WO2011143592A1 - Methods and compositions for the recombinant biosynthesis of propanol - Google Patents

Abstract

The invention provides methods for the biosynthesis of propanol using engineered host cells. Genes encoding enzymes for the conversion of metabolic intermediates to propanol, methods for optimizing expression of these enzymes in host cells, and methods for the production of propanol by these host cells are disclosed.

Description

METHODS AND COMPOSITIONS FOR THE

RECOMBINANT BIOSYNTHESIS OF PROPANOL

REFERENCE TO A SEQUENCE LISTING

[0001] This application includes a Sequence Listing submitted electronically as a text file named 18738PCT_CRF_sequencelisting.txt, created on May 13, 2011, with a size of 88 kb and comprising 20 sequences. The sequence listing is hereby incorporated by reference.

FIELD OF THE INVENTION

[0002] The present disclosure relates to methods for conferring propanol-producing properties to a heterotrophic or photoautotrophic host, such that the modified host can be used in the commercial production of propanol. The hosts described are useful for producing 1 -propanol and 2-propanol.

BACKGROUND OF THE INVENTION

[0003] Synthesis of propanol in biological systems represents an important method of production of propanol. Enzymatic activities can be assembled to complete a pathway from common metabolic intermediates, such as L-threonine, to propanol. Propanol can be separated into two isomeric forms, 1- propanol, or 2-propanol. 1 -propanol is a primary alcohol represented by the formula CH₃CH₂CH₂OH.

[0004] Enzymatic activities can be assembled to complete a pathway from common metabolic intermediates, such as L-threonine, to propanol. Either isoform of propanol can be further dehydrated for the synthesis of propylene, a versatile polymer and raw material for a wide variety of products.

SUMMARY OF THE INVENTION

[0005] The invention relates to engineered host cells and methods employing enzymatic pathways in engineered host cells in the production of propanol. Various microorganisms are genetically engineered to produce propanol via recombinant enzymes and pathways.

[0006] The invention, therefore, provides isolated polypeptides comprising or consisting of polypeptide sequences selected from the group consisting of sequences encoded by a threonine deaminase gene, a 2-keto-acid decarboxylase gene, a 1 -propanol dehydrogenase gene, a malonyl-CoA reductase gene, a propionyl-CoA synthase gene, a CoA-dependent aldehyde dehydrogenase gene, a succinate: Co A ligase gene, a methylmalonyl-CoA mutase gene, a methylmalonyl-CoA decarboxylase gene, a citramalate synthase gene, an isopropylmalate isomerase gene, an isopropylmalate dehydrognase gene, and related polypeptide sequences, fragments and fusions. These sequences are useful in the production of 1-propanol. The invention also provides isolated polypeptides comprising or consisting of polypeptide sequences selected from the group consisting of sequences encoded by an acetyl- CoA C-acetyltransferase (thiolase) gene, a succinyl-CoA:acetate CoA-transferase gene, an acetoacetyl-CoA transferase gene, an acetoacetate decarboxylase gene, an isopropanol dehydrogenase gene, and related polypeptide sequences, fragments and fusions. These sequences are useful in the production of 2-propanol. The invention further provides isolated polynucleotides comprising or consisting of nucleic acid sequences selected from the group consisting of coding sequences for a threonine deaminase gene, a 2-keto-acid decarboxylase gene, a 1-propanol dehydrogenase gene, a malonyl-CoA reductase gene, a propionyl-CoA synthase gene, a CoA-dependent aldehyde dehydrogenase gene, a succinate:CoA ligase gene, a methylmalonyl-CoA mutase gene, a methylmalonyl-CoA decarboxylase gene, a citramalate synthase gene, an isopropylmalate isomerase gene, an isopropylmalate dehydrogenase gene, an acetyl-CoA C-acetyltransferase (thiolase) gene, a succinyl-CoA:acetate CoA-transferase gene, an acetoacetyl-CoA transferase gene, an acetoacetate decarboxylase gene, an isopropanol dehydrogenase gene, codon/expression optimized variants for these nucleic acid sequences and related nucleic acid sequences and fragments. Also provided are vectors and host cells comprising these isolated polynucleotides.

[0007] Antibodies that specifically bind to the isolated polypeptides of the invention are also provided.

[0008] The invention also provides coding sequences for the a threonine deaminase gene, a 2-keto-acid decarboxylase gene, a 1-propanol dehydrogenase gene, a malonyl-CoA reductase gene, a propionyl-CoA synthase gene, a CoA-dependent aldehyde dehydrogenase gene, a succinate:CoA ligase gene, a methylmalonyl-CoA mutase gene, a methylmalonyl- CoA decarboxylase gene, a citramalate synthase gene, an isopropylmalate isomerase gene, an isopropylmalate dehydrogenase gene, an acetyl-CoA C-acetyltransferase (thiolase) gene, a succinyl-CoA:acetate CoA-transferase gene, an acetoacetyl-CoA transferase gene, an acetoacetate decarboxylase gene, an isopropanol dehydrogenase gene, nucleic acid sequences that are codon optimized coding sequences for these genes, and related nucleic acid sequences and fragments.

[0009] Accordingly, the invention described herein provides genes which can be expressed at high levels in a range of organisms that encode enzymes required for the production of propanol and other carbon based products of interest. When used in combination with other genes essential for the biosynthetic production of propanol, high levels of propanol production are achieved. Organisms such as bacterium (for example, cyanobacteria) are genetically modified to optimize production of propanol using light, water, and carbon dioxide.

[0010] Additional aspects of the invention are numbered below:

1. An engineered cyanobacterium, wherein said engineered cyanobacterium comprises one or more recombinant protein activities selected from the group consisting of malonyl- CoA reductase, propionyl-CoA synthase, and 1 -propanol dehydrogenase.

2. The engineered cyanobacterium of aspect 1, wherein two or more recombinant protein activities from the group consisting of malonyl-CoA reductase, propionyl-CoA synthase, and 1 -propanol dehydrogenase are selected.

3. The engineered cyanobacterium of aspect 1, comprising recombinant malonyl-CoA reductase, propionyl-CoA synthase, and 1 -propanol dehydrogenase activities.

4. The engineered cyanobacterium of any of aspects 1-3, further comprising a CoA- dependent aldehyde dehydrogenase protein activity.

5. The engineered cyanobacterium of any of aspects 1-4, wherein said malonyl-CoA reductase is at least 95% identical to SEQ ID NO: 4.

6. The engineered cyanobacterium of any of aspects 1-4, wherein said propionyl-CoA synthase is at least 95% identical to SEQ ID NO: 5.

7. The engineered cyanobacterium of any of aspects 1-4, wherein said 1 -propanol dehydrogenase is at least 95% identical to SEQ ID NO: 3

8. The engineered cyanobacterium of aspect 4, wherein said CoA-dependent aldehyde dehydrogenase is at least 95%> identical to SEQ ID NO: 6.

9. An engineered cyanobacterium, wherein said engineered cyanobacterium comprises malonyl-CoA reductase, propionyl-CoA synthase, 1 -propanol dehydrogenase, and CoA- dependent aldehyde dehydrogenase, wherein said malonyl-CoA reductase is at least 95% identical to SEQ ID NO: 4, wherein said propionyl-CoA synthase is at least 95% identical to SEQ ID NO: 5, wherein said 1 -propanol dehydrogenase is at least 95 %> identical to SEQ ID NO: 3, and wherein said CoA-dependent aldehyde dehydrogenase is at least 95 %> identical to SEQ ID NO: 6.

10. The engineered cyanobacterium of any of aspects 1-9, wherein said cyanobacterium is Synechococcus sp. PCC 7002.

11. The engineered cyanobacterium of any of aspects 1-9, wherein at least two of said recombinant genes are expressed under the control of promoters in opposite orientations. 12. The engineered cyanobacterium of aspect 11, wherein the sequences of said promoters are non-identical.

13. A method for producing 1 -propanol, comprising :

(i) culturing the engineered cyanobacterium of any of aspects 1-12 in a culture medium, and

(ii) exposing said engineered cyanobacterium to light and carbon dioxide, wherein said exposure results in the conversion of said carbon dioxide by said engineered cyanobacterium into 1-propanol.

14. The method of aspect 13, wherein said 1-propanol produced by said engineered cyanobacterium is greater than the amount of 1-propanol produced by an otherwise identical cyanobacterium, cultured under identical conditions, but lacking said recombinant protein activity.

15. An engineered host cell, comprising one or more recombinant protein activities selected from Tables 1-5, wherein said one or more recombinant protein activities facilitate the enzymatic synthesis of propanol or a precursor thereof.

16. An engineered host cell, comprising one or more recombinant protein activities selected from Tables 1-4, wherein said one or more recombinant protein activities facilitate the synthesis of 1-propanol or a precursor thereof.

17. An engineered host cell, comprising one or more recombinant protein activities selected from Table 5, wherein said one or more recombinant protein activities facilitate the synthesis of 2-propanol or a precursor thereof.

18. The host cell of aspect 15 or 16, wherein said host cell facilitates conversion of L- threonine to 1-propanol, and wherein said host cell comprises at least one recombinant protein activity selected from Table 1.

19. The host cell of aspect 15 or 16, wherein said host cell facilitates conversion of L- threonine to 1-propanol, and wherein said host cell comprises at least two recombinant protein activities selected from Table 1.

20. The host cell of aspect 15 or 16, wherein said host cell facilitates conversion of malonyl-CoA to 1-propanol, and wherein said host cell comprises at least one recombinant protein activity selected from Table 2.

21. The host cell of aspect 15 or 16, wherein said host cell facilitates conversion of malonyl-CoA to 1-propanol, and wherein said host cell comprises at least two recombinant protein activities selected from Table 2. 22. The host cell of aspect 15 or 16, wherein said host cell facilitates conversion of succinate to 1-propanol, and wherein said host cell comprises at least one recombinant protein activity selected from Table 3.

23. The host cell of aspect 15 or 16, wherein said host cell facilitates conversion of succinate to 1-propanol, and wherein said host cell comprises at least two recombinant protein activities selected from Table 3.

24. The host cell of aspect 15 or 16, wherein said host cell facilitates conversion of acetyl- CoA and pyruvate to 1-propanol, and wherein said host cell comprises at least one recombinant protein activity selected from Table 4.

25. The host cell of aspect 15 or 16, wherein said host cell facilitates conversion of acetyl-CoA and pyruvate to 1-propanol, and wherein said host cell comprises at least two recombinant protein activities selected from Table 4.

26. The host cell of aspect 15 or 17, wherein said host cell facilitates conversion of acetyl- CoA to 2-propanol via an acetoacetyl-CoA intermediate, and wherein said host cell comprises at least one recombinant protein activity selected from Table 5.

27. The host cell of aspect 15 or 17, wherein said host cell facilitates conversion of acetyl- CoA to 2-propanol via an acetoacetyl-CoA intermediate, and wherein said host cell comprises at least two recombinant protein activities selected from Table 5.

28. The host cell of any of aspects 15-27, comprising at least three recombinant protein activities wherein each of said three recombinant protein activities are listed in a single table selected from one of Tables 1-5.

29. The host cell of any of aspects 15-17 and 20-27, comprising at least four recombinant protein activities wherein each of said four recombinant protein activities are listed in a single table selected from one of Tables 2-5.

30. The host cell of any of aspects 15, 16, 18, and 19, comprising threonine deaminase, 2- keto-acid decarboxylase, and 1-propanol dehydrogenase protein activities.

31. The host cell of any of aspects 15,16, 20, and 21 , comprising malonyl-CoA reductase, propionyl-CoA synthase, CoA-dependent aldehyde dehydrogenase, and 1-propanol dehydrogenase protein activities.

32. The host cell of any of aspects 15, 16, 22, and 23, comprising succinate:CoA ligase, methylmalonyl-CoA mutase, methylmalonyl-CoA decarboxylase, CoA-dependent aldehyde dehydrogenase, and 1-propanol dehydrogenase protein activities. 33. The host cell of any of aspects 15, 16, 24, and 25, comprising citramalate synthase, isopropylmalate isomerase, isopropylmalate dehydrogenase, 2-keto-acid decarboxylase, and 1-propanol dehydrogenase protein activities.

34. The host cell of any of aspects 15, 17, 26, and 27, comprising acetyl-CoA C- acetyltransferase, succinyl-CoA:acetate CoA-transferase or acetoacetyl-CoA transferase, acetoacetate decarboxylase, and isopropanol dehydrogenase protein activities.

35. The host cell of any of aspects 15-34, wherein at least one of said recombinant protein activities are expressed by a gene under the control of an inducible promoter.

36. The host cell of aspect 35, wherein said inducible promoter is repressible by ammonia, indicible by nickel, or inducible by light.

37. The host cell of any of aspects 15-36, wherein said host cell is capable of

photosynthesis.

38. The host cell of any of aspects 15-36, wherein said host cell is a cyanobacterium.

39. The host cell of any of aspects 15-36, wherein said host cell is a gram-negative or gram-positive bacteria.

40. The host cell of any of aspects 15-36, wherein said host cell is an algae.

41. The host cell of any of aspects 15-40, wherein propanol is secreted into the medium.

42. An isolated or recombinant polypeptide comprising or consisting of an amino acid sequence selected from SEQ ID NO: 1-20.

43. An isolated or recombinant polynucleotide comprising or consisting of a nucleic acid sequence selected from the group consisting of:

a. a gene coding for an amino acid sequence of SEQ ID NO: 1-20;

b. a nucleic acid sequence that is a degenerate variant of a gene coding for an amino acid sequence of SEQ ID NO: 1-20;

c. a nucleic acid sequence at least 90%, at least 95%, at least 98%>, at least 99% or at least 99.9% identical to a gene coding for an amino acid sequence of SEQ ID NO: 1-20; and

d. a nucleic acid sequence that hybridizes under stringent conditions to any gene coding for an amino acid sequence of SEQ ID NO: 1-20.

44. The isolated polynucleotide of aspect 43, wherein the nucleic acid sequence is flanked by regions of 0.5kb or greater, wherein said flainking regions is homologous to the

Synechococcus sp. PCC 7002 genome or one of its endogenous plasmids, such that upon recombination with said genome or plasmid, the construct is inserted between a contiguous region of homology 45. The isolated polynucleotide of aspect 43, wherein the nucleic acid sequence and the sequence of interest are operably linked to one or more expression control sequences.

46. The isolated polynucleotide of aspect 45, wherein said expression control sequence is an inducible expression control sequence.

47. The isolated polynucleotide of aspect 46, wherein said inducible expression control sequence is a T7 promoter.

48. A vector comprising the isolated polynucleotide of any one of aspects 43-47.

49. A fusion protein comprising a polypeptide encoded by any one of the sequences recited in aspects 43-47, wherein said polypeptide is fused to a heterologous amino acid sequence.

50. A host cell comprising the isolated polynucleotide of any one of aspects 43-47.

51. A method for producing a host cell capable of producing propanol comprising genetically engineering an isolated or recombinant polynucleotide sequence encoding any one of the enzymes in Tables 1-5 into a host cell.

52. A method for producing propanol comprising culturing the host cell of any of aspects 15-41, 50, and 51 to produce propanol.

53. A method for synthesizing propanol in vitro, comprising: exposing any of the reactants or products listed in Tables 1-5 to the enzyme activities listed in Tables 1-5.

54. The method of any of aspects 51-53, wherein at least one of the chemical reactions listed in Tables 1-5 is an exogenous reaction.

55. The method of aspect 54, wherein said exogenous reaction is catalyzed by an enzymatic activity listed in Tables 1-5.

56. The method of aspect 54, wherein said exogenous reaction is performed by chemical synthesis.

57. A method for producing propylene, comprising dehydration of propanol synthesized by the method of any one of aspects 51-56.

58. A method for producing propylene, comprising dehydration of propanol synthesized by the host cell of any one of aspects 51-56.

DETAILED DESCRIPTION OF THE INVENTION

[0011] Unless otherwise defined herein, scientific and technical terms used in connection with the invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, biochemistry, enzymology, molecular and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The methods and techniques of the invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990); Taylor and Drickamer, Introduction to Glycobiology, Oxford Univ. Press (2003); Worthington Enzyme Manual, Worthington Biochemical Corp., Freehold, N.J.; Handbook of Biochemistry: Section A Proteins, Vol. I, CRC Press (1976); Handbook of Biochemistry: Section A Proteins, Vol. II, CRC Press (1976); Essentials of Glycobiology, Cold Spring Harbor Laboratory Press (1999).

[0012] The following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0013] The term "polynucleotide" or "nucleic acid molecule" refers to a polymeric form of nucleotides of at least 10 bases in length. The term includes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA or RNA containing non-natural nucleotide analogs, non-native inter- nucleoside bonds, or both. The nucleic acid can be in any topological conformation. For instance, the nucleic acid can be single-stranded, double-stranded, triple-stranded, quadruplexed, partially double-stranded, branched, hair-pinned, circular, or in a padlocked conformation.

[0014] Unless otherwise indicated, and as an example for all sequences described herein under the general format "SEQ ID NO:", "nucleic acid comprising SEQ ID NO: l" refers to a nucleic acid, at least a portion of which has either (i) the sequence of SEQ ID NO: 1 , or (ii) a sequence complementary to SEQ ID NO: 1. The choice between the two is dictated by the context. For instance, if the nucleic acid is used as a probe, the choice between the two is dictated by the requirement that the probe be complementary to the desired target.

[0015] An "isolated" or "substantially pure" nucleic acid or polynucleotide (e.g., an RNA, DNA or a mixed polymer) is one which is substantially separated from other cellular components that naturally accompany the native polynucleotide in its natural host cell, e.g., ribosomes, polymerases and genomic sequences with which it is naturally associated. The term embraces a nucleic acid or polynucleotide that (1) has been removed from its naturally occurring environment, (2) is not associated with all or a portion of a polynucleotide in which the "isolated polynucleotide" is found in nature, (3) is operatively linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature. The term "isolated" or "substantially pure" also can be used in reference to recombinant or cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are biologically synthesized by heterologous systems.

[0016] However, "isolated" does not necessarily require that the nucleic acid or polynucleotide so described has itself been physically removed from its native environment. For instance, an endogenous nucleic acid sequence in the genome of an organism is deemed "isolated" herein if a heterologous sequence is placed adjacent to the endogenous nucleic acid sequence, such that the expression of this endogenous nucleic acid sequence is altered. In this context, a heterologous sequence is a sequence that is not naturally adjacent to the endogenous nucleic acid sequence, whether or not the heterologous sequence is itself endogenous (originating from the same host cell or progeny thereof) or exogenous

(originating from a different host cell or progeny thereof). By way of example, a promoter sequence can be substituted (e.g., by homologous recombination) for the native promoter of a gene in the genome of a host cell, such that this gene has an altered expression pattern. This gene would now become "isolated" because it is separated from at least some of the sequences that naturally flank it.

[0017] A nucleic acid is also considered "isolated" if it contains any modifications that do not naturally occur to the corresponding nucleic acid in a genome. For instance, an endogenous coding sequence is considered "isolated" if it contains an insertion, deletion or a point mutation introduced artificially, e.g., by human intervention. An "isolated nucleic acid" also includes a nucleic acid integrated into a host cell chromosome at a heterologous site and a nucleic acid construct present as an episome. Moreover, an "isolated nucleic acid" can be substantially free of other cellular material or substantially free of culture medium when produced by recombinant techniques or substantially free of chemical precursors or other chemicals when chemically synthesized.

[0018] As used herein, the phrase "degenerate variant" of a reference nucleic acid sequence encompasses nucleic acid sequences that can be translated, according to the standard genetic code, to provide an amino acid sequence identical to that translated from the reference nucleic acid sequence. The term "degenerate oligonucleotide" or "degenerate primer" is used to signify an oligonucleotide capable of hybridizing with target nucleic acid sequences that are not necessarily identical in sequence but that are homologous to one another within one or more particular segments.

[0019] The term "percent sequence identity" or "identical" in the context of nucleic acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over a stretch of at least about nine nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides. There are a number of different algorithms known in the art which can be used to measure nucleotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. Pearson, Methods Enzymol. 183:63-98 (1990) (hereby incorporated by reference in its entirety). For instance, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOP AM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG Version 6.1, herein incorporated by reference. Alternatively, sequences can be compared using the computer program, BLAST (Altschul et al, J. Mol. Biol. 215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993); Madden et al, Meth. Enzymol. 266: 131-141 (1996); Altschul et al, Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), especially blastp or tblastn (Altschul et al, Nucleic Acids Res. 25:3389-3402 (1997)).

[0020] A particular, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is that of Karlin and Altschul (Proc. Natl. Acad. Sci. (1990) USA 87:2264-68; Proc. Natl. Acad. Sci. USA (1993) 90: 5873-77) as used in the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (J. Mol. Biol. (1990) 215:403-10). BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention.

BLAST polypeptide searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to polypeptide molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Research (1997) 25(17):3389-3402). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (http://www.ncbi.nlm.nih.gov). One skilled in the art may also use the ALIGN program incorporating the non-linear algorithm of Myers and Miller (Comput. Appl. Biosci. (1988) 4: 11-17). For amino acid sequence comparison using the ALIGN program one skilled in the art may use a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

[0021] The term "substantial homology" or "substantial similarity," when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, preferably at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above.

[0022] Alternatively, substantial homology or similarity exists when a nucleic acid or fragment thereof hybridizes to another nucleic acid, to a strand of another nucleic acid, or to the complementary strand thereof, under stringent hybridization conditions. "Stringent hybridization conditions" and "stringent wash conditions" in the context of nucleic acid hybridization experiments depend upon a number of different physical parameters. Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, solvents, the base composition of the hybridizing species, length of the complementary regions, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. One having ordinary skill in the art knows how to vary these parameters to achieve a particular stringency of

hybridization.

[0023] In general, "stringent hybridization" is performed at about 25 °C below the thermal melting point (T_m) for the specific DNA hybrid under a particular set of conditions. "Stringent washing" is performed at temperatures about 5 °C lower than the T_m for the specific DNA hybrid under a particular set of conditions. The T_m is the temperature at which 50%) of the target sequence hybridizes to a perfectly matched probe. See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), page 9.51, hereby incorporated by reference. For purposes herein, "stringent conditions" are defined for solution phase hybridization as aqueous hybridization (i.e., free of formamide) in 6xSSC (where 20xSSC contains 3.0 M NaCl and 0.3 M sodium citrate), 1% SDS at 65 °C for 8-12 hours, followed by two washes in 0.2xSSC, 0.1% SDS at 65°C for 20 minutes. It will be appreciated by the skilled worker that hybridization at 65 °C will occur at different rates depending on a number of factors including the length and percent identity of the sequences which are hybridizing.

[0024] A preferred, non-limiting example of stringent hybridization conditions includes hybridization in 4x sodium chloride/sodium citrate (SSC), at about 65-70 °C (or hybridization in 4x SSC plus 50% formamide at about 42-50 °C) followed by one or more washes in lx SSC, at about 65-70 °C. A preferred, non-limiting example of highly stringent hybridization conditions includes hybridization in lx SSC, at about 65-70 °C (or hybridization in lx SSC plus 50%) formamide at about 42-50 °C) followed by one or more washes in 0.3x SSC, at about 65-70 °C. A preferred, non-limiting example of reduced stringency hybridization conditions includes hybridization in 4x SSC, at about 50-60 °C (or alternatively hybridization in 6x SSC plus 50%> formamide at about 40-45 °C) followed by one or more washes in 2x SSC, at about 50-60 °C. Intermediate ranges e.g., at 65-70 °C or at 42-50 °C are also within the scope of the invention. SSPE (lx SSPE is 0.15 M NaCl, 10 mM NaH₂P0₄, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (lx SSC is 0.15 M NaCl and 15 mM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes each after hybridization is complete. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10 °C less than the melting temperature (T_m) of the hybrid, where T_m is determined according to the following equations. For hybrids less than 18 base pairs in length, T_m (°C)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs in length, T_m(°C)=81.5+16.6(logi₀[Na⁺]) +0.41 (% G+C)-(600/N), where N is the number of bases in the hybrid, and [Na⁺] is the concentration of sodium ions in the hybridization buffer ([Na⁺] for lx SSC=0.165 M).

[0025] The skilled practitioner recognizes that reagents can be added to hybridization and/or wash buffers. For example, to decrease non-specific hybridization of nucleic acid molecules to, for example, nitrocellulose or nylon membranes, blocking agents, including but not limited to, BSA or salmon or herring sperm carrier DNA and/or detergents, including but not limited to, SDS, chelating agents EDTA, Ficoll, PVP and the like can be used. When using nylon membranes, in particular, an additional, non-limiting example of stringent hybridization conditions is hybridization in 0.25-0.5M NaH₂P0₄, 7% SDS at about 65 °C, followed by one or more washes at 0.02M NaH₂P0₄, 1% SDS at 65 °C (Church and Gilbert (1984) Proc. Natl. Acad. Sci. USA 81 : 1991-1995,) or, alternatively, 0.2x SSC, 1% SDS.

[0026] The nucleic acids (also referred to as polynucleotides) of this invention may include both sense and antisense strands of R A, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. They may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, intemucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g.,

phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.) Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule. Other modifications can include, for example, analogs in which the ribose ring contains a bridging moiety or other structure such as the modifications found in "locked" nucleic acids.

[0027] The term "mutated" when applied to nucleic acid sequences means that nucleotides in a nucleic acid sequence may be inserted, deleted or changed compared to a reference nucleic acid sequence. A single alteration may be made at a locus (a point mutation) or multiple nucleotides may be inserted, deleted or changed at a single locus. In addition, one or more alterations may be made at any number of loci within a nucleic acid sequence. A nucleic acid sequence may be mutated by any method known in the art including but not limited to mutagenesis techniques such as "error-prone PCR" (a process for performing PCR under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product; see, e.g., Leung et al., Technique, 1 : 11-15 (1989) and Caldwell and Joyce, PCR Methods Applic. 2:28-33 (1992)); and "oligonucleotide-directed mutagenesis" (a process which enables the generation of site-specific mutations in any cloned DNA segment of interest; see, e.g., Reidhaar-Olson and Sauer, Science 241 :53-57 (1988)).

[0028] The term "derived from" is intended to include the isolation (in whole or in part) of a polynucleotide segment from an indicated source. The term is intended to include, for example, direct cloning, PCR amplification, or artificial synthesis from, or based on, a sequence associated with the indicated polynucleotide source.

[0029] The term "gene" as used herein refers to a nucleotide sequence that can direct synthesis of an enzyme or other polypeptide molecule (e.g., can comprise coding sequences, for example, a contiguous open reading frame (ORF) which encodes a polypeptide) or can itself be functional in the organism. A gene in an organism can be clustered within an operon, as defined herein, wherein the operon is separated from other genes and/or operons by intergenic DNA. Individual genes contained within an operon can overlap without intergenic DNA between the individual genes.

[0030] The term "expression" when used in relation to the transcription and/or translation of a nucleotide sequence as used herein generally includes expression levels of the nucleotide sequence being enhanced, increased, resulting in basal or housekeeping levels in the host cell, constitutive, attenuated, decreased or repressed.

[0031] The term "attenuate" as used herein generally refers to a functional deletion, including a mutation, partial or complete deletion, insertion, or other variation made to a gene sequence or a sequence controlling the transcription of a gene sequence, which reduces or inhibits production of the gene product, or renders the gene product non-functional. In some instances a functional deletion is described as a knockout mutation. Attenuation also includes amino acid sequence changes by altering the nucleic acid sequence, placing the gene under the control of a less active promoter, down-regulation, expressing interfering R A, ribozymes or antisense sequences that target the gene of interest, or through any other technique known in the art. In one example, the sensitivity of a particular enzyme to feedback inhibition or inhibition caused by a composition that is not a product or a reactant (non-pathway specific feedback) is lessened such that the enzyme activity is not impacted by the presence of a compound. In other instances, an enzyme that has been altered to be less active can be referred to as attenuated.

[0032] A "deletion" is the removal of one or more nucleotides from a nucleic acid molecule or one or more amino acids from a protein, the regions on either side being joined together.

[0033] A "knock-out" is a gene whose level of expression or activity has been reduced to zero. In some examples, a gene is knocked-out via deletion of some or all of its coding sequence. In other examples, a gene is knocked-out via introduction of one or more nucleotides into its open-reading frame, which results in translation of a non-sense or otherwise non- functional protein product.

[0034] The term "codon usage" is intended to refer to analyzing a nucleic acid sequence to be expressed in a recipient host organism (or acellular extract thereof) for the occurrence and use of preferred codons the host organism transcribes advantageously for optimal nucleic acid sequence transcription. The recipient host may be recombinantly altered with any preferred codon. Alternatively, a particular cell host can be selected that already has superior codon usage, or the nucleic acid sequence can be genetically engineered to change a limiting codon to a non-limiting codon (e.g., by introducing a silent mutation(s)).

[0035] The term "vector" as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Other vectors include cosmids, bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC), fosmids, phage and phagemids. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome (discussed in more detail below). Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome. Moreover, certain preferred vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as

"recombinant expression vectors" (or simply "expression vectors").

[0036] "Expression optimization" as used herein is defined as one or more optional modifications to the nucleotide sequence in the promoter and terminator elements resulting in desired rates and levels of transcription and translation into a protein product encoded by said nucleotide sequence. Expression optimization as used herein also includes designing an effectual predicted secondary structure (for example, stem-loop structures and termination sequences) of the messenger ribonucleic acid (mRNA) sequence to promote desired levels of protein production. Other genes and gene combinations essential for the production of a protein may be used, for example genes for proteins in a biosynthetic pathway, required for post-translational modifications or required for a heteromultimeric protein, wherein combinations of genes are chosen for the effect of optimizing expression of the desired levels of protein product. Conversely, one or more genes optionally may be "knocked-out" or otherwise altered such that lower or eliminated expression of said gene or genes achieves the desired expression levels of protein. Additionally, expression optimization can be achieved through codon optimization. Codon optimization, as used herein, is defined as modifying a nucleotide sequence for effectual use of host cell bias in relative concentrations of transfer ribonucleic acids (tRNA) such that the desired rate and levels of gene nucleotide sequence translation into a final protein product are achieved, without altering the peptide sequence encoded by the nucleotide sequence. [0037] The term "expression control sequence" as used herein refers to polynucleotide sequences which are necessary to affect the expression of coding sequences to which they are operatively linked. Expression control sequences are sequences which control the

transcription, post-transcriptional events and translation of nucleic acid sequences.

Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient R A processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence. The term "control sequences" is intended to include, at a minimum, all components whose presence is essential for expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

[0038] "Operatively linked" or "operably linked" expression control sequences refers to a linkage in which the expression control sequence is contiguous with the gene of interest to control the gene of interest, as well as expression control sequences that act in trans or at a distance to control the gene of interest.

[0039] The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell into which a recombinant vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. A recombinant host cell may be an isolated cell or cell line grown in culture or may be a cell which resides in a living tissue or organism.

[0040] The term "recombinant" refers to a biomolecule, e.g., a gene or protein, that (1) has been removed from its naturally occurring environment, (2) is not associated with all or a portion of a polynucleotide in which the gene is found in nature, (3) is operatively linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature. The term "recombinant" can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are biologically synthesized by heterologous systems, as well as proteins and/or mRNAs encoded by such nucleic acids. [0041] As used herein, an endogenous nucleic acid sequence in the genome of an organism (or the encoded protein product of that sequence) is deemed "recombinant" herein if a heterologous sequence is placed adjacent to the endogenous nucleic acid sequence, such that the expression of this endogenous nucleic acid sequence is altered. In this context, a heterologous sequence is a sequence that is not naturally adjacent to the endogenous nucleic acid sequence, whether or not the heterologous sequence is itself endogenous (originating from the same host cell or progeny thereof) or exogenous (originating from a different host cell or progeny thereof). By way of example, a promoter sequence can be substituted (e.g., by homologous recombination) for the native promoter of a gene in the genome of a host cell, such that this gene has an altered expression pattern. This gene would now become

"recombinant" because it is separated from at least some of the sequences that naturally flank it.

[0042] A nucleic acid is also considered "recombinant" if it contains any modifications that do not naturally occur to the corresponding nucleic acid in a genome. For instance, an endogenous coding sequence is considered "recombinant" if it contains an insertion, deletion or a point mutation introduced artificially, e.g., by human intervention. A "recombinant nucleic acid" also includes a nucleic acid integrated into a host cell chromosome at a heterologous site and a nucleic acid construct present as an episome.

[0043] The term "peptide" as used herein refers to a short polypeptide, e.g., one that is typically less than about 50 amino acids long and more typically less than about 30 amino acids long. The term as used herein encompasses analogs and mimetics that mimic structural and thus biological function.

[0044] The term "polypeptide" encompasses both naturally-occurring and non-naturally- occurring proteins, and fragments, mutants, derivatives and analogs thereof. A polypeptide may be monomeric or polymeric. Further, a polypeptide may comprise a number of different domains each of which has one or more distinct activities.

[0045] The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) exists in a purity not found in nature, where purity can be adjudged with respect to the presence of other cellular material (e.g., is free of other proteins from the same species) (3) is expressed by a cell from a different species, or (4) does not occur in nature (e.g., it is a fragment of a polypeptide found in nature or it includes amino acid analogs or derivatives not found in nature or linkages other than standard peptide bonds). Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be "isolated" from its naturally associated components. A polypeptide or protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art. As thus defined, "isolated" does not necessarily require that the protein, polypeptide, peptide or oligopeptide so described has been physically removed from its native environment.

[0046] An "isolated" or "purified polypeptide" is substantially free of cellular material or other contaminating polypeptides from the expression host cell from which the polypeptide is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. In one embodiment, an isolated or purified polypeptide has less than about 30% (by dry weight) of contaminating polypeptide or chemicals, more advantageously less than about 20% of contaminating polypeptide or chemicals, still more advantageously less than about 10% of contaminating polypeptide or chemicals, and most advantageously less than about 5% contaminating polypeptide or chemicals.

[0047] The term "polypeptide fragment" as used herein refers to a polypeptide that has a deletion, e.g., an amino-terminal and/or carboxy-terminal deletion compared to a full-length polypeptide. In a preferred embodiment, the polypeptide fragment is a contiguous sequence in which the amino acid sequence of the fragment is identical to the corresponding positions in the naturally-occurring sequence. Fragments typically are at least 5, 6, 7, 8, 9 or 10 amino acids long, preferably at least 12, 14, 16 or 18 amino acids long, more preferably at least 20 amino acids long, more preferably at least 25, 30, 35, 40 or 45, amino acids, even more preferably at least 50 or 60 amino acids long, and even more preferably at least 70 amino acids long.

[0048] A "modified derivative" refers to polypeptides or fragments thereof that are substantially homologous in primary structural sequence but which include, e.g., in vivo or in vitro chemical and biochemical modifications or which incorporate amino acids that are not found in the native polypeptide. Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labeling, e.g., with

radionuclides, and various enzymatic modifications, as will be readily appreciated by those skilled in the art. A variety of methods for labeling polypeptides and of substituents or labels useful for such purposes are well known in the art, and include radioactive isotopes such as

125 32 35 3

I, P, S, and H, ligands which bind to labeled antiligands (e.g., antibodies),

fluorophores, chemiluminescent agents, enzymes, and antiligands which can serve as specific binding pair members for a labeled ligand. The choice of label depends on the sensitivity required, ease of conjugation with the primer, stability requirements, and available instrumentation. Methods for labeling polypeptides are well known in the art. See, e.g., Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002) (hereby incorporated by reference).

[0049] The terms "thermal stability" and "thermostability" are used interchangeably and refer to the ability of an enzyme (e.g., whether expressed in a cell, present in an cellular extract, cell lysate, or in purified or partially purified form) to exhibit the ability to catalyze a reaction at least at about 20°C, preferably at about 25°C to 35°C, more preferably at about 37°C or higher, in more preferably at about 50°C or higher, and even more preferably at least about 60°C or higher.

[0050] The term "chimeric" refers to an expressed or translated polypeptide in which a domain or subunit of a particular homologous or non-homologous protein is genetically engineered to be transcribed, translated and/or expressed collinearly in the nucleotide and amino acid sequence of another homologous or non-homologous protein.

[0051] The term "fusion protein" refers to a polypeptide comprising a polypeptide or fragment coupled to heterologous amino acid sequences. Fusion proteins are useful because they can be constructed to contain two or more desired functional elements from two or more different proteins. A fusion protein comprises at least 10 contiguous amino acids from a polypeptide of interest, more preferably at least 20 or 30 amino acids, even more preferably at least 40, 50 or 60 amino acids, yet more preferably at least 75, 100 or 125 amino acids. Fusions that include the entirety of the proteins of the invention have particular utility. The heterologous polypeptide included within the fusion protein of the invention is at least 6 amino acids in length, often at least 8 amino acids in length, and usefully at least 15, 20, and 25 amino acids in length. Fusions that include larger polypeptides, such as an IgG Fc region, and even entire proteins, such as the green fluorescent protein ("GFP") chromophore- containing proteins, have particular utility. Fusion proteins can be produced recombinantly by constructing a nucleic acid sequence which encodes the polypeptide or a fragment thereof in frame with a nucleic acid sequence encoding a different protein or peptide and then expressing the fusion protein. Alternatively, a fusion protein can be produced chemically by crosslinking the polypeptide or a fragment thereof to another protein.

[0052] As used herein, the term "protomer" refers to a polymeric form of amino acids forming a subunit of a larger oligomeric protein structure. Protomers of an oligomeric structure may be identical or non-identical. Protomers can combine to form an oligomeric subunit, which can combine further with other identical or non-identical protomers to form a larger oligomeric protein.

[0053] As used herein, the term "antibody" refers to a polypeptide, at least a portion of which is encoded by at least one immunoglobulin gene, or fragment thereof, and that can bind specifically to a desired target molecule. The term includes naturally-occurring forms, as well as fragments and derivatives.

[0054] Fragments within the scope of the term "antibody" include those produced by digestion with various proteases, those produced by chemical cleavage and/or chemical dissociation and those produced recombinantly, so long as the fragment remains capable of specific binding to a target molecule. Among such fragments are Fab, Fab', Fv, F(ab')₂, and single chain Fv (scFv) fragments.

[0055] Derivatives within the scope of the term include antibodies (or fragments thereof) that have been modified in sequence, but remain capable of specific binding to a target molecule, including: interspecies chimeric and humanized antibodies; antibody fusions; heteromeric antibody complexes and antibody fusions, such as diabodies (bispecific antibodies), single-chain diabodies, and intrabodies (see, e.g., Intracellular Antibodies:

Research and Disease Applications (1998) Marasco,ed., Springer- Verlag New York, Inc.), the disclosure of which is incorporated herein by reference in its entirety).

[0056] As used herein, antibodies can be produced by any known technique, including harvest from cell culture of native B lymphocytes, harvest from culture of hybridomas, recombinant expression systems and phage display.

[0057] The term "non-peptide analog" refers to a compound with properties that are analogous to those of a reference polypeptide. A non-peptide compound may also be termed a "peptide mimetic" or a "peptidomimetic." See, e.g., Jones, Amino Acid and Peptide Synthesis, Oxford University Press (1992); Jung, Combinatorial Peptide and Nonpeptide Libraries: A Handbook, John Wiley (1997); Bodanszky et al., Peptide Chemistry— A

Practical Textbook, Springer Verlag (1993); Synthetic Peptides: A Users Guide, (Grant, ed., W. H. Freeman and Co., 1992); Evans et al., J. Med. Chem. 30:1229 (1987); Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger, Trends Neurosci., 8:392-396 (1985); and references sited in each of the above, which are incorporated herein by reference. Such compounds are often developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to useful peptides of the invention may be used to produce an equivalent effect and are therefore envisioned to be part of the invention. [0058] A "polypeptide mutant" or "mutein" refers to a polypeptide whose sequence contains an insertion, duplication, deletion, rearrangement or substitution of one or more amino acids compared to the amino acid sequence of a native or wild-type protein. A mutein may have one or more amino acid point substitutions, in which a single amino acid at a position has been changed to another amino acid, one or more insertions and/or deletions, in which one or more amino acids are inserted or deleted, respectively, in the sequence of the naturally-occurring protein, and/or truncations of the amino acid sequence at either or both the amino or carboxy termini. A mutein may have the same but preferably has a different biological activity compared to the naturally-occurring protein.

[0059] A mutein has at least 85% overall sequence homology to its wild-type counterpart. Even more preferred are muteins having at least 90% overall sequence homology to the wild- type protein.

[0060] In an even more preferred embodiment, a mutein exhibits at least 95% sequence identity, even more preferably 98%, even more preferably 99% and even more preferably 99.9%) overall sequence identity.

[0061] Sequence homology may be measured by any common sequence analysis algorithm, such as Gap or Bestfit.

[0062] Amino acid substitutions can include those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity or enzymatic activity, and (5) confer or modify other physicochemical or functional properties of such analogs.

[0063] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (Golub and Gren eds., Sinauer Associates, Sunderland, Mass., 2^nd ed. 1991), which is incorporated herein by reference. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, and other unconventional amino acids may also be suitable components for polypeptides of the invention. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, C-N,N,N- trimethyllysine, C -N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5 -hydroxy lysine, N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand end corresponds to the amino terminal end and the right-hand end corresponds to the carboxy-terminal end, in accordance with standard usage and convention. [0064] A protein has "homology" or is "homologous" to a second protein if the nucleic acid sequence that encodes the protein has a similar sequence to the nucleic acid sequence that encodes the second protein. Alternatively, a protein has homology to a second protein if the two proteins have "similar" amino acid sequences. (Thus, the term "homologous proteins" is defined to mean that the two proteins have similar amino acid sequences.) As used herein, homology between two regions of amino acid sequence (especially with respect to predicted structural similarities) is interpreted as implying similarity in function.

[0065] When "homologous" is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative

substitutions, the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson, 1994, Methods Mol. Biol.

24:307-331 and 25:365-389 (herein incorporated by reference).

[0066] The following six groups each contain amino acids that are conservative substitutions for one another: 1) Serine (S), Threonine (T); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0067] Sequence homology for polypeptides, which is also referred to as percent sequence identity, is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705. Protein analysis software matches similar sequences using a measure of homology assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as "Gap" and "Bestfit" which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and a mutein thereof. See, e.g., GCG Version 6.1. [0068] A preferred algorithm when comparing a particular polypeptide sequence to a database containing a large number of sequences from different organisms is the computer program BLAST (Altschul et al, J. Mol. Biol. 215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993); Madden et al, Meth. Enzymol. 266:131-141 (1996); Altschul et al, Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), especially blastp or tblastn (Altschul et al, Nucleic Acids Res. 25:3389-3402

(1997)).

[0069] Preferred parameters for BLASTp are: Expectation value: 10 (default); Filter: seg (default); Cost to open a gap: 11 (default); Cost to extend a gap: 1 (default); Max. alignments: 100 (default); Word size: 11 (default); No. of descriptions: 100 (default); Penalty Matrix: BLOWSUM62.

[0070] The length of polypeptide sequences compared for homology will generally be at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues. When searching a database containing sequences from a large number of different organisms, it is preferable to compare amino acid sequences. Database searching using amino acid sequences can be measured by algorithms other than blastp known in the art. For instance, polypeptide sequences can be compared using FASTA, a program in GCG Version 6.1. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. (Pearson, Methods Enzymol. 183:63-98 (1990) (herein incorporated by reference). For example, percent sequence identity between amino acid sequences can be determined using FASTA with its default parameters (a word size of 2 and the PAM250 scoring matrix), as provided in GCG Version 6.1, herein incorporated by reference.

[0071] To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes, and, if necessary, gaps can be introduced in the first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences as evaluated, for example, by calculating # of identical positions/total # of positions x 100.

Additional evaluations of the sequence alignment can include a numeric penalty taking into account the number of gaps and size of said gaps necessary to produce an optimal alignment. [0072] "Specific binding" refers to the ability of two molecules to bind to each other in preference to binding to other molecules in the environment. Typically, "specific binding" discriminates over adventitious binding in a reaction by at least two-fold, more typically by at least 10-fold, often at least 100-fold. Typically, the affinity or avidity of a specific binding reaction, as quantified by a dissociation constant, is about 10^"7 M or stronger (e.g., about 10^"8 M, 10^"9 M or even stronger).

[0073] The term "region" as used herein refers to a physically contiguous portion of the primary structure of a biomolecule. In the case of proteins, a region is defined by a contiguous portion of the amino acid sequence of that protein.

[0074] The term "domain" as used herein refers to a structure of a biomolecule that contributes to a known or suspected function of the biomolecule. Domains may be coextensive with regions or portions thereof; domains may also include distinct, non-contiguous regions of a biomolecule. Examples of protein domains include, but are not limited to, an Ig domain, an extracellular domain, a transmembrane domain, and a cytoplasmic domain.

[0075] As used herein, the term "molecule" means any compound, including, but not limited to, a small molecule, peptide, protein, sugar, nucleotide, nucleic acid, lipid, etc., and such a compound can be natural or synthetic.

[0076] The term "substrate affinity" as used herein refers to the binding kinetics or the kinetics of binding and catalytic turnover, K_m, the Michaelis-Menten constant as understood by one having skill in the art, for a substrate.

[0077] The term "sugar" as used herein refers to any carbohydrate endogenously produced from sunlight, carbon dioxide and water, any carbohydrate produced endogenously and/or any carbohydrate from any exogenous carbon source such as biomass, comprising a sugar molecule or pool or source of such sugar molecules.

[0078] The term "carbon source" as used herein refers to carbon dioxide, exogenous sugar, biomass, or inorganic carbon source.

[0079] Biomass refers to biological material produced by a biological system including material useful as a renewable energy source.

[0080] Threonine deaminase (E.C. 4.3.1.19) is a pyridoxal-phosphate protein that catalyzes the deamination of threonine to 2-ketobutyrate and ammonia. These enzymes are designated "IlvA." The genes encoding IlvA are designated "ilvA."

[0081] 2-keto-acid decarboxylase (E.C. 4.1.1.72) catalyzes the conversion of 2- ketobutyrate to propanal and carbon dioxide. These enzymes are designated "KivD." The genes encoding KivD are designated "kivD." [0082] 1-propanol dehydrogenase (E.C. 1.1.1.2) catalyzes the conversion of propanal to 1-propanol. These enzymes are designated "AdhA." The genes encoding AdhA are designated "adhA."

[0083] Malonyl-CoA reductase is a bifunctional enzyme which catalyzes the conversion of malonyl-CoA to 3-hydroxypropionate. These enzymes are designated "Mcr." The genes encoding Mcr are designated "mcr."

[0084] Propionyl-CoA synthase is a trifunctional enzyme which catalyzes the conversion of 3-hydroxypropionate to propionyl-CoA. These enzymes are designated "Pes." The genes encoding Pes are designated "pes."

[0085] CoA-dependent aldehyde dehydrogenase (E.C. 1.2.1.10) catalyzes the conversion of propionyl-CoA to propanal. These enzymes are designated "PduP." The genes encoding PduP are designated "pduP."

[0086] Succinate:CoA ligase (E.C. 6.2.1.5) catalyzes the conversion of succinate to succinyl-CoA. These enzymes are designated "SucCD." The genes encoding SucCD include "sucC and "sucD" (i.e. "sucCD" .

[0087] Methylmalonyl-CoA mutase (E.C. 5.4.99.2) catalyzes the isomerization of succinyl-CoA to methylmalonyl-CoA. These enzymes are designated "ScpA." The genes encoding ScpA are designated "scpA."

[0088] Methlymalonyl-CoA decarboxylase (E.C. 4.1.1.41) catalyzes the conversion of (R)-methylmalonyl-CoA to propionyl-CoA. These enzymes are designated "ScpB." The genes encoding ScpB are designated "scpB."

[0089] Citramalate synthase (E.C. 4.1.3.22) catalyzes the condensation of pyruvate and acetate to (S)-citramalate. These enzymes are designated "CimA." The genes encoding CimA are designated "cimA."

[0090] Isopropylmalate isomerase catalyzes the conversion of (S)-citramalate to erythro- β-methyl-D-malate. These enzymes are designated "LeuCD." The genes encoding LeuCD are designated "leuCD."

[0091] Isopropylmalate dehydrogenase catalyzes the conversion of erythro-P-methyl-D- malate to 2-oxobutanoate. These enzymes are designated "LeuB." The genes encoding LeuB are designated "leuB."

[0092] Acetyl-CoA C-acetyltransferase (thiolase) (E.C. 2.3.1.9) catalyzes the conversion of acetyl-CoA to acetoacetyl-CoA. These enzymes are designated "PhaA." The genes encoding PhaA are designated "phaA." [0093] Succinyl-CoA:acetate CoA-transferase (E.C. 2.8.3.8) catalyzes the conversion of acetoacetyl-CoA to acetoacetate. These enzymes are designated "AarC." The genes encoding AarC are designated "aarC."

[0094] Acetoacetyl-CoA transferase (E.C. 2.8.3.-) catalyze the conversion of acetoacetyl- CoA to acetoacetate. These enzymes are designated "AtoDA." The genes encoding AtoDA are designated "atoDA."

[0095] Acetoacetate decarboxylase (E.C. 4.1.1.4) catalyze the conversion of acetoacetate to acetone. These enzymes are designated "Ada" The genes encoding Adc are designated "ode."

[0096] Isopropanol dehydrogenase (E.C. 1.1.1.80) catalyze the conversion of acetone to 2-propanol. These enzymes are designated "Adh." The genes encoding Adh are designated "adh."

[0097] The term "catabolic" and "catabolism" as used herein refers to the process of molecule breakdown or degradation of large molecules into smaller molecules. Catabolic or catabolism refers to a specific reaction pathway wherein the molecule breakdown occurs through a single catalytic component or a multitude thereof or a general, whole cell process wherein the molecule breakdown occurs using more than one specified reaction pathway and a multitude of catalytic components.

[0098] The term "anabolic" and "anabolism" as used herein refers to the process of chemical construction of small molecules into larger molecules. Anabolic refers to a specific reaction pathway wherein the molecule construction occurs through a single catalytic component or a multitude thereof or a general, whole cell process wherein the molecule construction occurs using more than one specified reaction pathway and a multitude of catalytic components.

[0099] The term "correlated" in "correlated saturation mutagenesis" as used herein refers to altering an amino acid type at two or more positions of a polypeptide to achieve an altered functional or structural attribute differing from the structural or functional attribute of the polypeptide from which the changes were made.

[0100] Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice of the invention and will be apparent to those of skill in the art. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting. [0101] Throughout this specification and claims, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Nucleic Acid Sequences

[0102] The invention described herein provides various enzymes for the production of propanol. Specifically, the enzymes listed in Tables 1-5 are useful for synthesizing propanol. Propanol can then be isolated and used for other industrial applications.

[0103] In one embodiment, the nucleic acid molecules of the invention encode a polypeptide having any one of the amino acid sequences of Table 6. Also provided are nucleic acid molecules encoding a polypeptide sequence that is at least 50% identical to any one of the amino acid sequences of Table 6. Preferably, the nucleic acid molecule of the invention encodes a polypeptide sequence at least 55%, 60%, 65%, 70%>, 75%, 80%, 85%, 90% or 95% identical to any one of the amino acid sequences of Table 6, and the identity can even more preferably be 98%, 99%, 99.9% or even higher. In another embodiment, the nucleic acid molecule of the invention encodes a polypeptide sequence with a range of 80% to 85%), 85%o to 90%), or 90%> to 95% identity to any one of the amino acid sequences of Table 6.

[0104] The invention also provides nucleic acid molecules that hybridize under stringent conditions to the above-described nucleic acid molecules. As defined above, and as is well known in the art, stringent hybridizations are performed at about 25°C below the thermal melting point (T_m) for the specific DNA hybrid under a particular set of conditions, where the T_m is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe. Stringent washing can be performed at temperatures about 5°C lower than the T_m for the specific DNA hybrid under a particular set of conditions.

[0105] The nucleic acid molecule of the invention includes DNA molecules (e.g., linear, circular, cDNA, chromosomal DNA, double stranded or single stranded) and RNA molecules (e.g., tRNA, rRNA, mRNA) and analogs of the DNA or RNA molecules of the described herein using nucleotide analogs. The isolated nucleic acid molecule of the invention includes a nucleic acid molecule free of naturally flanking sequences (i.e., sequences located at the 5' and 3' ends of the nucleic acid molecule) in the chromosomal DNA of the organism from which the nucleic acid is derived. In various embodiments, an isolated nucleic acid molecule can contain less than about 10 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, 0.1 kb, 50 bp, 25 bp or 10 bp of naturally flanking nucleotide chromosomal DNA sequences of the microorganism from which the nucleic acid molecule is derived. [0106] In one embodiment, the invention provides a gene described in Tables 1-5 wherein said gene is separated from another gene or other genes by intergenic DNA (for example, an intervening or spacer DNA which naturally flanks the gene and/or separates genes in the chromosomal DNA of the organism).

[0107] Nucleic acid molecules comprising a fragment of any one of the above-described nucleic acid sequences are also provided. These fragments preferably contain at least 20 contiguous nucleotides. More preferably the fragments of the nucleic acid sequences contain at least 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or even more contiguous nucleotides.

[0108] In another embodiment, a nucleic acid molecule of the invention hybridizes to all or a portion of a nucleic acid molecule having a nucleotide sequence that encodes a polypeptide having any one of the amino acid sequences of Table 6. Such hybridization conditions are known to those skilled in the art (see, for example, Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, Inc. (1995); Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)).

[0109] The nucleic acid sequence fragments of the invention display utility in a variety of systems and methods. For example, the fragments may be used as probes in various hybridization techniques. Depending on the method, the target nucleic acid sequences may be either DNA or RNA. The target nucleic acid sequences may be fractionated (e.g., by gel electrophoresis) prior to the hybridization, or the hybridization may be performed on samples in situ. One of skill in the art will appreciate that nucleic acid probes of known sequence find utility in determining chromosomal structure (e.g., by Southern blotting) and in measuring gene expression (e.g., by Northern blotting). In such experiments, the sequence fragments are preferably detectably labeled, so that their specific hybridization to target sequences can be detected and optionally quantified. One of skill in the art will appreciate that the nucleic acid fragments of the invention may be used in a wide variety of blotting techniques not specifically described herein.

[0110] It should also be appreciated that the nucleic acid sequence fragments disclosed herein also find utility as probes when immobilized on microarrays. Methods for creating microarrays by deposition and fixation of nucleic acids onto support substrates are well known in the art. Reviewed in DNA Microarrays: A Practical Approach (Practical Approach Series), Schena (ed.), Oxford University Press (1999) (ISBN: 0199637768); Nature Genet. 21(l)(suppl): l-60 (1999); Microarray Biochip: Tools and Technology, Schena (ed.), Eaton Publishing Company/BioTechniques Books Division (2000) (ISBN: 1881299376), the disclosures of which are incorporated herein by reference in their entireties. Analysis of, for example, gene expression using microarrays comprising nucleic acid sequence fragments, such as the nucleic acid sequence fragments disclosed herein, is a well-established utility for sequence fragments in the field of cell and molecular biology. Other uses for sequence fragments immobilized on microarrays are described in Gerhold et al., Trends Biochem. Sci. 24: 168-173 (1999) and Zweiger, Trends Biotechnol. 17:429-436 (1999); DNA Microarrays: A Practical Approach (Practical Approach Series), Schena (ed.), Oxford University Press (1999) (ISBN: 0199637768); Nature Genet. 21(l)(suppl): l-60 (1999); Microarray Biochip: Tools and Technology, Schena (ed.), Eaton Publishing Company/BioTechniques Books Division (2000) (ISBN: 1881299376), the disclosures of each of which is incorporated herein by reference in its entirety.

[0111] As is well known in the art, enzyme activities are measured in various ways. For example, the pyrophosphorolysis of OMP may be followed spectroscopically. Grubmeyer et al, J. Biol. Chem. 268:20299-20304 (1993). Alternatively, the activity of the enzyme is followed using chromatographic techniques, such as by high performance liquid

chromatography. Chung and Sloan, J. Chromatogr. 371 :71-81 (1986). As another alternative the activity is indirectly measured by determining the levels of product made from the enzyme activity. More modern techniques include using gas chromatography linked to mass spectrometry (Niessen, W. M. A. (2001). Current practice of gas chromatography— mass spectrometry. New York, N.Y: Marcel Dekker. (ISBN: 0824704738)). Additional modern techniques for identification of recombinant protein activity and products including liquid chromatography-mass spectrometry (LCMS), high performance liquid chromatography (HPLC), capillary electrophoresis, Matrix- Assisted Laser Desorption Ionization time of flight-mass spectrometry (MALDI-TOF MS), nuclear magnetic resonance (NMR), near-infrared (NIR) spectroscopy, viscometry (Knothe, G., R.O. Dunn, and M.O. Bagby. 1997. "Biodiesel: The use of vegetable oils and their derivatives as alternative diesel fuels." Am. Chem. Soc. Symp. Series 666: 172-208), physical property-based methods, wet chemical methods, etc. are used to analyze the levels and the identity of the product produced by the organisms of the invention. Other methods and techniques may also be suitable for the measurement of enzyme activity, as would be known by one of skill in the art.

[0112] Another embodiment of the invention comprises mutant or chimeric nucleic acid molecules or genes. Typically, a mutant nucleic acid molecule or mutant gene is comprised of a nucleotide sequence that has at least one alteration including, but not limited to, a simple substitution, insertion or deletion. The polypeptide of said mutant can exhibit an activity that differs from the polypeptide encoded by the wild-type nucleic acid molecule or gene.

Typically, a chimeric mutant polypeptide includes an entire domain derived from another polypeptide that is genetically engineered to be collinear with a corresponding domain.

Preferably, a mutant nucleic acid molecule or mutant gene encodes a polypeptide having improved activity such as substrate affinity, improved thermostability, activity at a different pH, or optimized codon usage for improved expression in a host cell.

Vectors

[0113] The recombinant vector can be altered, modified or engineered to have different or a different quantity of nucleic acid sequences than in the derived or natural recombinant vector nucleic acid molecule. Preferably, the recombinant vector includes a gene or recombinant nucleic acid molecule of the invention operably linked to regulatory sequences including, but not limited to, promoter sequences, terminator sequences and/or artificial ribosome binding sites (RBSs), as defined herein.

[0114] Typically, the one or more copies of one or more of the genes of the invention are operably linked to regulatory sequence(s) in a manner which allows for the desired expression characteristics of the nucleotide sequence. Preferably one or more of the genes of the invention is transcribed and translated into a gene product encoded by the nucleotide sequence when the recombinant nucleic acid molecule is included in a recombinant vector, as defined herein, and is introduced into a microorganism.

[0115] The regulatory sequence may be comprised of nucleic acid sequences which modulate, regulate or otherwise affect expression of other nucleic acid sequences. In one embodiment, a regulatory sequence can be in a similar or identical position and/or orientation relative to a nucleic acid sequence of the invention as observed in its natural state, e.g., in a native position and/or orientation. For example, a gene of interest can be included in a recombinant nucleic acid molecule or recombinant vector operably linked to a regulatory sequence which accompanies or is adjacent to the gene of interest in the natural host cell, or can be adjacent to a different gene in the natural host cell, or can be operably linked to a regulatory sequence from another organism. Regulatory sequences operably linked to a gene of the invention can be from other bacterial regulatory sequences, bacteriophage regulatory sequences and the like.

[0116] In one embodiment, a regulatory sequence is a sequence which has been modified, mutated, substituted, derivated, deleted, including sequences which are chemically

synthesized. Preferably, regulatory sequences include promoters, enhancers, termination signals, anti-termination signals and other expression control elements that, for example, serve as sequences to which repressors or inducers bind or serve as or encode binding sites for transcriptional and/or translational regulatory polypeptides, for example, in the transcribed mRNA (see Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor

Laboratory Press, Cold Spring Harbor, N.Y., 1989). Regulatory sequences include promoters directing constitutive expression of a nucleotide sequence in a host cell, promoters directing inducible expression of a nucleotide sequence in a host cell and promoters which attenuate or repress expression of a nucleotide sequence in a host cell. Regulating expression of a gene of interest also can be done by removing or deleting regulatory sequences. For example, sequences involved in the negative regulation of transcription can be removed such that expression of a gene of interest is enhanced. In one embodiment, a recombinant nucleic acid molecule or recombinant vector of the invention includes a nucleic acid sequence or gene that encodes at least one bacterial gene product of the invention operably linked to a promoter or promoter sequence. Preferably, promoters of the invention include native promoters, surrogate promoters and/or bacteriophage promoters.

[0117] In one embodiment, a promoter is associated with a biochemical housekeeping gene or a promoter associated with a pathway related to propanol synthesis. In another embodiment, a promoter is a bacteriophage promoter. Other promoters include tef (the translational elongation factor (TEF) promoter) which promotes high level expression in Bacillus (e.g., Bacillus subtilis). Additional advantageous promoters, for example, for use in Gram positive microorganisms include, but are not limited to, the amyE promoter or phage SP02 promoters. Additional advantageous promoters, for example, for use in Gram negative microorganisms include, but are not limited to aph2, cl, cpcB, lacl-trc, EM7, tac, trp, tet, trp- tet, lpp, lac, lpp-lac, laclq, T7, T5, T3, gal, trc, ara, SP6, λ-ρ_β or -p_L, and nirA.

[0118] In another embodiment, a recombinant nucleic acid molecule or recombinant vector of the invention includes a transcription terminator sequence or sequences. Typically, terminator sequences refer to the regulatory sequences which serve to terminate transcription of a gene. Terminator sequences (or tandem transcription terminators) can further serve to stabilize mRNA (e.g., by adding structure to mRNA), for example, against nucleases.

[0119] In another embodiment, a recombinant nucleic acid molecule or recombinant vector of the invention has sequences allowing for detection of the vector containing sequences (i.e., detectable and/or selectable markers), for example, sequences that overcome auxotrophic mutations, for example, ura3 or ilvE, fluorescent markers, and/or calorimetric markers (e.g., lacZ/ -galactosidase), and/or antibiotic resistance genes (e.g., bla or tet). [0120] Also provided are vectors, including expression vectors, which comprise the above nucleic acid molecules of the invention, as described further herein. In a first embodiment, the vectors include the isolated nucleic acid molecules described above. In an alternative embodiment, the vectors of the invention include the above-described nucleic acid molecules operably linked to one or more expression control sequences.

Isolated Polypeptides

[0121] According to another aspect of the present invention, isolated polypeptides (including muteins, allelic variants, fragments, derivatives, and analogs) encoded by the nucleic acid molecules of the present invention are provided. In one embodiment, the isolated polypeptide comprises the polypeptide sequence corresponding to SEQ ID NOS: l-18. In an alternative embodiment of the present invention, the isolated polypeptide comprises a polypeptide sequence at least 85% identical to SEQ ID NOS: l-18. Preferably, the isolated polypeptide of the present invention has 90%, 95%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or even higher identity to SEQ ID NOS: l-18. More preferably, the isolated polypeptide of the present invention has 90%> to 95%, or 95% to 97% identity to SEQ ID NOS: l-18.

[0122] According to other embodiments of the present invention, isolated polypeptides comprising a fragment of the above-described polypeptide sequences are provided. These fragments preferably include at least 20 contiguous amino acids, more preferably at least 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or even more contiguous amino acids.

[0123] The polypeptides of the present invention also include fusions between the above- described polypeptide sequences and heterologous polypeptides. The heterologous sequences can, for example, include sequences designed to facilitate purification, e.g. histidine tags, and/or visualization of recombinantly-expressed proteins. Other non-limiting examples of protein fusions include those that permit display of the encoded protein on the surface of a phage or a cell, fusions to intrinsically fluorescent proteins, such as green fluorescent protein (GFP), and fusions to the IgG Fc region.

Host Cell Transformants

[0124] In another aspect of the invention, host cells transformed with the nucleic acid molecules or vectors of the invention, and descendants thereof, are provided. In some embodiments of the invention, these cells carry the nucleic acid sequences of the invention on vectors, which may but need not be freely replicating vectors. In other embodiments of the invention, the nucleic acids have been integrated into the genome of the host cells.

[0125] The host cell encoding at least one enzyme provided in Tables 1-5 can be a host cell wherein the enzyme coding gene is endogenous to the host cell, a host cell wherein the enzyme coding gene is exogenous to the host cell, or a host cell engineered to express an enzyme listed in Tables 1-5.

[0126] In a preferred embodiment, the host cell comprises one or more copies of at least one nucleic acid encoding at least one amino acid sequence in Table 6.

[0127] In an alternative embodiment, the host cells of the invention can be mutated by recombination with a disruption, deletion or mutation of the isolated nucleic acid of the invention so that the activity of any or all of the polypeptides in the host cell is reduced or eliminated compared to a host cell lacking the mutation.

[0128] In another aspect, the invention provides a method for expressing a polypeptide of the invention under suitable culture conditions and choice of host cell line for optimal enzyme expression, activity and stability (codon usage, salinity, pH, temperature, etc.).

Selected or Engineered Microorganisms For the Production of Carbon-Based Products of Interest

[0129] Microorganism: Includes prokaryotic and eukaryotic microbial species from the Domains Archaea, Bacteria and Eucarya, the latter including yeast and filamentous fungi, protozoa, algae, or higher Protista. The terms "microbial cells" and "microbes" are used interchangeably with the term microorganism.

[0130] A variety of host organisms can be transformed to produce a product of interest. Photoautotrophic organisms include eukaryotic plants and algae, as well as prokaryotic cyanobacteria, green-sulfur bacteria, green non-sulfur bacteria, purple sulfur bacteria, and purple non-sulfur bacteria.

[0131] The host cell can be a Gram-negative bacterial cell or a Gram-positive bacterial cell. A Gram-negative host cell of the invention can be, e.g., Gluconobacter, Rhizobium, Bradyrhizobium, Alcaligenes, Rhodobacter, Rhodococcus. Azospirillum, Rhodospirillum, Sphingomonas, Burkholderia, Desuifomonas, Geospirillum, Succinomonas, Aeromonas, Shewanella, Halochromatium, Citrobacter, Escherichia, Klebsiella, Zymomonas Zymobacter, or Acetobacter. A Gram-positive host cell of the invention can be, e.g., Fibrobacter,

Acidobacter, Bacteroides, Sphingobacterium, Actinomyces, Corynebacterium, Nocardia, Rhodococcus, Propionibacterium, Bifidobacterium, Bacillus, Geobacillus, Paenibacillus, Sulfobacillus, Clostridium, Anaerobacter, Eubacterium, Streptococcus, Lactobacillus, Leuconostoc, Enterococcus, Lactococcus, Thermobifida, Cellulomonas, or Sarcina.

[0132] Extremophiles are also contemplated as suitable organisms. Such organisms withstand various environmental parameters such as temperature, radiation, pressure, gravity, vacuum, desiccation, salinity, pH, oxygen tension, and chemicals. They include

hyperthermophiles, which grow at or above 80°C such as Pyrolobus fumarii; thermophiles, which grow between 60-80°C such as Synechococcus lividis; mesophiles, which grow between 15-60°C and psychrophiles, which grow at or below 15°C such as Psychrobacter and some insects. Radiation-tolerant organisms include Deinococcus radiodurans. Pressure- tolerant organisms include piezophiles or barophiles, which tolerate pressure of 130 MPa. Hypergravity- (e.g., >lg) hypogravity- (e.g., <lg) tolerant organisms are also contemplated. Vacuumtolerant organisms include tardigrades, insects, microbes and seeds. Dessicant- tolerant and anhydrobiotic organisms include xerophiles such as Artemia salina; nematodes, microbes, fungi and lichens. Salt-tolerant organisms include halophiles (e.g., 2-5 M NaCl) Halobacteriacea and Dunaliella salina. pH-tolerant organisms include alkaliphiles such as Natronobacterium, Bacillus firmus OF4, Spirulina spp. (e.g., pH > 9) and acidophiles such as Cyanidium caldarium, Ferroplasma sp. (e.g., low pH). Anaerobes, which cannot tolerate 0₂ such as Methanococcus jannaschii; microaerophils, which tolerate some 0₂ such as

Clostridium and aerobes, which require 0₂ are also contemplated. Gas-tolerant organisms, which tolerate pure C0₂ include Cyanidium caldarium and metal-tolerant organisms include metalotolerants such as Ferroplasma acidarmanus (e.g., Cu, As, Cd, Zn), Ralstonia sp. CH34 (e.g., Zn, Co, Cd, Hg, Pb). Gross, Michael. Life on the Edge: Amazing Creatures Thriving in Extreme Environments. New York: Plenum (1998) and Seckbach, J. "Search for Life in the Universe with Terrestrial Microbes Which Thrive Under Extreme Conditions." In Cristiano Batalli Cosmovici, Stuart Bowyer, and Dan Wertheimer, eds., Astronomical and Biochemical Origins and the Search for Life in the Universe, p. 511. Milan: Editrice Compositori (1997).

[0133] Plants include but are not limited to the following genera: Arabidopsis, Beta, Glycine, Jatropha, Miscanthus, Panicum, Phalaris, Populus, Saccharum, Salix, Simmondsia and Zea.

[0134] Algae and cyanobacteria include but are not limited to the following genera: Acanthoceras, Acanthococcus, Acaryochloris, Achnanthes, Achnanthidium, Actinastrum, Actinochloris, Actinocyclus, Actinotaenium, Amphichrysis, Amphidinium, Amphikrikos, Amphipleura, Amphiprora, Amphithrix, Amphora, Anabaena, Anabaenopsis, Aneumastus, Ankistrodesmus, Ankyra, Anomoeoneis, Apatococcus, Aphanizomenon, Aphanocapsa, Aphanochaete, Aphanothece, Apiocystis, Apistonema, Arthrodesmus, Artherospira,

Ascochloris, Asterionella, Asterococcus, Audouinella, Aulacoseira, Bacillaria, Balbiania, Bambusina, Bangia, Basichlamys, Batrachospermum, Binuclearia, Bitrichia, Blidingia, Botrdiopsis, Botrydium, Botryococcus, Botryosphaerella, Brachiomonas, Brachysira, Brachytrichia, Brebissonia, Bulbochaete, Bumilleria, Bumilleriopsis, Caloneis, Calothrix, Campylodiscus, Capsosiphon, Carteria, Catena, Cavinula, Centritr actus, Centronella, Ceratium, Chaetoceros, Chaetochloris, Chaetomorpha, Chaetonella, Chaetonema,

Chaetopeltis, Chaetophora, Chaetosphaeridium, Chamaesiphon, Chara, Characiochloris, Characiopsis, Characium, Charales, Chilomonas, Chlainomonas, Chlamydoblepharis, Chlamydocapsa, Chlamydomonas, Chlamydomonopsis, Chlamydomyxa, Chlamydonephris, Chlorangiella, Chlorangiopsis, Chlorella, Chlorobotrys, Chlorobrachis, Chlorochytrium, Chlorococcum, Chlorogloea, Chlorogloeopsis, Chlorogonium, Chlorolobion, Chloromonas, Chlorophysema, Chlorophyta, Chlorosaccus, Chlorosarcina, Choricystis, Chromophyton, Chromulina, Chroococcidiopsis, Chroococcus, Chroodactylon, Chroomonas, Chroothece, Chrysamoeba, Chrysapsis, Chrysidiastrum, Chrysocapsa, Chrysocapsella, Chrysochaete, Chrysochromulina, Chrysococcus, Chrysocrinus, Chrysolepidomonas, Chrysolykos,

Chrysonebula, Chrysophyta, Chrysopyxis, Chrysosaccus, Chrysophaerella,

Chrysostephanosphaera, Clodophora, Clastidium, Closteriopsis, Closterium, Coccomyxa, Cocconeis, Coelastrella, Coelastrum, Coelosphaerium, Coenochloris, Coenococcus,

Coenocystis, Colacium, Coleochaete, Collodictyon, Compsogonopsis, Compsopogon, Conjugatophyta, Conochaete, Coronastrum, Cosmarium, Cosmioneis, Cosmocladium, Crateriportula, Craticula, Crinalium, Crucigenia, Crucigeniella, Cryptoaulax, Cryptomonas, Cryptophyta, Ctenophora, Cyanodictyon, Cyanonephron, Cyanophora, Cyanophyta,

Cyanothece, Cyanothomonas, Cyclonexis, Cyclostephanos, Cyclotella, Cylindrocapsa, Cylindrocystis, Cylindrospermum, Cylindrotheca, Cymatopleura, Cymbella,

Cymbellonitzschia, Cystodinium Dactylococcopsis, Debarya, Denticula, Dermatochrysis, Dermocarpa, Dermocarpella, Desmatractum, Desmidium, Desmococcus, Desmonema, Desmosiphon, Diacanthos, Diacronema, Diadesmis, Diatoma, Diatomella, Dicellula, Dichothrix, Dichotomococcus, Dicranochaete, Dictyochloris, Dictyococcus,

Dictyosphaerium, Didymocystis, Didymogenes, Didymosphenia, Dilabifilum,

Dimorphococcus, Dinobryon, Dinococcus, Diplochloris, Diploneis, Diplostauron,

Distrionella, Docidium, Draparnaldia, Dunaliella, Dysmorphococcus, Ecballocystis, Elakatothrix, Ellerbeckia, Encyonema, Enteromorpha, Entocladia, Entomoneis,

Entophysalis, Epichrysis, Epipyxis, Epithemia, Eremosphaera, Euastropsis, Euastrum, Eucapsis, Eucocconeis, Eudorina, Euglena, Euglenophyta, Eunotia, Eustigmatophyta, Eutreptia, Fallacia, Fischerella, Fragilaria, Fragilariforma, Franceia, Frustulia, Curcilla, Geminella, Genicularia, Glaucocystis, Glaucophyta, Glenodiniopsis, Glenodinium,

Gloeocapsa, Gloeochaete, Gloeochrysis, Gloeococcus, Gloeocystis, Gloeodendron,

Gloeomonas, Gloeoplax, Gloeothece, Gloeotila, Gloeotrichia, Gloiodictyon, Golenkinia, Golenkiniopsis, Gomontia, Gomphocymbella, Gomphonema, Gomphosphaeria,

Gonatozygon, Gongrosia, Gongrosira, Goniochloris, Gonium, Gonyostomum,

Granulochloris, Granulocystopsis, Groenbladia, Gymnodinium, Gymnozyga, Gyrosigma, Haematococcus, Hafniomonas, Hallassia, Hammatoidea, Hannaea, Hantzschia,

Hapalosiphon, Haplotaenium, Haptophyta, Haslea, Hemidinium, Hemitonia, Heribaudiella, Heteromastix, Heterothrix, Hibberdia, Hildenbrandia, Hillea, Holopedium, Homoeothrix, Hormanthonema, Hormotila, Hyalobrachion, Hyalocardium, Hyalodiscus, Hyalogonium, Hyalotheca, Hydrianum, Hydrococcus, Hydrocoleum, Hydrocoryne, Hydrodictyon,

Hydrosera, Hydrurus, Hyella, Hymenomonas, Isthmochloron, Johannesbaptistia,

Juranyiella, Karayevia, Kathablepharis, Katodinium, Kephyrion, Keratococcus,

Kirchneriella, Klebsormidium, Kolbesia, Koliella, Komarekia, Korshikoviella, Kraskella, Lagerheimia, Lagynion, Lamprothamnium, Lemanea, Lepocinclis, Leptosira, Lobococcus, Lobocystis, Lobomonas, Luticola, Lyngbya, Malleochloris, Mallomonas, Mantoniella, Marssoniella, Martyana, Mastigocoleus, Gastogloia, Melosira, Merismopedia, Mesostigma, Mesotaenium, Micractinium, Micrasterias, Microchaete, Microcoleus, Microcystis,

Microglena, Micromonas, Microspora, Microthamnion, Mischococcus, Monochrysis, Monodus, Monomastix, Monoraphidium, Monostroma, Mougeotia, Mougeotiopsis,

Myochloris, Myromecia, Myxosarcina, Naegeliella, Nannochloris, Nautococcus, Navicula, Neglectella, Neidium, Nephroclamys, Nephrocytium, Nephrodiella, Nephroselmis, Netrium, Nitella, Nitellopsis, Nitzschia, Nodularia, Nostoc, Ochromonas, Oedogonium,

Oligochaetophora, Onychonema, Oocardium, Oocystis, Opephora, Ophiocytium, Orthoseira, Oscillatoria, Oxyneis, Pachycladella, Palmella, Palmodictyon, Pnadorina, Pannus, Paralia, Pascherina, Paulschulzia, Pediastrum, Pedinella, Pedinomonas, Pedinopera, Pelagodictyon, Penium, Peranema, Peridiniopsis, Peridinium, Peronia, Petroneis, Phacotus, Phacus, Phaeaster, Phaeodermatium, Phaeophyta, Phaeosphaera, Phaeothamnion, Phormidium, Phycopeltis, Phyllariochloris, Phyllocardium, Phyllomitas, Pinnularia, Pitophora, Placoneis, Planctonema, Planktosphaeria, Planothidium, Plectonema, Pleodorina, Pleurastrum, Pleurocapsa, Pleurocladia, Pleurodiscus, Pleurosigma, Pleurosira, Pleurotaenium,

Pocillomonas, Podohedra, Polyblepharides, Polychaetophora, Polyedriella, Polyedriopsis, Polygoniochloris, Polyepidomonas, Polytaenia, Polytoma, Polytomella, Porphyridium, Posteriochromonas, Prasinochloris, Prasinocladus, Prasinophyta, Prasiola, Prochlorphyta, Prochlorothrix, Protoderma, Protosiphon, Provasoliella, Prymnesium, Psammodictyon, Psammothidium, Pseudanabaena, Pseudenoclonium, Psuedocarteria, Pseudochate,

Pseudocharacium, Pseudococcomyxa, Pseudodictyosphaerium, Pseudokephyrion,

Pseudoncobyrsa, Pseudoquadrigula, Pseudosphaerocystis, Pseudostaurastrum,

Pseudostaurosira, Pseudotetrastrum, Pteromonas, Punctastruata, Pyramichlamys,

Pyramimonas, Pyrrophyta, Quadrichloris, Quadricoccus, Quadrigula, Radiococcus,

Radiofilum, Raphidiopsis, Raphidocelis, Raphidonema, Raphidophyta, Peimeria,

Rhabdoderma, Rhabdomonas, Rhizoclonium, Rhodomonas, Rhodophyta, Rhoicosphenia, Rhopalodia, Rivularia, Rosenvingiella, Rossithidium, Roya, Scenedesmus, Scherffelia, Schizochlamydella, Schizochlamys, Schizomeris, Schizothrix, Schroederia, Scolioneis, Scotiella, Scotiellopsis, Scourfieldia, Scytonema, Selenastrum, Selenochloris, Sellaphora, Semiorbis, Siderocelis, Diderocystopsis, Dimonsenia, Siphononema, Sirocladium,

Sirogonium, Skeletonema, Sorastrum, Spermatozopsis, Sphaerellocystis, Sphaerellopsis, Sphaerodinium, Sphaeroplea, Sphaerozosma, Spiniferomonas, Spirogyra, Spirotaenia, Spirulina, Spondylomorum, Spondylosium, Sporotetras, Spumella, Staurastrum,

Stauerodesmus, Stauroneis, Staurosira, Staurosirella, Stenopterobia, Stephanocostis, Stephanodiscus, Stephanoporos, Stephanosphaera, Stichococcus, Stichogloea, Stigeoclonium, Stigonema, Stipitococcus, Stokesiella, Strombomonas, Stylochrysalis, Stylodinium, Styloyxis, Stylosphaeridium, Surirella, Sykidion, Symploca, Synechococcus, Synechocystis, Synedra, Synochromonas, Synura, Tabellaria, Tabularia, Teilingia, Temnogametum, Tetmemorus, Tetrachlorella, Tetracyclus, Tetradesmus, Tetraedriella, Tetraedron, Tetraselmis,

Tetraspora, Tetrastrum, Thalassiosira, Thamniochaete, Thorakochloris, Thorea, Tolypella, Tolypothrix, Trachelomonas, Trachydiscus, Trebouxia, Trentepholia, Treubaria, Tribonema, Trichodesmium, Trichodiscus, Trochiscia, Tryblionella, Ulothrix, Uroglena, Uronema, Urosolenia, Urospora, Uva, Vacuolaria, Vaucheria, Volvox, Volvulina, Westella,

Woloszynskia, Xanthidium, Xanthophyta, Xenococcus, Zygnema, Zygnemopsis, and

Zygonium.

[0135] Green non-sulfur bacteria include but are not limited to the following genera: Chloroflexus, Chloronema, Oscillochloris, Heliothrix, Herpetosiphon, Roseiflexus, and Thermomicrobium.

[0136] Green sulfur bacteria include but are not limited to the following genera:

Chlorobium, Clathrochloris, and Prosthecochloris . [0137] Purple sulfur bacteria include but are not limited to the following genera:

Allochromatium, Chromatium, Halochromatium, Isochromatium, Marichromatium,

Rhodovulum, Thermochromatium, Thiocapsa, Thiorhodococcus, and Thiocystis,

[0138] Purple non-sulfur bacteria include but are not limited to the following genera: Phaeospirillum, Rhodobaca, Rhodobacter, Rhodomicrobium, Rhodopila,

Rhodopseudomonas, Rhodothalassium, Rhodospirillum, Rodovibrio, and Roseospira.

[0139] Aerobic chemolithotrophic bacteria include but are not limited to nitrifying bacteria such as Nitrobacteraceae sp., Nitrobacter sp., Nitrospina sp., Nitrococcus sp., Nitrospira sp., Nitrosomonas sp., Nitrosococcus sp., Nitrosospira sp., Nitrosolobus sp., Nitrosovibrio sp.; colorless sulfur bacteria such as, Thiovulum sp., Thiobacillus sp.,

Thiomicrospira sp., Thiosphaera sp., Thermothrix sp.; obligately chemolithotrophic hydrogen bacteria such as Hydrogenobacter sp., iron and manganese-oxidizing and/or depositing bacteria such as Siderococcus sp., and magnetotactic bacteria such as Aquaspirillum sp.

[0140] Archaeobacteria include but are not limited to methanogenic archaeobacteria such as Methanobacterium sp., Methanobrevibacter sp., Methanothermus sp., Methanococcus sp., Methanomicrobium sp., Methanospirillum sp., Methanogenium sp., Methanosarcina sp., Methanolobus sp., Methanothrix sp., Methanococcoides sp., Methanoplanus sp.; extremely thermophilic sulfur-metabolizers such as Thermoproteus sp., Pyrodictium sp., Sulfolobus sp., Acidianus sp. and other microorganisms such as, Bacillus subtilis, Saccharomyces cerevisiae, Streptomyces sp., Ralstonia sp., Rhodococcus sp., Corynebacteria sp., Brevibacteria sp., Mycobacteria sp., and oleaginous yeast.

[0141] HyperPhotosynthetic conversion requires extensive genetic modification; thus, in preferred embodiments the parental photoautotrophic organism can be transformed with exogenous DNA.

[0142] Preferred organisms for HyperPhotosynthetic conversion include: Arabidopsis thaliana, Panicum virgatum, Miscanthus giganteus, and Zea mays (plants), Botryococcus braunii, Chlamydomonas reinhardtii and Dunaliela salina (algae), Synechococcus sp PCC 7002, Synechococcus sp. PCC 7942, Synechocystis sp. PCC 6803, and Thermosynechococcus elongatus BP-1 (cyanobacteria), Chlorobium tepidum (green sulfur bacteria), Chloroflexus auranticus (green non-sulfur bacteria), Chromatium tepidum and Chromatium vinosum (purple sulfur bacteria), Rhodospirillum rubrum, Rhodobacter capsulatus, and

Rhodopseudomonas palusris (purple non-sulfur bacteria). [0143] Yet other suitable organisms include synthetic cells or cells produced by synthetic genomes as described in Venter et al. US Pat. Pub. No. 2007/0264688, and cell-like systems or synthetic cells as described in Glass et al. US Pat. Pub. No. 2007/0269862.

[0144] Still, other suitable organisms include microorganisms that can be engineered to fix carbon dioxide bacteria such as Escherichia coli, Acetobacter aceti, Bacillus subtilis, yeast and fungi such as Clostridium ljungdahlii, Clostridium thermocellum, Penicillium chrysogenum, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pseudomonas fluorescens, or Zymomonas mobilis.

[0145] A common theme in selecting or engineering a suitable organism is autotrophic fixation of C0₂ to products. This would cover photosynthesis and methanogenesis.

Acetogenesis, encompassing the three types of C0₂ fixation; Calvin cycle, acetyl-CoA pathway and reductive TCA pathway is also covered. The capability to use carbon dioxide as the sole source of cell carbon (autotrophy) is found in almost all major groups ofprokaryotes. The C0₂ fixation pathways differ between groups, and there is no clear distribution pattern of the four presently-known autotrophic pathways. Fuchs, G. 1989. Alternative pathways of autotrophic C0₂ fixation, p. 365-382. In H. G. Schlegel, and B. Bowien (ed.), Autotrophic bacteria. Springer- Verlag, Berlin, Germany. The reductive pentose phosphate cycle

(Calvin-Bassham-Benson cycle) represents the C0₂ fixation pathway in many aerobic autotrophic bacteria, for example, cyanobacteria.

Antibodies

[0146] In another aspect, the invention provides isolated antibodies, including fragments and derivatives thereof that bind specifically to the isolated polypeptides and polypeptide fragments of the invention or to one or more of the polypeptides encoded by the isolated nucleic acids of the invention. The antibodies of the invention may be specific for linear epitopes, discontinuous epitopes or conformational epitopes of such polypeptides or polypeptide fragments, either as present on the polypeptide in its native conformation or, in some cases, as present on the polypeptides as denatured, as, e.g., by solubilization in SDS. Among the useful antibody fragments provided by the instant invention are Fab, Fab', Fv, F(ab')₂, and single chain Fv fragments.

[0147] By "bind specifically" and "specific binding" is here intended the ability of the antibody to bind to a first molecular species in preference to binding to other molecular species with which the antibody and first molecular species are admixed. An antibody is said specifically to "recognize" a first molecular species when it can bind specifically to that first molecular species. [0148] As is well known in the art, the degree to which an antibody can discriminate as among molecular species in a mixture will depend, in part, upon the conformational relatedness of the species in the mixture; typically, the antibodies of the invention will discriminate over adventitious binding to unrelated polypeptides by at least two-fold, more typically by at least 5-fold, typically by more than 10-fold, 25-fold, 50-fold, 75-fold, and often by more than 100-fold, and on occasion by more than 500-fold or 1000-fold.

[0149] Typically, the affinity or avidity of an antibody (or antibody multimer, as in the case of an IgM pentamer) of the invention for a polypeptide or polypeptide fragment of the invention will be at least about lxl 0^"6 M, typically at least about 5x10^~7 M, usefully at least about lxl 0^"7 M, with affinities and avidities of lxl 0^"8 M, 5x10^~9 M, lxl 0^"10 M and even stronger proving especially useful.

[0150] The isolated antibodies of the invention may be naturally-occurring forms, such as IgG, IgM, IgD, IgE, and IgA, from any mammalian species. For example, antibodies are usefully obtained from species including rodents-typically mouse, but also rat, guinea pig, and hamster-lagomorphs, typically rabbits, and also larger mammals, such as sheep, goats, cows, and horses. The animal is typically affirmatively immunized, according to standard immunization protocols, with the polypeptide or polypeptide fragment of the invention.

[0151] Virtually all fragments of 8 or more contiguous amino acids of the polypeptides of the invention may be used effectively as immunogens when conjugated to a carrier, typically a protein such as bovine thyroglobulin, keyhole limpet hemocyanin, or bovine serum albumin, conveniently using a bifunctional linker. Immunogenicity may also be conferred by fusion of the polypeptide and polypeptide fragments of the invention to other moieties. For example, peptides of the invention can be produced by solid phase synthesis on a branched polylysine core matrix; these multiple antigenic peptides (MAPs) provide high purity, increased avidity, accurate chemical definition and improved safety in vaccine development. See, e.g., Tam et al, Proc. Natl. Acad. Sci. USA 85:5409-5413 (1988); Posnett et al, J. Biol. Chem. 263, 1719-1725 (1988).

[0152] Protocols for immunization are well-established in the art. Such protocols often include multiple immunizations, either with or without adjuvants such as Freund's complete adjuvant and Freund's incomplete adjuvant. Antibodies of the invention may be polyclonal or monoclonal, with polyclonal antibodies having certain advantages in immunohistochemical detection of the proteins of the invention and monoclonal antibodies having advantages in identifying and distinguishing particular epitopes of the proteins of the invention. Following immunization, the antibodies of the invention may be produced using any art-accepted technique. Host cells for recombinant antibody production-either whole antibodies, antibody fragments, or antibody derivatives-can be prokaryotic or eukaryotic. Prokaryotic hosts are particularly useful for producing phage displayed antibodies, as is well known in the art. Eukaryotic cells, including mammalian, insect, plant and fungal cells are also useful for expression of the antibodies, antibody fragments, and antibody derivatives of the invention. Antibodies of the invention can also be prepared by cell free translation.

[0153] The isolated antibodies, including fragments and derivatives thereof, can usefully be labeled. It is, therefore, another aspect of the invention to provide labeled antibodies that bind specifically to one or more of the polypeptides and polypeptide fragments of the invention. The choice of label depends, in part, upon the desired use. In some cases, the antibodies of the invention may usefully be labeled with an enzyme. Alternatively, the antibodies may be labeled with colloidal gold or with a fluorophore. For secondary detection using labeled avidin, streptavidin, captavidin or neutravidin, the antibodies of the invention may usefully be labeled with biotin. When the antibodies are used, e.g., for Western blotting

33 32 35 3 applications, they may usefully be labeled with radioisotopes, such as P, P, S, H and ¹²⁵I. As would be understood, use of the labels described above is not restricted to any particular application.

Methods for Designing Protein Variants

[0154] Increased propanol production can be achieved through the expression and optimization of one or more of the enzymes in Tables 1-5 in organisms well suited for modern genetic engineering techniques, that rapidly grow, are capable of thriving on inexpensive food resources, and from which isolation of a desired product is easily and inexpensively achieved. To increase the rate of propanol production, it would be

advantageous to design and select variants of the enzymes of the invention, including but not limited to, variants optimized for substrate affinity, substrate specificity, substrate catalytic conversion rate, improved thermostability, activity at a different pH and/or optimized codon usage for improved expression in a host cell. See, for example, amino acid changes correlated to alterations in the catalytic rate while maintaining similar affinities (RL Zheng and RG Kemp, J. Biol. Chem. (1994) Vol. 269: 18475-18479) or amino acid changes correlated with changes in the stability of the transition state that affect catalytic turnover (MA Phillips, et al, J. Biol. Chem., (1990) Vol. 265:20692-20698). It would be another advantage to design and select for enzymes altered to have substantially decreased reverse reaction activity in which enzyme-substrate products would be the result of energetically unfavorable bond formation or molecular re-configuration of the substrate or have improved forward reaction activity in which enzyme-substrate products would be the result of energetically favorable molecular bond reduction or molecular re-configuration. It would be yet another advantage to design and select for enzymes altered to have substantially decreased unwanted substrate by-product conversion reactions.

[0155] Accordingly, one method for the design of propanol pathway proteins of the invention utilizes computational and bioinformatic analysis to design and select for advantageous changes in primary amino acid sequences encoding enzyme activity.

Computational methods and bioinformatics provide tractable alternatives for rational design of protein structure and function. Recently, algorithms analyzing protein structure for biophysical character (for example, motional dynamics and total energy or Gibb's Free Energy evaluations) have become a commercially feasible methodology supplementing protein sequence analysis data that assess homology, identity and/or degree of sequence and domain conservation to improve upon or design the desirable qualities of a protein

(Rosetta++, University of Washington). For example, an in silico redesign of the

endonuclease I-Msol was based on computational evaluation of biophysical parameters of rationally selected changes to the primary amino acid sequence. Researchers were able to maintain wild-type binding selectivity and affinity yet improve the catalytic turnover by four orders of magnitude (Ashworth, et al., Nature (2006) vol. 441 :656-659).

[0156] In one embodiment of the invention, polypeptide sequences of the invention or related homologues in a complex with a substrate are obtained from the Protein Data Bank (PDB; HM Berman, et al., Nucleic Acids Research (2000) vol. 28:235-242) for computational analysis on steady state and/or changes in Gibb's free energy relative to the wild type protein. Substitutions of one amino acid residue for another are accomplished in silico interactively as a means for identifying specific residue substitutions that optimize structural or catalytic contacts between the protein and substrate using standard software programs for viewing molecules as is well known to those skilled in the art.

[0157] Specific amino acid substitutions are rationally chosen based on substituted residue characteristics that optimize, for example, Van der Waal's interactions,

hydrophobicity, hydrophilicity, steric non-interferences, pH-dependent electrostatics and related chemical interactions. The overall energetic change of the substitution protein model when unbound and bound to its substrate is calculated and assessed by one having skill in the art to be evaluated for the change in free energy for correlations to overall structural stability (e.g., Meiler, J. and D. Baker, Proteins (2006) 65:538-548). In addition, such computational methods provide a means for accurately predicting quaternary protein structure interactions such that in silico modifications are predictive or determinative of overall multimeric structural stability (Wollacott, AM, et al., Protein Science (2007) 16: 165-175; Joachimiak, LA, et al., J. Mol. Biol. (2006) 361 : 195-208).

[0158] Preferably, a rational design change to the primary structure of the protein sequences of the invention minimally alter the Gibb's free energy state of the unbound polypeptides and maintain a folded, functional and similar wild-type enzyme structure. More preferably a lower computational total free energy change of protein sequences of the invention is achieved to indicate the potential for optimized enzyme structural stability.

[0159] Although lower free energy of a protein structure relative to the wild type structure is an indicator of thermodynamic stability, the positive correlation of increased thermal stability to optimized function does not always exist. Therefore, preferably, optimal catalytic contacts between the modified protein structure and the substrate are achieved with a concomitant predicted favorable change in total free energy of the catabolic reaction, for example by rationally designing protein/substrate interactions that stabilize the transition state of the enzymatic reaction while maintaining a similar or favorable change in free energy of the unbound protein for a desired environment in which a host cell expresses the mutant protein.

Methods for Generating Protein Variants

[0160] Several methods well known to those with skill in the art are available to generate random nucleotide sequence variants for a corresponding polypeptide sequence using the Polymerase Chain Reaction ("PCR") (US Patent 4,683,202). In one embodiment of the invention is the generation of gene variants using the method of error prone PCR. (R.

Cadwell and G. Joyce, PCR Meth. Appl. (1991) Vol. 2:28-33; Leung, et al, Technique (1989) Vol. 1 : 11-15). Error prone PCR is achieved by the establishment of a chemical environment during the PCR experiment that causes an increase in unfaithful replication of a parent copy of DNA sought to be replicated. For example, increasing the manganese or magnesium ion content of the chemical admixture used in the PCR experiment, very low annealing temperatures, varying the balance among di-deoxy nucleotides added, starting with a low population of parent DNA templates or using polymerases designed to have increased inefficiencies in accurate DNA replication all result in nucleotide changes in progeny DNA sequences during the PCR replication process. The resultant mutant DNA sequences are genetically engineered into an appropriate vector to be expressed in a host cell and analyzed to screen and select for the desired effect on whole cell production of a product or process of interest. In one embodiment of the invention, random mutagenesis of nucleotide sequences of the invention is generated through error prone PCR using techniques well known to one skilled in the art. Resultant nucleotide sequences are analyzed for structural and functional attributes through clonal screening assays and other methods as described herein.

[0161] In another embodiment a specifically desired protein mutant is generated a using site-directed mutagenesis. For example, with overlap extension (An, et al., Appl. Microbiol. Biotech. (2005) vol. 68(6):774-778) or mega-primer PCR (E. Burke and S. Barik, Methods Mol. Bio. (2003) vol 226:525-532) one can use nucleotide primers that have been altered at corresponding codon positions in the parent nucleotide to yield DNA progeny sequences containing the desired mutation. Alternatively, one can use cassette mutagenesis (Kegler- Ebo, et al, Nucleic Acids Res. (1994) vol. 22(9): 1593-1599) as is commonly known by one skilled in the art.

[0162] Another embodiment of the invention is to select for a polypeptide variant for expression in a recipient host cell by comparing a first nucleic acid sequence encoding the polypeptide with the nucleic acid sequence of a second, related nucleic acid sequence encoding a polypeptide having more desirable qualities, and altering at least one codon of the first nucleic acid sequence to have identity with the corresponding codon of the second nucleic acid sequence, such that improved polypeptide activity, substrate specificity, substrate affinity, substrate catalytic conversion rate, improved thermostability, activity at a different pH and/or optimized codon usage for expression and/or structure of the altered polypeptide is achieved in the host cell.

[0163] In yet another embodiment of the invention, all amino acid residue variations are encoded at any desired, specified nucleotide codon position using such methods as site saturation mutagenesis (Meyers, et al., Science (1985) Vol. 229:242-247; Derbyshire, et al., Gene (1986) Vol. 46: 145-152; U.S. Patent 6,171,820). Whole gene site saturation

mutagenesis (K. Kretz, et al., Meth. Enzym. (2004) Vol. 388:3-11) is preferred wherein all amino acid residue variations are encoded at every nucleotide codon position. Both methods yield a population of protein variants differing from the parent polypeptide by one amino acid, with each amino acid substitution being correlated to structural/functional attributes at any position in the polypeptide. Saturation mutagenesis uses PCR and primers homologous to the parent sequence wherein one or more codon encoding nucleotide triplets is randomized. Randomization results in the incorporation of codons corresponding to all amino acid replacements in the final, translated polypeptide. Each PCR product is genetically engineered into an expression vector to be introduced into an expression host and screened for structural and functional attributes through clonal screening assays and other methods as described herein.

[0164] In one aspect of saturation mutagenesis, correlated saturation mutagenesis ("CSM") is used wherein two or more amino acids at rationally designated positions are changed concomitantly to different amino acid residues to engineer improved enzyme function and structure. Correlated saturation mutagenesis allows for the identification of complimentary amino acid changes having positive, synergistic effects on enzyme structure and function. Such synergistic effects include, but are not limited to, significantly altered enzyme stability, substrate affinity or catalytic turnover rate, independently or concomitantly increasing advantageously the production of propanol.

[0165] In one embodiment, mutational variants derived from the methods described herein are cloned. DNA sequences produced by saturation mutagenesis are designed to have restriction sites at the ends of the gene sequences to allow for excision and transformation into a host cell plasmid. Generated plasmid stocks are transformed into a host cell and incubated at optimal growth conditions to identify successfully transformed colonies.

[0166] Another embodiment utilizes gene shuffling (P. Stemmer, Nature (1994) Vol. 370:389-391) or gene reassembly (US 5,958,672) to develop improved protein

structure/function through the generation of chimeric proteins. With gene shuffling, two or more homologous nucleotide sequences, each encoding a gene listed in Tables 1-5 are treated with endonucleases at random positions, mixed together, heated until sufficiently melted and reannealed. Nucleotide sequences from homologues will anneal to develop a population of chimeric genes that are repaired to fill in any gaps resulting from the re-annealing process, expressed and screened for improved structure/function enzyme chimeras. Gene reassembly is similar to gene shuffling; however, nucleotide sequences for specific enzymatic domains are targeted and swapped with other homologous domains for reassembly into a chimeric gene. The genes are expressed and screened for improved structure/function enzyme chimeras.

[0167] In a further embodiment any and/or all sequences additionally are codon and expression optimized for the specific expression in the host cell.

Methods for Measuring Protein Variant Efficacy

[0168] Variations in expressed polypeptide sequences may result in measurable differences in the whole-cell rate of substrate conversion. It is desirable to determine differences in the rate of substrate conversion by assessing productivity in a host cell having a particular protein variant relative to other whole cells having a different protein variant. Additionally, it would be desirable to determine the efficacies of whole-cell substrate conversion as a function of environmental factors including, but not limited to, H, temperature nutrient concentration and salinity.

[0169] Therefore, in one embodiment, the biophysical analyses described herein on protein variants of the invention are performed to measure structural/functional attributes. Standard analyses of polypeptide activity are well known to one of ordinary skill in the art. Such analysis can require the expression and high purification of large quantities of polypeptide, followed by various physical methods (including, but not limited to, calorimetry, fluorescence, spectrophotometric, spectrometric, liquid chromatography (LC), mass spectrometry (MS), LC-MS, affinity chromatography, light scattering, nuclear magnetic resonance and the like) to assay function, function in a specific environment or functional differences among homologues.

[0170] In another embodiment, the polypeptides are expressed, purified and subject to the aforementioned analytical techniques to assess the functional difference among polypeptide sequence homologues, for example, the rate of substrate conversion specific for a particular enzyme function.

[0171] Batch culture (or closed system culture) analysis is well known in the art and can provide information on host cell population effects for host cells expressing genetically engineered genes. In batch cultures a host cell population will grow until available nutrients are depleted from the culture media.

[0172] In one embodiment, the polypeptides are expressed in a batch culture and analyzed for approximate doubling times, expression efficacy of the engineered polypeptide and end-point net product formation and net biomass production.

[0173] Turbidostats are well known in the art as one form of a continuous culture within which media and nutrients are provided on an uninterrupted basis and allow for non-stop propagation of host cell populations. Turbidostats allow the user to determine information on whole cell propagation and steady-state productivity for a particular biologically produced end product such as host cell doubling time, temporally delimited biomass production rates for a particular host cell population density, temporally delimited host cell population density effects on substrate conversion and net productivity of a host cell substrate conversion of, for example, L-threonine or acetyl-CoA to propanol. Turbidostats can be designed to monitor the partitioning of substrate conversion products to the liquid or gaseous state. Additionally, quantitative evaluation of net productivity of a carbon-based product of interest can be accurately performed due to the exacting level of control that one skilled in the art has over the operation of the turbidostat. These types of information are useful to assess the parsed and net efficacies of a host cell genetically engineered to produce a specific carbon-based product of interest.

[0174] In one embodiment, identical host cell lines differing only in the nucleic acid and expressed polypeptide sequence of a homologous enzyme are cultured in a uniform- environment turbidostat to determine highest whole cell efficacy for the desired carbon-based product of interest.

[0175] In another embodiment, identical host cell lines differing only in the nucleic acid and expressed polypeptide sequence of a homologous enzyme are cultured in a batch culture or a turbidostat in varying environments (e.g., temperature, pH, salinity, nutrient exposure) to determine highest whole cell efficacy for the desired carbon-based product of interest.

Pathways for the Enzymatic Synthesis of Propanol

[0176] Enzymes and enzymatic processes and pathways for the production of propanol using engineered cyanobacteria are represented in Tables 1-5. Tables 1-4 are enzymatic pathways directed to the synthesis of propanol- 1 from common intermediates in

cyanobacteria. Table 5 is an enzymatic pathway directed to the synthesis of 2-propanol from acetyl-CoA via acetoacetyl-CoA in cyanobacteria.

[0177] 1 -propanol and 2-propanol can be converted to propylene via dehydration.

(Rivard, J., et al., (2001) The Canadian Journal of Chemical Engineering 79(4), 517-523, Dias, J., et al, (2001) J. Chem. Soc, Dalton Trans., 228-231, Yadav, G.D., and Murkute A.D., (2004) Langmuir 20(26), 11607-19).

EXAMPLE 1: 1-PROPANOL VIA L-THREONINE

Table 1 shows an enzymatic pathway for 1-propanol synthesis in an engineered

cyanobacterium from L-threonine. Expression of ilvA, kivD, and adhA can be optimized in a strain such as Synechococcus sp. PCC 7002 by using a divergent construct with various promoters. For example, a construct can be synthesized in which a promoterless kivD gene is in one orientation and a promoterless UvA-adhA operon is in the opposite orientation, and in which the kivD and ilvA genes originate divergently from a common locus. Furthermore, one or more restriction sites can be included at the common locus such that divergent promoter sequences can be inserted between the kivD and ilvA genes, thus subjecting kivD to control of one of the promoters and the UvA-adhA operon to control of the other promoter. The construct is designed such that each of the ilvA, kivD, and adhA genes is preceded by an appropriate ribosome binding site. Furthermore, a selectable marker with constitutive promoter, such as a kanamycin-resistance gene, can be placed downstream of kivD to allow selection of recombinants. TABLE 1. Pathway from L-threonine to 1-propanol.

EXAMPLE 2: 1-PROPANOL VIA MALONYL-COA

[0178] The green- sulfur bacterium Chloroflexus aurantiacus has been shown to synthesize propionyl-CoA from malonyl-CoA (an intermediate already present in

cyanobacteria) as part of a carbon fixation cycle (Alber, B.E., and Fuchs, G. (2002) J. Biol. Chem. 277, 12137-43). Along with two Chloroflexus genes from this cycle {mcr and pes) two other genes, pduP and adhA, are used to complete a pathway from malonyl-CoA to 1- propanol in cyanobacteria. An example of such a pathway is shown in Table 2.

[0179] Expression of mcr, pes, pduP, and adhA can be optimized in a strain such as Synechococcus sp. PCC 7002 by using a divergent construct with various promoters. For example, a construct can be synthesized in which a promoterless pcs-adhA operon is in one orientation and a promoterless mcr-pduP operon is in the opposite orientation, and in which the pes and mcr genes originate divergently from a common locus. Furthermore, one or more restriction sites can be included at the common locus such that divergent promoter sequences can be inserted between the pes and mcr genes, thus subjecting the pcs-adhA operon to control of one of the promoters and the mcr-pduP operon to control of the other promoter. The construct is designed such that each of the mcr, pes, pduP, and adhA genes is preceded by an appropriate ribosome binding site. Furthermore, a selectable marker with constitutive promoter, such as a kanamycin-resistance gene, can be placed downstream of adhA to allow selection of recombinants.

[0180] Enhanced 1-propanol production can be achieved by using techniques used to enhance fatty acid production in other organisms. Enhancement of the intracellular malonyl- CoA concentration has been shown to increase fatty acid flux in other systems (Davis, M.S., et al, (2000) J. Biol. Chem. 275, 28593-98).

EXAMPLE 3: 1-PROPANOL VIA SUCCINYL-COA

[0181] E. coli contains a pathway from succinate to propionate encoded by three genes (Haller, T., et al. (2000) Biochemistry 39, 4622-29). Two of the genes from this pathway (scpA and scpB) can be combined with two other genes to construct a pathway in

cyanobacteria from endogenous succinyl-CoA to 1-propanol, as shown in an example in Table 3. Succinyl-CoA production can be augmented by overexpression and/or deregulation of TCA-cycle genes, glyoxylate-shunt genes, and PEP carboxylase.

[0182] Expression of scpAB, pduP, adhA, and sucCD can be optimized in a strain such as Synechococcus sp. PCC 7002 by using a divergent construct with various promoters. For example, a construct can be synthesized in which a promoterless scpAB-adhA operon is in one orientation and a promoterless pduP-sucCD operon is in the opposite orientation, and in which the scpA and pduP genes originate divergently from a common locus. Furthermore, one or more restriction sites can be included at the common locus such that divergent promoter sequences can be inserted between the scpA and pduP genes, thus subjecting the scpAB-adhA operon to control of one of the promoters and the pduP-sucCD operon to control of the other promoter. The construct is designed such that each of the scpA, scpB, pduP, adhA, sucC, and sucD genes is preceded by an appropriate ribosome binding site.

Furthermore, a selectable marker with constitutive promoter, such as a kanamycin-resistance gene, can be placed downstream of adhA to allow selection of recombinants.

EXAMPLE 4: 1-PROPANOL VIA SUCCINYL-COA

[0183] G. sulfurreducens contains an alternative pathway for isoleucine biosynthesis (Risso, C, et al. (2008) J. Bacteriol. 190, 2266-74). Three genes from this alternative pathway can be combined with two other genes to construct a pathway from endogenous pyruvate to 1-propanol in cyanobacteria, as shown in an example in Table 4.

[0184] Expression of cimA, leuBCD, kivD, and adhA can be optimized in a strain such as Synechococcus sp. PCC 7002 by using a divergent construct with various promoters. For example, a construct can be synthesized in which a promoterless cimA-leuB-adhA operon is in one orientation and a promoterless kivD-leuCD operon is in the opposite orientation, and in which the cimA and kivD genes originate divergently from a common locus. Furthermore, one or more restriction sites can be included at the common locus such that divergent promoter sequences can be inserted between the cimA and kivD genes, thus subjecting the cimA-leuB-adhA operon to control of one of the promoters and the kivD-leuCD operon to control of the other promoter. The construct is designed such that each of the cimA, leuB, leuC, leuD, kivD, and adhA genes is preceded by an appropriate ribosome binding site.

Furthermore, a selectable marker with constitutive promoter, such as a kanamycin-resistance gene, can be placed downstream of adhA to allow selection of recombinants.

*As described in Risso, C, Van Dien, S.J., Orloff, A., Lovley, D.R., and Coppi, M.V. Elucidation of an alternate isoleucine biosynthesis pathway in Geobacter sulfurreducens. J. Bacteriol. 190 (7): 2266-74 (2008). Gene IDs are: cimA = GSU1798, leuC = GSU1902, leuD = GSU1903, leuB = GSU2879.

EXAMPLE 5: OPTIMAL PRODUCTION OF 1-PROPANOL

[0185] To facilitate optimal production, any of at least one of the added genes in any of Tables 1-4 can be placed under control of an inducible promoter, such as one repressible by ammonia, inducible by nickel, inducible by certain ranges of intensity or wavelengths of light, etc. The overall construct (e.g., any of the constructs described in Examples 1-4, above) can be flanked by regions of 0.5 kb or greater, each of which is homologous to the Synechococcus sp. PCC 7002 genome or one of its endogenous plasmids, such that upon recombination with said genome or plasmid, the construct is inserted between the contiguous regions of homology. Such recombination may or may not displace chromosomal elements, depending upon what homology regions are selected. For example, if a region of the chromosome can be represented by contiguous sequences A-B-C, then a construct A-insert- B will result in a strain with chromosomal sequence A-insert-B-C, while a construct A- insert-C will result in a strain with chromosomal sequence A-insert-C (having deleted B through homologous recombination). After transformation of the host strain by natural transformation, conjugation, electroporation (or any other suitable method), the host is permitted to recover for 24 h on a minimal agar plate under illumination at 37°C, after which the selective agent is added underneath the agar. After a few days, colonies are selected and transformation confirmed by PCR or other means. Transformants are streaked several times under selective pressure to segregate transformed genes such that all copies of the chromosome or plasmid contain the recombinant genes. Segregated transformants are then grown for several days under illumination in test tubes or flasks in minimal liquid medium under ambient or augmented CO₂, and the supernatant of these cultures is subsequently tested for the presence of 1-propanol. The gene integrations may alternatively be carried out with more than one recombination event at separate loci if desired, using more than one selectable marker and separate divergent or nondivergent promoters, wherein each recombination event integrates a subset of the genes of interest.

EXAMPLE 6: 2-PROPANOL VIA ACETOACETYL-COA

[0186] 2-propanol can be produced in E. coli by assembling and expressing a pathway from acetyl-CoA via acetoacetyl-CoA (Hanai, T., et al. (2007) Appl. Environ. Microbiol. 73, 7814-18). Acetyl-CoA is a ubiquitous metabolite in bacteria, participating in many essential pathways. A pathway for 2-propanol production in cyanobacteria is shown in Table 5.

[0187] Expression of phaA, aarC, atoDA, adc, and adh can be optimized in a strain such as Synechococcus sp. PCC 7002 by using a divergent construct with various promoters. For example, a construct can be synthesized in which a promoterless atoDA-adh operon (or alternatively, a promoterless aarC-adh operon) is in one orientation and a promoterless phaA- adc operon is in the opposite orientation, and in which the atoD and phaA genes originate divergently from a common locus. Furthermore, one or more restriction sites can be included at the common locus such that divergent promoter sequences can be inserted between the atoD and phaA genes, thus subjecting the atoDA-adh operon to control of one of the promoters and the phaA-adc operon to control of the other promoter. The construct is designed such that each of the phaA, adc, adh, atoD, and atoA genes (or aarC in place of atoD and atoA) is preceded by an appropriate ribosome binding site. Furthermore, a selectable marker with constitutive promoter, such as a kanamycin-resistance gene, can be placed downstream of adh to allow selection of recombinants.

[0188] To facilitate optimal production of 2-propanol, at least one of the added genes can be placed under control of an inducible promoter, such as one repressible by ammonia, inducible by nickel, inducible by certain ranges of intensity or wavelengths of light, etc. The overall construct can be flanked by regions of 0.5 kb or greater, each of which is homologous to the Synechococcus sp. PCC 7002 genome or one of its endogenous plasmids, such that upon recombination with said genome or plasmid, the construct is inserted between the contiguous regions of homology. Such recombination may or may not displace chromosomal elements, depending upon what homology regions are selected. For example, if a region of the chromosome can be represented by contiguous sequences A-B-C, then a construct A- insert-B will result in a strain with chromosomal sequence A-insert-B-C, while a construct A-insert-C will result in a strain with chromosomal sequence A-insert-C (thus having deleted B). After transformation of the host strain by natural transformation, conjugation, electroporation, or other method, the host is permitted to recover for 24 h on a minimal agar plate under illumination at 37°C, after which the selective agent is added underneath the agar. After a few days, colonies are selected and transformation confirmed by PCR or other means. Transformants are streaked several times under selective pressure to segregate transformed genes such that all copies of the chromosome or plasmid contain the recombinant genes. Segregated transformants are then grown for several days under illumination in test tubes or flasks in minimal liquid medium under ambient or augmented C0₂, and the supernatant of these cultures is subsequently tested for the presence of 2-propanol. The gene integrations may alternatively be carried out with more than one recombination event at separate loci if desired, using more than one selectable marker and separate divergent or nondivergent promoters, wherein each recombination event integrates a subset of the genes of interest.

[0189] Hanai et al. (2007) selected atoDA, which utilizes acetate as the CoA acceptor. aarC can be used as an alternative, which utilizes succinate for this purpose.

[0190] In addition to the in vivo production of propanol discussed above, the proteins encoded by the genes listed in Tables 1-5 can be purified. When incubated in vitro with appropriate reagents and conditions, the proteins will catalyze the enzymatic synthesis of propanol in vitro from appropriate starting materials {e.g., an L-threonine or acetyl-CoA).

INFORMAL SEQUENCE LISTING

Claims

CLAIMS What is claimed is:

1. An engineered cyanobacterium, wherein said engineered cyanobacterium comprises one or more recombinant protein activities selected from the group consisting of malonyl- CoA reductase, propionyl-CoA synthase, and 1-propanol dehydrogenase.

2. The engineered cyanobacterium of claim 1, wherein two or more recombinant protein activities from the group consisting of malonyl-CoA reductase, propionyl-CoA synthase, and 1-propanol dehydrogenase are selected.

3. The engineered cyanobacterium of claim 1, comprising recombinant malonyl-CoA

reductase, propionyl-CoA synthase, and 1-propanol dehydrogenase activities.

4. The engineered cyanobacterium of any of claims 1-3, further comprising a CoA- dependent aldehyde dehydrogenase protein activity.

5. The engineered cyanobacterium of any of claims 1-4, wherein said malonyl-CoA

reductase is at least 95% identical to SEQ ID NO: 4.

6. The engineered cyanobacterium of any of claims 1-4, wherein said propionyl-CoA

synthase is at least 95% identical to SEQ ID NO: 5.

7. The engineered cyanobacterium of any of claims 1-4, wherein said 1-propanol

dehydrogenase is at least 95% identical to SEQ ID NO: 3

8. The engineered cyanobacterium of claim 4, wherein said CoA-dependent aldehyde

dehydrogenase is at least 95 %> identical to SEQ ID NO: 6.

9. An engineered cyanobacterium, wherein said engineered cyanobacterium comprises

malonyl-CoA reductase, propionyl-CoA synthase, 1-propanol dehydrogenase, and CoA- dependent aldehyde dehydrogenase, wherein said malonyl-CoA reductase is at least 95%> identical to SEQ ID NO: 4, wherein said propionyl-CoA synthase is at least 95% identical to SEQ ID NO: 5, wherein said 1-propanol dehydrogenase is at least 95 %> identical to SEQ ID NO: 3, and wherein said CoA-dependent aldehyde dehydrogenase is at least 95% identical to SEQ ID NO: 6.

10. The engineered cyanobacterium of any of claims 1-9, wherein said cyanobacterium is Synechococcus sp. PCC 7002.

11. The engineered cyanobacterium of any of claims 1-9, wherein at least two of said

recombinant genes are expressed under the control of promoters in opposite orientations.

12. The engineered cyanobacterium of claim 11, wherein the sequences of said promoters are non-identical.

13. A method for producing 1-propanol, comprising:

(i) culturing the engineered cyanobacterium of any of claims 1-12 in a culture medium, and

(ii) exposing said engineered cyanobacterium to light and carbon dioxide, wherein said exposure results in the conversion of said carbon dioxide by said engineered cyanobacterium into 1-propanol.

14. The method of claim 13, wherein said 1-propanol produced by said engineered

cyanobacterium is greater than the amount of 1-propanol produced by an otherwise identical cyanobacterium, cultured under identical conditions, but lacking said recombinant protein activity.

15. An engineered host cell, comprising one or more recombinant protein activities selected from Tables 1-5, wherein said one or more recombinant protein activities facilitate the enzymatic synthesis of propanol or a precursor thereof.

16. An engineered host cell, comprising one or more recombinant protein activities selected from Tables 1-4, wherein said one or more recombinant protein activities facilitate the synthesis of 1-propanol or a precursor thereof.

17. An engineered host cell, comprising one or more recombinant protein activities selected from Table 5, wherein said one or more recombinant protein activities facilitate the synthesis of 2-propanol or a precursor thereof.

18. The host cell of claim 15 or 16, wherein said host cell facilitates conversion of L- threonine to 1-propanol, and wherein said host cell comprises at least one recombinant protein activity selected from Table 1.

19. The host cell of claim 15 or 16, wherein said host cell facilitates conversion of L- threonine to 1-propanol, and wherein said host cell comprises at least two recombinant protein activities selected from Table 1.

20. The host cell of claim 15 or 16, wherein said host cell facilitates conversion of malonyl- CoA to 1-propanol, and wherein said host cell comprises at least one recombinant protein activity selected from Table 2.

21. The host cell of claim 15 or 16, wherein said host cell facilitates conversion of malonyl- CoA to 1-propanol, and wherein said host cell comprises at least two recombinant protein activities selected from Table 2.

22. The host cell of claim 15 or 16, wherein said host cell facilitates conversion of succinate to 1-propanol, and wherein said host cell comprises at least one recombinant protein activity selected from Table 3.

23. The host cell of claim 15 or 16, wherein said host cell facilitates conversion of succinate to 1-propanol, and wherein said host cell comprises at least two recombinant protein activities selected from Table 3.

24. The host cell of claim 15 or 16, wherein said host cell facilitates conversion of acetyl- CoA and pyruvate to 1-propanol, and wherein said host cell comprises at least one recombinant protein activity selected from Table 4.

25. The host cell of claim 15 or 16, wherein said host cell facilitates conversion of acetyl- CoA and pyruvate to 1-propanol, and wherein said host cell comprises at least two recombinant protein activities selected from Table 4.

26. The host cell of claim 15 or 17, wherein said host cell facilitates conversion of acetyl- CoA to 2-propanol via an acetoacetyl-CoA intermediate, and wherein said host cell comprises at least one recombinant protein activity selected from Table 5.

27. The host cell of claim 15 or 17, wherein said host cell facilitates conversion of acetyl- CoA to 2-propanol via an acetoacetyl-CoA intermediate, and wherein said host cell comprises at least two recombinant protein activities selected from Table 5.

28. The host cell of any of claims 15-27, comprising at least three recombinant protein

activities wherein each of said three recombinant protein activities are listed in a single table selected from one of Tables 1-5.

29. The host cell of any of claims 15-17 and 20-27, comprising at least four recombinant protein activities wherein each of said four recombinant protein activities are listed in a single table selected from one of Tables 2-5.

30. The host cell of any of claims 15, 16, 18, and 19, comprising threonine deaminase, 2- keto-acid decarboxylase, and 1-propanol dehydrogenase protein activities.

31. The host cell of any of claims 15,16, 20, and 21, comprising malonyl-CoA reductase, propionyl-CoA synthase, CoA-dependent aldehyde dehydrogenase, and 1-propanol dehydrogenase protein activities.

32. The host cell of any of claims 15, 16, 22, and 23, comprising succinate:CoA ligase, methylmalonyl-CoA mutase, methylmalonyl-CoA decarboxylase, CoA-dependent aldehyde dehydrogenase, and 1-propanol dehydrogenase protein activities.

33. The host cell of any of claims 15, 16, 24, and 25, comprising citramalate synthase,

isopropylmalate isomerase, isopropylmalate dehydrogenase, 2-keto-acid decarboxylase, and 1-propanol dehydrogenase protein activities.

34. The host cell of any of claims 15, 17, 26, and 27, comprising acetyl-CoA C- acetyltransferase, succinyl-CoA:acetate CoA-transferase or acetoacetyl-CoA transferase, acetoacetate decarboxylase, and isopropanol dehydrogenase protein activities.

35. The host cell of any of claims 15-34, wherein at least one of said recombinant protein activities are expressed by a gene under the control of an inducible promoter.

36. The host cell of claim 35, wherein said inducible promoter is repressible by ammonia, indicible by nickel, or inducible by light.

37. The host cell of any of claims 15-36, wherein said host cell is capable of photosynthesis.

38. The host cell of any of claims 15-36, wherein said host cell is a cyanobacterium.

39. The host cell of any of claims 15-36, wherein said host cell is a gram-negative or gram- positive bacteria.

40. The host cell of any of claims 15-36, wherein said host cell is an algae.

41. The host cell of any of claims 15-40, wherein propanol is secreted into the medium.

42. An isolated or recombinant polypeptide comprising or consisting of an amino acid

sequence selected from SEQ ID NO: 1-20.

43. An isolated or recombinant polynucleotide comprising or consisting of a nucleic acid sequence selected from the group consisting of:

a. a gene coding for an amino acid sequence of SEQ ID NO: 1-20;

b. a nucleic acid sequence that is a degenerate variant of a gene coding for an amino acid sequence of SEQ ID NO: 1-20;

c. a nucleic acid sequence at least 90%, at least 95%, at least 98%>, at least 99% or at least 99.9% identical to a gene coding for an amino acid sequence of SEQ ID NO: 1- 20; and

d. a nucleic acid sequence that hybridizes under stringent conditions to any gene

coding for an amino acid sequence of SEQ ID NO: 1-20.

44. The isolated polynucleotide of claim 43, wherein the nucleic acid sequence is flanked by regions of 0.5kb or greater, wherein said flainking regions is homologous to the

Synechococcus sp. PCC 7002 genome or one of its endogenous plasmids, such that upon recombination with said genome or plasmid, the construct is inserted between a contiguous region of homology

45. The isolated polynucleotide of claim 43, wherein the nucleic acid sequence and the

sequence of interest are operably linked to one or more expression control sequences.

46. The isolated polynucleotide of claim 45, wherein said expression control sequence is an inducible expression control sequence.

47. The isolated polynucleotide of claim 46, wherein said inducible expression control

sequence is a T7 promoter.

48. A vector comprising the isolated polynucleotide of any one of claims 43-47.

49. A fusion protein comprising a polypeptide encoded by any one of the sequences recited in claims 43-47, wherein said polypeptide is fused to a heterologous amino acid sequence.

50. A host cell comprising the isolated polynucleotide of any one of claims 43-47.

51. A method for producing a host cell capable of producing propanol comprising genetically engineering an isolated or recombinant polynucleotide sequence encoding any one of the enzymes in Tables 1-5 into a host cell.

52. A method for producing propanol comprising culturing the host cell of any of claims 15- 41, 50, and 51 to produce propanol.

53. A method for synthesizing propanol in vitro, comprising: exposing any of the reactants or products listed in Tables 1-5 to the enzyme activities listed in Tables 1-5.

54. The method of any of claims 51-53, wherein at least one of the chemical reactions listed in Tables 1-5 is an exogenous reaction.

55. The method of claim 54, wherein said exogenous reaction is catalyzed by an enzymatic activity listed in Tables 1-5.

56. The method of claim 54, wherein said exogenous reaction is performed by chemical synthesis.

57. A method for producing propylene, comprising dehydration of propanol synthesized by the method of any one of claims 51-56.

58. A method for producing propylene, comprising dehydration of propanol synthesized by the host cell of any one of claims 51-56.