTPGPLAWDGGAGFTSEDGRGGITLRVAVANGLGNAKKLITKMQAGEAKYDFVEIMACPAGCV GGGGQPRSTDKAITQKRQAALYNLDEKSTLRRSHENPSIRELYDTYLGEPLGHKAHELLHTHYVAGGVEE KDEKK
[54] Chlamydomonas reinhardtii iron-hydrogenase HydA2 (hydA2) is encodedbythe followingmRNA sequence (NCBIAccessionNo. AY055756 and SEQ ID NO: 18): 1 tgacaaacaa gacccacaac aattcccctg aagactattc gtcgcaggca caaccgagcg
61 aaccgcggca acactgtccc tggccccgac tcagcctact tgttctcaat tagctagtgt
121 ctgcggatag attagcaggA TGgcgcttgg tcttcttgcc gagctgcgcg cgggtcaggc
181 cgtggcatgt gctcgccgca ccaatgcccc tgctcaccct gcggcagtgg tgccctgcct
241 gcctagccgt gcgggcaagt tcttcaatct aagtcagaag gtcccttcgt cgcagtctgc 301 gcgcggctcg accattcgtg tggcagcgac cgcaactgat gctgttccgc actggaagct 361 cgcgctcgag gagcttgaca agcccaagga tggcggccgc aaggtcttga tcgcgcaagt 421 ggccccggcc gtccgcgttg ccattgctga gtcattcggc ctggccccgg gtgccgtgtc 481 gccgggcaaa ctagccaccg gcttgcgtgc cctcggcttc gaccaggtgt ttgacacgct 541 gttcgcggcc gacctgacca tcatggagga gggcacggag ctgctgcacc gcctcaagga 601 gcacctggag gcccacccgc actccgatga gccgctgccc atgttcacca gctgctgccc 661 cggctgggtc gccatgatgg agaagtccta ccctgagctc attcccttcg tcagctcctg 721 caagagtccc cagatgatga tgggcgccat ggtgaagacc tacctgtccg agaagcaggg 781 catccccgcc aaggacatcg tcatggtctc cgtcatgcct tgcgtgcgca agcagggcga 841 ggctgaccgc gagtggttct gtgtgagcga gccgggcgtg cgcgacgtgg accacgtaat 901 caccaccgcc gagctgggca acatcttcaa ggagcgtggc atcaacctgc ccgagctgcc
961 cgacagtgac tgggaccagc cgctgggcct gggctccggc gccggcgtac tgttcggcac
1021 caccggcggc gtcatggagg cggcgctgcg cacggcctac gagatagtga ccaaggagcc
1081 cctgccgcgc ctcaacctga gcgaggtgcg cggcttggac ggcatcaagg aggcgtccgt
1141 gacgctggtc cccgctccgg gctccaagtt cgccgagctg gtggcggagc gcctggcgca 1201 caaggtcgag gaggcggccg cggctgaggc ggcggcggcg gtggagggcg ccgtgaagcc
1261 gcccatcgcg tacgacggcg gccagggttt ctccacggat gacggcaagg gcggcctgaa 1321 gctgcgggtg gcggtggcga acggcctggg caacgccaag aagctgatcg gcaagatggt
1381 atctggcgag gccaagtacg acttcgtaga aatcatggcc tgccctgccg gctgcgtggg 1441 cggcggcggc cagccccgct ccaccgacaa gcagatcacc cagaagcggc aggcggctct 1501 gtacgacctg gacgagcgca acacgctgcg ccgcagccac gaaaacgagg cggtcaacca 1561 gctgtacaag gagttcctgg gcgagcccct gtcccaccgc gcccacgagc tgctgcacac 1621 ccactacgtg ccaggcggcg ccgaggccga tgcttagggc acagctcttg cgtggtatgt 1681 gtaaacttat gcgtccgagt gagccttgga cttggagctg cggaagccga ctgccgaggc 1741 ggggagcgcc cgtgcggagc atatgcgatg tatggtggta gtgggaggcg gctgtggggg 1801 atcactatga cacggacact tcgaataagg ttgcgcagct agcgctaagg ggtggtgcgc 1861 cctggttcaa tacacataca tccccacgcg gcaggtaggg gcatgttata agtgagatca 1921 tcatgttttg ctcggagttc agatacttaa ggactgaaag gttggattat tgctttgaac 1981 ggaacaggag gtcgggtgga ttacaattgt cctggaacgg tgcggattta gcatcttgac 2041 gtacacagaa tgttacatct agtggacacg cgcggcactg agcgcataca tcatgcgcat
2101 gtgcgccttc gcctgcacgc acgtacgtaa ccgacggcgg tattcgtcgc gaagtgtgtg
2161 tgtcattagg tttgtgtttg tcgtgatgtg gtggtggcaa aactgggact catgtacggt
2221 acggtatggg ctgtgccacc gtacatgtgg cgatgtgatt gtgagtctcc tgctgtctca 2281 cgcgcagggg gaggtgcaac cacccgttgt gcgagccctc tttatataat aagctaagcg
2341 tcctttcaaa gctgagtttt cgaatggtgt ggcgccaggc cgccagctgt ttttggtgtg
2401 aggtaggtac gggcccatgg gctttgacag tgcctggaag tgctcacgcg cgtgtgtgac
2461 cagttgacca ccggcaggcg caacatggcg cacagacagt gtcctatgac ctgtaacact
2521 gccctgcctt aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
[55] Chlamydomonas reinhardtii iron-hydrogenase HydA2 (hydA2) is encoded by the following amino acid sequence (NCBI Accession No. AAL23573.1 and SEQ ID NO: 19):
MALGLLAELRAGQAVACARRTNAPAHP AAWPCLPSRAGKFFNLSQKVPSSQSARGSTIRVAATATDAVP HWKLALEELDKPKDGGRKVLIAQVAPAVRVAIAESFGLAPGAVSPGKLATGLRALGFDQVFDTLFAADLTI MEEGTELLHRLKEHLEAHPHSDEPLPMFTSCCPGWVAMMEKSYPELIPFVSSCKSPQMMMGAMVKTYLSE KQGπ>AKDIV]VrVS\ηVff CVRKQGEADREWFCVSEPGVRD
GLGSGAGVLFGTTGGVMEAALRTAYEIVTKEPLPRLNLSEVRGLDGIKEASVTLVPAPGSKFAELVAERLA HKVEEAAAAEAAAAVEGAVKPPIAYDGGQGFSTDDGKGGLKLRVAVANGLGNAKKLIGKMVSGEAKYD FVEIMACP AGCVGGGGQPRSTDKQITQKRQAALYDLDERNTLRRSHENEAVNQLYKEFLGEPLSHRAHEL LHTHYVPGGAEADA
[56] Chlamydomonas reinhardtii iron hydrogenase assembly protein (HYDEF) is encoded by the following mRNA sequence (NCBI Accession No. XM_001691413 and SEQ ID NO: 20):
1 gctatttacc aaacttctga aatagccgaa cgaacccacg gtgacaaagg cacaacgaag , 61 caaattgtgt agcaacacca ccactaccgc cacacgggcg cgcaaagctt taaaacaaca 121 aacttagaca gtgaactcat aagctgatac tgttacgccc tgcgagtact ttttaccccc 181 gaatccggta ctctatacca tatttcacac ggccgctgcg cgaggcgcta gcggccgctg 241 acgacgcgAT Ggcgcacagc ctcagcgcac acagccgtca ggctggtgac agaaagcttg 301 gcgcgggcgc ggcctcgtcg cgcccgagct gtccctcgcg ccgcattgtg cgcgtcgccg 361 cgcatgcgag cgcctccaag gccacgcccg acgtcccggt cgatgacctg cctcccgctc 421 atgcccgcgc tgccgtcgcg gccgccaacc gccgcgctcg tgccatggct tcggctgagg 481 ccgccgcgga gaccctgggt gacttcctgg ggctcggcaa gggcgggctt tcgcccgggg 541 ccaccgccaa cctggacagg gaacaggtac tgggtgtgct ggaggcggtg tggcgccgcg 601 gcgacctcaa cctggagcgc gcgctgtaca gccacgccaa cgccgtcacc aacaagtact 661 gcggcggcgg tgtgtattac cgcggcctgg tggagttctc caacatctgc cagaacgact 721 gcagctactg cggcatccgc aacaaccaga aggaggtgtg gcgctacacc atgccggtgg 781 aggaggtggt ggaggtggcc aagtgggcgc tggagaacgg catccgcaac atcatgctgc 841 agggcggcga gctcaaaacg gagcagcgcc tggcgtatct ggaggcgtgc gtgcgcgcca 901 tccgcgagga gaccacccag ctggacctgg agatgcgcgc gcgcgccgcc tccaccacca 961 cagctgaggc cgccgcctcc gcgcaggcgg acgcagaggc caagaggggc gagccggagc 1021 taggcgtggt ggtgtcgctg agtgtgggcg agctgcccat ggagcagtac gagcggctgt 1081 tcagggctgg cgcgcggcgc tacctgatcc gcatcgagac ctccaacccc gacctgtacg 1141 ctgcgctgca ccccgagccc atgagctggc acgcgcgcgt ggagtgcctg cgcaacctca
1201 agaaggccgg ctacatgctg ggcactggcg tgatggtggg gctgccgggc cagacgctgc 1261 acgacctggc gggcgacgtc atgttcttcc gcgacatcaa ggccgacatg atcggcatgg
1321 gccccttcat cacgcagccg ggcacgcccg ccaccgacaa gtggacggcg ctatacccca
1381 acgccaacaa gaacagccac atgaagtcca tgttcgacct cacaaccgcc atgaacgcgc 1441 tggtgcgaat caccatgggc aacgtcaaca tcagcgccac caccgcgctg caggccatca 1501 tccccaccgg ccgcgagatt gcgctggagc gcggcgccaa tgtggtgatg cccatcctca 1561 cgcccaccca gtaccgcgag tcctaccagc tgtacgaggg caagccctgc atcaccgaca 1621 ccgccgtgca gtgccggcgc tgcctggaca tgcgcctgca cagcgtgggc aagacctccg 1681 ccgcgggcgt gtggggcgac cccgcctcct tcctgcaccc catcgtgggc gtgcccgtgc 1741 cgcacgacct gtccagcccc gcgctggccg ccgccgcctc cgccgacttc cacgaggtgg 1801 gagccggccc ctggaacccc atccgactgg agcgattggt ggaggtgccg gaccgctacc 1861 ccgaccccga taaccatggc cgcaagaagg ccggggccgg caagggcggc aaggcccacg 1921 actcccacga cgacggcgac cacgacgacc accaccacca ccacggcgcc gcgcccgcgg 1981 gcgccgcggc tggcaagggc accggtgccg ccgcgatcgg tggcggcgcc ggcgcttcgc 2041 gccagcgcgt ggcaggcgct gcggcggcct ctgcgcggct gtgtgcgggc gcgcgccgcg 2101 ctgggcgcgt ggtggcgtcg ccgctgcggc cggcggcggc gtgccgcggc gtggcagtga 2161 aggcggcggc ggcggcggct ggcgaggatg cgggcgcggg caccagtggc gtgggcagca 2221 acattgtgac cagccccggc atcgccagca ccaccgctca cggtgtgccg cgaatcaaca 2281 tcggcgtgtt cggagtcatg aatgcgggca agtcgacgct ggtgaacgct ctggcgcagc 2341 aggaggcgtg catcgtggac tccacgcccg gcaccaccgc cgacgtcaag acggtgcttc 2401 tagagttgca cgcgctgggc ccggccaagc tgctggacac tgcggggctg gacgaggtgg 2461 gcgggctggg cgacaagaag cggcgcaagg cgctcaacac cctcaaggag tgcgacgtgg 2521 cggtgctggt cgtggacacg gacacggcgg cggcggccat caagtccggc cgcctggcgg 2581 aggcgctgga gtgggagtcc aaggtgatgg agcaggcgca caagtacaac gtcagcccag 2641 tgctgctgct caacgtcaag agccgggggc taccggaggc gcaggcagcg tccatgctgg 2701 aggcggtggc aggcatgctg gacccaagca agcagattcc ccgcatgtcg ctggacctgg 2761 ccagcacgcc gctgcacgag cgctccacca tcacctcggc cttcgtcaag gagggcgccg 2821 tgcgctccag ccgctacggc gcgccgctgc caggctgcct gccgcgctgg agcctgggcc 2881 gcaacgccag gctgctcatg gtcatcccca tggacgccga gacccccggc ggccgcctgc 2941 tgcgcccaca ggcgcaggtc atggaggagg ccatccggca ctgggccacg gtactgagcg 3001 tgcgcctgga cctggacgcg gcgcgcggca agctaggacc cgaggcgtgc gagatggagc 3061 gccagcgctt tgacggcgtc atcgcaatga tggagaggaa cgacggcccc acgctggtgg 3121 tcaccgactc gcaggctatc gacgtggtgc acccctggac tctggaccgc tcctccgggc 3181 ggccgctggt gcccatcacc accttctcca tcgccatggc ctaccagcag aacggcgggc 3241 ggctggaccc ctttgtggag gggctggagg cgctagagac gctgcaggac ggcgaccgcg 3301 tgctgatctc ggaggcgtgc aaccacaacc gcatcacctc cgcctgcaac gacatcggca 3361 tggtgcagat ccccaacaag ctggaggcgg cgctgggcgg caagaagctg cagatcgagc 3421 acgccttcgg ccgcgagttc ccggagcttg agtcgggcgg tatggacggt ctgaagctgg 3481 ccattcactg cggcggctgc atgattgacg cccagaagat gcagcagcgc atgaaggacc 3541 tgcacgaggc aggcgtgccc gtcaccaact acggcgtgtt cttctcttgg gccgcctggc 3601 ccgacgccct gcgccgcgcg ctggagccct ggggtgtcga gccgcccgta ggcactcccg 3661 ccacgcccgc cgccgcgccg gctaccgcag ccagcggcgt gtaagattgc cagcgtggta 3721 ggaccaaaag cggcttatga ttggctttgg ccccccttca tgcaactggc acgcaagctg 3781 agcgtgcttg gtgcttgaag gaaaggtaca tgctagattg ctcggtggtt gaaatgtttc 3841 aacccatgtt cagagctaca catgttcaca cggtattctt tggttcctta gttgcatgct 3901 cacgccgaat tcgctgactg cataaagcgc cgagcctaag gctggtgagc atacaagtat 3961 gatatggctg atgttggtcg ggctcctagc tttactgaac gcttgcgggc ctgtcagttt 4021 acttcgagtg ggcaaggctt gtgcacagag tgtaccatta gctgctatgg tgcttggcag 4081 ccaattgcgc aaggcaccac tcaataatgc agtacgtgtc ggacttcaga ctggtgcatg 4141 ctgctgtttg gggtgcaccc tgcagatgca cccctgccac tcatttacag tggtattgta 4201 ctgcggatgg catggccgtg cctgacgtgc tccgccaatt gctgctgatc gaaatacata 4261 ataattaata agggcacctt gcagcacgcg acactactga ggatgtgtga cgcgtgcgtg 4321 gcagtacagg cgaggacgga cgcatggcgg catgtgccct gtaattcacc cgccccg
[57] Chlamydomonas reinhardtii iron hydrogenase assembly protein (HYDEF) is encoded by the following amino acid sequence (NCBI Accession No. XP_001691465.1 and SEQ ID NO:
21):
MAHSLSAHSRQAGDRKLGAGAASSRPSCPSRIm^VAAHASASKATPDWXT)DLPPAHARAAVAAANRR ARAMASAEAAAETLGDFLGLGKGGLSPGATANLDREQVLGVLEAVWRRGDLNLERALYSHANAVTNKY
CGGGVYYRGLVEFSNicQNDCSYCGiRNNQKEVWRYTMP VΈEWEVAKWALENGIRNIMLQGGELKTEQ
RLAYLEACVRAIREETTQLDLEMRARAASTTTAEAAASAQAD AEAKRGEPELGVWSLSVGELPMEQ YER LFRAGARRYLIRIETSNPDLYAALHPEPMSWHARVECLRNLKKAGYMLGTGVMVGLPGOTLHDLAGDVM FFRDIKADMIGMGPFITQPGTPATDKWTALYPNANKNSHMKSMFDLTTAMNALVRITMGNVNISATTALQ AIIPTGREIALERGALSRVVMPILTPTQYRESYQLYEGKPCITDTAVQCRRCLDMRLHSVGKTSAAGVWGDPA SFLHPIVGVPVPHDLSSPALAAAASADFHEVGAGPWNPIRLERLVEVPDRYPDPDNHGRKKAGAGKGGKA HDSHDDGDHDDHHHHHGAAPAGAAAGKGTGAAAIGGGAGASRQRVAGAAAAVMNAGKSTLVNALAQ
QEACΓVDSTPGTTAD VKTVLLELHALGP AKLLDTAGLDEVGGLGDKKRRKALNTLKECDVAVLVVDTDT AAAAIKSGRLAEALEWESKVMEQAHKYNVSPVLLLNVKSRGLPEAQAASMLEAVAGMLDPSKQIPRMSL DLASTPLHERSTITSAFVKEGAVRSSRYGAPLPGCLPRWSLGRNARLLMVTPMDAETPGGRLLRPQAQVME EAIRHWATVLSVRLDLD AARGKLGPEACEMERQRFDGVIAMMERNDGPTLWTDSQAID WHPWTLDRS
SGRPLVPITΓFSIAMAYQQNGGRLDPFVEGLEALETLQDGDRVLISEACNHNRITSACNDIGMVQIPNKLEA ALGGKKXQIEHAFGREFPELESGGMDGLKLAIHCGGCMROAQKMQQRMKDLHEAGVPVTNYGVFFSWAA WPDALRRALEPWGVEPPVGTPATPAAAPATAASGV
[58] Chlamydomonas reinhardtii Fe-hydrogenase assembly protein (HydG) is encoded by the following mRNA sequence (NCBI Accession No. AY582740 and SEQ ID NO: 22):
1 ATGtcggtac ctctgcagtg caatgcgggg cgcctgctcg cgggccagcg gccctgcggc 61 gtccgcgccc ggctgaatcg tcgcgtttgt gtcccagtca ccgcgcacgg caaggcctct 121 gcgacccgcg aatatgctgg tgacttcctt cccggcacta ccatttcaca cgcgtggagt 181 gtcgagcgtg agacgcacca caggtaccgc aaccccgccg agtggatcaa cgaggccgct 241 attcacaagg cgctggagac ctccaaggcg gacgcccagg acgccggacg ggtgcgcgag 301 atcctggcca aggccaagga aaaggccttc gtcaccgagc atgcgcccgt caacgccgag 361 tccaagtccg agttcgtgca aggcctgacg ctggaggagt gcgctacgct catcaacgtg 421 gactcgaaca acgtcgagct gatgaatgag atcttcgaca cggccctggc catcaaggag 481 cgcatctacg ggaaccgtgt ggtgctcttc gcgccgcttt acatcgccaa tcactgcatg 541 aacacctgca cctactgcgc cttccgctcc gccaacaagg gcatggagcg ctccatcctc 601 accgacgacg acctacgcga ggaggtagcg gcgctgcagc gccagggcca ccgccgcatc 661 ctggcgctca ccggcgagca ccccaagtac acctttgaca acttcctgca cgccgtgaac 721 gtgatcgcat ctgtcaagac ggagccggag ggcagcatcc gccgcatcaa tgtggagatt 781 ccgcccctat cggtgtcgga catgcgccgc ctgaagaaca cggacagcgt gggcacgttc 841 gtgctgttcc aggagaccta ccaccgcgac accttcaagg tcatgcaccc ctccggccca 901 aagtccgact tcgacttccg cgtgctgacg caggaccggg ccatgcgcgc cggccttgac 961 gacgtgggca tcggcgccct gttcggactg tacgactacc gctacgaggt gtgcgcgatg 1021 ttgatgcaca gcgagcacct ggagcgcgag tacaacgccg gcccgcacac catcagcgtg 1081 cctcgcatgc gccctgccga cggctccgag ctgtccattg cgccgccgta cccggtcaat 1141 gacgctgact tcatgaaact ggtggcggtg ctgcgcatcg cggtgccgta caccggcatg
1201 atcctgtcca ccagggagtc gcccgagatg cgctctgcgc tgctcaagtg cggcatgagc 1261 cagatgagcg cgggcagccg cacggacgtg ggcgcctacc acaaggacca cacgctgtca
1321 accgaggcca acctgtccaa gctggcgggt cagttcacgc tgcaggacga gcgccccacc
1381 aacgagatcg tcaagtggct gatggaggag ggctacgtgc ccagctggtg cacggcctgc 1441 taccgccagg gccgcaccgg cgaggacttc atgaacatct gcaaggccgg cgacatccac 1501 gacttctgcc accccaactc gctgctcacg ctccaggagt acctgatgga ctacgccgac 1561 cccgacctgc gcaagaaggg cgagcaggtg attgcgcgcg agatgggccc cgacgcctcg 1621 gagccgctgt cggcgcagag ccgcaagcga ctggagcgca agatgaagca ggtgctggag 1681 ggcgagcacg acgtgtacct gtaa
[59] Chlamydomonas reinhardtiiFe-hydrogenase assemblyprotein (HydG) is encodedbythe following amino acid sequence (NCBIAccessionNo. AAS92602.1 and SEQ ID NO: 23):
MSVPLQCNAGRLLAGQRPCGVRARLNRRVCVPVTAHGKASATREYAGDFLPGTTISHAWSVERETHHRY RNPAEWINEAAfflKALETSKADAQDAGRVREILAKAKEKAFVTEHAPVNAESKSEFVQGLTLEECATLINV DSNNVELMNEIFDTALAIKERIYGNRVVLFAPLYIANHCMNTCTYCAFRSANKGMERSILTDDDLREEVAA LQRQGHRRILALTGEHPKYTFDNFLHAVNVIASλ^TEPEGSIRRINVEIPPLSVSDMRRLKNTDSVGTFVLFQ ETYHRDTFKVMHPSGPKSDFDFRVLTQDRAMRAGLDDVGIGALFGLYDYRYEVCAMLMHSEHLEREYNA GPHTISVPRMRPADGSELSIAPPYPVNDADFMKLVAVLRIAVPYTGMILSTRESPEMRSALLKCGMSQMSA GSRTDVGAYHKDHTLSTEANLSKLAGQFTLQDERPTNEIVKWLMEEGYVPSWCTACYRQGRTGEDFMNI CKAGDIHDFCHPNSLLTLQEYLMDYADPDLRKKGEQVIAREMGPDASEPLSAQSRKRLERKMKQVLEGEH DVYL
[60] Chlamydomonas reinhardtii Fe-hydrogenase assembly protein (HydEF) is encoded by the following mRNA sequence (NCBI Accession No. AY582739 and SEQ ID NO: 24): i ATGgcgcaca gcctcagcgc acacagccgt caggctggtg acagaaagct tggcgcgggc
61 gcggcctcgt cgcgcccgag ctgtccctcg cgccgcattg tgcgcgtcgc cgcgcatgcg
121 agcgcctcca aggccacgcc cgacgtcccg gtcgatgacc tgcctcccgc tcatgcccgc
181 gctgccgtcg cggccgccaa ccgccgcgct cgtgccatgg cttcggctga ggccgccgcg
241 gagaccctgg gtgacttcct ggggctcggc aagggcgggc tttcgcccgg ggccaccgcc
301 aacctggaca gggaacaggt actgggtgtg ctggaggcgg tgtggcgccg cggcgacctc
361 aacctggagc gcgcgctgta cagccacgcc aacgccgtca ccaacaagta ctgcggcggc
421 ggtgtgtatt accgcggcct ggtggagttc tccaacatct gccagaacga ctgcagctac
481 tgcggcatcc gcaacaacca gaaggaggtg tggcgctaca ccatgccggt ggaggaggtg
541 gtggaggtgg ccaagtgggc gctggagaac ggcatccgca acatcatgct gcagggcggc
601 gagctcaaaa cggagcagcg cctggcgtat ctggaggcgt gcgtgcgcgc catccgcgag
661 gagaccaccc agctggacct ggagatgcgc gcgcgcgccg cctccaccac cacagctgag
721 gccgccgcct ccgcgcaggc ggacgcagag gccaagaggg gcgagccgga gctaggcgtg
781 gtggtgtcgc tgagtgtggg cgagctgccc atggagcagt acgagcggct gttcagggct
841 ggcgcgcggc gctacctgat ccgcatcgag acctccaacc ccgacctgta cgctgcgctg
901 caccccgagc ccatgagctg gcacgcgcgc gtggagtgcc tgcgcaacct caagaaggcc
961 ggctacatgc tgggcactgg cgtgatggtg gggctgccgg gccagacgct gcacgacctg
1021 gcgggcgacg tcatgttctt ccgcgacatc aaggccgaca tgatcggcat gggccccttc
1081 atcacgcagc cgggcacgcc cgccaccgac aagtggacgg cgctataccc caacgccaac
1141 aagaacagcc acatgaagtc catgttcgac ctcacaaccg ccatgaacgc gctggtgcga
1201 atcaccatgg gcaacgtcaa catcagcgcc accaccgcgc tgcaggccat catccccacc
1261 ggccgcgaga ttgcgctgga gcgcggcgcc aatgtggtga tgcccatcct cacgcccacc
1321 cagtaccgcg agtcctacca gctgtacgag ggcaagccct gcatcaccga caccgccgtg
1381 cagtgccggc gctgcctgga catgcgcctg cacagcgtgg gcaagacctc cgccgcgggc
1441 gtgtggggcg accccgcctc cttcctgcac cccatcgtgg gcgtgcccgt gccgcacgac
1501 ctgtccagcc ccgcgctggc cgccgccgcc tccgccgact tccacgaggt gggagccggc
1561 ccctggaacc ccatccgact ggagcgattg gtggaggtgc cggaccgcta ccccgacccc
1621 gataaccatg gccgcaagaa ggccggggcc ggcaagggcg gcaaggccca cgactcccac
1681 gacgacggcg accacgacga ccaccaccac caccacggcg ccgcgcccgc gggcgccgcg
1741 gctggcaagg gcaccggtgc cgccgcgatc ggtggcggcg ccggcgcttc gcgccagcgc
1801 gtggcaggcg ctgcggcggc ctctgcgcgg ctgtgtgcgg gcgcgcgccg cgctgggcgc
1861 gtggtggcgt cgccgctgcg gccggcggcg gcgtgccgcg gcgtggcagt gaaggcggcg
1921 gcggcggcgg ctggcgagga tgcgggcgcg ggcaccagtg gcgtgggcag caacattgtg
1981 accagccccg gcatcgccag caccaccgct cacggtgtgc cgcgaatcaa catcggcgtg
2041 ttcggagtca tgaatgcggg caagtcgacg ctggtgaacg ctctggcgca gcaggaggcg
2101 tgcatcgtgg actccacgcc cggcaccacc gccgacgtca agacggtgct tctagagttg
2161 cacgcgctgg gcccggccaa gctgctggac actgcggggc tggacgaggt gggcgggctg
2221 ggcgacaaga agcggcgcaa ggcgctcaac accctcaagg agtgcgacgt ggcggtgctg
2281 gtcgtggaca cggacacggc ggcggcggcc atcaagtccg gccgcctggc ggaggcgctg
2341 gagtgggagt ccaaggtgat ggagcaggcg cacaagtaca acgtcagccc agtgctgctg
2401 ctcaacgtca agagccgggg gctaccggag gcgcaggcag cgtccatgct ggaggcggtg
2461 gcaggcatgc tggacccaag caagcagatt ccccgcatgt cgctggacct ggccagcacg
2521 ccgctgcacg agcgctccac catcacctcg gccttcgtca aggagggcgc cgtgcgctcc
2581 agccgctacg gcgcgccgct gccaggctgc ctgccgcgct ggagcctggg ccgcaacgcc
2641 aggctgctca tggtcatccc catggacgcc gagacccccg gcggccgcct gctgcgccca
2701 caggcgcagg tcatggagga ggccatccgg cactgggcca cggtactgag cgtgcgcctg
2761 gacctggacg cggcgcgcgg caagctagga cccgaggcgt gcgagatgga gcgccagcgc
2821 tttgacggcg tcatcgcaat gatggagagg aacgacggcc ccacgctggt ggtcaccgac
2881 tcgcaggcta tcgacgtggt gcacccctgg actctggacc gctcctccgg gcggccgctg
2941 gtgcccatca ccaccttctc catcgccatg gcctaccagc agaacggcgg gcggctggac 3001 ccctttgtgg aggggctgga ggcgctagag acgctgcagg acggcgaccg cgtgctgatc 3061 tcggaggcgt gcaaccacaa ccgcatcacc tccgcctgca acgacatcgg catggtgcag 3121 atccccaaca agctggaggc ggcgctgggc ggcaagaagc tgcagatcga gcacgccttc 3181 ggccgcgagt tcccggagct tgagtcgggc ggtatggacg gtctgaagct ggccattcac 3241 tgcggcggct gcatgattga cgcccagaag atgcagcagc gcatgaagga cctgcacgag 3301 gcaggcgtgc ccgtcaccaa ctacggcgtg ttcttctctt gggccgcctg gcccgacgcc 3361 ctgcgccgcg cgctggagcc ctggggtgtc gagccgcccg taggcactcc cgccacgccc 3421 gccgccgcgc cggctaccgc agccagcggc gtgtaa [61] Chlamydomonas reinhardtii Fe-hydrogenase assembly protein (HydEF) is encoded by the following amino acid sequence (NCBI Accession No. AAS92601.1 and SEQ ID NO: 25):
MAHSLSAHSRQAGDRKLGAGAASSRPSCPSRRΓVRVAAHASASKATPDVP VDDLPPAHARAAVAAANRR ARAMASAEAAAETLGDFLGLGKGGLSPGATANLDREQVLGVLEAVWRRGDLNLERALYSHANAVTNKY CGGGVYYRGLVEFSMCQNDCSYCGIRNNQKEVWRYTMPVEEX^EVAKWALENGIRNIMLQGGELKTEQ RLAYLEACVRAIREETTQLDLEMRARAASTTTAEAAASAQAD AEAKRGEPELGVWSLSVGELPMEQ YER LFRAGAΛRYLIRIETSNPDLYAALHPEPMSWHARVΈCLRNLKKAGYMLGTGVMVGLPGQTLHDLAGDVM FFRDIKADMIGMGPFITQPGTPATDKWTALYPNANK^SHMKSMFDLTTAMNALVRITMGNVNISATTALQ ALFFTGREIALERGANVVMPILTPTQYRESYQLYEGKPCITDTAVQCRRCLDMRLHSVGKTSAAGVWGDPA SFLHPIVGVPVPHDLSSPALAAAASADFHEVGAGPWNPIRLERLVEVPDRYPDPDNHGRKKAGAGKGGKA HDSHDDGDHDDHHHHHGAAP AGAAAGKGTGAAAIGGGAGASRQRVAGAAAASARLCAGARRAGRVVA SPLRP AAACRGVAVKAAAAAAGEDAGAGTSGVGSNIVTSPGIASTTAHGVPRINIGVFGVMNAGKSTLVN ALAQQEACIΛΩSTPGTTADVKTVLLELHALGPAKLLDTAGLDEVGGLGDKKRRKALNTLKECDVAVLVV DTDTAAAAIKSGRLAEALEWESKVMEQAHKYNVSPVLLLNVKSRGLPEAQAASMLEAVAGMLDPSKQIP RMSLDLASTPLHERSTITSAFVKEGAVRSSRYGAPLPGCLPRWSLGRNARLLMVIPMDAETPGGRLLRPQA QVMEEAIRHWATΛΑSVRLDLDAARGKLGPEACEMERQRFDGΛΗDYVIMERNDGPTLVVTDSQAIDVVHPWT LDRSSGRPLΛΦITTFSIAMAYQQNGGRLDPFVEGLEALETLQDGDRVLISEAΑ^JHNRITSACNDIGMVQIPN KLEAALGGKKLQIEHAFGREFPELESGGMDGLKLAFFLCGGCMROAQKMQQRMKDLHEAGVPVTNYGVFF SWAAWPDALRRALEPWGVEPPVGTPATPAAAPATAASGV Ferredoxins
[62] Ferredoxins are iron-sulfur proteins that mediate electron transfer in a range of metabolic reactions. Ferredoxins are small proteins containing iron and sulfur atoms organized as iron- sulfur clusters. These proteins act like capacitors because they can accept or discharge electrons. As a result of this ability, the oxidation states of the iron atoms are changed (usually, by +2 or +3). As such, ferredoxin acts as an electron transfer agent in biological redox reactions.
Ferredoxins are classified according to the nature of their iron-sulfur clusters and by sequence similarity.
[63] One group of ferredoxins, originally found in chloroplast membranes, has been termed
"chloroplast-type" or "plant-type". The active center is a [Fe2S2] cluster, where the iron atoms are tetrahedrally coordinated both by inorganic sulfur atoms and by sulfurs provided by four conserved cysteine (Cys) residues. In chloroplasts, Fe2S2 ferredoxins function as electron carriers in the photosynthetic electron transport chain and as electron donors to various cellular proteins, such as glutamatε synthase, nitrate reductase and sulfite reductase. [64] The [Fe4S4] ferredoxins are subdivided into low-potential (bacterial-type) and high- potential (HiPIP) ferredoxins. A group OfFe4S4 ferredoxins, originally found in bacteria, has been termed "bacterial-type". Bacterial-type ferredoxins are further subdivided into groups, based on their sequence properties. Most contain at least one conserved domain, including four cysteine residues that bind to a [Fe4S4] cluster, hi Pyrococcus fuήosus Fe4S4 ferredoxin, one of the conserved Cys residues is substituted with aspartic acid.
[65] During the evolution of bacterial-type ferredoxins, intrasequence gene duplication, transposition and fusion events occurred, resulting in the appearance of proteins with multiple iron-sulfur centers, hi some bacterial ferredoxins, one of the duplicated domains has lost one or more of the four conserved Cys residues. These domains have either lost their iron-sulfur binding property, or bind to a [Fe3S4] cluster instead of a [Fe4S4] cluster.
[66] 3-D structures are known for a number of monocluster and dicluster bacterial-type ferredoxins. The fold belongs to the α+β class, with 2-7 α-helices and four β-strands forming a barrel-like structure, and an extruded loop containing three "proximal" Cys ligands of the iron- sulfur cluster.
[67] High potential iron-sulfur proteins (HiPIPs) form a unique family OfFe4S4 ferredoxins that function in anaerobic electron transport chains. Several HiPIPs have so far been characterized structurally, their folds belonging to the α+β class. As in other bacterial ferredoxins, the [Fe4S4] cluster adopts a cubane-like conformation and is ligated to the protein via four Cys residues.
[68] Compositions and methods of the invention encompass polynucleotide and polypeptide sequences that encode for exogenous ferredoxin enzymes. For example, the invention provides methods of introducing a ferredoxin gene, or any portion thereof, isolated from Spinacia oleracea into a cell from E.coli. [69] Ferredoxin gene sequences of all species are encompassed by the invention. Ferredoxin genes of photosynthetic plants and bacteria are preferred. In a particularly preferred embodiment, ferredoxin I of Spinacia olearcea is used.
[70] Ferredoxin sequences, or any portion thereof, having 50%, 60%, 70%, 80%, 90%, or 100% identity to the sequences provided below, or any point in between, are encompassed by the invention. [71] Ferredoxin I (Spinach chloroplast ferredoxin, unprocessed form), is encodedbythe following amino acid sequence (NCBI AccessionNo.1704156A and SEQ ID NO: 26):
MAATTTTMMG]VL^TTFVPKPQAPPMMAALPSNTGRSLFGLKTGSRGGRMTMAAYKVTLVTPTGNVEFQC PDDVYILDAAEEEGEDLPYSCRAGSCSSCAGKLKTGSLNQDDQSFLDDDQIDEGWVLTCAAYPVSDVTIET HKEEELTA
[72] Spinacia oleracea Ferredoxin I is encoded bythe following mRNA sequence (NCBI AccessionNo. M35660 and SEQ ID NO: 27):
1 ttacaaaatt gtaaaaATGg cagcaaccac cacaacaatg atgggcatgg ccaccacctt 61 tgtcccaaaa ccccaagcac caccaatgat ggcggcgctt ccatccaaca ccggccgctc
121 tttgttcgga ctcaagaccg gtagccgtgg cggaaggatg acaatggctg cctacaaggt
181 aaccttggta acacccaccg gtaacgtaga gtttcaatgc ccagacgatg tttacatctt
241 ggatgctgct gaagaagaag gcattgactt gccttactca tgcagagctg ggtcgtgctc
301 ttcatgcgcc ggaaagctta agacaggtag tcttaaccaa gatgatcaga gttttttgga 361 tgacgatcag atcgatgaag gatgggttct tacctgtgct gcttaccctg ttagtgatgt
421 tactattgag acccacaagg aagaggagct tactgcctaa ttcatttttt tttatttttt
481 attattttgt tctcatttga gagggaattg aaagactaaa aaagagtaaa tgcagcgagg
541 agtttttact tcttcgatct gtggtttgta attgtgtatt atcatgttaa tcaattgctc
601 ttataagctt aattactacg taatatatat at
[73] Spinacia oleracea Ferredoxin I is encoded bythe following amino acid sequence (NCBI
AccessionNo. AAA34028.1 and SEQ ID NO: 28):
MAATTTTMMGMATTFVPKPQAPPMMAALPSNTGRSLFGLKTGSRGGRMTMAAYKVTLVTPTGNVEFQC PDDVYILDAAEEEGIDLPYSCRAGSCSSCAGKLKTGSLNQDDQSFLDDDQE)EGWVLTCAAYPVSDVTIET HKEEELTA
[74] Spinacia oleracea FerredoxinNADP+ oxidoreductase precursor (FNR) is encoded bythe following mRNA sequence (NCBI AccessionNo. X07981 and SEQ ID NO: 29):
1 ttttttgcat aaacttatct tcatagttgc cactccaatt tgctccttga atctcctcca 61 cccaatacat aatccactcc tccatcaccc acttcactac taaatcaaac ttaactctgt
121 ttttctctct cctcctttca tttcttattc ttccaatcat cgtactccgc cATGaccacc
181 gctgtcaccg ccgctgtttc tttcccctct accaaaacca cctctctctc cgcccgaagc
241 tcctccgtca tttcccctga caaaatcagc tacaaaaagg ttcctttgta ctacaggaat
301 gtatctgcaa ctgggaaaat gggacccatc agggcccaga tcgcctctga tgtggaggca 361 cctccacctg ctcctgctaa ggtagagaaa cattcaaaga aaatggagga aggcattaca
421 gtgaacaagt ttaagcctaa gaccccttac gttggaagat gtcttcttaa caccaaaatt
481 actggggatg atgcacccgg agagacctgg cacatggttt tttcccatga aggagagatc
541 ccttacagag aagggcaatc cgttggggtt attccagatg gggaagacaa gaatggaaag
601 ccccataagt tgagattgta ctcgatcgcc agcagtgctc ttggtgattt tggtgatgct 661 aaatctgttt cgttgtgtgt aaaacgactc atctacacca atgacgctgg agagacgatc
721 aagggagtct gctccaactt cttgtgtgac ttgaaacccg gtgctgaagt gaagttaaca
781 ggaccagttg gaaaggagat gctcatgccc aaagacccta acgcgacaat tatcatgctt
841 ggaactggaa ctgggattgc tcctttccgt tcattcttgt ggaagatgtt cttcgaaaag
901 catgatgatt acaagtttaa cggcttggct tggcttttct tgggtgtacc cacaagcagt 961 tctcttctct acaaagagga atttgagaag atgaaggaaa aggctccaga caacttcagg
1021 ctggattttg cagtgagcag agagcaaact aacgagaaag gggagaagat gtacattcaa
1081 acccgaatgg cacaatacgc agttgagcta tgggaaatgt tgaagaaaga taatacttat
1141 ttctacatgt gtggtctcaa gggaatggaa aagggaattg acgacattat ggtttcattg
1201 gctgctgcag aaggcattga ttggattgaa tacaagaggc agttgaagaa ggcagaacaa 1261 tggaacgttg aagtctacta ataactcttg tacaaagatc tcttattcct ttttgtgaag 1321 catcttctat atttatctat tgccattatt atctcactgc accttgtaga taggtgggta 1381 agtttttttc cccatattgt aatgacgata agccaattgc attttagcac catagtataa 1441 agctcttgtt ggaagactac tttttcagct aatatataca tggttgacag tc
[75] Spinacia oleracea Ferredoxin NADP+ oxidoreductase precursor (FNR) is encoded by the following amino acid sequence (NCBI Accession No. CAA30791.1 and SEQ ID NO: 30):
MTTAVTAAVSFPSTKTTSLSARSSSVISPDKISYKKVPLYYRNVSATGKMGPIRAQIASDVEAPPPAPAKVEKHSKK MEEGITVNKFKPKTPYVGRCLLNTKITGDDAPGETWHMVFSHEGEIPYREGQSVGVIPDGEDKNGKPHKLRLYSIAS FRSFLWKMFFEKHDDYKFNGLAWLFLGVPTSSSLLYKEEFEKMKEKAPDNFRLDFAVSREQTNEKGEKMYIQTRMAQ YAVELWEMLKKDNTYFYMCGLKGMEKGIDDIMVSLAAAEGIDWIEYKRQLKKAEQWNVEVY
[76] Spinacia oleracea Ferredoxinis encodedbythe followingmRNA sequence (NCBI AccessionNo. X64351 and SEQ ID NO: 31): 1 atttgcagta atgtttgatg tttgatttga gtgatgaatg ttgctggaga tatcaagtac
Sl gttactggat gaagctggag atatcaagaa cttagggtcc ttttcgatct ccttaccaga
121 ccgcctatca atcttgctca gaagctcaga aaacttaaca acattgtgag aggtgtggca
181 gtccagcact ggggtgtaac cattttcgat ctgtgcaggg tggttcatga ttatgacttg
241 ggcaataaag ttagctgcct ccttagcagg gtcattcttg gagtgagatg ccatgtattc 301 acgcttgatt tccttgacag aaacattctt tacattgaaa ccaacattgt caccagggag
361 agcctctgga agggactcat ggtgcatctc aattctcaac tgattttact tcagcagtca
421 gtccagtagg accgaaggta acaagcatac caggcttcaa cacaccagtc acgtcctaca
481 agcacggttc caatacctcg aaggctccaa tctttaacct taattttttt aatatgaact
541 ttgtgaacat atttgtatct aattttatat ttattggttg taatacaatt taaagttttt 601 atataatccc gcgtgcatat aagactaact ttatataaac aaaatctgat atcacgttgg
661 aattaggcat atagatacaa agattaggat ggaagcttgg gatcacgatt aatgaatatc
721 tactggtttt aaaaaaaaaa tcaaagagat gacaagaaca attaaccaca aaaaccattg
781 aaggccaata aaaagggacc catgaatgaa aaccttagtc taacttacac tataaaacac
841 atgaaaaaaa aaaaaaaaaa aaaaaagtga tggcagattt tttttgcata aacttatctt 901 catagttgcc actccaattt gctccttgaa tctcctccac ccaatacata atccactcct
961 ccatcaccca cttcactact aaatcaaact taactctgtt tttctctctc ctcctttcat
1021 ttcttattct tccaatcatc gtactccgcc ATGaccaccg ctgtcaccgc cgctgtttct
1081 ttcccctcta ccaaaaccac ctctctctcc gcccgaagct cctccgtcat ttcccctgac
1141 aaaatcagct acaaaaaggt gattcccaat ttcactgtgt tttttattaa taatttgtta 1201 ttttgatgat gagatgatta atttgggtgc tgcaggttcc tttgtactac aggaatgtat
1261 ctgcaactgg gaaaatggga cccatcaggg cccagatcgc ctctgatgtg gaggcacctc
1321 cacctgctcc tgctaaggta gagaaacatt caaagaaaat ggaggaaggc attacagtga
1381 acaagtttaa gcctaagacc ccttacgttg gaagatgtct tcttaacacc aaaattactg
1441 gggatgatgc acccggagag acctggcaca tggttttttc ccatgaaggt aatttctata 1501 ttactatccg gccacacttt gatcatatga ttaagcactt tcgagtaaga taattattta
1561 agaattc
[77] Spinacia oleracea Ferredoxin is encoded by the following amino acid sequence (NCBI Accession No. CAA45703.1 and SEQ ID NO: 32): MTTAVTAAVSFPSTKTTSLSARSSSVISPDKISYKKWLYΎRISΓVSATGKMGPIRAQIASDVEAPPPAPAKVEK HSKKMEEGiTVNKFKPKTP YVGRCLLNTKITGDDAPGETWΉMVFSHE
[78] Spinacia oleracea Ferredoxin is encoded by the following mRNA sequence (NCBI Accession No. X74881 and SEQ ID NO: 33): 1 gaattcggca cgagatcaat tttacaacaa aaattaatat accATGaaag ctcttcaagc
61 ttcaatagct tacagcttcc ccatttcttc tcctgcagct tctcccagac gtttctcccg
121 tgtaattcgt gctcaagcgg atccttctga caaatctatg gaagtaatga ggaaattttc
181 tgagcagttc tgtcgtaagt cagatacata cttttgtgtt gataaaagtg tcactgccgt 241 tgttatcaag ggattagcag atcacaggga cacattaggt gcaccccttt gcccatgtcg
301 gcattatgat gacaaagaag ctgaagcaaa acagggtttt tggaattgcc catgtgtgcc
361 aatgagggag aggaaggagt gccactgtat gctgtttctg acccctgaca atgattttgc
421 tggcaaggag cagactatca cattggatga aattcgagaa gttacatcaa atatgtaaaa
481 ttgtgatctg aagcctctta tgcgtagtaa cagtaggcct gtacattacg tactatcacg 541 aggatgccaa taactgttgt actgttatat gttgtatata ctcttattct ggagagcttt
601 gtcatctcta actcggcttt atcaaggtat atagccatac tcgtatctga gtggtgtatt
661 tggttttatg atactccgta tcatataata ctgagtgtta aagaaagaaa aggtcaagaa
721 tgaagtacta caaaaaaa [79] Spinacia oleracea Ferredoxin is encoded by the following amino acid sequence (NCBI Accession No. CAA52867.1 and SEQ ID NO: 34):
MKALQASIAYSFPISSPAASPRRFSRVIRAQADPSDKSMEV^ DHRDTLGAPLCPCRHYDDKEAEAKQGFWNCPCVPMRERKECHCMLFLTPDNDFAGKEQTITLDEIREVTS
NM
Sulfite Reductase
[80] Engineerred E. coli of the invention contain at least one mutation in a sulfite reductase gene. Moreover, engineerred E. coli of the invention contain at least one exogenous sulfite reductase gene. In combination with other exogenous genes and their products, e.g. hydrogenase, hydrogenase maturation factor(s), and ferredoxin, the following reaction is performed:
H2SO4 + 3H2 → H2S + 3H2O
[81] The product of this reaction, H2S, is used by the E. coli to generate cysteine and methionine, which are essential for cell growth and survival. Thus, if any component of the artificial pathway is non-functional, the reaction will not proceed, H2S will not be produced, and therefore, sufficient levels of cyteine will not be generated by the engineered E. coli.
Consequently, the failure of any component of this artifical pathway to function leads to the inability of the E. coli to survive. As such, E. coli survival under selective pressure is an indicator that the exogenous hydrogenase is functional and possess at least one desired biological property. [82] Compositions and methods of the invention encompass polynucleotide and polypeptide sequences that encode for a sulfite reductase enzyme. Specifically, compositions and methods of the invention encompass polynucleotide and polypeptide sequences that include a mutation. This mutation is any insertion, deletion, inversion, translocation, transposition, excision, truncation, frameshift, or point mutation in a polynucleotide or polypeptide sequence encoding for a sulfite reductase enzyme that causes theresulting geneproductto be non-functional. Furthermore, the mutation is also anyRNA orDNAmediated silencingtechnologythatprevents transcription of the gene ortranslation ofthe mRNA into apolypeptide. Mutations ofthe invention occur at any pointwithinthe sequence encoding forthe alpha subunit, beta component, orbothregions ofthe sulfitereductase enzyme. Exemplarysequences areprovidedbelow.
[83] Moreover, compositions andmethods ofthe invention encompass polynucleotide and polypeptide sequences that encode forexogenous sulfitereductase enzymes. For example, the inventionprovides methods ofintroducing a sulfitereductase gene, or anyportion thereof, isolated fromZea mays into acell fromE.coli. [84] Contemplated sequences include sulfite reductase genes, or anyportionthereof, from any species ofbacteria orphotosynthetic organism. Specifically, sequenceshaving 50%, 60%, 70%, 80%, 90%, or 100% identityto the sequences providedbelow, oranypoint inbetween, are encompassedbythe invention. [85] E. coli sulfitereductase is encodedbythe followingmRNA sequence (NCBI Accession No. NZ_AAJV02000023, REGION: 73896..75608 and SEQ ID NO: 35) (reverse complement shown):
1 ATGagcgaaa aacatccagg gcctttagtg gtcgaaggaa aactgacaga cgccgagcgc
61 atgaagcttg aaagcaacta cctgcgcggc accattgcgg aagatttaaa cgacggtctg
121 accggcggct ttaagggcga taacttcctg ctgatccgct tccacggcat gtatcagcag 181 gatgaccgcg acatccgcgc cgaacgtgct gaacagaagc tggagccgcg ccacgcgatg
241 ctgcttcgct gtcgtctgcc gggtggggtg attaccacta aacagtggca ggcgatcgac
301 aaatttgccg gtgaaaacac catctatggc agcattcgcc tgaccaaccg ccagacgttt
361 cagttccacg gcattctgaa aaagaacgtc aaaccggtgc accagatgct gcactcggtc
421 ggtcttgatg cgctggcgac agctaacgac atgaaccgta acgtactctg cacctcgaac 481 ccttacgagt cgcagctgca cgcggaagcg tacgagtggg cgaagaagat ttctgagcat
541 ctgttgcctc gtacccgcgc gtatgcggag atctggctcg accaggaaaa agtcgccact
601 actgatgaag aaccgatcct tggtcagacc tacctgccgc gtaagttcaa aaccacggta
661 gtgatcccgc cgcagaacga tatcgatctg catgccaacg acatgaactt cgtggcaatc
721 gccgaaaacg gcaagctggt aggcttcaac ctgctggtgg gcggtgggct ttctatcgaa 781 cacggcaaca agaaaaccta cgcccgcacg gcgagcgagt ttggctatct gccgctggag
841 catacgctgg cggtggcgga agccgtcgtg acaactcagc gtgactgggg taaccgaacc
901 gatcgtaaaa atgccaaaac caaatacacg ctggagcgcg tgggggttga gacgtttaaa
961 gcggaagtgg agcgtcgcgc ggggatcaaa tttgaaccga tccgtccata tgagttcacc
1021 ggacgaggcg atcgtattgg ctgggttaag ggcattgatg ataactggca cctgacgctg 1081 tttatcgaaa atggtcgcat ccttgattat ccggggcgtc cgctgaaaac cggcctgctg
1141 gagatcgcga agatccacaa aggcgatttc cgcattacgg cgaaccagaa tctgatcatc
1201 gccggtgtac cggaaagcga gaaagcgaag atcgagaaga tcgccaaaga gagcggttta
1261 atgaatgccg tcacgccgca gcgtgaaaac tcgatggcct gcgtgtcatt cccgacttgc
1321 ccgctggcga tggcggaagc agagcgtttc ctgccgtctt ttatcgacaa catcgataat 1381 ttaatggcga aacatggtgt cagcgatgag catatcgtga tgcgtgtaac aggctgcccg
1441 aacggttgtg gtcgcgcgat gctggcggaa gtgggcctgg tgggtaaagc accgggtcgc
1501 tacaatctgc atcttggcgg caaccgcatt gggacacgta tcccacggat gtataaagaa
1561 aacatcaccg agccggaaat cctggcgtcg cttgatgaac tgatagggcg ctgggcgaaa
1621 gagcgcgaag cgggtgaagg cttcggcgac tttacggtgc gtgcgggcat cattcgcccg 1681 gtgctcgatc cggcgcgcga tttgtgggat taa
[86] E. coli sulfite reductase, alpha subunit, cysJ, is encoded by the following amino acid sequence (NCBI Accession No. ZP_03046139.1 and SEQ ID NO: 36): MTTQVPPSALLPLNPEQLARLQAATTDLTPTQLAWVSGYFWGVLNQQPAALAATPAPAAEMPGITIISASQ TGNAJ^VAEALRDDLLAAKINVKL\ΠSΓAGDYKFKQ]A.SEKLLIVYTSTQGEGEPPEEAVALHKFLFSKKAP KLENTAFAVFSLGDSSYEFFCQSGKDFDSKLAELGGERLLDRVD ADVEYQAAASEWRARWD ALKSRAPV AAPSQSVATGAVNEIHSSPYSKDAPLVASLSVNQKITGRNSEKDVRHIEIDLGDSGLRYQPGDALGVWYQN DPALVKELVELLWLKGDEPVTVEGKTLPLNEALQWHFELTVNTANIVENYATLTRSETLLPLVGDKAKLQ HYAATTPIVDMVRFSPAQLD AEALINLLRPLTPRLYSIASSQAEVENEVHVTVGWRYD VEGRARAGGASS FLADRVEEEGEVRVFIEHNDNFRLP ANPETP VIMIGPGTGIAPFRAFMQQRAADEAPGKNWLFFGNPHFTED FLYQVEWQRYVKDGVLTRroLAWSRDQKEKVYVQDKLREQGAELWRWINDGAHrrVCGDANRMAKDV EQALLEVIAEFGGMDTEAADEFLSELRVERRYQRDVY [87] E. coli sulfite reductase, beta component, cysl, is encoded by the following amino acid sequence (NCBI Accession No. ZP_03046100.1 and SEQ ID NO: 37):
MSEKHPGPLWEGKLTDAERMKLESNYLRGTIAEDLNDGLTGGFKGDNFLLIRFHGMYQQDDRDIRAERA EQKLEPRHAMLLRCRLPGGλαTTKQWQAroKFAGENTIYGSIRLTNRQTFQFHGILKICm^KP VHQMLHSVG LDALATANDMNRNVLCTSNP YESQLHAEAYEWAKKISEHLLPRTRAYAEIWLDQEKVATTDEEPILGQTY LPRKFKTTVVIPPQNDIDLHANDMNFVAIAENGKLVGFNLLVGGGLSIEHGNKKTYARTASEFGYLPLEHT LAVAEAVVTTQRDWGNRTDRKNAKTKYTLERVGVETFKAEVERRAGIKFEPIRPYEFTGRGDRIGWVKGI DDNWHLTLFIENGWLDYPGRPLKTGLLEIAKIHKGDFRITANQNLIIAGVPESEKAKIEKIAKESGLMNAVT PQRENSMACVSFPTCPLAMAEAERFLPSFIDNIDNLMAKHGVSDEHIVMRVTGCPNGCGRAMLAEVGLVG KAPGRYNLHLGGNWGTRIPRMYKENITEPEILASLDELIGRWAKEREAGEGFGDFTVRAGIIRPVLDPARDL WD
[88] ZeaMays ferredoxin-sulfite reductaseprecursor(sir), is encodedbythe following mRNA sequence (NCBIAccessionNo. NM_001111832 and SEQ ID NO: 38):
1 tgcgaacgag caaaggacaa atatcctcct cgctcgcagt cgcagctagt acgccgccgc 61 acctccgccc gccaccttct tcccggctcc agcgATGtcg ggggcgattg ggggcgccga
121 ggtccacggc ttccggggcg cagcggcgca gctcccgcgg tcgcgggtgc tcgggaggcc
181 gatccgggtg gcgccacccg ccgcggcccg gccgggcggc gcgtccgcgg gtagcatacg
241 cgccgtctcc gcgcctgcga agaaggatgc ttctgaagtt aagcgaagca aggttgagat
301 aatcaaggaa aagagcaact tcctccggta ccctttgaac gaggagttgg tctcagaggc 361 cccaaatatc aacgagagtg ctgttcagct gatcaagttt catggaagct accagcaaac
421 tgaccgagat gtccgtgggc agaagaatta ctcgtttatg ctccggacaa agaacccttg
481 tgggaaagtt ccaaaccaac tttatttagc tatggataca ctagccgatg agtttggcat
541 cggaacactc cgcctgacga ccaggcagac attccagttg cacggtgttc ttaagaagaa
601 cttgaagact gttctaagca ctgtcataaa gaatatgggc tcaacattgg gtgcttgtgg 661 cgatctcaac aggaatgtac ttgctcctgc agcgccttat gtcaaaaaag atatcctttt
721 tgctcaacaa acagcagaga acattgcagc acttcttaca ccacagtccg gggcttatta
781 tgacctgtgg gtggatggcg agaagataat gtcagctgaa gagcctcctg aggtgacaaa
841 agcccgcaat gacaactcgc atggaacgaa cttccctgac tctccggaac caatctatgg
901 cacccagtat ctaccaagga agttcaaggt tgcggttacc gcggctggtg ataactctgt 961 tgatattctg accaatgaca tcggtgttgt tgttgtttca gatgatgcag gagaacctat
1021 tggctttaac atctatgttg gtggtggcat gggaaggaca caccgagtgg aaactacatt
1081 ccctcggctg gctgatccat tgggttatgt tccaaaagaa gatatattat atgctataaa
1141 ggccattgtc gtcacacaga gggaaaatgg aagaagggat gaccgcaagt atagtaggat
1201 gaagtatatg attgaccgtt ggggaataga taggtttcgg gctgaagttg aaaaatatta 1261 cgggaagaag tttgaaagtt tccgaccatt gccagagtgg cagtttaaca gctaccttgg 1321 ctggcaagaa cagggtgatg ggaaattatt ctatggagtg catgttgata atggtcgtgt 1381 tggtgggcaa gcaaagaaaa ctctacgaga gataattgag aagtataatt tggatgttag 1441 tattacccca aaccaaaatc ttattttatg tgggattgat caagcatgga gagaacccat 1501 aactacagct cttgcacaag ctggcctgct ggaaccgaag gatgtcgacc ccttgaattt 1561 aactgccatg gcatgccctg ccttgccact gtgccctttg gcacaaacag aagctgaacg 1621 ggggatctta cccattctta aacgaattag agcagtcttc aataaggttg gtatcaagga 1681 ttcggagtct gtggttgtga ggataactgg atgccctaat ggatgcgcta gaccatatat 1741 ggcagagctt ggtttcgttg gtgatggccc aaaaagttac cagatctggc tgggtggaac 1801 accaaaccag agtacgctag cagaatcatt tatggacaag gtgaagcttg atgacatcga 1861 gaaggttttg gagcctctct ttacctattg gaatggcaca cgccaggaag gcgaatcttt 1921 tggaagcttc acaaaccgaa caggattcga caaattgaaa gaggtagtga ataagtgggc 1981 agagtcaccg tcagccgcat gaagatttcg ctttcgttgg ataaaatccc aggctcgcag 2041 cacaattttg gccacgggat taattatatg acgaaggaaa tccagataag ccgagaaatg 2101 gaaaaaggaa gactcgggtt gtgagttgct atgtttgttg ggttgagtga tcgatttttt 2161 tttgtgtgtg ctctcaagga gacctctgtg aacttgaata aactccacac ggagttgtgc 2221 caaattagac cgaggcgtcc gtgaacttga atcagttcct cgggatgttg tgcctgagaa 2281 taaatccgac cgaggcgtct gtgaacttaa ttcaattcgt caggaagctg tgccttagaa 2341 taaattcgac gggtgtccaa ttttcttgtc
[89] Zea Mays ferredoxin-sulfite reductase precursor (sir), is encoded by the following amino acid sequence (NCBI Accession No. NIM)Ol 105302.1 and SEQ ID NO: 39):
MSGAIGGAEΛΗGFRGAAAQLPRSRVLGRPIRVAPPAAARPGGASAGSIRAVSAPAKKDASEVKRSKVEIIKE
KSNFLRYPLNEELVSEAPNINESAVQLIKFHGSYQQTDRDVRGQKNYSFMLRTKNPCGKVPNQLYLAMDT
LADEFGIGTLRLTTRQTFQLHGVLKKNLKTX^STVIKNMGSTLGACGDLNRNVLAPAAPYVKKDILFAQQT AEMAALLTPQSGAYYDLWVDGEKJMSAEEPPEVTKARNDNSHGTNFPDSPEPIYGTQYLPRKFKVAVTAA GDNSVDILTNDIGWVTVSDDAGEPIGFMYVGGGMGRTHRVETCFPRLA^ ENGRRDDRKYSRMKYMIDRWGRORFRAEΛΕKYYGKKFESFRPLPEWQFNSYLGWQEQGDGKLFYGVHV DNGRVGGQAKKTLREIFFIKYNLDVSITPNQNLILCGIDQAWREPITTALAQAGLLEPKDVDPLNLTAMACPA LPLCPLAQTEAERGILPILKRIRAVFNKVGIKDSESVVVRITGCPNGCARP YMAELGFVGDGPKSYQIWLGG TPNQSTLAESFMDKVΈXDDIEKVLEPLFTYWNGTRQEGESFGSFTNRTGFDKXKEVVNKWAESPSAA
Nitrite Reductase
[90] Engineerred E. coli of the invention contain at least one mutation in a nitrite reductase gene. Moreover, engineerred E. coli of the invention contain at least one exogenous nitrite reductase gene.
[91] Compositions and methods of the invention encompass polynucleotide and polypeptide sequences that encode for a sulfite reductase enzyme. Specifically, compositions and methods of the invention encompass polynucleotide and polypeptide sequences that include a mutation. This mutation is any insertion, deletion, inversion, translocation, transposition, excision, truncation, frameshift, or point mutation in a polynucleotide or polypeptide sequence encoding for a sulfite reductase enzyme that causes the resulting gene product to be non-functional. Furthermore, the mutation is also any RNA or DNA mediated silencing technology that prevents transcription of the gene or translation of the mRNA into a polypeptide. Exemplary sequences are provided below.
[92] Moreover, compositions and methods of the invention encompass polynucleotide and polypeptide sequences that encode for exogenous nitrite reductase enzymes. For example, the invention provides methods of introducing a nitrite reductase gene, or any portion thereof, isolated from a green plant or an algae into a cell from E.coli.
[93] Contemplated sequences include nitrite reductase genes, or any portion thereof, from any species of bacteria or photosynthetic organism. Specifically, sequences having 50%, 60%, 70%, 80%, 90%, or 100% identity to the sequences provided below, or any point in between, are encompassed by the invention.
[94] E. coli nitrite reductase, nirA, is encoded by the following mRNA sequence (NCBI Accession No. NC_000913, Region 1396798..1397550 and SEQ E) NO: 40)(reverse complement shown):
1 ATGatcccgg aaaagcgaat tatacggcgc attcagtctg gcggttgtgc tatccattgc 61 caggattgca gcatcagcca gctttgcatc ccgttcacac tcaacgaaca tgagcttgat
121 cagcttgata atatcattga gcggaagaag cctattcaga aaggccagac gctgtttaag
181 gctggtgatg aacttaaatc gctttatgcc atccgctccg gtacgattaa aagttatacc
241 atcactgagc aaggcgacga gcaaatcact ggtttccatt tagcaggcga cctggtggga
301 tttgacgcca tcggcagcgg ccatcacccg agcttcgcgc aggcgctgga aacctcgatg 361 gtatgtgaaa tcccgttcga aacgctggac gatttgtccg gtaaaatgcc gaatctgcgt
421 cagcagatga tgcgtctgat gagcggtgaa atcaaaggcg atcaggacat gatcctgctg
481 ttgtcgaaga aaaatgccga ggaacgtctg gctgcattca tctacaacct gtcccgtcgt
541 tttgcccaac gcggcttctc ccctcgtgaa ttccgcctga cgatgactcg tggcgatatc
601 ggtaactatc tgggcctgac ggtagaaacc atcagccgtc tgctgggtcg cttccagaaa 661 agcggcatgc tggcagtcaa aggtaaatac atcaccatcg aaaataacga tgcgctggcc
721 cagcttgctg gtcatacgcg taacgttgcc tga
[95] E. coli nitrite reductase, nirA, is encoded by the following amino acid sequence (NCBI Accession No. NP_415850.1 and SEQ ID NO: 41) MIPEKMIRMQSGGCAIHCQDCSISQLCIPFTLNEHELDQLDNIIERKKPIQKGQTLFKAGDELKSLYAIRSGTI KSYTITEQGDEQITGFHLAGDLVGFD AIGSGHHPSFAQALETSMVCEIPFETLDDLSGKMPNLRQQMMRLM SGEIKGDQDMILLLSKKNAEERLAAFlYNLSRRFAQRGFSPREFRLTMTRGDIGNYLGLTVETISRLLGRFQK SGMLAVKGKYITIENNDALAQLAGHTRNVA [96] E. colinitritereductase, nirB, is encodedbythe followingmRNA sequence (NCBI
AccessionNo. NC_000913, Region3492033..3494576 and SEQ IDNO: 42):
1 ATGagcaaag tcagactcgc aattatcggt aacggtatgg tcggccatcg ctttatcgaa
61 gatcttcttg ataaatctga tgcggccaac tttgatatta ccgttttctg tgaagaaccg
121 cgcatcgctt atgaccgcgt acacctctcg tcttacttct ctcaccacac cgccgaagag 181 ctgtcgctgg tgcgcgaagg cttctacgag aaacacggca tcaaagttct ggtcggcgaa
241 cgcgctatca ccatcaaccg tcaggagaag gtgattcact ccagcgccgg acgtaccgtt
301 ttttatgaca agctgatcat ggcaaccggt tcctacccgt ggatcccgcc aatcaaaggt 361 tctgatactc aggactgctt tgtctatcgc actattgaag acctcaacgc cattgaatcc
421 tgcgcccgtc gcagcaaacg cggtgccgtt gttggtggcg gcctgttagg tctggaagcc
481 gcaggcgcgc tgaaaaactt aggtattgaa acccacgtta tcgaatttgc ccctatgctg
541 atggcagaac agcttgatca gatgggcggc gagcagctgc gtcgcaaaat cgaaagtatg
601 ggcgtgcgcg ttcacaccag caaaaacacc cttgagattg tgcaggaagg tgttgaagcg
661 cgtaaaacca tgcgttttgc cgacggcagc gaactggaag tcgactttat cgtcttctct
721 accggtatcc gtccgcgcga taagctggca acccagtgtg gtctggacgt tgctccgcgt
781 gggggtattg tcattaatga ttcctgccag acttccgatc cggatatcta cgccatcggt
841 gaatgcgcaa gctggaacaa ccgtgtattt ggtctggtag cacctggcta caaaatggcg
901 caggtcgccg ttgaccatat tctcggtagc gaaaacgcct ttgaaggtgc tgaccttagc
961 gccaagctga aactgctggg cgtagacgta ggcggtattg gtgatgcgca cggtcgcacg
1021 cctggcgcac gtagctacgt ttacctcgac gaaagtaaag agatctacaa acgcctgatt
1081 gtcagcgaag acaacaaaac cctgctcggt gcggtactgg tgggcgatac cagcgactac
1141 ggtaacctgc tgcaactggt gctgaacgct atcgaactgc cggaaaaccc ggattccctg
1201 atcctgccag cacactcggg tagcggcaag ccgtctatcg gtgttgataa actgccggac
1261 agcgcgcaaa tctgctcctg cttcgacgtc accaaaggtg atctgattgc tgccatcaac
1321 aaaggctgcc acacagttgc ggcgctgaaa gctgaaacca aagcgggtac tggctgcggt
1381 ggctgtatcc cgctggtcac tcaggtactg aacgcggaac tggcgaaaca gggcatcgaa
1441 gttaacaaca acctgtgcga acactttgct tattcgcgtc aggaactgtt ccatttgatc
1501 cgcgttgaag gcattaaaac cttcgaagaa ctgctggcga aacacggcaa aggctacggt
1561 tgtgaagttt gtaaaccaac cgtcggttcg ctgctggcct cctgctggaa cgaatacatt
1621 ctgaagccgg aacatactcc gctgcaggat tctaacgaca acttcctcgc taacatccag
1681 aaagacggca cctactcggt gatcccgcgt tctccgggcg gtgaaatcac cccggaaggg
1741 ctgatggcgg taggtcgtat cgcgcgtgaa tttaatctct acaccaagat cactggctcc
1801 cagcgtctgg cgatgtttgg cgcacagaaa gacgatctgc cggagatctg gcgtcagctg
1861 attgaagccg gcttcgaaac cggtcatgcc tatgcgaaag cactgcgtat ggcgaaaacc
1921 tgcgtgggta gcacctggtg ccgctacggc gttggcgaca gcgtcggcct cggcgtggaa
1981 ctggaaaacc gctacaaagg catccgtacg ccgcacaaaa tgaagttcgg tgtctccggc
2041 tgtacccgtg aatgttcaga agctcagggt aaagacgtgg gtattatcgc cactgaaaaa
2101 ggctggaacc tgtatgtttg cggtaacggc ggcatgaaac cgcgtcatgc ggatctgctg
2161 gcggcggata tcgatcgcga aacgctgatc aaatatctcg accgcttcat gatgttctac
2221 atccgtactg ccgacaaact gacgcgtacc gcaccgtggt tagaaaacct cgaaggcggc
2281 atcgattacc tgaaagcagt gatcattgac gacaaactgg ggctgaacgc acatctggaa
2341 gaagagatgg cgcgcctgcg tgaagcggta ctgtgtgagt ggactgaaac ggtcaatacg
2401 ccgtctgcgc agactcgctt caaacacttc atcaacagcg acaagcgtga cccgaacgtg
2461 cagatggtgc cagagcgcga acagcaccgt ccggcaacgc cgtatgaacg tatcccagta
2521 actctggtgg aggacaacgc atga
[97] E. coli nitrite reductase, nirB, is encoded by the following amino acid sequence (NCBI Accession No. NP_417824.1 and SEQ ID NO: 43):
MSKVRLAIIGNGMVGHRFIEDLLDKSDAANFDITVFCEEPRIAYDRVHLSSYFSHHTAEELSLVREGFYEKH GIKVLVGERMTINRQEKVIHSSAGRTVFYDKLIMATGSYPWIPPIKGSDTQDCFVYRTIEDLNAIESCARRSK RGAWGGGLLGLEAAGALKNLGIETHVIEFAPMLMAEQLDQMGGEQLRRKIESMGVRVHTSKNTLEIVQE GVEARKTMRFADGSELEVDFIVFSTGIRPRDKLATQCGLDVAPRGGIVINDSCQTSDPDIYAIGECASWNNR VFGLVAPGYKMAQVAVDHILGSENAFEGADLSAKLKLLGVDVGGIGDAHGRTPGARSYVYLDESKEIYKR LIVSEDNKTLLGAVLVGDTSDYGNLLQLVLNAIELPENPDSLILPAHSGSGKPSIGVDKLPDSAQICSCFDVT KGDLIAAINKGCHTVAALKAETKAGTGCGGCffLVTQVLNAELAKQGIEVNNNLCEHFAYSRQELFHLIRV EGIKTFEELLAKHGKGYGCEVCKPTVGSLLASCWNEYILKPEHTPLQDSNDNFLANIQKDGTYSVIPRSPGG EITPEGLMAVGRIAREFNLYTKITGSQRLAMFGAQKDDLPEIWRQLIEAGFETGHAYAKALRMAKTCVGST WCRYGVGDSVGLGVELENRYKGIRTPHKMKFGVSGCTRECSEAQGKDVGIIATEKGWNLYVCGNGGMKP
LREAVLCEWTETVNTPSAQTRFKHF]NSDKRDPNVQMVPEREQHRPATPYERIPVTLVEDNA [98] E. coli nitrite reductase, nirD, is encoded by the following mRNA sequence (NCBI Accession No. NC_000913, Region 3494573..3494899 and SEQ ID NO: 44):
1 ATGagccagt ggaaagacat ctgcaaaatc gatgacatcc tgcctgaaac cggcgtctgc
61 gcgctgttag gtgacgagca ggtcgcgatt ttccgcccgt atcacagcga tcaggtgttt 121 gcgatcagca acatcgaccc gttcttcgag tccagcgtgc tgtcacgcgg actgattgcg
181 gaacaccagg gcgagctgtg ggtcgccagc ccgctgaaaa aacagcgttt tcgcttaagc
241 gacggcttgt gcatggaaga cgaacagttt tccgtcaaac attacgaagc gcgagtgaaa
301 gacggcgtgg tgcagctgcg cggttaa [99] E. coli nitrite reductase, nirD, is encoded by the following amino acid sequence (NCBI Accession No. NP_417825.1 and SEQ ID NO: 45):
MSQWKDICKIDDILPETGVCALLGDEQVAIFRPYHSDQVFAISNIDPFFESSVLSRGLIAEHQGELWVASPLK KQRFRLSDGLCMEDEQFSVKHYEARVKDGVVQLRG Glutamate Synthase
[100] Engineerred E. coli of the invention contain at least one mutation in a glutamate synthase gene. Moreover, engineerred E. coli of the invention contain at least one exogenous glutamate synthase gene. [101] Compositions and methods of the invention encompass polynucleotide and polypeptide sequences that encode for a sulfite reductase enzyme. Specifically, compositions and methods of the invention encompass polynucleotide and polypeptide sequences that include a mutation. This mutation is any insertion, deletion, inversion, translocation, transposition, excision, truncation, frameshift, or point mutation in a polynucleotide or polypeptide sequence encoding for a sulfite reductase enzyme that causes the resulting gene product to be non-functional. Furthermore, the mutation is also any RNA or DNA mediated silencing technology that prevents transcription of the gene or translation of the mRNA into a polypeptide. Exemplary sequences are provided below.
[102] Moreover, compositions and methods of the invention encompass polynucleotide and polypeptide sequences that encode for exogenous glutamate synthase enzymes. For example, the invention provides methods of introducing a glutamate synthase gene, or any portion thereof, isolated from a green plant or an algae into a cell from E. coli.
[103] Contemplated sequences include glutamate synthase genes, or any portion thereof, from any species of bacteria or photosynthetic organism. Specifically, sequences having 50%, 60%, 70%, 80%, 90%, or 100% identity to the sequences provided below, or any point in between, are encompassed by the invention. [104] E. coli glutamate synthase,gltB, is encoded by the following mRNA sequence (NCBI Accession No. NC_000913, Region 3352654..3357207 and SEQ ID NO: 46): i ATGacacgca aaccccgtcg ccacgctctt tctgtgcccg tgcgcagcgg ttcggaagtg
61 gggttcccgc agagcctggg ggaggttcac gatatgttgt acgataaatc ccttgagagg
121 gataactgtg gtttcggcct gatcgcccac atagaaggcg aacctagcca caaggtagtg
181 cgtactgcaa tacacgcact ggcccgcatg cagcaccgtg gcgcgattct cgccgatggt
241 aaaaccggcg acggttgcgg cttgctgtta caaaaaccgg atcgcttttt tcgcatcgtt
301 gcgcaggagc gcggctggcg tttagcaaaa aactacgctg tcgggatgct cttcctgaat
361 aaagatcctg aactcgccgc tgccgcacgc cgcatcgttg aagaagaact gcaacgcgaa
421 accttgtcga ttgtgggctg gcgtgatgtc cccactaacg aaggcgtgct gggtgaaatc
481 gccctctcct ctctgccacg cattgagcaa atttttgtga acgccccggc aggctggcgt
541 ccacgcgata tggagcgccg tctgtttatc gcccgccgcc gcattgaaaa gcgtctcgaa
601 gccgacaaag acttctacgt ctgtagcctg tcgaatctgg tgaacatcta taaaggtctg
661 tgtatgccga cggatctgcc gcgcttttat ctggatcttg cggacctgcg tctggaatcg
721 gccatttgcc tgttccacca gcgcttctcc actaacaccg taccgcgctg gccgctggcg
781 caaccgttcc gctatctggc gcataacggt gaaatcaaca ccatcaccgg taaccgccaa
841 tgggcgcgtg cgcgtactta taaattccag acaccgctta tccctgacct gcacgacgcc
901 gcaccgttcg tcaacgaaac cggctctgac tccagttcga tggataacat gctggaactg
961 ctgctggcag gcgggatgga tatcatccgc gccatgcgtc tattagtacc acccgcctgg
1021 cagaacaacc cggatatgga cccggaactg cgtgccttct ttgactttaa ctccatgcat
1081 atggagccgt gggatggccc ggcgggcatc gtgatgtccg acggtcgttt tgccgcctgt
1141 aacctcgacc gtaacggtct gcgtccggcg cgctacgtca tcaccaaaga taagctcatc
1201 acctgcgcct ctgaagtcgg tatctgggat taccagcctg acgaagtggt cgaaaaaggc
1261 cgcgtcgggc caggcgaact gatggttatc gacacccgca gtgggcgtat tctgcactcg
1321 gcagaaaccg atgacgatct gaaaagccgc catccatata aagagtggat ggagaaaaac
1381 gtccgccgac tggtaccgtt tgaagatctg cccgatgaag aagtgggtag ccgcgaactg
1441 gacgacgaca cgcttgccag ctaccagaaa cagtttaact acagcgcgga agagctggac
1501 tccgtaattc gcgtactggg cgaaaacggt caggaagcgg tcggttcgat gggcgatgat
1561 accccattcg ccgtgctctc cagtcagccg cgcattattt acgactactt ccgccagcag
1621 tttgcccagg tgactaaccc gccaatcgac ccgctgcgtg aagcgcatgt tatgtcgctc
1681 gccaccagta tcggtcgtga aatgaacgtc ttttgcgaag cagagggcca ggcgcaccgt
1741 ttaagcttta aatcgccgat tctgctctac tccgatttca aacagctcac gacgatgaaa
1801 gaggagcact accgcgcaga tacgctggat atcacctttg acgtcactaa aaccacgctc
1861 gaagcgacag tcaaagagct gtgcgacaaa gccgaaaaaa tggtacgtag cggcaccgtg
1921 ctgctggtgc tctccgaccg gaatatcgct aaagatcgcc tgccggttcc agccccgatg
1981 gcggttggcg cgatccagac ccgtctggtc gatcaaagcc tgcgttgcga tgccaacatc
2041 atcgtcgaaa ccgccagcgc ccgcgatccg caccacttcg ccgtgttgct gggcttcggc
2101 gcgacggcta tttatccata ccttgcctat gaaacgctgg gccgcctggt agacacccat
2161 gcgattgcca aagattatcg taccgtgatg ctcaactacc gtaacggcat caacaaaggc
2221 ttgtacaaaa tcatgtccaa aatgggcatc tccaccatcg cctcttaccg ctgctcgaaa
2281 ctgtttgaag cggtcggtct acacgatgat gtagtgggcc tgtgcttcca gggggcggtc
2341 agccgcattg gtggagcaag ctttgaagac ttccagcagg atctgctgaa tctgtcgaaa
2401 cgtgcctggc tggcgcgtaa gcccatcagc cagggcggtc tgctgaaata cgtccacggc
2461 ggcgaatacc acgcctacaa cccggacgtg gtgcgcacgc tgcaacaagc ggtacaaagc
2521 ggcgagtaca gcgactatca ggaatacgcg aagctggtta atgagcgtcc ggcaaccacg
2581 ctgcgcgatc tgctggcaat tacgccgggt gaaaacgcgg tcaacattgc tgatgttgaa
2641 ccggcaagcg aactgtttaa acgctttgat accgccgcga tgtctatcgg cgcgttaagc
2701 ccggaagccc acgaggcgct ggcggaagcg atgaacagca tcggcggtaa ttcgaactcc
2761 ggtgaaggcg gcgaagaccc ggcgcgctac ggcaccaaca aagtgtcgcg catcaagcag
2821 gtggcttccg gtcgctttgg ggttactccg gcgtatctgg tcaatgccga cgtcattcag
2881 attaaagtcg cccagggcgc gaagccaggc gaaggcggtc agttgccggg tgataaagtc
2941 acgccttaca tcgccaaact gcgctattcg gtgcccggag tgacgctgat ctccccgccg
3001 ccgcaccacg atatctactc tatcgaggac ttagcgcagc tcattttcga cctcaagcag
3061 gttaacccga aagcgatgat ctccgtgaag ctggtttccg aaccgggagt aggcaccatc
3121 gcgactggcg tggcaaaagc ttatgcggac ttgatcacca tcgcaggcta tgacggcggc 3181 accggcgcaa gtccgctttc atcggtgaaa tacgcaggct gtccgtggga gctggggctt 3241 gttgaaaccc agcaggcgct ggttgctaac ggcttgcgtc acaagatccg tttgcaggtc 3301 gatggcggcc tgaaaacggg tgtcgatatc atcaaggcgg cgattctcgg cgcagaaagc 3361 ttcggcttcg gcactggccc gatggtggcg ctcggctgta aatatctacg tatttgccat 3421 ctgaacaact gcgcaacggg tgtagcaact caggatgaca aactgcgtaa gaaccactat 3481 cacggcctgc cattcaaggt gacgaattac tttgagttta tcgcccgtga aacccgcgag 3541 ctgatggcac agcttggcgt aacacgtctg gtggatctga ttggtcgcac cgacctgctg 3601 aaagagctgg acggtttcac cgccaaacag cagaagctgg cgctgtcgaa gctgctggag 3661 actgccgaac cgcatccagg taaggcactc tactgcaccg aaaacaaccc gccgtttgat 3721 aacggcctgc tgaacgcgca gttgctgcaa caggcgaaac cgtttgtcga tgagcgccag 3781 agcaaaacct tctggttcga tattcgcaac accgaccgtt ctgtcggcgc gtcgctttca 3841 ggctatatcg cccagacgca cggcgatcag gggctggcag ccgatcctat caaagcgtac 3901 ttcaacggca ccgcaggcca gagcttcggc gtgtggaacg cgggcggcgt ggaactgtac 3961 ctgaccggtg atgccaacga ctatgtcggt aaaggcatgg cgggcggctt aatcgccatt 4021 cgtcctccgg ttggttccgc cttccgcagc catgaagcaa gcattatcgg caacacctgc 4081 ctgtatggcg cgaccggtgg tcgtctgtat gccgcaggcc gcgcgggtga acgtttcggc 4141 gtgcgtaact ccggtgctat caccgtggta gaaggcattg gcgacaacgg ttgtgaatat 4201 atgacgggtg gtatcgtctg cattctgggt aaaaccggcg ttaacttcgg tgcgggcatg 4261 accggcggtt tcgcttacgt tctcgatgaa agcggcgatt tccgcaaacg cgttaacccg 4321 gaactggtcg aggtcttaag cgttgacgct ctggcgatcc atgaagagca tctgcgtggt 4381 cttatcaccg agcatgtgca gcataccggc tctcagcgcg gtgaagagat tctggcgaac 4441 tggtcaacct tcgccactaa atttgcgctg gttaaaccga agtccagtga tgtaaaagca 4501 ctgctgggtc accgtagtcg tagcgcagct gagttgcgcg tgcaggcgca gtaa
[105] E. coli glutamate synthase,gltB, is encoded by the following amino acid sequence (NCBI Accession No. NP_417679.1 and SEQ ID NO: 47):
MTREPRRHALSVPVRSGSEVGFPQSLGEVHDMLYDKSLERDNCGFGLIAHIEGEPSHKVVRTAIHALARMQ HRGAILADGKTGDGCGLLLQKPDRFFMVAQERGWTTLAKNYAVGMLFLNKDPELAAAARRIVEEELQRET LSIVGWRDVPTNEGVLGEIALSSLPRIEQIFVNAPAGWRPRDMERRLFIARRRIEKΫEU. MYKGLCIVFFTDLPRFYLDLADLRLESAICLFHQRFSTNTVPRWLVPPAWQNNPDMDPELRAFFDFNSMHM EPΛVDGPAGIVMSDGRFAACNLDRNGLRPARYVITKDKLITCASEVGIWDYQPDEVVEKGRVGPGELMVID TRSGRILHSAETDDDLKSRHPYKEWMEKNVRRLVPFEDLPDEEVGSRELDDDTLASYQKQFNYSAEELDS XORVLGENGQEAVGSMGDDTPFAVLSSQPRIIYDYFRQQFAQVTTSΓPPIDPLREAHVMSLATSIGREMNVFCE AEGQAHRLSFKSPILLYSDFKQLTTMKΕEHYRADTLDITFDVTKTTLEATΛ^KELCDKAEKMVRSGTVLLVL SDRNIAKDRLPWAPMAVGAIQTRLVDQSLRCDANIIVETASARDPHHFAVLLGFGATAIYPYLAYETLGRL VDTHAIAKDYRTXNV[LNYRNGINKGLYKIMSKMGISTIASYRCSKLFEAVGLHDDVVGLCFQGAVSRIGGAS FEDFQQDLLNLSKRAWLARKPISQGGLLKYVHGGEYHAYNPDWRTLQQAVQSGEYSDYQEYAKLVNER PATTLRDLLAITPGENAVNIADVEPASELFKRFDTAAMSIGALSPEAHEALAEAMNSIGGNSNSGEGGEDPA RYGTNKVSRIKQVASGRFGVTPAYLVNADVIQIKVAQGAKPGEGGQLPGDKVTPYIAKLRYSVPGVTLISP PPHHDR^SFFIDLAQLIFDLKQVNPKAMISVKLVSEPGVGTIATGVAKAYADLITIAGYDGGTGASPLSSVKY AGCPWELGLVETQQALVANGLRHKIRLQVDGGLKTGVDIIKAAILGAESFGFGTGPMVALGCKYLRICHLN NCATGVATQDDKLRKNHYHGLPFKVTNYFEFIARETRELMAQLGVTRLVDLIGRTDLLKELDGFTAKQQK LALSKLLETAEPHPGKALYCTENNPPFDNGLLNAQLLQQAKPFVDERQSKTFWFDIRNTDRSVGASLSGYI AQTHGDQGLAADPIKAYFNGTAGQSFGVWNAGGVELYLTGDANDYVGKGMAGGLIAIRPPVGSAFRSHE ASIIGNTCLYGATGGRLYAAGRAGERFGVRNSGAITWEGIGDNGCEYMTGGIVCILGKTGVNFGAGMTG GFAYVLDESGDFRKRVNPELVEVLSVDALAIHEEHLRGLITEHVQHTGSQRGEEILANWSTFATKFALVKP KSSDVKALLGHRSRSAAELRVQAQ
[106] E. coli glutamate synthase, gltD, is encoded by the following mRNA sequence (NCBI Accession No. NC_000913, Region 3357220..3358638 and SEQ ID NO: 48):
1 ATGagtcaga atgtttatca atttatcgac ctgcagcgcg ttgatccgcc aaagaaaccg 61 ctgaagatcc gcaaaattga gtttgttgaa atttacgagc cgttttccga aggccaggcc
121 aaagcgcagg ctgaccgctg cctgtcgtgc ggcaacccat actgcgagtg gaaatgcccg
181 gtacacaact acatcccgaa ctggctgaag ctcgccaacg aggggcgtat ttttgaagcg
241 gcggaactgt cgcaccagac caacaccctg ccggaagttt gcggacgagt ctgcccgcaa 301 gaccgtctgt gcgaaggttc ctgcactctg aacgatgagt ttggcgcggt gaccatcggc
361 aacattgagc gctatatcaa cgataaagcg ttcgagatgg gctggcgtcc ggatatgtct
421 ggtgtgaaac agaccggtaa aaaagtggcg attatcggcg caggcccggc aggtctggcg
481 tgtgcggatg tcctgacgcg taacggcgta aaagccgttg tcttcgaccg tcatccagaa
541 attggcgggc tgctgacctt cggtattccg gccttcaagc tggaaaaaga ggtaatgacg 601 cgtcgccgtg aaatcttcac cggcatgggt attgaattca aactcaatac cgaagtgggc
661 cgcgacgtac agctggacga tctgctgagt gattacgatg ccgtgttcct tggcgtcggg
721 acttatcagt caatgcgcgg cgggctggaa aacgaagacg ccgatggcgt gtacgcagcg
781 ctgccgttcc tcatcgccaa caccaaacag ttaatgggct ttggtgaaac ccgcgacgaa
841 ccgttcgtca gcatggaagg caaacgcgtg gtggtccttg gcggtggcga cactgcgatg 901 gactgcgtgc gtacgtccgt gcgccaggga gcgaagcacg ttacctgtgc ctatcgtcgt
961 gatgaagaga acatgccggg ttcccgccgc gaagtgaaaa acgcgcggga agaaggcgta
1021 gagttcaaat tcaacgtcca gccgctgggt attgaagtga acggtaacgg caaagtcagc
1081 ggcgtaaaaa tggtgcgtac cgaaatgggc gaaccggacg ccaaaggccg tcgccgcgcg
1141 gagatcgttg caggttccga acatatcgtt ccggcagatg cggtgatcat ggcgtttggt 1201 ttccgtccac acaacatgga atggctggca aaacacagcg tcgagctgga ttcacaaggc
1261 cgcatcatcg ccccggaagg cagcgacaac gccttccaga ccagcaaccc gaaaatcttt
1321 gctggcggcg atatcgtccg tggttccgat ctggtggtga ccgctattgc cgaaggtcgt
1381 aaggcggcag acggtattat gaactggctg gaagtttaa [107] E. coli glutamate synthase, gltD, is encoded by the following amino acid sequence (NCBI Accession No. NP_417680.1 and SEQ ID NO: 49): MSQNWQFroLQRVDPPKKPLKIRKJEFVEire^
AISΠEGRIFEAAELSHQTNTLPEVCGRVCPQDRLCEGSCTLNDEFGAVTIGMERYINDKAFEMGWRPDMSGV KQTGKKVAIIGAGP AGLACAD VLTRNGVKAVYFDRHPEIGGLLTFGIPAFKLEKEVMTRRREIFTGMGIEFK LNTEVGRDVQLDDLLSDYDAVFLGVGTYQSMRGGLENEDADGVYAALPFLIANTKQLMGFGETRDEPFV SMEGKRVVVLGGGDTAMDCΛ^TSVRQGAKHVTCAYRRDEENMPGSRREVKNAREEGVEFKFNVQPLGIE VNGNGKVSGVKMVRTEMGEPDAKGRRRAEIVAGSEHIVPADAVIMAFGFRPHNMEWLAKHSVELDSQGR IIAPEGSDNAFQTSNPKFFAGGDIVRGSDLVVTAIΛEGRKAADGIMNWLEV Oxidation and Reduction
[108] As used herein, the term "oxidation" describes the loss of electrons by a molecule, atom or ion. Alternatively, "oxidation" describes an increase in oxidation number. Conversely, the term "reduction" as used herein describes the gain of electrons by a molecule, atom or ion. Alternatively, "reduction" describes a decrease in oxidation number. The term "redox" as used herein describes a pair, series, or plurality of reduction and oxidation reactions occurring wherein the oxidation state of at least one molecule, atom, or ion changes.
Photosvnthetic Organisms
[109] Polynucleotides and polypeptides of the invention are isolated from photosvnthetic organisms of the invention. [110] All photosynthetic organisms are encompassed by the present invention. Exemplary organisms include, but are not limited to, green plants and bacteria. In one embodiment, the organism is Spinacia olearcea. Other contemplated plants encompassed by the invention include, but are not limited to, Arabidopsis and all species of corn. [111] In a preferred embodiment, the organism is an aquatic phototroph. Aquatic phototrophs encompassed by the invention include, but are not limited to, the eukaryotic Chlorophyta division comprising the Chlamydomonas and Chlorella genera, as well as the bacterial Phyla Cyanobacteria, Chloroflexi, Chlorobi, and the Alpha Proteohacteria. [112] In another preferred embodiment, the photosynthetic organism is Chlamydomonas reinhardtii. In another preferred embodiment, the photosynthetic organism is Clostridium acetobutylicum. In a third preferred embodiment, the photosynthetic organism is Clostridium saccharobutylicum.
[113] Alternatively, or in addition, the organism is cyanobacteria, also known as blue-green algae, blue-green bacteria, or cyanophyta. Cyanobacteria of the invention are isolated directly from fresh or salt water. Moreover, cyanobacteria of the invention are isolated from nature or cultured. Exemplary cyanobacteria include, but are not limited to, those of the following genera, Anabaena, Oscillataria, Halospirulina, Planktothricoides, Prochlorococcus, Prochloron, and Prochlorothrix.
Non-Photosvnthetic Organisms
[114] Polynucleotides and polypeptides of the invention are isolated from non-photosynthetic organisms of the invention. Moreover, endogenous and exogenous polynucleotide and polypeptides of the invention are inserted into cells of non-photosynthetic organisms. [115] Alternatively, or in addition, a non-photosynthetic bacterium is preferred. In this embodiment, Escherichia coli (E. coli) are used for directed evolution of one or more polypeptide species of an engineered hydrogenase.
[116] Other bacteria encompassed by the invention include, but are not limited to, species of the following genera, Clostridium (specifically, C. pasteurianum), Ralstonia (specifically, R. eutrophia), Thermosynechococcus (specifically, T. longates), Thermotoga (specifically, T. neapolitana), and Desulfovibrio (specifically, D. gigas, D.desulfuricans, and D. baculatum). [117] Finally, all yeast are encompassed by the invention. In a preferred embodiment, the invention comprises the use of all strains of Saccharomyces cerevisiae.
Definitions [118] As used herein, the term "endogenous gene" is meant to describe a gene that exists in the genome of the host cell. Conversely, the term "exogenous gene" is meant to describe a gene that does not exist within the genome of a host cell. The term, "transgenic" is meant to describe any polynucleotide or polypeptide sequence, gene, or cell that is isolated from one species and introduced into another species. [119] As used herein, the term "isolated" is meant to describe a polynucleotide sequence, polypeptide sequence, or gene, that is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. An isolated polynucleotide is, for example, a recombinant DNA or RNA molecule, provided one of the nucleic acid sequences normally found immediately flanking that recombinant RNA molecule in a naturally-occurring molecule is removed or absent. Thus, isolated polynucleotides include, without limitation, a recombinant DNA or RNA that exists as a separate molecule (e.g., a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences as well as recombinant DNA or RNA that is incorporated into a vector, an autonomously replicating plasmid, or into the genomic DNA or RNA of a prokaryote or eukaryote. hi addition, an isolated polynucleotide can include a recombinant DNA or RNA molecule that is part of a hybrid, chimeric, or fusion polynucleotide.
[120] Isolated polypeptides of the invention are encoded by isolated polynucleotides. Alternatively, or in addition, isolated polypeptides are segregated away from other polypeptides and/or cellular material by art-recognized methods. Isolated polypeptides of the invention exist as homomeric or heteromeric monomers, dimers, tetramers, oligomers. Isolated polypeptides of the invention are folded, misfolded, or denatured. Furthermore, isolated polypeptides of the invention contain one or more subunits. [121] Isolated cells of the invention contain polynucleotides and polypeptides of the invention. As such, an isolated cell of the invention is engineered, altered, or manipulated. Isolated cells of the invention are derived from any species of organism. Alternatively, or in addition, isolated cells are unicellular organisms.
[122] Endogenous and exogenous polypeptides and polynucleotides of the invention are introduced into cells. As used herein, the term "introduced" is meant to describe any process by which a polynucleotide or polypeptide sequence crosses a plasma membrane of a host cell. Exemplary methods of introducing polynucleotide or polypeptide sequences into host cells include, but are not limited to, electroporation, heat shock, magnetofection, gene gun, endocytosis, vesicle fusion, and lipofection. Alternatively, or in addition, polynucleotides and polypeptides of the invention are bound, coupled, operably linked, fused, or tethered, to compounds that facilitate transportation of these sequences into a cell. For example, a polynucleotide or polypeptide is bound to a cationic polymer, a nanoparticle, or calcium phosphate.
[123] As used herein, the term "biological property" is meant to describe a property of a enzyme, for instance, a hydrogenase enzyme, that is affected by the cellular or extracellular environment. For example, a hydrogenase enzyme catalyzes the reversible oxidation of molecular hydrogen (2H+ <→ H2). However, the presence of certain environmental factors including, but not limited to, atmospheric molecular oxygen, high temperature, low temperature, oxidizing agents, and heavy metals. Moreover, the term "biological property" is meant to describe operational parameters of the enzyme such as catalytic rate. The term catalytic rate is equivalent to kinetic rate. The term "catalytic rate" is meant to describe the rate, speed, or efficiency (speed with minimal energy or starting material of the reaction, e.g. 2H+ or H2 ) at which a hydrogenase converts 2H+→ H2 or H2 — » 2H+.
[124] As used herein, the term "ferredoxin-dependent enzyme" is meant to describe any enzyme that requires the presence of a functional ferredoxin polypeptide in order to fulfill any of its functions. Exemplary ferredoxin-dependent enzymes of E. coli include, but are not limited, to sulfite reductase (cysl, cysJ), nitrite reductase (nirAB), and glutamate synthetase (gltBD). As used hererin, the term "homolog" is meant to describe a ferredoxin-dependent enzyme from any species or an enzyme with a polypeptide sequence that that is 50%, 60%, 70%, 80%, 90%, or 100% (or any point in between) identical to a ferredoxin-dependent enzyme. Moreover, the term "homolog" is meant to describe a ferredoxin-dependent enzyme from any species or an enzyme encoded by a polynucleotide sequence that is 50%, 60%, 70%, 80%, 90%, or 100% (or any point in between) identical to the polynucleotide sequence that encodes for a ferredoxin-dependent enzyme.
[125] As used herein, the term "selective pressure" is meant to describe an environmental condition in which an engineered E. coli of the invention is grown that challenges the ability of the E. coli to survive. Selective pressure is specific to the type of biological property of a hydrogenase enzyme that is desired. For example, if an oxygen tolerant hydrogenase enzyme is desired, then the engineered E. coli is grown in the presence of atmospheric oxygen, wherein the atmospheric oxygen applies selective pressure upon the survival of the E. coli. Similarly, to select for a thermostable hydrogense, the engineered E. coli is grown at higher or lower temperatures than the hydrogenase can normally tolerate and be catalytically active.
Alternatively, or in addition, the range of temperatures include those temperatures, high or low, at which the E. coli would normally be unable to grow. In this scenario, the selective pressure is inceased or decreased temperature. To select for an increased catalytic rate, the expression levels of the exogenous hydrogenase gene are decreased, such that each hydrogenase expressed within the engineered E. coli must catalyze reactions at a faster rate for the engineered E. coli to survive. In this third scenario, the selective pressure is decreased hydrogenase expression. [126] As used herein, the term "survival" of an engineered E. coli is meant to describe either the absolute or relative survival of an engineered E. coli compared to a wild type, or unengineered, E. coli. In one aspect of the invention, engineered E. coli containing hydrogenase enzymes having the desired biology property survive whereas engineered E. coli that contain hydrogenase enzymes without the desired biological property die. Alternatively, or in addition, engineered E. coli containing hydrogenase enzymes having the desired biology property survive for a longer period of time than engineered E. coli that contain hydrogenase enzymes without the desired biological property. In another scenario, engineered E. coli containing hydrogenase enzymes having the desired biology property proliferate at a faster rate, and survive better or thrive, compared to engineered E. coli that contain hydrogenase enzymes without the desired biological property.
[127] As used herein, the term "functional" enzyme is meant to describe an enzyme, e.g. hydrogenase, ferredoxin, sulfite reductase, etc., that catalyzes a reaction. Alternatively, or in addition, a functional enzyme catalyzes a given reaction at a particular rate. As such, in certain aspect of the invention, a non-functional enzyme catalyzes a reaction at a rate that is too slow to produce a required product for a cell or to maintain survival.
EXAMPLES Example 1 : Experimental Test Systems and Methods
[128] In this example, a selection for oxygen-resistance is performed, however, a number of different characteristics can be selected. An E. coli strain of the genotype Δcysl [pSIR-Fd (Zea sulfite reductase, Spinacea Ferredoxiη); pHydAl (Clostridium acetobutylicum hydrogenase active subunit); pHydEF, HydG (Chlamydomonas reinhardtii hydrogenase maturation factors)/ is mutagenized by a standard procedure with, for example, N-nitrosoguanidine, ethylmethane sulfonate, or ultraviolet light. For desciptions of protocols for use of these and other mutagens, see Miller, Experiments in Molecular Genetics (Cold Spring Harbor Press, Cold Spring Harbor, New York. [129] The mutagenized bacteria are plated on selective agar plates containing M9 minimal medium with added ferrous iron in the form Of(FeSO4 at 10 μM, or M63 medium, or another minimal medium. The medium is lacking in cysteine, methionine, and any other form of reduced sulfur but may optionally contain other amino acids, vitamins, and nutrients depending on other features of the genotype of the E. coli strain background. The plates are poured according to standard procedures, but are incubated for at least 4 days at room temperature in anaerobic conditions, for example, in an MBRAUN anaerobic glove box (M. Braun Group).
[130] After mutagenesis, the bacteria are grown for many generations to allow fixation of any mutations, particularly mutations that are linked to a high-copy plasmid and that are recessive. The bacteria are spun down and the pellet is transferred to an anaerobic chamber. In the chamber, the pellet is resuspended in a buffer that has been stored in the chamber for several days in a loose-capped container so that any dissolved oxygen will be removed. The bacteria are resuspended to a final concentration of about 107 cells/ml and 0.1 mis is spread on a selective plate. The mutagenized bacteria are titered on selective and non-selective plates. Antibiotics corresponding to the drug resistance markers on the various plasmids are optionally included in the plates, although the nature of the genetic selection generally demands the presence of each of the plasmids. [131] Mutants are then characterized according to the genetic location of the causative mutation or mutations. As an initial step, mutant isolates are first tested as to whether the mutant phenotype co-segregates with one of the plasmids in the strain. Each mutant can be tested in two ways. In the first method, recipient bacterial strains are generated that are identical to the non- mutagenized parent strain except that each recipient is lacking one of the plasmids and, is thus, sensitive to the corresponding antibiotic. Plasmid DNA is prepared from each mutant. The recipient strains are transformed with this DNA and selected for resistant to the relevant antibiotic. The resulting transformed strains are tested for the mutant phenotype. [132] Li the second method, a mutant strain is grown in the absence of antibiotic selection for a long period of time, and a set of segregants are identified that lack each one of the antibiotic resistance markers. Such segregants are identified, for example, by replica-plating or by toothpicking. The rate of formation of such segregants is enhanced by growth in the presence of acridine orange according to standard procedures (Miller, Experiments in Molecular Genetics). If the mutant phenotype appears to co-segregate with a given plasmid, the gene or genes that contribute to the artificial hydrogenase-dependent pathway are sequenced. If multiple mutations are identified, then these are tested individually by total gene synthesis of the relevant gene containing the single mutation being tested, followed by placement of the mutant gene into an otherwise non-mutagenized host. [133] In some cases, the mutation causing the mutant phenotype will not be linked to a plasmid but will instead be in the host chromosome and can be mapped. Such methods are well known in the art of bacterial genetics, and include Hfr crosses and Pl transductions. Because of the complexity of the mutant phenotype being assayed and the possible variations in strain background, the method of linked transposon mutagenesis (Roth) is used, in which drug-resistant transposon insertions linked to a mutation of interest are generated, and then the drug-resistance phenotype is mapped.
Example 2: Construction of a Bacterial Strain Whose Growth is Conditionally Dependent on the Activity of an Exogenous FeFe Hydrogenase Using Cysteine Auxotrophv as a Phenotype [134] To construct a bacterial strain whose growth depends on the activity of an FeFe hydrogenase, the following plasmids were constructed:
1. pCaHydA (Hydrogenase from C. acetobutylicum) 2. pCrHydAl (Hydrogenase from C. reinhardtii)
3. pCrHydEF-CrHydG (Hydrogenase maturation factors from C. reinhardtii)
4. pSoFd-ZmSIR (Ferredoxin from S. oleracea and sulfite reductase from Z. mays) [135] Plasmids were produced using the Novagen Duet backbone vectors (Merck KGaA, Darmstadt Germany), an integrated system of compatible vectors used for expressing multiple genes simultaneously in E coli.
Table 2
Figure imgf000043_0001
[136] Maps oftheplasmids described in Table 2 are found inFigures 2-5.
[137] The following sequences encodetheplasmids describedinTable2, andrepresentedby the schematic diagrams ofFigures 2-5.
[138] CaHydA (HydA from Clostridium acetobutylicum) is encodedbythe following polynucleotide sequence and SEQ ID NO: 50 (PCRproduct from aconstructprovidedbyMatt Posewitz): atgggcgcggccgcatctagaatgggcaaaacaataatcttaaatggcaatgaagtgcatacagataaagatattac tatccttgagctagcaagagaaaataatgtagatatcccaacactctgctttttaaaggattgtggcaattttggaa aatgcggagtctgtatggtagaggtagaaggcaagggctttagagctgcttgtgttgccaaagttgaagatggaatg gtaataaacacagaatccgatgaagtaaaagaacgaatcaaaaaaagagtttcaatgcttcttgataagcatgaatt taaatgtggacaatgttctagaagagaaaattgtgaattccttaaacttgtaataaagacaaaagcaaaagcttcaa aaccatttttaccagaagataaggatgctctagttgataatagaagtaaggctattgtaattgacagatcaaaatgt gtactatgcggtagatgcgtagctgcatgtaaacagcacacaagcacttgctcaattcaatttattaaaaaagatgg acaaagggctgttggaactgttgatgatgtttgtcttgatgactcaacatgcttattatgcggtcagtgtgtaatcg cttgtcctgttgctgctttaaaagaaaaatcccatatagaaaaagttcaagaagctcttaatgaccctaaaaaacat gtcattgttgcaatggctccatcagtaagaactgctatgggcgaattattcaaaatgggatatggaaaagatgtaac aggaaaactatatactgcacttagaatgttaggctttgataaagtatttgatataaactttggtgcagatatgacta taatggaagaagctactgaacttttaggcagagttaaaaataatggcccattccctatgtttacatcttgctgtcct gcatgggtaagattagctcaaaattatcatcctgaattattagataatctttcatcagcaaaatcaccacaacaaat atttggtactgcatcaaaaacttactatccttcaatttcaggaatagctccagaagatgtttatacagttactatca tgccttgtaatgataaaaaatatgaagcagatattcctttcatggaaactaacagcttaagagatattgatgcatcc ttaactacaagagagcttgcaaaaatgattaaagatgcaaaaattaaatttgcagatcttgaagatggtgaagttga tcctgctatgggtacttacagtggtgctggagctatctttggtgcaaccggtggcgttatggaagctgcaataagat cagctaaagactttgctgaaaataaagaacttgaaaatgttgattacactgaagtaagaggctttaaaggcataaaa gaagcggaagttgaaattgctggaaataaactaaacgttgctgttataaatggtgcttctaacttcttcgagtttat gaaatctggaaaaatgaacgaaaaacaatatcactttatagaagtaatggcttgccctggtggatgtataaatggtg gaggtcaacctcacgtaaatgctcttgatagagaaaatgttgattacagaaaactaagagcatcagtattatacaac caagataaaaatgttctttcaaagagaaagtcacatgataatccagctattattaaaatgtatgatagctactttgg aaaaccaggtgaaggacttgctcacaaattactacacgtaaaatacacaaaagataaaaatgtttcaaaacatgaag gcgcgccgagcgcgtggagccatccgcagtttgaaaaaactagttaa [139] CaHydA (HydA from Clostridium acetobutylicum) is encoded by the following amino acid sequence and SEQ ID NO: 51 : mgaaasrmgktiilngnevhtdkditilelarennvdiptlcflkdcgnfgkcgvcmvevegkgfraacvakvedgm vintesdevkerikkrvsmlldkhefkcgqcsrrenceflklviktkakaskpflpedkdalvdnrskaividrskc vlcgrcvaackqhtstcsiqfikkdgqravgtvddvclddstcllcgqcviacpvaalkekshiekvqealndpkkh vivamapsvrtamgelfkmgygkdvtgklytalrmlgfdkvfdinfgadmtimeeatellgrvknngpfpmftsccp awvrlaqnyhpelldnlssakspqqifgtasktyypsisgiapedvytvtimpcndkkyeadipfmetnslrdidas lttrelakmikdakikfadledgevdpamgtysgagaifgatggvmeaairsakdfaenkelenvdytevrgfkgik eaeveiagnklnvavingasnffefmksgkmnekqyhfievmacpggcingggqphvnaldrenvdyrklrasvlyn qdknvlskrkshdnpaiikmydsyfgkpgeglahkllhvkytkdknvskhegapsawshpqfekts
[140] CrHydA (HydAl from Chlamydomonas reinhardtiϊ) is encoded by the following polynucleotide sequence and SEQ ID NO: 52 (Synthetic construct designed to express the C. reinhardii protein while removing the >GC bias that complicates exogenous expression): atgggcgcggccgcatctagagctgcaccagccgcagaagctcctttgtctcatgttcaacaggccttagccgagct tgcaaaaccaaaggatgaccctactagaaaacacgtatgtgtccaagtggccccagctgttagggtagcaattgctg aaacacttggtttggcccctggagcaaccactccaaagcagttagctgagggcctaagaaggcttggttttgatgaa gtgttcgacacattgtttggagccgatttaaccataatggaagagggctcagaattgttacatagactaactgaaca ccttgaggcacatcctcactccgacgaaccattgcctatgttcacaagttgctgtccaggttggatcgctatgttag aaaaaagctatcctgatctaattccatacgtgagctcatgcaagtcccctcaaatgatgttggccgcaatggttaaa agttatttagctgagaagaaaggtatagccccaaaggatatggtaatggtcagcatcatgccatgtaccagaaaaca atctgaagcagacagggattggttttgcgttgacgctgatcctactcttagacagttggatcatgtgattacaaccg ttgagttaggaaatatattcaaggaaagaggcatcaacctagccgaacttccagagggtgaatgggacaatcctatg ggagtaggttcaggcgcaggtgtcttgtttggaactacaggcggcgtgatggaagctgctttaaggactgcctacga gctattcaccggtacaccattgcctagattatcccttagtgaagttaggggaatggatggtattaaagaaactaaca ttaccatggtaccagcacctggctctaagtttgaggaattgttaaaacatagagctgccgcaagagctgaagccgca gctcacggaacaccaggtcctctagcatgggacggcggtgctggattcactagcgaggatggtaggggcggcataac attgagagtcgccgttgcaaatggattaggtaacgctaaaaagcttatcaccaaaatgcaagccggcgaagcaaagt atgattttgtggagattatggcttgtccagccggatgtgttggtggaggcggacaacctagatcaactgacaaagca ataacacagaagaggcaagctgccctatacaatttggatgaaaaatccactttaagaagaagtcatgaaaacccatc tatcagggagctttatgacacctacttgggtgaacctttaggtcacaaggcacatgaactattgcacacacattatg tagctggcggagtcgaggaaaaagatgaaaagaaataa [141] CrHydA (HydAl from Chlamydomonas reinhardtii) is encodedbythe following amino acid sequence and SEQ ID NO: 53:
Mgaaasraapaaeaplshvqqalaelakpkddptrkhvcvqvapavrvaiaetlglapgattpkqlaeglrrlgfde vfdtlfgadltimeegsellhrltehleahphsdeplpmftsccpgwiamleksypdlipyvssckspqmmlaamvk sylaekkgiapkdmvmvsimpctrkqseadrdwfcvdadptlrqldhvittvelgnifkerginlaelpegewdnpm gvgsgagvlfgttggvmeaalrtayelftgtplprlslsevrgmdgiketnitmvpapgskfeellkhraaaraeaa ahgtpgplawdggagftsedgrggitlrvavanglgnakklitkmqageakydfveimacpagcvggggqprstdka itqkrqaalynldekstlrrshenpsirelydtylgeplghkahellhthyvaggveekdekk
[142] CrHydEF (from Chlamydomonas reinhardtii) is encodedbythe followingpolynucleotide sequence and SEQ ID NO: 54 (Synthetic construct designedto express the C. reinhardiiprotein whileremovingthe >GC bias that complicates exogenous expression): atgggcgcggccgcatctagagctgcacatgcctctgcttcaaaagcaactccagatgttcctgtagacgatcttcc acctgcccacgctagagcagccgtcgccgcagctaataggagagccagggcaatggcttccgccgaagcagctgccg agacattaggtgactttctaggacttggcaagggtggattgagtccaggcgcaaccgctaacttagatagagaacaa gtgctaggtgttcttgaggccgtatggagaaggggtgacttgaatttagaaagagcattgtatagccatgctaacgc cgtcactaataaatactgtggaggcggtgtgtattacagaggattagttgagttctctaacatttgccagaatgatt gttcatattgcggtataaggaacaatcaaaaggaggtatggagatacacaatgcctgtcgaagaagttgtggaggtt gcaaaatgggccctagaaaacggcatcaggaatattatgcttcagggtggagaacttaagaccgagcaaagattagc ttacctagaagcctgtgtaagagcaataagggaggaaactacacaattggatttagaaatgagagctagagccgcat ccaccactacagctgaggccgcagctagtgcacaggctgacgccgaagcaaaaaggggtgaaccagagcttggcgtc gtggttagcttgtctgtaggtgaattacctatggaacaatacgagagactatttagagctggagccaggagatatct tatcaggattgaaacctcaaatccagatttgtacgcagctttacaccctgaaccaatgtcctggcatgccagagtcg agtgcctaagaaacttgaagaaagcaggttatatgttaggcactggagttatggtgggccttcctggccaaacattg cacgacttagctggtgatgttatgttctttagggatataaaggccgacatgatcggaatgggtccattcattactca gcctggcaccccagcaacagataaatggactgctctatacccaaatgctaacaagaatagtcatatgaaatctatgt ttgacttgaccacagccatgaacgcattagtaagaattactatgggtaatgtcaacataagcgctacaaccgccctt caagcaatcattcctactggaagagaaatagccctagagaggggtgccaatgtggttatgccaatcttgacacctac tcagtatagagaatcataccaattatatgaaggcaagccatgtattaccgatacagcagtacaatgtagaaggtgcc ttgatatgagattgcattccgtcggaaaaaccagtgctgccggtgtttggggtgaccctgcatctttcttacaccca atagtgggcgttcctgtaccacatgatctatcatctcctgctttggccgcagctgccagcgcagactttcacgaggt cggagctggtccatggaaccctatcaggttagaaagacttgttgaagtgccagatagataccctgatccagacaatc atggtaggaaaaaggccggcgcaggaaaaggcggcaaggctcacgattcccatgacgatggagatcatgacgatcac catcaccatcatggtgccgcaccagctggtgccgcagctggcaaaggaaccggtgccgcagctattggcggcggagc cggtgctagcagacagagagtagctggcgccgcagctgcctcagcaaggttgtgtgctggagccagaagagcaggta gggtcgttgcttctcctctaagaccagccgcagcttgcaggggtgtggccgttaaggcagctgctgccgcagctggc gaggacgccggagcaggtacaagcggtgtaggctccaatattgtcaccagtcctggaatagcttcaaccacagccca cggtgttccaagaatcaacattggcgtgttcggagtaatgaatgcaggtaaatctactttagtcaacgctttggccc aacaagaagcatgtatagttgatagcacccctggtacaactgctgacgtcaagaccgttcttttagaactacatgca ttgggcccagctaaattacttgatacagccggattggatgaggtaggtggtctaggcgacaagaaaagaaggaaggc attaaatactttgaaagaatgcgatgtcgctgttcttgtggtagacaccgatacagccgcagctgccatcaaatccg gaagattagcagaggccctagaatgggaaagtaaggtcatggagcaggctcacaaatacaacgtttcacctgtgttg ttattgaatgtaaagagcagaggccttccagaagcccaagcagccagcatgctagaagccgttgcaggcatgttaga tccttccaaacagattccaaggatgtcattggacttagcttctactcctcttcatgagagaagtacaataactagcg cctttgtcaaggaaggagcagttaggtcctcaagatacggtgctccactacctggttgtttgccaagatggtcttta ggcaggaacgccagattgcttatggtgattccaatggatgcagaaacccctggaggtagactattaaggccacaagc tcaagtaatggaggaagccatcagacactgggcaacagtcttgagtgttagattagacttggatgctgccaggggta aacttggccctgaagcatgtgagatggaaagacagaggttcgatggagtaattgctatgatggagagaaatgacggt ccaactctagttgtgaccgattctcaagccatagacgtcgttcatccttggacattagatagatcctcaggcaggcc attggtgcctatcactacctttagtattgcaatggcttatcaacagaacggaggtagacttgatccatttgtagaag gcctagaagccttagagacattgcaagacggcgatagagtcttaatatctgaagcatgcaatcataataggatcact tcagcttgtaacgacattggaatggttcaaatacctaataagttggaagctgcacttggtggtaaaaagctacagat tgagcacgctttcggcagagaatttccagaattagagtctggaggtatggatggcttgaaacttgccatccattgcg gaggttgtatgattgatgcacaaaagatgcagcaaagaatgaaagacctacacgaagctggtgtacctgttaccaac tatggcgtgttctttagctgggccgcatggccagatgctttaaggagagccttggaaccttggggagtcgagcctcc agttggtacacctgcaactccagctgccgcacctgctaccgccgcatccggtgtgtaa
[143] CrHydEF (from Chlamydomonas reinhardtiϊ) is encodedbythe following amino acid sequence and SEQ ID NO: 55:
Mgaaasraahasaskatpdvpvddlppaharaavaaanrraramasaeaaaetlgdflglgkgglspgatanldreq vlgvleavwrrgdlnleralyshanavtnkycgggvyyrglvefsnicqndcsycgirnnqkevwrytmpveewev akwalengirnimlqggelkteqrlayleacvraireettqldlemraraastttaeaaasaqadaeakrgepelgv wslsvgelpmeqyerlfragarrylirietsnpdlyaalhpepmswhairveclrnlkkagymlgtgvmvglpgqtl hdlagdvmffrdikadmigmgpfitqpgtpatdkwtalypnanknshmksmfdlttamnalvritmgnvnisattal qaiiptgreialerganwmpiltptqyresyqlyegkpcitdtavqcrrcldmrlhsvgktsaagvwgdpasflhp ivgvpvphdlsspalaaaasadfhevgagpwnpirlerlvevpdrypdpdnhgrkkagagkggkahdshddgdhddh hhhhgaapagaaagkgtgaaaigggagasrqrvagaaaasarlcagarragrwasplrpaaacrgvavkaaaaaag sdagagtsgvgsnivtspgiasttahgvprinigvfgvmnagkstlvnalaqqeacivdstpgttadvktvllelha lgpaklldtagldevgglgdkkrrkalntlkecdvavlwdtdtaaaaiksgrlaealeweskvmeqahkynvspvl llnvksrglpeaqaasmleavagmldpskqiprmsldlastplherstitsafvkegavrssrygaplpgclprwsl grnarllmvipmdaetpggrllrpqaqvmeeairhwatvlsvrldldaargklgpeacemerqrfdgviammerndg ptlwtdsqaidwhpwtldrssgrplvpittf siamayqqnggrldpfveglealetlqdgdrvliseacnhnrit sacndigmvqipnkleaalggkklqiehafgrefpelesggmdglklaihcggcmidaqkmqqrmkdlheagvpvtn ygvf f swaavφdalrralepwgveppvgtpatpaaapataasgv
[144] CrHydG (from Chlamydomonas reinhardtii) is encoded by the following polynucleotide sequence and SEQ ID NO: 56 (Synthetic construct designed to express the C. reinhardii protein while removing the >GC bias that complicates exogenous expression): atggaattcgcggccgcatctagaactgctcatggtaaagcatctgccacaagagaatatgctggagattttttgcc aggcaccactatttcacacgcatggtccgttgagagggaaacacatcacagatacaggaatcctgccgagtggataa acgaagctgcaatccataaggccttagaaaccagtaaagctgacgcacaagatgctggtagagtaagagagattcta gccaaggcaaaagaaaaggctttcgtcactgaacacgccccagtgaatgcagagagcaaatctgaatttgttcaggg acttacattggaagagtgtgctaccttaataaacgtagactcaaataacgtcgaactaatgaatgagatcttcgata ctgcccttgcaattaaggaaaggatatatggcaacagagtggttttgtttgctcctttatacatcgccaatcattgc atgaacacatgtacctattgcgcattcagatccgctaataaaggtatggaaaggagtattttgactgacgatgattt aagagaggaagtagccgcactacaaaggcagggtcatagaaggattcttgctttgacaggagaacacccaaagtaca cttttgacaatttcttacatgctgtcaacgttatagccagcgtgaaaaccgagcctgaaggctctatcaggagaatt aatgttgaaatcccacctctatcagtatccgatatgagaaggttgaagaacacagacagtgtcggtacttttgtgtt attccaagagacctatcacagagatacatttaaagttatgcatccatctggacctaagagcgatttcgactttagag tacttactcaagatagggcaatgagagctggtttggacgatgtcggcatcggtgccttatttggactatacgattat aggtacgaagtttgtgcaatgcttatgcactcagaacatttggagagagaatataatgctggtccacatacaatttc cgtgcctagaatgaggccagccgacggcagtgagttatctatagcacctccatacccagttaacgatgctgacttca tgaagctagtagcagtcttgagaatcgctgtgccttataccggtatgattttatcaactagagaatctccagaaatg aggagcgcccttttgaaatgcggaatgtcccagatgagtgcaggttcaagaacagatgttggcgcttaccacaagga tcatactttatctaccgaggccaatctaagcaaattggcaggacaatttacattacaagacgaaagacctactaacg aaattgtaaagtggcttatggaggaaggttatgtcccatcctggtgtaccgcttgttacaggcagggcagaacaggt gaagatttcatgaatatatgcaaagccggagacatccacgatttttgtcatcctaacagtctattgactttacaaga gtatcttatggattacgcagacccagatttgaggaagaaaggtgaacaggttattgctagagagatgggccctgacg cctcagaaccattatctgcacaaagcagaaagaggctagaaagaaaaatgaagcaagtgttggagggtgaacatgat gtttatttaactagtagcggccgctgcagaaacctaggctgctgccaccgctga
[145] CrHydG (from Chlamydomonas reinhardtii) is encodedbythe following amino acid sequence and SEQ ID NO: 57: Mefaaasrtahgkasatreyagdflpgttishawsverethhryrnpaewineaaihkaletskadaqdagrvreil akakekafvtehapvnaesksefvqgltleecatlinvdsnnvelmneifdtalaikeriygnrwlfaplyianhc mntctycafrsankgmersiltdddlreevaalqrqghrrilaltgehpkytfdnflhavnviasvktepegsirri nveipplsvsdmrrlkntdsvgtfvlfqetyhrdtfkvmhpsgpksdfdfrvltqdramraglddvgigalfglydy ryevcamlmhsehlereynagphtisvprmrpadgselsiappypvndadfmklvavlriavpytgmilstrespem rsallkcgmsqmsagsrtdvgayhkdhtlsteanlsklagqftlqderptneivkwlmeegyvpswctacyrqgrtg edfmnickagdihdfchpnslltlqeylmdyadpdlrkkgeqviaremgpdaseplsaqsrkrlerkmkqvlegehd vyltssgrcrnlgcchr
[146] SoFD (fromSpinacea oleracea) is encodedbythe followingpolynucleotide sequence and SEQ IDNO: 58 (Synthetic construct, codon optimized): atggctgcatataaagttactttggtaacaccaaccggtaatgtcgaatttcaatgtcctgatgacgtgtacatttt agacgccgctgaggaagagggaatagatctaccatattcttgcagagcaggctcatgttccagttgcgccggtaagc ttaaaactggaagcttgaaccaggatgaccaatctttcttagatgatgaccagatcgatgaaggctgggttctaaca tgtgctgcataccctgtatcagacgtcaccattgaaactcataaggaggaagaacttacagcctaa [147] SoFD (from Spinacea oleracea) is encoded by the following amino acid sequence and
SEQ ID NO: 59: maaykvtlvtptgnvefqcpddvyildaaeeegidlpyscragscsscagklktgslnqddqsfIdddqidegwvlt caaypvsdvtiethkeeelta
[148] zmSIR (from Zea mays) is encoded by the following polynucleotide sequence and SEQ
ID NO: 60 (RT-PCR product from corn husk tissue): atggcgaagaaggatgcttctgaagttaagcgaagcaaggttgagataatcaaggaaaagagcaacttcctccggta ccctttgaacgaggagttggtctcagaggccccaaatatcaacgagagtgctgttcagctgatcaagtttcatggaa gctaccagcaaactgaccgagatgtccgtgggcagaagaattactcgtttatgctccggacaaagaacccttgtggg aaagttccaaaccaactttatttagctatggatacactagccgatgagtttggcatcggaacactccgcctgacgac caggcagacattccagttgcacggtgttcttaagaagaacttgaagactgttctaagcactgtcataaagaatatgg gctcaacattgggtgcttgtggcgatctcaacaggaatgtacttgctcctgcagcgccttatgtcaaaaaagatatc ctttttgctcaacaaacagcagagaacattgcagcacttcttacaccacagtccggggcttattatgacctgtgggt ggatggcgagaagataatgtcagctgaagagcctcctgaggtgacaaaagcccgcaatgacaactcgcatggaacga acttccctgactctccggaaccaatctatggcacccagtatctaccaaggaagttcaaggttgcggttaccgcggct ggtgataactctgttgatattctgaccaatgacatcggtgttgttgttgtttcagatgatgcaggagaacctattgg ctttaacatctatgttggtggtggcatgggaaggacacaccgagtggaaactacattccctcggctggctgatccat tgggttatgttccaaaagaagatatattatatgctataaaggccattgtcgtcacacagagggaaaatggaagaagg gatgaccgcaagtatagtaggatgaagtatatgattgaccgttggggaatagataggtttcgggctgaagttgaaaa atattacgggaagaagtttgaaagtttccgaccattgccagagtggcagtttaacagctaccttggctggcaagaac agggtgatgggaaattattctatggagtgcatgttgataatggtcgtgttggtgggcaagcaaagaaaactctacga gagataattgagaagtataatttggatgttagtattaccccaaaccaaaatcttattttatgtgggattgatcaagc atggagagaacccataactacagctcttgcacaagctggcctgctggaaccgaaggatgtcgaccccttgaatttaa ctgccatggcatgccctgccttgccactgtgccctttggcacaaacagaagctgaacgggggatcttacccattctt aaacgaattagagcagtcttcaataaggttggtatcaaggattcggagtctgtggttgtgaggataactggatgccc taatggatgcgctagaccatatatggcagagcttggtttcgttggtgatggcccaaaaagttaccagatctggctgg gtggaacaccaaaccagagtacgctagcagaatcatttatggacaaggtgaagcttgatgacatcgagaaggttttg gagcctctctttacctattggaatggcacacgccaggaaggcgaatcttttggaagcttcacaaaccgaacaggatt cgacaaattgaaagaggtagtgaataagtgggcagagtcaccgtcagccgcatga
[149] zmSIR (from Zea mays) is encoded by the following polynucleotide sequence and SEQ ID NO: 61: makkdasevkrskveiikeksnf lryplneelvseapninesavqlikfhgsyqqtdrdvrgqknysfmlrtknpcg kvpnqlylamdtladefgigtlrlttrqtfqlhgvlkknlktvlstviknmgstlgacgdlnrnvlapaapyvkkdi
If aqqtaeniaalltpqsgayydlwvdgekimsaeeppevtkarndnshgtnfpdspepiygtqylprkfkvavtaa gdnsvdiltndigwwsddagepigfniyvgggmgrthrvettfprladplgyvpkedilyaikaiwtqrengrr ddrkysrmkymidrwgidrfraevekyygkkfesfrplpewqfnsylgwqeqgdgklfygvhvdngrvggqakktlr eiiekynldvsitpnqnlilcgidqawrepittalaqagllepkdvdplnltaraacpalplcplaqteaergilpil kriravfnkvgikdsesvwritgcpngcarpymaelgfvgdgpksyqiwlggtpnqstlaesfmdkvklddiekvl eplf tywngtrqegesfgsftnrtgfdklkewnkwaespsaa
Example 3: Bacterial Growth Conditions in an Environment Lacking Oxygen and Containing Hydrogen [150] Bacteria were plated on standard petri dishes containing M9 minimal agar media as described above. Petri dishes were enclosed in airtight Vacu-Quik jars (Almore International), and a defined atmosphere was established in the jars using the method of serial evacuation and replacement with customized gas mixtures. Anaerobic hydrogen atmospheres contained 5% hydrogen and the balance nitrogen by volume.
Example 4: Mutagenesis of a Bacterial Strain Whose Growth is Conditionally Dependent on the Activity of an Exogenous FeFe Hydro genase and Iisolation of Mutants with Enhanced Oxygen Resistance.
[151] An E. coli strain BL21(DE3) with the genotype Δcysl [pCaHydA, pCrHydEF-CrHydG, pSoFD-ZmSIR] was exposed to ultraviolet light at an intensity empirically determined to kill 90% of growing cells (ImJ). Cells were allowed to recover for 4 hours in the dark, in selective media, before being plated out on minimal media and exposed to a customized atmosphere. In the case of selections for oxygen tolerance, the final atmosphere contained, by partial pressure, 20% oxygen, 5% hydrogen and the balance nitrogen. Colonies visible after 4 days were recultured in oxygen-rich conditions in both the presence or absence of hydrogen gas, to assay the hydrogen-dependence of the observed growth phenotype.
Example 5: Construction of a Bacterial Strain Whose Growth is Conditionally Dependent on the
Activity of an Exogenous FeFe Hydrogenase Using Reduced Nitrosen Auxotrophy as a
Phenotype.
[152] An E. coli strain of the genotype AnirB [pNIR-Fd (Zea nitrite reductase, Spinacea Ferredoxiηj; pHydAl (Clostridium acetobutylicum hydrogenase active subunit); pHydEF, HydG (Chlamydomonas reinhardtii hydrogenase matruation factors)/ is mutagenized by a standard procedure with, for example, N-nitrosoguanidine, ethylmethane sulfonate, or ultraviolet light. Miller, Experiments in Molecular Genetics (Cold Spring Harbor Press, Cold Spring Harbor, New York) describes protocols for use of these and other mutagens. The mutagenized bacteria are plated on selective agar plates containing minimal medium with added ferrous iron in the form of (FeSO4 at 10 μM, or M63 medium, or another minimal medium. The media contains an oxidized nitrogen source such as nitrate or nitrite, but no reduced nitrogen in the form of ammonia or amino acids. Vitamins and nutrients that without metabolically accessilble reduced nitrogen may be added, depending on other features of the genotype of the E. coli strain background. The plates are poured according to standard procedures, but are incubated for at least 4 days at room temperature in anaerobic conditions, for example in a an MBRAUN anerobic glove box (M. Braun Group). Mutagenesis procedures and the isolation and characterization of desired mutations proceeds otherwise as in the example above.
Example 6: Construction of a Bacterial Strain Whose Growth is Conditionally Dependent on the Activity of an Exogenous FeFe Hydro genase Using Reduced Glutamate Auxotrophv as a Phenotype
[153] An E. coli strain of the genotype AgdhA AgItB [pGlsF-Fd (Glutamate synthase from Synechocystis sp. PCC 6308, Spinacea Ferredoxiη); pHydAl (Clostridium acetobutylicum hydrogenase active subunit); pHydEF, HydG (Chlamydomonas reinhardtii hydrogenase matruation factors)/ is mutagenized by a standard procedure with, for example, N- nitrosoguanidine, ethylmethane sulfonate, or ultraviolet light. Miller, Experiments in Molecular Genetics (Cold Spring Harbor Press, Cold Spring Harbor, New York) describes protocols for use of these and other mutagens. The mutagenized bacteria are plated on selective agar plates containing minimal medium with added ferrous iron in the form Of(FeSO4 at 10 μM, or M63 medium, or another minimal medium. The media contains no glutamate or glutamine, which must be synthesized by the exogenous pathway to allow cell survival. Vitamins and nutrients that without metabolically accessible glutamate maybe added, depending on other features of the genotype of the E. coli strain background. The plates are poured according to standard procedures, but are incubated for at least 4 days at room temperature in anaerobic conditions, for example in a an MBRAUN anerobic glove box (M. Braun Group). Mutagenesis procedures and the isolation and characterization of desired mutations proceeds otherwise as in the example above.
Example 7: Hydro genase-dependent growth [154] An engineered strain of E. coli that required the function of a hydrogenase enzyme for viability under certain conditions was generated and isolated. Such strains and methods to isolate strains with such desirable properties are useful as a tool to perform directed evolution experiments on hydrogenase enzymes. A number of hydrogenase properties are engineered using this system, e.g., oxygen-tolerance. The property of oxygen tolerance greatly increases the commercial utility of the hydrogenase enzyme. [155] The behavior of the system has been characterized and quantified in several ways. The oxygen-tolerance of the hydrogenase in the system has been quantified. When E. coli growth is supported by the hydrogenase, cells are only viable in atmospheres with reduced levels of oxygen. Normal air is 21% oxygen. This strain is only viable in atmospheres 8-10% oxygen. [156] Using the methods described herein, mutants with improved oxygen tolerance were identified and isolated. The mutants obtained are able to grow consistently in atmospheres of 12% oxygen. These data indicate that the methods are effective and useful to identify and isolate new hydrogenases with with desirable properties.
[157] Using a directed evolution approach, the spontaneous mutation rate of a strain was quantified. Random mutations in the E. coli genome could rescue oxygen-tolerant growth independently of changes in the hydrogenase. The frequency at which this occurs is relevant to the commercial application of the system, because these random mutations create false positive mutants that complicate and slow the directed evolution process. This false positive rate was measured and found to be quite low. The methods and systems described herein have been used to reliably to perform directed evolution and yield the target properties sought. [158] Oxygen-tolerance of the hydrogenase in the system has been measured, and the system has been used to successfully identify and isolate oxygen-tolerant mutants. Figure 6 shows hydrogenase-dependent growth of E. coli. The data shown in Figure 6 indicate that the cells require hydrogenase function to grow, and that hydrogenase function is destroyed by oxygen. Bacterial strains were constructed with instances of the selection system employing the C. acetobutylicum hydrogenase and ferredoxin from either Z. mays or S. oleracea. To evaluate oxygen tolerance, cells were plated on minimal media and grown in atmospheres composed of hydrogen, nitrogen, and differing percentages of oxygen. Colony area was measured by quantitative image analysis after 72 hours of growth. Strains not expressing the hydrogenase showed no growth. Expression of the hydrogenase rescued growth, but only at permissive oxygen levels. No growth was observed at oxygen levels above 12 percent using the strains tested. A strong link between hydrogenase function and cell growth enables engineering of the hydrogenase through directed evolution. Error bars are standard deviations. N = 50.
OTHER EMBODIMENTS
[159] The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. AU other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.
[160] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

What is claimed is:CLAIMS
1. A microorganism comprising an oxygen-tolerant FeFe hydrogenase enzyme, wherein viability of said microorganism is dependent upon function of said hydrogenase enzyme.
2. The microorganism of claim 1, wherein said microorganism is viable in an atmosphere comprising greater than 10% oxygen.
3. The microorganism of claim 1 , wherein said microorganism is viable in an atmosphere comprising greater than 12% oxygen.
4. The microorganism of claim 1, wherein said microorganism is E. coli.
5. The microorganism of claim 1 , wherein said microorganism comprises a mutation in an NAD(P)H- or ferrodoxin-dependent enzyme gene rendering a corresponding gene product absent or inactive, a first nucleic acid encoding a heterologous ferrodoxin-dependent enzyme, and a second nucleic acid encoding a heterologous FeFe hydrogenase.
6. The microorganism of claim 5, wherein said microorganism further comprises a heterologous errodoxin-encoding sequence.
7. The microorganism of claim 5, wherein said microorganism further comprises one or more nucleic acids encoding a heterologous hydrogenase maturation factor.
8. The microorganism of claim 5, wherein said microorganism is an enteric bacteria.
9. The microorganism of claim 5, microorganism comprises Escherichia coli (E. coli) or Salmonella typhimurium.
10. The microorganism of claim 5, wherein said NAD(P)H-dependent enzyme is a bacterial sulfite reductase and wherein said heterologous ferrodoxin-ependent enzyme is plant-derived sulfite reductase.
11. An E. coli comprising a mutation in an endogenous sulfite reductase gene, an exogenous sulfite reductase gene, an exogenous ferredoxin gene, an exogenous hydrogenase gene, and at least one exogenous hydrogenase maturation cofactor gene.
12. The E. coli of claim 11, wherein said mutation in an endogenous sulfite reductase gene is a deletion of the cysl open reading frame.
13. The E. coli of claim 11, wherein said exogenous sulfite reductase gene is isolated from Zea mays.
14. The E. coli of claim 11 , wherein said exogenous ferredoxin gene is isolated from Spinacia oleracea.
15. The E. coli of claim 11, wherein said exogenous hydrogenase gene comprises a catalytic hydrogenase subunit isolated from Clostridium acetobutylicum.
16. The E. coli of claim 11, wherein said exogenous hydrogenase maturation cofactor gene is isolated from Chlamydomonas reinhardtii.
17. The E. coli of claim 11, wherein said exogenous hydrogenase gene comprises nucleotide sequences isolated from one species.
18. The E. coli of claim 11, wherein said exogenous hydrogenase gene comprises nucleotide sequences isolated from more than one species.
19. The E. coli of claim 11, wherein said exogenous hydrogenase gene encodes a [FeFe] hydrogenase.
20. A method of selecting for a biological property of a hydrogenase enzyme, comprising: (a) providing an engineered E. coli comprising a mutation in a gene encoding a ferredoxin-dependent enzyme; (b) introducing into said engineered E. coli an exogenous gene encoding said ferredoxin- dependent-enzyme or a homolog thereof, an exogenous ferredoxin gene, an exogenous hydrogenase gene, and at least one exogenous hydrogenase maturation factor; and
(c) growing said engineered E. coli in media lacking the product of said ferredoxin- dependent enzyme and under selective pressure; wherein the survival of said engineered E. coli under selective pressure indicates the presence of a functional hydrogenase enzyme, thereby identifying those hydrogenase enzymes having the desired biological property.
21. The method of claim 20, wherein said ferredoxin-dependent enzyme is sulfite reductase, nitrite reductase, or glutamate synthase.
22. The method of claim 20, wherein said ferredoxin-dependent enzyme is sulfite reductase.
23. The method of claim 20, wherein said mutation in a gene encoding a ferredoxin- dependent enzyme is a deletion of the cysl open reading frame of sulfite reductase.
24. The method of claim 20, wherein said exogenous gene encoding said ferredoxin- dependent-enzyme or a homolog thereof is the sulfite reductase gene.
25. The method of claim 20, wherein said sulfite reductase gene is isolated from Zea mays.
26. The method of claim 20, wherein said exogenous ferredoxin gene is isolated from Spinacia oleracea.
27. The method of claim 20, wherein said exogenous hydrogenase gene comprises a catalytic hydrogenase subunit isolated from Clostridium acetobutylicum.
28. The method of claim 20, wherein said exogenous hydrogenase maturation cofactor gene is isolated from Chlamydomonas reinhardtii.
29. The method of claim 20, wherein said exogenous hydrogenase gene comprises nucleotide sequences isolated from one species.
30. The method of claim 20, wherein said exogenous hydrogenase gene comprises nucleotide sequences isolated from more than one species.
31. The method of claim 20, wherein said biological property is oxygen tolerance, thermostability, or resistance to oxidizing agents or poisons.
32. The method of claim 20, wherein said hydrogenase enzyme is a [FeFe] hydrogenase.
33. The method of claim 20, wherein said product of said ferredoxin-dependent-enzyme is cysteine.
34. The method of claim 20, wherein said selective pressure is oxygen, high temperature, low temperature, hydrogen peroxide, nitrous oxide, an oxidizing agent, a heavy metal, cyanide, or any compound known to interfere with hydrogenase activity.
35. The method of claim 20, wherein said selection occurs in vivo.
36. A method of selecting for oxygen tolerance of a [FeFe] hydrogenase enzyme, comprising:
(a) providing an engineered E. coli comprising a mutation in a sulfite reductase gene;
(b) introducing into said engineered E. coli an exogenous sulfite reductase gene or a homolog thereof, an exogenous ferredoxin gene, an exogenous hydrogenase gene, and at least one exogenous hydrogenase maturation factor; and (c) growing said engineered E. coli in media lacking cysteine and in the presence of oxygen; wherein the survival of said engineered E. coli in the presence of oxygen indicates the presence of a functional hydrogenase enzyme, thereby identifying those hydrogenase enzymes having oxygen tolerance.
37. A method of selecting for increased catalytic rate of a [FeFe] hydrogenase enzyme, comprising:
(a) providing an engineered E. coli comprising a mutation in a sulfite reductase gene; (b) introducing into said engineered E. coli an exogenous sulfite reductase gene or a homolog thereof, an exogenous ferredoxin gene, an exogenous hydrogenase gene, and at least one exogenous hydrogenase maturation factor, wherein the expression level of said hydrogenase gene is low; and
(c) growing said engineered E. coli in media lacking cysteine; wherein the survival of said engineered E. coli indicates the presence of a functional hydrogenase enzyme, thereby identifying those hydrogenase enzymes having an increased catalytic rate.
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