Oxygen tolerant hydrogenase by mutating electron supply pathway

Abstract

Compositions and methods are provided for an O.sub.2 tolerant FeFe hydrogenase. The hydrogenases of the invention comprise specific amino acid substitutions relative to the native, or wild-type enzymes.

Claims

1. A modified FeFe hydrogenase comprising at least one amino acid substitution relative to the wild-type sequence, said modified FeFe hydrogenase has [i] at least 80% sequence identity to SEQ ID NO: 1, and at least one amino acid substitution at L192, G194, T356 or S357; [ii] at least 96% sequence identity to SEQ ID NO: 2, and at least one amino acid substitution at L191, G193, N355 and/or D356; [iii] at least 80% sequence identity to SEQ ID NO: 3, and at least one amino acid substitution at L192, G194, T356 or A357; [iv] at least 80% sequence identity to SEQ ID NO: 4, and at least one amino acid substitution at L192, G194, T356 or A357; or [v] at least 80% sequence identity to SEQ ID NO: 5, and at least one amino acid substitution at L191, G193, N355 or D356; wherein the modified enzyme retains at least 10% of the initial specific activity following exposure to 0.01 atm. O.sub.2 for 5 minutes.

2. The modified hydrogenase of claim 1, wherein the FeFe hydrogenase is derived from a Clostridium species.

3. The modified hydrogenase of claim 1, wherein the amino acid substitution is selected from A156C, M166C, G194C, Q195C, I197C, A156C+L191C, G158C+I197C, N160C+T161C, N160C+A165C, N160C+L192C, N160C+Q195C, N160C+I197C, T161C+G194C, T161C+I197C, T163C+N189C, T163C+Q195C, T163C+I197C, Y164C+Q195C, A165C+N189C, A165C+L192C, A165C+Q195C, A165C+I197C, M166C+Q195C, M166C+I197C, F185C+I197C, N189C+G194C, N189C+I197C, L191C+L192C, L191C+I197C, Q195C+I197C, L192F, L192W, L192S, L192D, L192G, P301C, T356C, S357C, P301C+T356C, P301C+A498C, P301C+G502C, G302C+T356C, G302C+S357C, G302C+A498C, W303C+G507C, P354C+G508C, T356C+S357C, S357C+A498C, S357C+N505C, T356V, T356I, T356L, T356P, S357A, S357V, S357I, S357L, S357T, S357P, T356V+S357T, T356V+S357V, T356V+S357P, where numbering is made relative to SEQ ID NO:1.

4. The modified hydrogenase of claim 3, wherein the amino acid substitutions is one or more of L192G, G194C, T356V, S357T.

5. The modified hydrogenase of claim 4, wherein the hydrogenase comprises the amino acid sequence set forth in SEQ ID NO:6.

6. The modified hydrogenase of claim 3, wherein the at least one amino acid substitutions that provides for faster H.sub.2 generation is selected from A156C, G158C, M166C, L192C, Q195C, G185C+I197C, N160C+L192C, T163C+Y164C, Y164C+I197C, A165C+L191C, L192E, L192G, P301C, T356C, M498C, N505C+P301C, N505C, T356C+S357C, S357A, S357V, S357I, S357P, T356V+S357T.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

(2) FIG. 1: The general concept of photosynthetic H.sub.2 production illustrating one obstacle: O.sub.2 inactivation of the hydrogenase.

(3) FIG. 2: Cpl published structure with key FeS clusters labeled.

(4) FIG. 3: Two reaction series used for evaluating O.sub.2 sensitivity of the hydrogenases during H.sub.2 production (PGL=phosphogluconolactone, G6P=glucose 6-phosphate, G6PD=glucose 6-phosphate dehydrogenase, FNR=ferredoxin NADP reductase, and Fd=ferredoxin)

(5) FIG. 4: H.sub.2 production activity and O.sub.2 tolerance of Cpl mutants with single Cys replacements near the (A) Proximal Delivery Center (PDC) or (B) the Proximal Catalytic Center (PCC). The dotted line represents the aerobic H.sub.2 yield by the WT Cpl (7.5% of the anaerobic yield over the same time period).

(6) FIG. 5: H.sub.2 production activity and O.sub.2 tolerance of Cpl mutants with double Cys replacements near the (A) PDC or (B) PCC. The upper and lower tile for each mutant represent the anaerobic H.sub.2 production activity and the aerobic H.sub.2 yield, respectively.

(7) FIG. 6: H.sub.2 production activity and O.sub.2 tolerance of Cpl saturation mutagenesis library at 4 selected sites. The upper and lower tile for each mutant represent the anaerobic H.sub.2 production activity and the aerobic H.sub.2 yield, respectively.

(8) FIG. 7: Double amino acid replacement of T356V S357T confers higher O.sub.2 tolerance during NADPH-driven H.sub.2 production.

(9) FIG. 8: (A) Electron pathway for PSI-mediated, light dependent H.sub.2 production, (B) Early results showing light dependent H.sub.2 production.

(10) FIG. 9: PSI-mediated, light dependent H.sub.2 production with the WT Cpl versus the T356V S357T mutant in the presence of 5% O.sub.2.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(11) Specific amino acid positions in hydrogenase have been identified that, when altered from the WT residues, increase O.sub.2 tolerance of FeFe hydrogenase.

(12) Throughout this application, various publications, patents, and published patent applications are referred to by an identifying citation. The disclosures of these publications, patents, and published patent specifications referenced in this application are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains.

(13) The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook, Fritsch, and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, (F. M. Ausubel et al. eds., 1987); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.); PCR 2: A PRACTICAL APPROACH (M. J. McPherson, B. D. Hames and G. R. Taylor eds., 1995); and ANIMAL CELL CULTURE (R. I. Freshney. Ed., 1987); Transgenic Animal Technology: A Laboratory Handbook, by Carl A. Pinkert, (Editor) First Edition, Academic Press; ISBN: 0125571658.

(14) All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

(15) As used in this specification and the appended claims, the singular forms a, an and the include plural references unless the content clearly dictates otherwise. Thus, for example, reference to a polypeptide includes a mixture of two or more such agents.

Definitions

(16) Hydrogenase. Hydrogenases catalyse the reversible oxidation/production of molecular hydrogen (H.sub.2) and play a vital role in anaerobic metabolism. Metal containing hydrogenases are subdivided into three classes: FeFe hydrogenases, NiFe hydrogenases, and Fe hydrogenases. H.sub.2 oxidation is coupled to the reduction of electron acceptors such as O.sub.2, nitrate, sulphate, CO.sub.2 and fumarate, whereas proton reduction (H.sub.2 evolution) is coupled to molecules such as ferredoxin. The methods of the invention may be applied to any of the FeFe hydrogenases.

(17) In one embodiment, the term hydrogenase as used herein refers to an enzyme that meets one or more of the criteria provided herein. Using these criteria, one of skill in the art can determine the suitability of a candidate enzyme for use in the methods of the invention. Many enzymes will meet multiple criteria, including two, three, four or more of the criteria, and some enzymes will meet all of the criteria. The terms hydrogenase can refer to a full length enzyme or fragment thereof with the capability of catalyzing H.sub.2 oxidation/production.

(18) Hydrogenases of the invention include enzymes having at least about 20% sequence identity at the amino acid level, more usually at least about 40% sequence identity, and preferably at least about 70% sequence identity to one of the following hydrogenases: Chlamydomonas reinhardtii iron-iron-hydrogenase (Genbank accession AY055756); Clostridium pasteurianum hydrogenase (Genbank accession AAA23248.1); Megasphaera elsdenii hydrogenase (Genbank accession AF120457); Desulfovibrio vulgaris hydrogenase (Genbank accession CAA26266.1). For example, see Forestier et al. (2003) Eur. J. Biochem. 270 (13), 2750-2758; Meyer et al. (1991) Biochemistry 30:9697-9704; Voordouw et al. (1985) Eur. J. Biochem. 148:515-520; Atta et al. (2000) Biochim Biophys Acta. 1476(2):368-71; Fauque et al. (1988) FEMS Microbiol. Rev. 4, 299-344; Cammack et al. (1994) Methods Enzymol. 243, 43-68; and de Lacey et al. (1997) J. Am. Chem. Soc. 119, 7181-7189, each herein incorporated by reference.

(19) Homology-based identification (for example, by a PILEUP sequence analysis) of enzymes can be routinely performed by those of skill in the art upon contemplation of this disclosure to identify those suitable for use in the methods of the present invention. Such enzymes are usually produced in microorganisms, particularly bacteria. Hydrogenases of the invention also include an enzyme belonging to the enzyme classifications EC 1.12.7.2 and EC 1.12.2.1.

(20) The nucleic acid sequences encoding the above hydrogenases may be accessed from public databases as previously cited. Identification of additional hydrogenases is accomplished by conventional screening methods of DNA libraries or biological samples for DNA sequences having a high degree of similarity to known hydrogenase sequences.

(21) Iron-iron hydrogenase. The hydrogenases containing no other metal than Fe and containing an active site H-cluster consisting of a 4Fe-4S subcluster joined by a cysteine residue to a 2Fe-2S cluster are called FeFe hydrogenases (FeFe H.sub.2ases). Two families of FeFe H.sub.2ases have been described. Cytoplasmic, soluble, monomeric FeFe H.sub.2ases are found in strict anaerobes such as Clostridium pasteurianum and Megasphaera elsdenii. They are extremely sensitive to inactivation by O.sub.2 and catalyze both H.sub.2 evolution and uptake. Periplasmic, heterodimeric (FeFe) H.sub.2ases from Desulfovibrio spp., can be purified aerobically but catalyze mainly H.sub.2 oxidation.

(22) 3-D structures of H.sub.2-evolving FeFe H.sub.2ase I from Clostridium pasteurianum (Cpl) and Desulfovibrio desulfuricans uptake hydrogenase (DdH) are known. The overall structure of Cpl resembles a mushroom consisting of four domains: the large active site domain forms cap and three smaller domains form stem. The stem domains bind four FeS clusters and are termed FS4A-FS4B, FS4C and FS2. The N-terminal FS2 domain binds a 2Fe-2S cluster and shares the overall fold with plant-type ferredoxins. The FS4A-FS4B domain is adjacent to the active site domain; it contains two 4Fe-4S clusters and has the overall fold similar to that of bacterial type ferredoxins. The FS4C domain is placed between the FS2 and FS4A-FS4B domains and consists of two -helices linked by a loop that binds a single 4Fe-4S cluster via one His and three Cys residues. The large subunit of DdH lacks FS4C and FS2 clusters and corresponding domains. The small subunit of DdH has an unusual fold consisting of alternating random coil and four -helices that form a belt around the large subunit.

(23) The active site domain of the FeFe H.sub.2ases contains an unusual FeS cluster termed the H-cluster. H-cluster consists of the 4Fe-4S subcluster bridged via the Cys thiolate to the FeFe (binuclear iron-iron) subcluster. The two FeFe atoms are designated Fe1 and Fe2 (proximal and distal with respect to the 4Fe-4S subcluster) and are 2.6 apart. With the exception of the bridging Cys, the FeFe subcluster is coordinated by non-protein ligands. In Cpl, both FeFe atoms are octahedrally coordinated to five CO/CN ligands, three S ligands and one water molecule. Fe1 and Fe2 are bridged by two S atoms and one CO or CN ligand. The two bridging sulphurs themselves are bridged by atom(s) of unknown identity. In DdH, Fe1 and Fe2 are bridged by a small molecule that has been modelled as 1,3-propanedithiol (PDT). Fe1 is octahedrally coordinated while Fe2 has square pyramidal coordination geometry.

(24) In some embodiments of the invention, the FeFe hydrogenase is derived from a Clostridium species, for example as shown in the appended sequences of SEQ ID NO:1, 2, 3, 4 and 5. Hydrogenases of interest include, without limitation, those found in the species Clostridium botulinum; Clostridium tyrobutyricum; Clostridium perfringens; Clostridium butyricum; Clostridium saccharobutylicum; Clostridium novyi; Clostridium pasteurianum; Clostridium acetobutylicum; Clostridium cellulovorans; Clostridium paraputrificum; Clostridium kluyveri DSM 555; Clostridium papyrosolvens, etc. and the related species Alkaliphilus metalliredigens, Eubacterium acidaminophilum, Anaerocellum thermophilum, Caldicellulosiruptor saccharolyticus etc.

(25) O.sub.2 tolerance. As used herein, the term O.sub.2 tolerance refers to the ability of a hydrogenase enzyme to retain H.sub.2 production activity in the presence of molecular oxygen. As is known in the art, even trace levels of O.sub.2 are sufficient to inactivate known hydrogenases. Enzymes that have been mutated to increase tolerance have at least about 10% activity, at least about 20% activity, at least about 30% activity or more after a solution of the protein is exposed to 0.01 atm O.sub.2 for at least about 5 minutes, at least about 15 minutes, at least about 30 minutes, at least about 45 minutes, at least about 1 hour, or more. The enzymes may be more resistant to O.sub.2 when the enzyme is present in a microbial host cell (relative to a solution).

(26) Ferredoxin-NADP-reductase (FNR), EC 1.18.1.2, may be obtained from any suitable source, including E. coli, Anaebaena sp., and the like, including FNR from photosynthetic organisms such as higher plants, e.g. Spinacea oleracea (spinach).

(27) In photosynthetic organisms, FNR is the last enzyme in the transfer of electrons during photosynthesis from photosystem I to NADPH. In such organisms it is a soluble protein that is found both free in the chloroplast stroma and bound to the thylakoid membrane. This binding occurs opposite to the active site of the enzyme and does not seem to affect the structure of the active site or have a significant impact on the enzyme's activity. In the plant-like family of FNRs, selective evolutionary pressure has led to differences in the catalytic rate enhancements of FNRs in photosynthetic and non-photosynthetic organisms. Electron transfer by FNR is a rate limiting step in photosynthesis, so the plastidic FNRs in plants have evolved to catalyze higher electron transfer rates. These plastidic FNRs are 20-100 fold more active than bacterial FNRs.

(28) In non-photosynthetic organisms, the FNR primarily works in reverse to provide reduced ferredoxin for various metabolic pathways. These pathways include N.sub.2 fixation, terpenoid biosynthesis, steroid metabolism, oxidative stress response, and FeS protein biogenesis.

(29) For the purposes of the present invention, an active fragment of FNR, i.e. a fragment that confers substantially all of the enzymatic activity of the native protein, e.g. at least about 50% of the activity, at least about 75%, at least about 80%, at least about 90%, at least about 95%, when measured under standard conditions, may be included in a cell or cell-free system for generation of H.sub.2.

(30) Ferredoxin. Ferredoxins of interest include, without limitation, Clostridium pasteurianum ferredoxin; Synechocystis ferredoxin, E. coli ferredoxin, Spinacia oleracea ferredoxin; Anabaena ferredoxin, derivatives; variants; homologs; mutants; and the like. Included, without limitation, are 2Fe-2S, and 4Fe-4S ferredoxins. A candidate ferredoxin may be assayed for H.sub.2 production with a hydrogenase and/or FNR of interest, and may be evolved to increase electron transfer rates within such pathways. The ferredoxin may be synthesized in a cell with the hydrogenase.

(31) Sugar. As used herein, the term refers to a number of carbohydrates, such as monosaccharides, disaccharides, oligosaccharides, and polysaccharides, usually pentose or hexose sugars or polymers thereof. Monosaccharides that find use include, without limitation, fructose, arabinose, lyxose, ribose, xylose, ribulose, xylulose, deoxyribose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose. Disaccharides may include sucrose, lactose, maltose, etc. Polysaccharides may include starches, glycogen, cellulose, pectin, peptidoglycan, lipopolysaccharides, capsules, exopolysaccharides, and the like. Sugars may be phosphorylated, e.g. glucose-6-phosphate, etc. Sugars may be included in the reaction mix at a concentration sufficient to provide energy for H.sub.2 evolution, e.g. from about 1 mM to about 1000 mM, and may be about 5 mM, 10 mM, 25 mM, 50 mM, 75 mM, 100 mM, 250 mM, 500 mM, 750 mM, 1000 mM, and may also be supplied by continuous addition.

(32) Cell-free reaction mix: as used herein refers to a reaction mixture comprising an O.sub.2 tolerant hydrogenase as described herein, and such components as are required for the generation of H.sub.2. The hydrogenase may be capable of catalyzing the synthesis of H.sub.2 from sugar, which sugar may be a phosphorylated or non-phosphorylated sugar. A reaction mixture of interest comprises extracts from bacterial cells, and the synthesis is performed under conditions in which some O.sub.2 may be present, e.g. up to about 1 vol. % O.sub.2 in the reaction headspace; up to about 2 vol. % O.sub.2, up to about 3 vol. % O.sub.2, up to about 4 vol. % O.sub.2, up to about 5 vol. % O.sub.2, or more.

(33) The volume percent of cell extract in the reaction mix will vary, where the extract is usually at least about 10% of the total volume; more usually at least about 20%; and in some instances may provide for additional benefit when provided at least about 50%; at least about 60%; or at least 75% of the total volume. In certain industrial applications the volume percent of extract may be around about 90% or higher.

(34) The reaction mixture may be further supplemented with one or more of niacin, nicotinamide, NAD, etc., usually at a concentration of from about 0.1 mM to 10 mM, e.g. at about 0.5 mM, about 1.0 mM, about 4 mM, etc. as a precursor or source of NAD and NADP; a nuclease, particularly a ribonuclease, which may be used at a conventional dose for example from about 0.5 g/ml to about 50 g/ml or higher, to break down nucleic acids and generate adenine; and an agent to inactivate the endogenous microbial cell glycolytic pathway and thus maximize conversion yields.

(35) The terms nucleic acid molecule and polynucleotide are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.

(36) A coding sequence or a sequence which encodes a selected polypeptide, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide, for example, in a cell-free polypeptide synthesis reaction; or in vivo when placed under the control of appropriate regulatory sequences (or control elements). The boundaries of the coding sequence are typically determined by a start codon at the 5 (amino) terminus and a translation stop codon at the 3 (carboxy) terminus. A transcription termination sequence may be located 3 to the coding sequence. Other control elements may also be associated with a coding sequence.

(37) Operably linked refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given promoter that is operably linked to a coding sequence (e.g., a reporter expression cassette) is capable of effecting the expression of the coding sequence when the proper enzymes are present. The promoter or other control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. For example, intervening un-translated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered operably linked to the coding sequence.

(38) A vector is capable of transferring gene sequences to target cells. Typically, vector construct, expression vector, and gene transfer vector, mean any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells. Thus, the term includes cloning and expression vehicles, as well as integrating vectors.

(39) Purified polynucleotide refers to a polynucleotide of interest or fragment thereof which is essentially free, e.g., contains less than about 50%, preferably less than about 70%, and more preferably less than about 90%, of the protein with which the polynucleotide is naturally associated. Techniques for purifying polynucleotides of interest are well-known in the art and include, for example, disruption of the cell containing the polynucleotide with a chaotropic agent and separation of the polynucleotide(s) and proteins by ion-exchange chromatography, affinity chromatography and sedimentation according to density.

(40) A polypeptide is used in it broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics. The subunits may be linked by peptide bonds or by other bonds, for example ester, ether, etc. As used herein, the term amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.

(41) Before describing the present invention in detail, it is to be understood that this invention is not limited to particular formulations or method parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

(42) Although a number of methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein.

Modified Hydrogenase Polypeptides

(43) Modified hydrogenase polypeptides are provided, which modifications provide for increased O.sub.2 tolerance in the active protein, as described above. The amino acid substitutions may also be combined with other amino acid substitutions that enhance, or that do not adversely affect the H.sub.2 production activity. The modified hydrogenase polypeptides have a tolerance to O.sub.2 for at least about 2.5 minutes, at least about 5 minutes, at least about 7.5 minutes, at least about 10 minutes, or more. The specific activity after exposure to O.sub.2 may be, relative to the activity in the absence of O.sub.2, at least about 20% of the specific activity, at least about 30%, at least about 40%, at least about 50% or more, following exposure to 0.01 atm. O.sub.2 for 5 minutes.

(44) In addition to the above modifications, the sequence of hydrogenase polypeptides may be altered in various ways known in the art to generate targeted changes in sequence. The sequence changes may be substitutions, insertions or deletions, e.g. truncations at the amino or carboxy terminus. Such alterations may be used to alter properties of the protein, by affecting the stability, specificity, etc. Techniques for in vitro mutagenesis of cloned genes are known. Examples of protocols for scanning mutations may be found in Gustin et al., Biotechniques 14:22 (1993); Barany, Gene 37:111-23 (1985); Colicelli et al., Mol Gen Genet 199:537-9 (1985); and Prentki et al., Gene 29:303-13 (1984). Methods for site specific mutagenesis can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, CSH Press 1989, pp. 15.3-15.108; Weiner et al., Gene 126:35-41 (1993); Sayers et al., Biotechniques 13:592-6 (1992); Jones and Wnistorfer, Biotechniques 12:528-30 (1992); Barton et al., Nucleic Acids Res 18:7349-55 (1990); Marotti and Tomich, Gene Anal Tech 6:67-70 (1989); and Zhu Anal Biochem 177:120-4 (1989). In addition, such techniques as QuikChange (Invitrogen) can be employed or targeted mutations can be introduced by total or partial synthesis of the gene from chemically synthesized oligonucleotides by overlap extension PCR techniques.

(45) The peptides may be joined to a wide variety of other oligopeptides or proteins for a variety of purposes. By providing for expression of the subject peptides, various post-expression modifications may be achieved. For example, by employing the appropriate coding sequences, one may provide farnesylation or prenylation. The peptides may also be combined with other proteins; with ligands or receptors of interest; with viral proteins, transmembrane localization sequences etc., or with specific binding agents including other polypeptides.

(46) The hydrogenase of the invention may be produced in eukaryotic or prokaryotic cells, may be synthesized in vitro, or synthesized in a cell free synthetic system. Expression in photosynthetic cells is of particular interest, e.g. plants cells, photosynthetic microorganisms, and the like.

(47) Modifications of interest that do not alter primary sequence include chemical derivatization of polypeptides, e.g., acetylation, amidation, carboxylation, etc. Also embraced are sequences that have phosphorylated amino acid residues, e.g. phosphotyrosine, phosphoserine, or phosphothreonine.

(48) Also included in the subject invention are peptides that have been modified using ordinary molecular biological techniques and synthetic chemistry so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a catalyst. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g. D-amino acids or non-naturally occurring synthetic amino acids. D-amino acids may be substituted for some or all of the amino acid residues.

(49) The subject peptides may be prepared by cell-free synthesis, using conventional methods as known in the art. Cell-free synthesis of polypeptides utilizes a reaction mix comprising biological extracts and/or defined reagents. The reaction mix will comprise a template for production of the macromolecule, e.g. DNA, mRNA, etc.; monomers for the macromolecule to be synthesized, e.g. amino acids, nucleotides, etc., and such co-factors, enzymes and other reagents that are necessary for the synthesis and activation, e.g. ribosomes, tRNA, polymerases, transcriptional factors, maturases, etc. For example, a cell-free system may be used as described in U.S. Pat. No. 7,351,563 using a cell-extract containing 3 maturases expressed from genes obtained from Shewanella oneidensis. Such synthetic reaction systems are well-known in the art, and have been described in the literature. The cell free synthesis reaction may be performed as batch, continuous flow, or semi-continuous flow, as known in the art.

(50) The polypeptides may also be isolated and purified in accordance with conventional methods of recombinant synthesis. A lysate may be prepared of the expression host and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. For the most part, these compositions will comprise at least 20% by weight of the desired product, more usually at least about 75% by weight, and preferably at least about 95% by weight, in relation to contaminants related to the method of preparation of the product and its purification. Usually, the percentages will be based upon total protein.

(51) In one embodiment of the invention, the hydrogenase consists essentially of a polypeptide sequence of at least 100 amino acids, at least 200, amino acids, at least 300 amino acids, at least 400 amino acids, up to the full length of any of SEQ ID NO:1-5 and further comprising at least one amino acid substitution as described herein. By consisting essentially of in the context of a polypeptide described herein, it is meant that the polypeptide is composed of the hydrogenase sequence, which sequence may be flanked by one or more amino acid or other residues that do not materially affect the basic characteristic(s) of the polypeptide. Also included are fusions of this sequence with a binding partner of interest.

(52) Amino acid substitutions (numbering relative to SEQ ID NO:1) that provide for faster H.sub.2 production include A156C, G158C, M166C, L192C, Q195C, G185C+I197C, N160C+L192C, T163C+Y164C, Y164C+I197C, A165C+L191C, L192E, L192G, P301C, T356C, M498C, N505C+P301C, N505C, T356C+S357C, S357A, S357V, S357I, S357P, T356V+S357T. One or more of these substitutions may be combined with amino acid substitutions that provide for greater O.sub.2 tolerance.

(53) Amino acid substitutions (numbering relative to SEQ ID NO:1) that provide for greater O.sub.2 tolerance include A156C, M166C, G194C, Q195C, I197C, A156C+L191C, G158C+I197C, N160C+T161C, N160C+A165C, N160C+L192C, N160C+Q195C, N160C+I197C, T161C+G194C, T161C+I197C, T163C+N189C, T163C+Q195C, T163C+I197C, Y164C+Q195C, A165C+N189C, A165C+L192C, A165C+Q195C, A165C+I197C, M166C+Q195C, M166C+I197C, F185C+I197C, N189C+G194C, N189C+I197C, L191C+L192C, L191C+I197C, Q195C+I197C, L192F, L192W, L192S, L192D, L192G, P301C, T356C, S357C, P301C+T356C, P301C+A498C, P301C+G502C, G302C+T356C, G302C+S357C, G302C+A498C, W303C+G507C, P354C+G508C, T356C+S357C, S357C+A498C, S357C+N505C, T356V, T356I, T356L, T356P, S357A, S357V, S357I, S357L, S357T, S357P, T356V S357T, T356V+S357V, T356V+S357P.

(54) The invention includes nucleic acids encoding the peptides of the invention. Hydrogenase coding sequences can be generated by methods known in the art, e.g. by in vitro synthesis, recombinant methods, etc. to provide a coding sequence that corresponds to a hydrogenase polypeptide that can serve as an intermediate in the production of the hydrogenase peptide. Using the known genetic code, one can produce a suitable coding sequence. Double or single stranded fragments can be obtained from the DNA sequence by chemically synthesizing oligonucleotides in accordance with conventional methods, by restriction enzyme digestion, by PCR amplification, etc.

(55) Hydrogenase encoding nucleic acids can be provided as a linear molecule or within a circular molecule, and can be provided within autonomously replicating molecules (vectors) or within molecules without replication sequences. Expression of the nucleic acids can be regulated by their own or by other regulatory sequences known in the art. The nucleic acids can be introduced into suitable host cells using a variety of techniques available in the art, such as by conjugation, transfection with naked or encapsulated nucleic acids, liposome-mediated DNA transfer, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, gene gun, calcium phosphate-mediated transfection, and the like.

(56) Expression vectors may be used to introduce a hydrogenase coding sequence into a cell. Such vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences. Transcription cassettes may be prepared comprising a transcription initiation region, the target gene or fragment thereof, and a transcriptional termination region. The transcription cassettes may be introduced into a variety of vectors, e.g. plasmid; retrovirus, e.g. lentivirus; adenovirus; and the like, where the vectors are able to transiently or stably be maintained in the cells, usually for a period of at least about one day, more usually for a period of at least about several days to several weeks.

(57) Methods of transferring genetic material into bacterial cells are well known in the art, including transfection, e.g. by Ca.sup.++, electroporation, etc.; infection with viral vectors; conjugative mating, etc. Any of these methods may be used as appropriate for the desired host cell.

(58) For example, genes and transposons are routinely transferred to organisms such as filamentous cyanobacteria by conjugal transfer of plasmids from Escherichia coli or another suitable host. Suitable methods for such transfer are described, for example, by Elhai and Wolk P (1988) Methods in Enzymology. 167: 747-754; and Elhai et al. (1997) J Bacteriol 179:1998-2005, each herein incorporated by reference.

(59) The expression cassettes of the present invention may be introduced into the genome of plant or microorganism, including, for example, photosynthetic microorganisms in order to produce transgenic microorganisms and plants for purposes of H.sub.2 production methods of the present invention. A variety of transformation techniques are well known in the art. Those methods include, but are not limited to, direct microinjection into nuclei, electroporation; calcium phosphate precipitation, liposomes, protoplast fusion, viral infection, and the like.

(60) The amount of protein produced in a translation reaction can be measured in various fashions. One method relies on the availability of an assay that measures the activity of the particular protein being translated. Examples of assays for measuring protein activity are the methyl viologen assay described in the examples. These assays measure the amount of functionally active protein produced from the translation reaction. Activity assays will not measure full length protein that is inactive due to improper protein folding or lack of other post translational modifications necessary for protein activity.

(61) An alternative assay for the hydrogenase activity is one that demonstrates actual evolution of H.sub.2, as many useful applications of hydrogenase synthesis require the production of H.sub.2. To produce H.sub.2, a reaction must contain a source of electrons, a source of protons, active hydrogenase protein, and an electron carrier that can deliver electrons to hydrogenase. The electron source may be provided as a reduced carrier, e.g. reduced methyl viologen; reduced ferrodoxin; etc. A suitable buffering agent may serve as a source of protons. The candidate synthesis product serves as a source of hydrogenase. H.sub.2 is evolved as electrons are donated from the reduced carrier to hydrogenase. Where the carrier provides for a colorimetric change, such as with methyl viologen, the results may be read spectrophotometrically. Alternatively, gas chromatography or other methods may be used to detect the presence of H.sub.2 evolved from the reaction.

(62) In addition, the invention includes kits comprising the polypeptides, nucleic acids, and vectors described herein. The kits can also include a substrate, and instructions for use in methods described herein.

(63) Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

(64) Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

(65) Methods recited herein may be carried out in any order of the recited events which is logically possible, as well as the recited order of events.

(66) Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

(67) All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

(68) It must be noted that as used herein and in the appended claims, the singular forms a, an, and the include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as solely, only and the like in connection with the recitation of claim elements, or use of a negative limitation.

(69) The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

Experimental

(70) FIG. 1 illustrates a long term objective to engineer a photosynthetic bacterium to efficiently produce H.sub.2 from sunlight and water. The solar energy activates the photosystem to split water and a new pathway uses a small protein, ferredoxin, to direct the electrons to a prolific H.sub.2-producing enzyme, an FeFe hydrogenase. The first technical barrier was that the hydrogenase was rapidly inactivated by O.sub.2, a side product of water splitting. However, recent progress has been made toward reengineering the FeFe hydrogenase, Cpl, to be O.sub.2-tolerant by mutating residues near the proximal catalytic and/or proximal delivery FeS clusters (FIG. 2)

(71) The more complex type of FeFe hydrogenases has a complex H-cluster active site composed of a catalytic 2Fe-2S cluster with additional adducts. A neighboring 4Fe-4s cluster, bridged by a cysteinyl thiol, directly supplies the electrons required to reduce protons to then form H.sub.2. This adjacent cluster is referred to herein as the proximal catalytic cluster (PCC). These electrons are supplied from a ferredoxin protein by way of an electron conduction pathway within the enzyme composed of three 4Fe-4S clusters and one 2Fe-2S cluster. The conduction cluster nearest the proximal catalytic cluster supplies the electrons into the enzyme's active site. This conduction cluster is referred to herein as the proximal delivery cluster (PDC). O.sub.2 tolerance of the enzyme is improved by substituting residues close to the Fe or S atoms of either the PDC or PCC.

(72) To facilitate a search for the desired O.sub.2 tolerance (during H.sub.2 production), we developed two types of reaction series (FIG. 3). The top one uses a strong reducing agent, dithionite, to reduce ferredoxin (Fd) as the source of electrons for the hydrogenase (H.sub.2ase). This reaction is not compatible with O.sub.2 presence because dithionite rapidly reduces O.sub.2 to water. However, it is useful for determining residual activity after long O.sub.2 exposures. The bottom reaction uses a more natural electron source, Glucose 6-phosphate (G6P). G6P dehydrogenase (G6PD) transfers two electrons from G6P to reduce NADP.sup.+, and Ferredoxin NADP.sup.+ Reductase (FNR) then transfers the electrons to Fd so they can be accepted by the H.sub.2ase.

(73) Developing O.sub.2 tolerance by directed mutation. FIG. 2 shows the structure of Cpl. The yellow spheres are sulfur atoms and the smaller red and blue spheres indicate the CO and CN adducts on the catalytic 2Fe-2S cluster. The adjacent 4Fe-4S cluster is labeled as the proximal catalytic center, and the next one, that directly provides electrons to the H-cluster, we call the proximal delivery center. In our first Cpl screen using the H.sub.2 oxidation assay, the mutations most influential for increasing O.sub.2 tolerance were located adjacent to the proximal delivery center. However, these changes only increased O.sub.2 tolerance when the enzyme was consuming H.sub.2 and did not confer O.sub.2 tolerance during H.sub.2 production. More recently, work by others with a NiFe hydrogenase that primarily consumes H.sub.2 indicated that two extra Cys residues surrounding the FeS center nearest the enzyme's active site were required for O.sub.2 tolerance. Such cysteines are not present in the FeFe hydrogenases; and motivated by the NiFe hydrogenase structure, we have been evaluating the introduction of Cys and non-Cys near the proximal delivery and catalytic clusters in Cpl.

(74) We first replaced all residues within 6.5 of the PDC or PCC with Cys to see which sites have high influence on the anaerobic H.sub.2 production activity and aerobic H.sub.2 yield. We then combined single Cys replacements around each cluster to create Cpl mutants with double Cys replacements. We did not discover any double cysteine replacement mutants that showed a higher aerobic yield, i.e. O.sub.2 tolerance, than the best single Cys replacement mutant, Cpl.sup.G194C (FIGS. 4 and 5; all amino acid residue locations referred to in this section are based on SEQ ID NO: 1). However, we did see a few combinations that resulted in higher O.sub.2 tolerance than the improvement from either of the individual Cys replacements that were combined. Next, we selected two sites around each proximal cluster that showed the highest improvement in the anaerobic H.sub.2 production activity or O.sub.2 tolerance, and saturation mutagenesis libraries were created for these 4 sites. When evaluating these libraries, we observed non-Cys replacements, e.g., L192G that improved O.sub.2 tolerance more than the highest level obtained with Cys replacements (FIG. 6). Furthermore, several combinations of non-Cys replacements at T356 and S357 synergistically improved O.sub.2 tolerance. These two sites had previously shown synergistic enhancement in O.sub.2 tolerance of Cpl when replaced with Cys. The most promising mutant from our library was identified with T356V and S357T substitutions. FIG. 7 compares the WT Cpl to this mutant using the NADPH-driven H.sub.2 production reaction series (FIG. 3). The first 10 minutes indicated baseline activities, and 5% O.sub.2 (chosen to mimic expected production conditions) was then introduced. The mutant retains about 12 times more activity after nearly an hour of O.sub.2 exposure. This is by far the most O.sub.2 tolerant FeFe hydrogenase ever observed.

(75) It is important to recognize that these mutations are highly non-obvious relative to recognized and suggested mechanisms for oxygen tolerance in hydrogenases. The best studied oxygen tolerance example is for the [NiFe] hydrogenase from Ralstonia eutropha. This hydrogenase has been shown to be oxygen tolerant because of two extra cysteines around the iron sulfur center near the catalytic center. This unique center therefore has six cysteines that either help to comprise or that surround the FeS center. These extra cysteines serve as an additional reservoir for electrons to allow oxygen molecules that adsorb to the active site to be completely reduced to water. Removing these cysteines causes oxygen sensitivity. Another theory is that oxygen diffusion channels within the protein structure allow the oxygen to reach the active site of [FeFe] hydrogenase, and that closing these channels would increase oxygen tolerance. It is clear that a single cysteine replacement mutants would not be capable of providing either advantage, and further that the combined T356V and S357T substitutions could contribute to neither of the recognized mechanisms. Therefore, the oxygen tolerance observed with the present invention could not have been predicted based on prior observations and the data presented herein demonstrates that oxygen tolerance for [FeFe] hydrogenases is obtained by a new and unpredicted mechanism.

(76) Developing tools for measuring light-dependent electron flux: In photosynthetic organisms, the PSII complex first splits water to produce electrons, protons, and O.sub.2. The electrons are then delivered to PSI (using several electron carriers including the protein, plastocyanin) where another photon boosts their energetic content. These electrons are then typically transferred to a native ferredoxin which then selectively delivers the electrons to FNR in order to provide reducing equivalents in the form of NADPH to drive carbon fixation, biosynthesis, and other essential metabolic processes.

(77) A goal is to use most of the available reducing equivalents for producing H.sub.2 while channeling only those required for cell viability to the FNR. In order to study this more carefully, we have been developing in vitro protocols for light dependent electron flux measurements. Following the lead of Kubota et al., we attached purification tags to PSI subunit proteins in the photosynthetic bacterium, Synechocystis sp. PCC 6803. This enabled PSI isolation from cell extracts. In parallel, plastocyanin was also purified. These were combined with the Synechocystis ferredoxin and Cpl to produce the system diagrammed in FIG. 8A. Ascorbic acid is the initial electron source, but light energy is required to boost the energy level to that needed for ferredoxin reduction and H.sub.2 production (FIG. 8B)

(78) Test with O.sub.2-tolerant FeFe hydrogenase. Finally we evaluated the O.sub.2 tolerant FeFe hydrogenase in the light-driven, PSI-mediated reaction series (FIG. 9). It was compared to the WT Cpl by expressing and maturing both the WT and the mutant in vivo and then purifying the hydrogenases. Under 5.0 vol % O.sub.2, Cpl.sup.T356V S357T produced H.sub.2 that is equivalent to 70% of the amount expected anaerobically. This is very promising and suggests that light driven H.sub.2 production catalyzed by the mutated Cpl could continue for several hours in the presence of O.sub.2.

(79) The mutation sites within Cpl replaced with Cys were chosen based on the distance to the closest Fe or S atom of the proximal 4Fe-4S clusters. The cutoff distance was 6.5 angstrom and we used a software called Chimera in calculating the distance for each site. 17 sites were identified in vicinity of the PDC, and 18 around the PCC. 4 were within the cutoff distance to both proximal clusters.

(80) The genes for these 31 single Cys replacement mutants were created by using a site-directed mutagenesis protocol called QuikChange. A plasmid from our group called pK7 sCpl that contains the gene for Cpl WT was used as the DNA template. Once the plasmids for single Cys replacement mutants were obtained, the same protocol was repeated with these plasmids as the template in creating genes for double Cys replacement mutants.

(81) In expressing the mutants, the cell-free protein synthesis technique was first used that had been previously developed. 350 ng of the mutated pK7 sCpl plasmid was mixed in 20 uL of the cell-free (CF) reaction mixture and incubated overnight inside an anaerobic glovebox. 50-200 ug/mL of mutant proteins were obtained in the cell-free buffer after 12-16 hours. As explained before, the most promising mutant, Cpl.sup.T356V S357T and 7 other variants were also expressed and matured in vivo and then purified using a C-terminal strep tag for the more thorough analysis shown in FIGS. 7 and 9.

(82) O.sub.2 tolerance of these mutant proteins was first evaluated in the following manner. H.sub.2 production reaction mixtures were prepared inside an anaerobic glovebox with an atmosphere of 100% N.sub.2. The final concentrations of each reagent (unless otherwise stated) was added in the following order: 50 mM Tris-HCl pH 7.0, 10 mM G6P, 1.0 U G6PD, 5 mM NADPH, 50 M RrFNR (rice root FNA), 5 M SynFd (Synechocystis ferredoxin). 10 L of the cell-free reaction product solution that contained Cpl mutant proteins was added last. The total reaction volume was 200 L which was placed at the bottom of 2.0 mL target screw thread vials. Before sealing the vials with rubber septa, magnetic stir bars were put to enable mixing during O.sub.2 exposure. After sealing, the samples were removed from the glovebox. The H.sub.2 production reactions were incubated at room temperature while mixing at 250 rpm. A 23-gauge needle syringe was used to sample 200 L of the headspace to measure H.sub.2 and O.sub.2 concentrations using gas chromatography with a Hewlett Packard 6890 Gas Chromatograph and a ShinCarbon ST 100/120 mesh column. 5.0 vol % O.sub.2 was introduced to the headspace of vials by injecting 0.56 mL of air (21% O.sub.2) and removing the same volume afterwards. All experiments were done in duplicate and error bars in the figures represent standard deviation.

(83) Aerobic H.sub.2 production by the WT and mutant Cpl proteins in the light-driven, PSI-mediated reaction series was conducted similarly with the following two differences. First, the reaction mixture contained the following reagents instead: 20 mM ascorbate, 5 M DCIP, 250 g/mL plastocyanin, 50 g/mL PSI, 2 M SynFd, 100 nM RrFNR, 100 nM Cpl (expressed in vivo, maturated and purified), 1 mM NADPH in 50 mM HEPES buffer pH 7.0 with 1 M sucrose, 5 mM CaCl.sub.2 & MgCl.sub.2. Second, the reactor vials were first covered with aluminum foil for the first 10 minutes of analysis (FIG. 9) to block light from the light bulb generating about 50 mol photons m.sup.2 s.sup.1).

(84) TABLE-US-00001 Residues in the Residues in the vicinity of vicinity of the proximal delivery the proximal catalytic 4Fe4S cluster 4Fe4S cluster Distance Distance between between residue residue and the and the FeS FeS Sequence Amino cluster Sequence Amino cluster number acid () number acid () 140 leu 4.6 191 leu 6.0 156 ala 6.0 192 leu 6.0 158 gly 6.0 195 gln 6.0 160 asn 6.0 301 pro 4.9 161 thr 4.2 302 gly 5.3 163 thr 4.1 303 trp 5.8 164 tyr 6.0 354 pro 5.8 165 ala 4.1 356 thr 6.0 166 met 4.7 357 ser 4.2 185 phe 4.6 358 lys 4.7 189 asn 6.0 497 met 4.2 191 leu 4.0 498 ala 5.0 192 leu 6.0 502 gly 6.0 194 gly 4.5 504 val 6.0 195 gln 6.0 505 asn 6.0 197 ile 6.0 506 gly 5.8 356 thr 6.0 507 gly 6.0 508 gly 6.0
Table 1 indicates the residues evaluated for substituting the native residue with a Cys residue. The numbers indicate the approximate distance between the new residue and the closest Fe or S atom in the neighboring proximal 4Fe-4S cluster. Note that some residues appear in both tables.

(85) TABLE-US-00002 TABLE 2 Mutations around the PDC Mutations around the PCC Faster H.sub.2 Higher O.sub.2 Faster H.sub.2 Higher O.sub.2 production tolerance production tolerance A156C, A156C, P301C, P301C, G158C, M166C, T356C, T356C, M166C, G194C, M498C, S357C L192C, Q195C, N505C P301C T356C, Q195C, I197C, P301C N505C, P301C A498C, G185C I197C, A156C L191C T356C S357C, P301C G502C, N160C L192C, G158C I197C S357A, G302C T356C, T163C Y164C, N160C T161C, S357V, G302C S357C, Y164C I197C, N160C A165C, S357I, G302C A498C, A165C L191C, N160C L192C, S357P, W303C G507C, L192E, N160C Q195C, T356V S357T, P354C G508C, L192G, N160C I197C, T356C S357C, T161C G194C, S357C A498C, T161C I197C, S357C N505C, T163C N189C, T356V, T163C Q195C, T356I, T163C I197C, T356L, Y164C Q195C, T356P, A165C N189C, S357A, A165C L192C, S357V, A165C Q195C, S357I, A165C I197C, S357L, M166C Q195C, S357T, M166C I197C, S357P, F185C I197C, T356V S357T, N189C G194C, T356V S357V, N189C I197C, T356V S357P L191C L192C, L191C I197C, Q195C I197C, L192F, L192W, L192S, L192D, L192G,
Table listing all mutations around the PDC or PCC that significantly improved either the anaerobic H.sub.2 production activity or O.sub.2 tolerance of Cpl.

(86) Sites for amino acid substitution within Cpl were chosen based on the distance to the closest Fe or S atom of the proximal 4Fe-4S cluster. A cutoff distance of about 6.5 angstrom was used. 17 sites were identified in vicinity of the PDC, and 18 around the PCC. 4 sites (L191, L192, Q195, and T356) were within the cutoff distance to both clusters.

(87) Changes in O.sub.2 tolerance were determined by mutating the residues surrounding the PDC or PCC into Cys. These single Cys replacement mutations around the PDC or PCC were combined to create Cpl mutants with double Cys replacements. A few combinations of single Cys replacements caused synergistic improvement in O.sub.2 tolerance. This information was used in selecting the sites for combining non-Cys replacements around the PDC or PCC as well for further improvement in O.sub.2 tolerance.

(88) Non-Cys replacements, e.g., L192G, were found that improved O.sub.2 tolerance more than the highest level obtained with Cys replacements. Combinations of non-Cys replacements at T356 and S357 synergistically improved O.sub.2 tolerance. The mutant with greatest O.sub.2 tolerance had T356V and S357T substitutions.