WO2006105082A2 - Oxydation d'alcanes par des hydroxylases modifiees - Google Patents

Oxydation d'alcanes par des hydroxylases modifiees Download PDF

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Publication number
WO2006105082A2
WO2006105082A2 PCT/US2006/011273 US2006011273W WO2006105082A2 WO 2006105082 A2 WO2006105082 A2 WO 2006105082A2 US 2006011273 W US2006011273 W US 2006011273W WO 2006105082 A2 WO2006105082 A2 WO 2006105082A2
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Prior art keywords
amino acid
seq
polypeptide
acid residue
residues
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PCT/US2006/011273
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English (en)
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WO2006105082A3 (fr
Inventor
Frances Arnold
Peter Meinhold
Matthew W. Peters
Rudi Fasan
Mike M. Y. Chen
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The California Institute Of Technology
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Application filed by The California Institute Of Technology filed Critical The California Institute Of Technology
Priority to EP06748800A priority Critical patent/EP1899471A4/fr
Publication of WO2006105082A2 publication Critical patent/WO2006105082A2/fr
Priority to US11/697,404 priority patent/US8715988B2/en
Publication of WO2006105082A3 publication Critical patent/WO2006105082A3/fr
Priority to US14/270,268 priority patent/US9074178B2/en
Priority to US14/788,365 priority patent/US9404096B2/en
Priority to US15/224,900 priority patent/US9963720B2/en
Priority to US15/942,001 priority patent/US10648006B2/en
Priority to US16/872,275 priority patent/US11214817B2/en
Priority to US17/560,753 priority patent/US20220112524A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)

Definitions

  • This invention relates to modified hydroxylases.
  • the invention further relates to cells expressing such modified hydroxylases and methods of producing hydroxylated alkanes by contacting a suitable substrate with such cells. Also included are modified hydroxylases comprising unique regioselectivities .
  • Exemplary monooxygenases include the cytochrome P450 monooxygenases (“P450s”) .
  • P450s are a group of widely-distributed heme-containing enzymes that insert one oxygen atom from diatomic oxygen into a diverse range of hydrophobic substrates, often with high regio- and stereoselectivity. Their ability to catalyze these reactions with high specificity and selectivity makes P450s attractive catalysts for chemical synthesis and other applications, including oxidation chemistry.
  • polypeptides that convert alkanes to alcohols.
  • nucleic acid molecules that encode such polypeptides, cells expressing such polypeptides, and methods of synthesizing alcohols from a suitable alkane substrate. Accordingly, in various embodiments, isolated or recombinant polypeptides comprising residues 1 to about 455 of the amino acid sequence set forth in SEQ ID N0:l, 2, 3, 4, 5 or 6, are provided.
  • the polypeptides include up to 50, 25, 10, or 5 conservative amino acid substitutions excluding residues: (a) 47, 78, 82, 94, 142, 175, 184, 205, 226, 236, 252, 255, 290, 328, and 353 of SEQ ID N0:l; (b) 48, 79, 83, 95, 143, 176, 185, 206, 227, 238, 254, 257, 292, 330, and 355 of SEQ ID N0:2 or SEQ ID NO:3; (c) 49, 80, 84, 96, 144, 177, 186, 207, 228, 239, 255, 258, 293, 331, and 357 of SEQ ID NO:4; (d) 54, 85, 89, 101, 149, 182, 191, 212, 232, 243, 259, 262, 297, 336, and 361 of SEQ ID N0:5; and (e) 51, 82, 86, 98, 146,
  • a Zl amino acid residue at positions: (a) 47, 82, 142, 205, 236, 252, and 255 of SEQ ID NO:1; (b) 48, 83, 143, 206, 238, 254, and 257 of SEQ ID NO:2 or SEQ ID NO:3; (c) 49, 84, 144, 207, 239, 255, and 258 of SEQ ID NO:4; (d) 54, 89, 149, 212, 243, 259, and 262 of SEQ ID N0:5; and (e) 51, 86, 146, 208, 242, 258, and 262 of SEQ ID NO:6;
  • a Zl amino acid residue includes glycine (G) , asparagine (N) , glutamine (Q) , serine (S) , threonine (T) , tyrosine (Y) , or cysteine (C) .
  • a Z2 amino acid residue includes alanine (A) , valine (V) , leucine (L) , isoleucine (I) , proline (P) , or methionine (M) .
  • a Z3 amino acid residue includes lysine (K) , or arginine (R) .
  • a Z4 amino acid residue includes tyrosine (Y), phenylalanine (F), tryptophan (W), or histidine (H) .
  • polypeptides provided herein display hydroxylase activity that converts an alkane to an alcohol.
  • the polypeptide further includes a Z3 amino acid residue at position: (a) 285 of SEQ ID N0:l; (b) 287 of SEQ ID N0:2 or 3; (c) 288 of SEQ ID N0:4; (d) 292 of SEQ ID N0:5; and (e) 291 of SEQ ID NO: 6.
  • a Z3 amino acid residue includes lysine (K) , arginine (R) , or histidine (H) .
  • the amino acid residue at this position is an arginine (R) .
  • alkanes include methane (CH 4 ) , ethane (C 2 H6) , propane (CaH 8 ) , butane (C4H10) , pentane (C5H12) , hexane (C6H14) , heptane (C 7 Hi6) , octane (CsHis) , nonane (C9H20) 1 decane (C10H22) , undecane (CnH 24 ) , and dodecane (C12H26) •
  • alcohols include methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, nonanol, decanol, undecanol, and dodecanol .
  • amino acid sequence of the polypeptide includes residues at the following positions:
  • V valine
  • I isoleucine
  • amino acid sequence of the polypeptide includes residues at the following positions:
  • S serine residue at position: (a) 82, 142, and 255 of SEQ ID NO:1; (b) 83, 143 and 257 of SEQ ID N0:2 or SEQ ID N0:3; (c) 84, 144, and 258 of SEQ ID NO:4; (d) 89, 149, and 262 of SEQ ID N0:5; (e) 86, 146 and 262 of SEQ ID N0:6;
  • G glycine (G) amino acid residue at position: (a) 252 of SEQ ID NO:1; (b) 254 of SEQ ID NO:2 or SEQ ID N0:3; (c) 255 of SEQ ID NO:4; (d) 259 of SEQ ID NO:5; (e) 258 of SEQ ID NO:
  • V valine (V) amino acid residue at position: (a) 184, 290 and 353 of SEQ ID N0:l; (b) 185, 292, and 355 of SEQ ID NO:2 or SEQ ID NO:3; (c) 186, 293 and 357 of SEQ ID NO:4; (d) 191, 297, and 361 of SEQ ID N0:5; (e) 188, 296, and 363 of SEQ ID NO: 6;
  • an isolated or recombinant polypeptide that includes residues 1 to about 455 of the amino acid sequence set forth in SEQ ID N0:l, 2, 3, 4, 5 or 6 is provided.
  • the polypeptide includes amino acid residues in the amino acid sequence that correspond to the following positions, at least 80%, 85%, 90%, or 95% of the amino acid residues in the amino acid sequence at the positions specified in (a) - (h) are as follows:
  • S serine residue at position: (a) 82, 142, and 255 of SEQ ID NO:1; (b) 83, 143 and 257 of SEQ ID NO:2 or SEQ ID NO:3; (c) 84, 144, and 258 of SEQ ID NO:4; (d) 89, 149, and 262 of SEQ ID N0:5; (e) 86, 146 and 262 of SEQ ID NO: 6;
  • G glycine (G) amino acid residue at position: (a) 252 of SEQ ID NO:1; (b) 254 of SEQ ID NO:2 or SEQ ID NO:3; (c) 255 of SEQ ID NO:4; (d) 259 of SEQ ID N0:5; (e) 258 of SEQ ID NO:
  • V valine amino acid residue at position: (a) 184, 290 and 353 of SEQ ID N0:l; (b) 185, 292, and 355 of SEQ ID NO:2 or SEQ ID NO:3; (c) 186, 293 and 357 of ' SEQ ID N0:4; (d) 191, 297, and 361 of SEQ ID N0:5; (e) 188, 296, and 363 of SEQ ID NO: 6;
  • the polypeptide further includes a Z3 amino acid residue at position: (a) 285 of SEQ ID NO:1; (b) 287 of SEQ ID N0:2 or 3; (c) 288 of SEQ ID N0:4; (d) 292 of SEQ ID N0:5; and (e) 291 of SEQ ID NO: 6.
  • a Z3 amino acid residue includes lysine (K) , arginine (R) , or histidine (H) .
  • the amino acid residue at this position is an arginine (R) .
  • an isolated or recombinant polypeptide that includes residues 456-1048 of SEQ ID NO:1, 456-1059 of SEQ ID NO:2, 456-1053 of SEQ ID NO:3, 456-1064 of SEQ ID NO:4, 456-1063 of SEQ ID N0:5, or 456-1077 of SEQ ID NO: 6, is provided.
  • the polypeptide includes an amino acid sequence with up to 65, 40, 25, or 10 conservative amino acid substitutions excluding residues: (a) 464, 631, 645, 710 and 968 of SEQ ID N0:l; (b) 475, 641, 656, 721 and 980 of SEQ ID NO:2; (c) 467, 634, 648, 713 and 972 of SEQ ID N0:3; (d) 477, 644, 659, 724 and 983 of SEQ ID NO:4; (e) 472, 640, 656, 723 and 985 of SEQ ID NO:5; and (f) 480, 648, 664, 733 and 997 of SEQ ID NO: 6.
  • the amino acid sequence includes the following residues: (1) a Zl, Z3, Z4, or Z5 amino acid residue at position: (a) 464 of SEQ ID N0:l; (b) 475 of SEQ ID N0:2; (c) 467 of SEQ ID N0:3; (d) 477 of SEQ ID N0:4; (e) 472 of SEQ ID N0:5; and (f) 480 of SEQ ID NO: 6;
  • Zl is an amino acid residue selected from the group consisting of glycine (G) , asparagine (N) , glutamine (Q) , serine (S) , threonine (T) , tyrosine (Y) , and cysteine (C) ;
  • Z2 is an amino acid residue selected from the group consisting of alanine (A) , valine (V) , leucine (L) , isoleucine (I) , proline (P) , and methionine (M) ;
  • Z3 is an amino acid residue selected from the group consisting of lysine (K) , and arginine (R) ;
  • Z4 is an amino acid residue selected from the group consisting of tyrosine (Y) , phenylalanine (F) , tryptophan (W) , and histidine (H) ; and is an amino acid residue selected from the group consisting of threonine (T), va
  • T threonine amino acid residue at position: (a) 710 of SEQ ID N0:l; (b) 721 of SEQ ID NO:2; (c) 713 of SEQ ID N0:3; (d) 724 of SEQ ID N0:4; (e) 723 of SEQ ID N0:5; and (f) 733 of SEQ ID NO: 6; and
  • an isolated or recombinant polypeptide that includes the amino acid sequence set forth in SEQ ID N0:7 with up to 75, 50, 25, or 10 conservative amino acid substitutions excluding residues 47, 78, 82, 94, 142, 175, 184, 205, 226, 236, 252, 255, 290, 328, 353, 464, and 710, is provided.
  • the polypeptide can display hydroxylase activity that converts an alkane to an alcohol.
  • an isolated or recombinant polypeptide that includes the amino acid sequence set forth in SEQ ID NO: 8 with up to 75, 50, 25, or 10 conservative amino acid substitutions excluding residues 47, 78, 82, 94, 142, 175, 184, 205, 226, 236, 252, 255, 290, 328, 353, 464, and 710, is provided.
  • an isolated or recombinant polypeptide that includes the amino acid sequence set forth in SEQ ID NO: 9 with up to 75, 50, 25, or 10 conservative amino acid substitutions excluding residues 47, 78, 82, 94, 142, 175, 184, 205, 226, 236, 252, 255, 290, 328, 353, 464, and
  • an isolated or recombinant polypeptide that includes the amino acid sequence set forth in
  • SEQ ID NO: 10 with up to 75, 50, 25, or 10 conservative amino acid substitutions excluding residues 47, 78, 82, 94, 142,
  • polypeptides of SEQ ID NO:7, 8, 9 or 10 further optionally include an arginine (R) at amino acid residue position 285.
  • polypeptides provided herein can display hydroxylase activity that converts an alkane to an alcohol.
  • an alkane includes methane (CFU), ethane
  • an alcohol includes methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, nonanol, decanol, undecanol, and dodecanol .
  • an isolated or recombinant polypeptide that includes the amino acid sequence set forth in
  • an isolated or recombinant polypeptide that includes the amino acid sequence set forth in
  • SEQ ID NO: 12 with up to 75, 50, 25, or 10 conservative amino acid substitutions excluding residues 47, 78, 82, 94, 142,
  • an isolated or recombinant polypeptide that includes the amino acid sequence set forth in SEQ ID NO: 13 with up to 75, 50, 25, or 10 conservative amino acid substitutions excluding residues 47, 78, 82, 94, 142, 175, 184, 205, 226, 236, 252, 255, 290, 328, 353, 464, 631, and 710, is provided.
  • isolated or recombinant polypeptides of the invention include: (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 7; (b) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 7; (c) a polypeptide comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, or 98% sequence identity to the amino acid sequence set forth in SEQ ID N0:7 excluding amino acid residues 47, 78, 82, 94, 142, 175, 184, 205, 226, 236, 252, 255, 290, 328, 353, 464, and 710 of SEQ ID NO: 7; or (d) a polypeptide comprising an amino acid sequence that can be optimally aligned with the sequence of SEQ ID NO: 7 to generate a similarity score of at least 1830, using the BLOSUM62 matrix, a gap existence penalty of 11, and a gap extension penalty of 1, excluding amino acid residues 47
  • isolated or recombinant polypeptides of the invention include: (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8; (b) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 8; or (c) a polypeptide comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, or 98% sequence identity to the amino acid sequence set forth in SEQ ID NO:8 excluding amino acid residues 47, 78, 82, 94, 142, 175, 184, 205, 226, 236, 252, 255, 290, 328, 353, 464, and 710 . of SEQ ID NO: 8.
  • isolated or recombinant polypeptides of the invention include: (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 9; (b) a polypeptide consisting of the amino acid sequence set rortn m suy l ⁇ NU:y; or (c) a polypeptide comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, or 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9 excluding amino acid residues 47, 78, 82, 94, 142, 175, 184, 205, 226, 236, 252, 255, 290, 328, 353, 464, and 710 of SEQ ID NO: 9.
  • isolated or recombinant polypeptides of the invention include: (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 10; (b) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 10; or (c) a polypeptide comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, or 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10 excluding amino acid residues 47, 78, 82, 94, 142, 175, 184, 205, 226, 236, 252, 255, 290, 328, 353, 464, and 710 of SEQ ID NO:10.
  • isolated or recombinant polypeptides of the invention include: (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 11; (b) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 11; or (c) a polypeptide comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, or 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11 excluding amino acid residues 47, 78, 82, 94, 142, 175, 184, 205, 226, 236, 252, 255, 285, 290, 328, 353, 464, and 710 of SEQ ID NO: 11.
  • isolated or recombinant polypeptides of the invention include: (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 12; (b) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 12; or (c) a polypeptide comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, or 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12 excluding amino acid residues 47, 78, 82, 94, 142, l/b, ⁇ «4, 2Ub, 2I'O 1 236, 252, 255, 290, 328, 353, 464, 645, and 710 of SEQ ID N0:12.
  • isolated or recombinant polypeptides of the invention include: (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 13;
  • polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 13; or (c) a polypeptide comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, or 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13 excluding amino acid residues 47, 78, 82, 94, 142, 175, 184, 205, 226, 236, 252, 255, 290, 328, 353, 464, 631, and 710 of SEQ ID NO:13.
  • nucleic acid molecules include:
  • nucleic acid molecule which encodes a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:7, 8, 9, 10, 11, 12, or 13;
  • nucleic acid molecule which encodes a polypeptide comprising residues 1 to about 455 of the amino acid sequence set forth in SEQ ID NO: 7 or 11;
  • nucleic acid molecule which encodes a polypeptide comprising residues about 456 to about 1088 of the amino acid sequence set forth in SEQ ID NO:7, 8,
  • nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO: 15;
  • nucleic acid molecule consisting of the nucleotide sequence set forth in SEQ ID NO: 15;
  • nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO: 17;
  • nucleic acid molecule consisting of the nucleotide sequence set forth in SEQ ID NO: 17.
  • nucleic acid molecules of the invention include: (a) a nucleic acid molecule which encodes a polypeptide comprising residues 1 to about 455 of the amino acid sequence set forth in SEQ ID NO: 2 or 3 with the following amino acid residues: 48 and 206 are cysteine (C); 79 and 330 are phenylalanine (F) ; 83, 143, and 257 are serine (S) ; 95 and 176 are isoleucine (I); 185, 292, and 355 are valine (V); 227 is arginine (R) ; 238 is glutamine (Q) ; and 254 is glycine (G) ;
  • nucleic acid molecule which encodes a polypeptide consisting of residues 1 to about 455 of the amino acid sequence set forth in SEQ ID NO: 2 or 3 with the following amino acid residues: 48 and 206 are cysteine (C); 79 and 330 are phenylalanine (F) ; 83, 143, and 257 are serine (S) ; 95 and 176 are isoleucine (I); 185, 292, and 355 are valine (V); 227 is arginine (R) ; 238 is glutamine (Q) ; and 254 is glycine (G) ;
  • nucleic acid molecule which encodes a polypeptide comprising residues 1 to about 455 of the amino acid sequence set forth in SEQ ID NO: 4 with the following amino acid residues: 49 and 207 are cysteine (C); 80 and 331 are phenylalanine (F); 84, 144, and 258 are serine (S); 96 and 177 are isoleucine (I); 186, 293, and 357 are valine (V); 228 is arginine (R) ; 239 is glutamine (Q) ; and 255 is glycine (G) ;
  • nucleic acid molecule which encodes a polypeptide consisting of residues 1 to about 455 of the amino acid sequence set forth in SEQ ID NO: 4 with the following amino acid residues: 49 and 207 are cysteine (C); 80 and 331 are phenylalanine (F); 84, 144, and 258 are serine (S); 96 and 177 are isoleucine (I); 186, 293, and 357 are valine (V); 228 is arginine (R) ; 239 is glutamine (Q) ; and 255 is glycine (G) ;
  • nucleic acid molecule which encodes a polypeptide comprising residues 1 to about 455 of the amino acid sequence set forth in SEQ ID NO: 5 with the following amino acid residues: 54 and 212 are cysteine (C); 85 and 336 are phenylalanine (F); 89, 149, and 262 are serine (S); 101 and 182 are isoleucine (I); 191, 297, and 361 are valine (V); 232 is arginine (R) ; 243 is glutamine (Q) ; and 259 is glycine (G) ;
  • nucleic acid molecule which encodes a polypeptide consisting o ⁇ residues 1 to about 455 of the amino acid sequence set forth in SEQ ID NO: 5 with the following amino acid residues: 54 and 212 are cysteine (C); 85 and 336 are phenylalanine (F) ; 89, 149, and 262 are serine (S) ; 101 and 182 are isoleucine (I); 191, 297, and 361 are valine (V); 232 is arginine (R) ; 243 is glutamine (Q) ; and 259 is glycine (G) ;
  • a nucleic acid molecule which encodes a polypeptide comprising residues 1 to about 455 of the amino acid sequence set forth in SEQ ID NO: 6 with the following amino acid residues: 51 and 208 are cysteine (C); 82 and 337 are phenylalanine (F); 86, 146, and 262 are serine (S); 98 and 179 are isoleucine.
  • (I); 188, 296, and 363 are valine (V); 231 is arginine (R) ; 243 is glutamine (Q) ; and 258 is glycine (G) ; and (h) a nucleic acid molecule which encodes a polypeptide consisting of residues 1 to about 455 of the amino acid sequence set forth in SEQ ID NO: 6 with the following amino acid residues: 51 and 208 are cysteine (C); 82 and 337 are phenylalanine (F); 86, 146, and 262 are serine (S); 98 and 179 are isoleucine (I); 188, 296, and 363 are valine (V); 231 is arginine (R) ; 243 is glutamine (Q) ; and 258 is glycine (G) .
  • nucleic acid molecules of the invention include: (a) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 with the following amino acid residues: 48 and 206 are cysteine (C) ; 79 and 330 are phenylalanine (F) ; 83, 143, and 257 are serine (S); 95 and 176 are isoleucine (I); 185, 292, and 355 are valine (V); 227, 287, and 656 are arginine
  • vectors that include a nucleic acid molecule of the invention are provided.
  • host cells transfected with a nucleic acid molecule of the invention, or a vector that includes a nucleic acid molecule of the invention are provided.
  • Host cells include eucaryotic cells such as yeast cells, insect cells, or animal cells.
  • Host cells also include procaryotic cells such as bacterial cells.
  • methods for producing a cell that converts an alkane to alcohol generally include: (a) transforming a cell with an isolated nucleic acid molecule encoding a polypeptide that includes an amino acid sequence set forth, in SEQ ID NO: 7, 8, 9, 10, 11, 12, or 13; (b) transforming a cell with an isolated nucleic acid molecule encoding a polypeptide of the invention; or (c) transforming a cell with an isolated nucleic acid molecule of the invention.
  • methods for selecting a cell that converts an alkane to an alcohol are provided.
  • the methods generally include: (a) providing a cell containing a nucleic acid construct that includes a nucleotide sequence 73 that encodes a modified cytochrome P450 polypeptide, the nucleotide sequence selected from: (i) a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 7, 8, 9, 10, 11, 12, or 13; (ii) a nucleic acid molecule encoding a polypeptide of the invention; or (iii) a nucleic acid molecule of the invention.
  • the methods further include (b) culturing the cell in the presence of a suitable alkane and under conditions where the modified cytochrome P450 is expressed at a level sufficient to convert an alkane to an alcohol. Such conditions are met when an alcohol is produced at a level detectable by a method provided herein, or a method known to one skilled in the art of enzymology .
  • methods ' for producing an alcohol include: (a) providing a cell containing a nucleic acid construct comprising a nucleotide sequence that encodes a modified cytochrome P450 polypeptide, the nucleotide sequence selected from: (i) a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 7, 8, 9, 10, 11, 12, or 13; (ii) a nucleic acid molecule encoding a polypeptide of the invention; or (iii) a nucleic acid molecule of the invention.
  • the methods further include (b) culturing the cell in the presence of a suitable alkane and under conditions where the modified cytochrome P450 is expressed at an effective level; and (c) producing an alcohol by hydroxylation of the suitable alkane such as methane (CH4) , ethane (C 2 H 6 ) , propane (C 3 H 8 ) , butane (C 4 Hi 0 ) , pentane (C 5 Hi 2 ) , hexane (C 6 Hi 4 ) , heptane (C 7 Hi 6 ) 1 octane (C ⁇ Hi ⁇ ) , nonane (C9H20) , decane (C10H22) , undecane (C 11 H 2 4) , and dodecane (C12H26) •
  • a suitable alkane such as methane (CH4) , ethane (C 2 H 6 ) , propane (C 3 H 8 ) , butane (C 4 Hi
  • methods for producing a cytochrome P450 variant that hydroxylates an alkane include selecting a parent cytochrome P450 polypeptide and modifying at least one amino acid residue positioned in or near the active site of the heme domain of the parent cytochrome P450 polypeptide. In general the modification reduces the volume of the active site.
  • the method further includes contacting the polypeptide comprising the modified amino acid with at least one alkane under conditions suitable for hydroxylation of the alkane and detecting a hydroxylated alkane.
  • the modification may include a substitution of the parent amino acid for a different amino acid or it may include modifying the parent amino acid to include an additional group.
  • substitutions can include a phenylalanine, tyrosine, histidine, or serine for the parent amino acid residue positioned in or near the active site of the heme domain of the parent cytochrome P450 polypeptide.
  • Figure 1 depicts the reduction of substrate size and increase in C-H bond dissociation energy achieved by directed evolution. Directed evolution was used to convert wild-type P450 BM-3 stepwise from a fatty acid hydroxylase into an enzyme capable of activating the higher-energy C-H bond of ethane .
  • the figure shows the substrate range of wild-type P450 BM-3 and its mutant 53-5H and, for comparison, the range of substrates of other alkane monooxygenases .
  • Figure 2 depicts a gas chromatogram of ethanol reaction mixtures using variant 53-5H as the catalyst. Prior to analysis, ethanol was derivatized to form ethyl nitrite for detection via an electron capture detector (ECD) .
  • ECD electron capture detector
  • Figure 3 depicts the conversion of ethane to ethanol using 200 nM protein corresponds to 50 and 250 turnovers per enzyme active site of 53-5H and 35-E11, respectively. Halving or doubling the enzyme concentration yielded approximately correspondingly lower or higher product concentrations, respectively. Control reactions, either without ethane or with inactivated protein, did not contain ethanol concentrations above the background level of 1 ⁇ M. Error bars are the standard deviation of three experiments.
  • Figure 4 depicts 13 C-NMR spectrum of singly 13 C- labeled ethanol incubated with 53-5H and NADPH regeneration system. No 13 C-labeled carbonyl peaks due to overoxidation of the ethanol were detectable.
  • Figure 5 depicts an absorption spectrum of 53-5H.
  • the ferric heme iron is in low-spin form. A shift to high- spin ferric iron upon addition of ethane could not be detected. Addition of octane did not induce an increase at 390 nm.
  • the spectrum of 35-E11 is substantially similar.
  • Figures 6A - 6F depict an amino acid sequence alignment of 6 CYP102 homologs and BM-3 mutant 35-E11. Corresponding mutations in homologs are provided in Table 4.
  • Figure 7 depicts the spectra of methane reactions of BM-3 variant 35-E11 and the variant 35-E11-E464R after treatment with alcohol oxidase and Purpald. The peak at 550 nm corresponds to methanol produced. Solutions containing all reactants except methane do not absorb in this region when similarly treated.
  • Figure 8 depicts SEQ ID NO: 1 (CYP102A1 from Bacillus megaterium) .
  • Figure 9 depicts SEQ ID NO: 2 (CYP102A2 from Bacillus megaterium).
  • Figure 10 depicts SEQ ID NO: 3 (CYP102A3 from
  • Bacillus subtilis 58% identity to CYP102A1) .
  • FIG. 11 depicts SEQ ID NO: 4 (CYP102A5 from
  • Figure 12 depicts SEQ ID NO: 5 (CYP102E1 from
  • Figure 13 depicts SEQ ID NO: 6 (CYP102A6 from
  • Bradyrhizobium japonicum 46% identity to CYP102A1 .
  • Figure 14 depicts SEQ ID NO: 7 (35-E11) .
  • Figure 15 depicts SEQ ID NO: 8 (35-E11-E464R) .
  • Figure 16 depicts SEQ ID NO: 9 (35-E11-E464Y) .
  • Figure 17 deoicts SEQ ID NO: 10 (35-E11-E464T) .
  • Figure 18 depicts SEQ ID NO: 11 (20-D3) .
  • Figure 19 depicts SEQ ID NO: 12 (23-1D) .
  • Figure 20 depicts SEQ ID NO: 13 (21-4G) .
  • Figure 21 depicts SEQ ID NO: 15 (53-5H) .
  • Figures 22A and 22B depict SEQ ID NO: 16 (expression plasmid pCWORI-53-H containing the heme and reductase domain of variant 53-5H) .
  • Figure 23 depicts SEQ ID NO: 17 (nucleic acid sequence encoding variant 35-E11) .
  • Figures 24A and 24B depict SEQ ID NO: 18 (expression plasmid pCWORI-35-Ell containing heme and reductase domain of variant 35-E11) .
  • Figure 25 depicts alkyl methyl ether assays.
  • Panel a) shows hydroxylation of the propane surrogate dimethyl ether produces formaldehyde which can easily be detected using the dye Purpald.
  • Panel b) shows hexyl methyl ether can be used as an octane surrogate.
  • Panel c) shows a typical screening plate with P450-containing cell lysate, which, upon hydroxylation of the methoxy group of DME or HME and addition of Purpald, forms a purple color.
  • Each well represents a different BM-3 variant .
  • Figure 26 depicts standard curves for formaldehyde ( ⁇ ) and hexanal ( ⁇ ) using Purpald. The assay was performed in a 96-well microtiter plate and the final volume was 250 ⁇ L per well .
  • Figure 27 depicts a colorimetric reaction after addition of Purpald to reaction mixtures containing BM-3 mutants, chloromethane and cofactor regeneration system. Panel A and Panel B refer to independent experiments.
  • Figure 28 depicts total turnover numbers for the reaction of CM dehalogenation catalyzed by BM-3 mutants.
  • Figure 29 depicts a colorimetric response for the reaction BM-3 variants substituted at position A328.
  • Figure 30 depicts the chemical structures of products from the hydroxylation/dehalogenation reaction catalyzed by P450 mutants on different kinds of halogenated alkanes .
  • Figures 31A - 31C depict an amino acid sequence alignment of the heme domain (residues 1-455) of 6 CYP102 homologs and BM-3 mutant 35-Ell. Corresponding mutations in homologs are provided in Table 4.
  • Figure 32 depicts protein similarity scores for polypeptides provided herein.
  • Panel A shows exemplary similarity scores for full length polypeptides (e.g., SEQ ID NOs: 1 - 7) .
  • Panel B shows exemplary similarity scores for residues 1-455 of of SEQ ID NOs: 1 - 7 corresponding to the heme domain of P450 polypeptides.
  • Figure 33 depicts protein percent (%) identities for polypeptides provided herein.
  • Panel A shows exemplary percent (%) identities for full length polypeptides (e.g., SEQ ID NOs: 1 - 7) .
  • Panel B shows exemplary percent (%) identities for residues 1-455 of of SEQ ID NOs: 1 - 7 corresponding to the heme domain of P450 polypeptides.
  • Like reference symbols in the various drawings indicate like elements .
  • polypeptides of the invention may contain one or more modified amino acids.
  • modified amino acids may be advantageous in, for example, (a) increasing polypeptide in vivo half-life, (b) reducing or increasing polypeptide antigenicity, and (c) increasing polypeptide storage stability.
  • Amino acid(s) are modified, for example, co-translationally or post- translationally during recombinant production (e.g., N-linked glycosylation at N—X—S/T motifs during expression in mammalian cells) or modified by synthetic means.
  • a "mutant", “variant” or “modified” protein, enzyme, polynucleotide, gene, or cell means a protein, enzyme, polynucleotide, gene, or cell, that has been altered or derived, or is in some way different or changed, from a parent protein, enzyme, polynucleotide, gene, or cell.
  • a mutant or modified protein or enzyme is usually, although not necessarily, expressed from a mutant polynucleotide or gene.
  • a “mutation” means any process or mechanism resulting in a mutant protein, enzyme, polynucleotide, gene, or cell. This includes any mutation in which a protein, enzyme, polynucleotide, or gene sequence is altered, and any detectable change in a cell arising from such a mutation. Typically, a mutation occurs in a polynucleotide or gene sequence, by point mutations, deletions, or insertions of single or multiple nucleotide residues.
  • a mutation includes polynucleotide alterations arising within a protein-encoding region of a gene as well as alterations in regions outside of a protein-encoding sequence, such as, but not limited to, regulatory or promoter sequences .
  • a mutation in a gene can be "silent", i.e., not reflected in an amino acid alteration upon expression, leading to a "sequence-conservative" variant of the gene. This generally arises when one amino acid corresponds to more than one codon.
  • Non-limiting examples of a modified amino acid include a glycosylated amino acid, a sulfated amino acid, a prenlyated (e.g., farnesylated, geranylgeranylated) amino acid, an acetylated amino acid, an acylated amino acid, a pegylated amino acid, a biotinylated amino acid, a carboxylated amino acid, a phosphorylated amino acid, and the like.
  • the polypeptides may be produced by direct peptide synthesis using solid-phase techniques (e.g., Stewart et al . (1969) Solid- Phase Peptide Synthesis (WH Freeman Co, San Francisco) ; and Merrifield (1963) J. Am. Chem. Soc. 85: 2149-2154). Peptide synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City, Calif.) in accordance with the instructions provided by the manufacturer.
  • solid-phase techniques e.g., Stewart et al . (1969) Solid- Phase Peptide Synthesis (WH Freeman Co, San Francisco) ; and Merrifield (1963) J. Am. Chem. Soc. 85: 2149-2154.
  • Peptide synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (
  • Cytochrome P450 monooxygenase or "P450 enzyme” means an enzyme in the superfamily of P450 haem-thiolate proteins, which are widely distributed in bacteria, fungi, plants and animals. The enzymes are involved in metabolism of a plethora of both exogenous and endogenous compounds . Usually, they- act as terminal oxidases in multicomponent electron transfer chains, called here P450-containing monooxygenase systems. The unique feature which defines whether an enzyme is a cytochrome P450 enzyme is traditionally considered to be the characteristic absorption maximum ("Soret band") near 450 nm observed upon binding of carbon monoxide (CO) to the reduced form of the heme iron of the enzyme.
  • Soret band characteristic absorption maximum
  • Heme domain refers to an amino acid sequence within an oxygen carrier protein, which sequence is capable of binding an iron-complexing structure such as a porphyrin. Compounds of iron are typically complexed in a porphyrin (tetrapyrrole) ring that may differ in side chain composition.
  • Heme groups can be the prosthetic groups of cytochromes and are found in most oxygen carrier proteins .
  • Exemplary heme domains include that of P450 BM-3 (P450. sub .BM-P) , SEQ ID NO: 3, as well as truncated or mutated versions of these that retain the capability to bind the iron-complexing structure.
  • P450 BM-3 P450. sub .BM-P
  • SEQ ID NO: 3 SEQ ID NO: 3
  • the skilled artisan can readily identify the heme domain of a specific protein using methods known in the art.
  • a "protein” or "polypeptide”, which terms are used interchangeably herein, comprises one or more chains of chemical building blocks called amino acids that are linked together by chemical bonds called peptide bonds.
  • an “enzyme” means any substance, preferably composed wholly or largely of protein, that catalyzes or promotes, more or less specifically, one or more chemical or biochemical reactions.
  • the term “enzyme” can also refer to a catalytic polynucleotide (e.g., RNA or DNA).
  • a "native” or “wild-type” protein, enzyme, polynucleotide, gene, or cell means a protein, enzyme, polynucleotide, gene, or cell that occurs in nature.
  • the polypeptides include up to 50, 25, 10, or 5 conservative amino acid substitutions excluding residues: (a) 47, 78, 82, 94, 142, 175, 184, 205, 226, 236, 252, 255, 290, 328, and 353 of SEQ ID NO:1; (b) 48, 79, 83, 95, 143, 176, 185, 206, 227, 238, 254, 257, 292, 330, and 355 of SEQ ID NO:2 or SEQ ID N0:3; (c) 49, 80, 84, 96, 144, 177, 186, 207, 228, 239, 255, 258, 293, 331, and 357 of SEQ ID N0:4; (d) 54, 85, 89, 101, 149, 182, 191, 212, 232, 243, 259, 262, 297, 336, and 361 of SEQ ID N0:5; and (e) 51, 82, 86, 98, 146,
  • the polypeptide further includes a Z3 amino acid residue at position: (a) 285 of SEQ ID NO:1; (b) 287 of SEQ ID N0:2 or 3; (c) 288 of SEQ ID N0:4;
  • a Z3 amino acid residue includes lysine (K) , arginine (R) , or histidine
  • the amino acid residue at this position is an arginine (R) .
  • amino acid sequence is a polymer of amino acids (a protein, polypeptide, etc.) or a character string representing an amino acid polymer, depending on context.
  • protein polypeptide
  • peptide amino acid sequence
  • Constant amino acid substitution or, simply, “conservative variations” of a particular sequence refers to the replacement of one amino acid, or series of amino acids, with essentially identical amino acid sequences.
  • substitutions, deletions or additions which alter, add or delete a single amino acid or a percentage of amino acids in an encoded sequence result in "conservative variations” where the alterations result in the deletion of an amino acid, addition of an amino acid, or substitution of an amino acid with a chemically similar amino acid.
  • Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • one conservative substitution group includes Alanine (A) , Serine (S) , and Threonine (T) .
  • Another conservative substitution group includes Aspartic acid (D) and Glutamic acid (E) .
  • Another conservative substitution group includes Asparagine (N) and Glutamine (Q) .
  • Yet another conservative substitution group includes Arginine (R) and Lysine (K) .
  • Another conservative substitution group includes Isoleucine, (I) Leucine (L), Methionine (M), and Valine (V).
  • Another conservative substitution group includes Phenylalanine (F) , Tyrosine (Y) , and Tryptophan (W) .
  • “conservative amino acid substitutions” of a listed polypeptide sequence include substitutions of a percentage, typically less than 10%, of the amino acids of the polypeptide sequence, with a conservatively selected amino acid of the same conservative substitution group. Accordingly, a conservatively substituted variation of a polypeptide of the invention can contain 100, 75, 50, 25, or 10 substitutions with a conservatively substituted variation of the same conservative substitution group.
  • a conservatively substituted variation of a polypeptide of the invention can contain 100, 75, 50, 25, or 10 substitutions with a conservatively substituted variation of the same conservative substitution group.
  • the "activity" of an enzyme is a measure of its ability to catalyze a reaction, i.e., to "function", and may be expressed as the rate at which the product of the reaction is produced.
  • enzyme activity can be represented as the amount of product produced per unit of time or per unit of enzyme (e.g., concentration or weight), or in terms of affinity or dissociation constants.
  • cytochrome P450 activity refers to an activity exerted by a cytochrome P450 protein, polypeptide or nucleic acid molecule on a cytochrome P450 polypeptide substrate, as determined in vivo, or in vitro, according to standard techniques. The biological activity of cytochrome P450 is described herein.
  • nucleic acid constructs which are disclosed yield a functionally identical construct.
  • substitutions i.e., substitutions in a nucleic acid sequence which do not result in an alteration in an encoded polypeptide
  • conserve amino acid substitutions in one or a few amino acids in an amino acid sequence are substituted with different amino acids with highly similar properties, are also readily identified as being highly similar to a disclosed construct.
  • conservative variations of each disclosed sequence are a feature of the polyeptides provided herein.
  • Constant variants are proteins or enzymes in which a given amino acid residue has been changed without altering overall conformation and function of the protein or enzyme, including, but not limited to, replacement of an amino acid with one having similar properties, including polar or non-polar character, size, shape and charge.
  • Amino acids other than those indicated as conserved may differ in a protein or enzyme so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and can be, for example, at least 30%, at least 50%, at least 70%, at least 80%, or at least 90%, as determined according to an alignment scheme.
  • sequence similarity means the extent to which nucleotide or protein sequences are related.
  • sequence identity herein means the extent to which two nucleotide or amino acid sequences are invariant.
  • Sequence alignment means the process of lining up two or more sequences to achieve maximal levels of identity (and, in the case of amino acid sequences, conservation) for the purpose of assessing the degree of similarity. Numerous methods for aligning sequences and assessing similarity/identity are known in the art such as, for example, the Cluster Method, wherein similarity is based on the MEGALIGN algorithm, as well as BLASTN, BLASTP, and FASTA (Lipman and Pearson, 1985; Pearson and Lipman, 1988) .
  • Non-conservative modifications of a particular polypeptide are those which substitute any amino acid not characterized as a conservative substitution.
  • any substitution which crosses the bounds of the six groups set forth above include substitutions of basic or acidic amino acids for neutral amino acids, (e.g., Asp, GIu, Asn, or GIn for VaI, lie, Leu or Met) , aromatic amino acid for basic or acidic amino acids (e.g., Phe, Tyr or Trp for Asp, Asn, GIu or GIn) or any other substitution not replacing an amino acid with a like amino acid.
  • Basic side chains include lysine (K) , arginine (R) , histidine (H) ; acidic side chains include aspartic acid (D) , glutamic acid (E) ; uncharged polar side chains include glycine (G) , asparagine (N) , glutamine (Q) , serine (S) , threonine (T) , tyrosine (Y) , cysteine (C) ; nonpolar side chains include alanine (A) , valine (V) , leucine (L) , isoleucine (I) , proline (P) , phenylalanine (F) , methionine (M) , tryptophan (W) ; beta-branched side chains include threonine (T), valine (V), isoleucine (I); aromatic side chains include tyrosine (Y) , phenylalanine (F) , tryptophan (
  • an arginine (R) to cysteine (C) substitution is made in SEQ ID N0:l at position 47.
  • This substitution is generally not considered a "conservative" substitution.
  • Similar substitutions are made throughout the various sequences at the indicated positions in order to modify the activity of the polypeptide. Such modifications are made in order to generate polypeptides that are active on substrates such as alkanes.
  • a parent polypeptide e.g., SEQ ID N0:l, 2, 3, 4, 5 or 6
  • SEQ ID N0:l, 2, 3, 4, 5 or 6 can be mo ⁇ irie ⁇ to include an amino acid substitution at a particular residue by modifying the nucleic acid sequence that encodes the parent polypeptide (see below for additional information regarding modifying nucleic acid sequences) .
  • the region of P450 modified is identified on the left side of Table 4.
  • the specific substitutions are identified throughout Table 4. Accordingly, "Z" groups are identified as follows: a Zl amino acid residue includes glycine (G) , asparagine (N) , glutamine
  • a Z2 amino acid residue includes alanine (A) , valine (V) , leucine (L), isoleucine (I), proline (P), or methionine (M);
  • a Z3 amino acid residue includes lysine (K) , or arginine (R) ;
  • a Z4 amino acid residue includes tyrosine (Y) , phenylalanine
  • a Z5 amino acid residue includes threonine (T), valine (V), and isoleucine (I)
  • a Z6 amino acid residue includes aspartic acid (D) and glutamic acid (E) .
  • a polyeptide provided herein can include amino acids that are "restricted” to particular amino acid substitutions.
  • residues 47, 78, 82, 94, 142, 175, 184, 205, 226, 236, 252, 255, 290, 328, and 353 can be restricted to substitutions set forth in a "Z" group as defined below and throughout the specification. It is understood that not all of the identified restricted residues need be altered in the same polyeptide.
  • the invention encompasses polyeptides where only about 80%, 85%, 90% or 95% of the restricted amino acid residues are altered in a given polyeptide.
  • each "Z” group can be included at the appropriate position in a designated polypeptide. Accordingly, in other embodiments, the amino acid sequence of the polypeptide includes residues at the following positions: (1) a glycine (G), glutamine (Q), serine (S), threonine
  • T cysteine amino acid residue at position: (a) 47, 82, 142, 205, 236, 252, and 255 of SEQ ID N0:l; (b) 48, 83, 143, 206, 238, 254, and 257 of SEQ ID N0:2 or SEQ ID N0:3; (c) 49, 84, 144, 207, 239, 255, and 258 of SEQ ID N0:4; (d) 54, 89, 149, 212, 243, 259, and 262 of SEQ ID N0:5; and (e) 51, 86, 146, 208, 242, 258, and 262 of SEQ ID N0:6;
  • V valine
  • I isoleucine
  • F phenylalanine
  • H histidine
  • substrate or “suitable substrate” means any substance or compound that is converted or meant to be converted into another compound by the action of an enzyme catalyst.
  • the term includes alkanes, and includes not only a single compound, but also combinations of compounds, such as solutions, mixtures and other materials which contain at least one substrate.
  • Substrates for hydroxylation using the cytochrome P450 enzymes of the invention include para- nitrophenoxycarboxylic acids (“pNCAs”) such as 12-pNCA, as well as decanoic acid, myristic acid, lauric acid, and other fatty acids and fatty acid-derivatives.
  • pNCAs para- nitrophenoxycarboxylic acids
  • alkane/alkene- substrates propane, propene, ethane, ethene, butane, butene, pentane, pentene, hexane, hexene, cyclohexane, octane, octene, styrene, p-nitrophenoxyoctane (8-pnpane) , and various derivatives thereof, can be used.
  • derivative refers to the addition of one or more functional groups to a substrate, including, but not limited, alcohols, amines, halogens, thiols, amides, carboxylates, etc.
  • a "parent" protein, enzyme, polynucleotide, gene, or cell is any protein, enzyme, polynucleotide, gene, or cell, from which any other protein, enzyme, polynucleotide, gene, or cell, is derived or made, using any methods, tools or techniques, and whether or not the parent is itself native or mutant.
  • a parent polynucleotide or gene encodes for a parent protein or enzyme .
  • amino acid sequence of the polypeptide includes residues at the following positions:
  • G glycine (G) amino acid residue at position: (a) 252 of SEQ ID N0:l; (b) 254 of SEQ ID N0:2 or SEQ ID N0:3; (c) 255 of SEQ ID N0:4; (d) 259 of SEQ ID N0:5; (e) 258 of SEQ ID
  • SEQ ID N0:l includes the amino acid sequence of cytochrome P450 BM-3 isolated from Bacillus megaterium. This wild-type P450 BM-3 is also designated CYP102A1.
  • SEQ ID NO: 2 provides the amino acid sequence of wild-type cytochrome P450 from Bacillus subtilis strain IAl.15. This wild-type P450 is also designated CYP102A2 and shares 59% amino acid sequence identity to CYP102A1 (SEQ ID NO:1) .
  • SEQ ID NO: 3 includes the amino acid sequence of wild-type cytochrome P450 from Bacillus subtilis strain IAl.15) .
  • This wild-type P450 is also designated CYP102A3 and shares 58% amino acid sequence identity to CYP102A1 (SEQ ID N0:l) .
  • SEQ ID NO: 4 includes the amino acid sequence of wild- type cytochrome P450 from Bacillus cereus.
  • This wild-type P450 is also designated CYP102A5 and shares 60% amino acid sequence identity to CYP102A1 (SEQ ID NO:1).
  • SEQ ID NO:5 includes the amino acid sequence of wild-type cytochrome P450 from Ralstonia metallidurans .
  • This wild-type P450 is also designated CYP102E1 and shares 38% amino acid sequence identity to CYP102A1 (SEQ ID N0:l).
  • SEQ ID N0:6 includes the amino acid sequence of wild-type cytochrome P450 from Bradyrhizobium japonicum. This wild-type P450 is also designated CYP102A6 and shares 38% amino acid sequence identity to CYP102A1 (SEQ ID N0:l).
  • a polynucleotide, polypeptide, or other component is “isolated” when it is partially or completely separated from components with which it is normally associated (other proteins, nucleic acids, cells, synthetic reagents, etc.).
  • a nucleic acid or polypeptide is "recombinant” when it is artificial or engineered, or derived from an artificial or engineered protein or nucleic acid.
  • a polynucleotide that is inserted into a vector or any other heterologous location, e.g., in a genome of a recombinant organism, such that it is not associated with nucleotide sequences that normally flank the polynucleotide as it is found in nature is a recombinant polynucleotide.
  • a protein expressed in vitro or in vivo from a recombinant polynucleotide is an example of a recombinant polypeptide.
  • a polynucleotide sequence that does not appear in nature for example a variant of a naturally occurring gene, is recombinant.
  • the cytochromes P450 set forth in SEQ ID NOs : 1 - 6 are closely related to one another and show a high degree of sequence identity.
  • the sequences can be aligned based on the sequence homology.
  • the alignment provided in Figure 6A - 6F identifies "equivalent positions" in the sequences.
  • An equivalent position denotes a position which, on the basis of the alignment of the sequence of the parent cytochrome P450 in question with the "reference" cytochrome P450 amino acid sequence in question (e.g. SEQ ID NO: 1) so as to achieve juxtapositioning of amino acid residues which are common to both, corresponds most closely to a particular position in the reference sequence in question. This process can cause gaps or insertions to appear in the sequences.
  • the "linker” region generally includes those residues from positions about 450 to about 480 of, for example, SEQ ID N0:l - 6.
  • the "reductase” domain generally includes those residues from positions about 470 to about 1050 of SEQ ID NOs: 1-6.
  • polypeptides that include amino acid substitutions at these residues are provided. These substitutions can be made in conjunction with substitutions in the "heme” domain or they can be made in isolation from those in the heme domain.
  • an isolated or recombinant polypeptide that includes residues 456-1048 of SEQ ID N0:l, 456-1059 of SEQ ID N0:2, 456-1053 of SEQ ID N0:3, 456-1064 of SEQ ID NO:4, 456-1063 of SEQ ID NO:5, or 456-1077 of SEQ ID NO: 6, is provided.
  • the polypeptide includes an amino acid sequence with up to 65, 40, 25, or 10 conservative amino acid substitutions excluding residues: (a) 464, 631, 645, 710 and 968 of SEQ ID N0:l; (b) 475, 641, 656, 721 and 980 of SEQ ID N0:2; (c) 467, 634, 648, 713 and 972 of SEQ ID NO:3; (d) 477, 644, 659, 724 and 983 of SEQ ID NO:4; (e) 472, 640, 656, 723 and 985 of SEQ ID NO:5; and (f) 480, 648, 664, 733 and 997 of SEQ ID NO: 6.
  • the amino acid sequence includes the following residues:
  • a Zl, Z3, Z4, or Z5 amino acid residue at position (a) 464 of SEQ ID NO:1; (b) 475 of SEQ ID NO:2; (c) 467 of SEQ ID NO:3; (d) 477 of SEQ ID NO:4; (e) 472 of SEQ ID NO:5; and (f) 480 of SEQ ID NO: 6;
  • Zl is an amino acid residue selected from the group consisting of glycine (G) , asparagine (N) , glutamine (Q) , serine (S) , threonine (T) , tyrosine (Y) , and cysteine (C) ;
  • Z2 is an amino acid residue selected from the group consisting of alanine (A), valine (V), leucine (L), isoleucine (I), proline
  • Z3 is an amino acid residue selected from the group consisting of lysine (K) , and arginine (R)
  • Z4 is an amino acid residue selected from the group consisting of tyrosine (Y) , phenylalanine (F) , tryptophan (W) , and histidine
  • H is an amino acid residue selected from the group consisting of threonine (T) , valine (V) , and isoleucine (I) .
  • amino acid sequence of the polypeptide includes residues at the following positions:
  • T threonine amino acid residue at position: (a) 710 of SEQ ID NO:1; (b) 721 of SEQ ID N0:2; (c) 713 of SEQ ID N0:3; (d) 724 of SEQ ID N0:4; (e) 723 of SEQ ID N0:5; and (f) 733 of SEQ ID NO: 6; and
  • polypeptides provided herein generally include a "heme domain" which includes amino acid residues 1 to about 455 of the various P450 sequences provided in, for example, SEQ ID N0:l through SEQ ID NO: 13.
  • heme domain refers to a catalytically functional region of the polypeptide that exhibits monooxygenase/hydroxylase activity.
  • a heme domain is a redox domain capable of binding an iron-complexing structure such as a porphyrin. Compounds of iron are typically complexed in a porphyrin (tetrapyrrole) ring that may differ in side chain composition.
  • Heme groups can be the prosthetic groups of cytochromes and are found in most oxygen carrier proteins.
  • Exemplary heme domains include that of P450 BM-3 (e.g., amino acid residues 1 to about 455 of SEQ ID N0:l), as well as truncated or mutated versions of these that retain the capability to bind the iron-complexing structure.
  • P450 BM-3 e.g., amino acid residues 1 to about 455 of SEQ ID N0:l
  • truncated or mutated versions of these that retain the capability to bind the iron-complexing structure.
  • the skilled artisan can readily identify the heme domain of a specific protein using methods known in the art.
  • amino acid residues 1 to "about” or “approximately” 430 means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system.
  • “about” can mean a range of up to 20%, up to 10%, up to 5%, up to 1% of a given value, such as the number of amino acid residues in a heme domain or a reductase domain.
  • hydroxylase or “monooxygenase” should be considered to include any enzyme that can insert one oxygen atom from diatomic oxygen into a substrate.
  • exemplary enzymes include the cytochrome P450 monooxygenases .
  • Cytochrome P450 monooxygenase or “P450 enzyme” means an enzyme in the superfamily of P450 heme- thiolate proteins, which are widely distributed in bacteria, fungi, plants and animals.
  • the unique feature which defines whether an enzyme is a cytochrome P450 enzyme is traditionally considered to be the characteristic absorption maximum ("Soret band") near 450 nm observed upon binding of carbon monoxide (CO) to the reduced form of the heme iron of the enzyme.
  • Soret band characteristic absorption maximum
  • Reactions catalyzed by cytochrome P450 enzymes include epoxidation, N-dealkylation, O-dealkylation, S-oxidation and hydroxylation.
  • the most common reaction catalyzed by P450 enzymes is the monooxygenase reaction, i.e., insertion of one atom of oxygen into a substrate while the other oxygen atom is reduced to water.
  • the invention envisions the synthesis of chimeric polypeptides that include a heme catalytic domain derived from a first source and an electron transfer domain (e.g., a reductase domain) derived from a second source.
  • the first source and the second source may differ by the genus, or the species from which they are derived, or they may be derived from the same species as one another but from different organelles or compartments in the same species.
  • the electron transfer domain is a heme reductase domain.
  • chimeric polypeptides that comprise: 1) a variant heme domain isolated from a first organism and modified to include a new activity; and 2) a variant reductase domain isolated from a second organism and modified to include a new activity or an activity that a complements the heme domain, are provided.
  • Methods for engineering a chimeric polypeptide of the invention are disclosed in U.S. Patent Application Publication Number 20050124025, the contents of which are incorporated herein by reference .
  • an isolated or recombinant polypeptide that includes residues 1 to about 455 of the amino acid sequence set forth in SEQ ID N0:l, 2, 3, 4, 5 or 6 is provided.
  • the polypeptide includes amino acid residues in the amino acid sequence that correspond to the following positions, at least 80%, 85%, 90%, or 95% of the amino acid residues in the amino acid sequence at the positions specified in (a) - (h) are as follows:
  • G glycine (G) amino acid residue at position: (a) 252 of SEQ ID NO:1; (b) 254 of SEQ ID N0:2 or SEQ ID NO:3; (c) 255 of SEQ ID NO:4; (d) 259 of SEQ ID N0:5; (e) 258 of SEQ ID NO:
  • V valine (V) amino acid residue at position: (a) 184, 290 and 353 of SEQ ID NO:1; (b) 185, 292, and 355 of SEQ ID N0:2 or SEQ ID N0:3; (c) 186, 293 and 357 of SEQ ID NO:4; (d) iyi, ZV/, and 361 or SKQ ID N0:5; (e) 188, 296, and 363 of SEQ ID NO: 6;
  • the polypeptide further includes a Z3 amino acid residue at position: (a) 285 of SEQ ID N0:l; (b) 287 of SEQ ID N0:2 or 3; (c) 288 of SEQ ID N0:4; (d) 292 of SEQ ID N0:5; and (e) 291 of SEQ ID NO: 6.
  • a Z3 amino acid residue includes lysine (K) , arginine (R) , or histidine (H) .
  • the amino acid residue at this position is an arginine (R) .
  • a polypeptide provided herein includes amino acid residue substitutions that correspond to positions in a particular sequence at least 80%, 85%, 90%, or 95% of the time.
  • the invention encompasses polypeptides that contain the recited amino acid substitutions at 80%, 85%, 90%, or 95% of the recited positions in a given sequence. The skilled artisan will recognize that not every substitution from a group of substitutions is necessary to obtain a modified polypeptide that is active on an alkane substrate.
  • an isolated or recombinant polypeptide that includes the amino acid sequence set forth in SEQ ID NO: 7, is provided.
  • SEQ ID NO: 7 (see Figure 14) provides an amino acid sequence of a P450 BM-3 variant. This variant is also designated 35-E11.
  • the polypeptide may contain up to 75, 50, 25, or 10 conservative amino acid substitutions excluding residues 47, 78, 82, 94, 142, 175, 184, 205, 226, 236, 252, 255, 290, 328, 353, 464, and 710.
  • an isolated or recombinant polypeptide that includes the amino acid sequence set forth in SEQ ID NO: 8 is provided.
  • SEQ ID NO: 8 (see Figure 15) provides an amino acid sequence of a P450 BM-3 variant. This variant is also designated 35-E11-E464R.
  • the polypeptide may contain up to 75, 50, 25, or 10 conservative amino acid substitutions excluding residues 47, 78, 82, 94, 142, 175, 184, 205, 226, 236, 252, 255, 290, 328, 353, 464, and 710.
  • an isolated or recombinant polypeptide that includes the amino acid sequence set forth in SEQ ID NO: 9 is provided.
  • SEQ ID NO: 9 provides an amino acid sequence of a P450 BM-3 variant. This variant is also designated 35-E11-E464Y.
  • the polypeptide may contain up to 75, 50, 25, or 10 conservative amino acid substitutions excluding residues 47, 78, 82, 94, 142, 175, 184, 205, 226, 236, 252, 255, 290, 328, 353, 464, and 710.
  • an isolated or recombinant polypeptide that includes the amino acid sequence set forth in SEQ ID NO: 10 is provided.
  • SEQ ID NO: 10 (see Figure 17) provides an amino acid sequence of a P450 BM-3 variant. This variant is also designated 35-E11-E464T .
  • the polypeptide may contain up to 75, 50, 25, or 10 conservative amino acid substitutions excluding residues 47, 78, 82, 94, 142, 175, 184, 205, 226, 236, 252, 255, 290, 328, 353, 464, and 710.
  • the polypeptides of SEQ ID NO:7, 8, 9 or 10 further optionally include an arginine (R) at amino acid residue position 285.
  • an isolated or recombinant polypeptide that includes the amino acid sequence set forth in SEQ ID NO:11 is provided.
  • SEQ ID N0:ll provides an amino acid sequence of a P450 BM-3 variant. This variant is also designated 20-D3.
  • the polypeptide may contain up to 75, 50, 25, or 10 conservative amino acid substitutions excluding residues 47, 78, 82, 94, 142, 175, 184, 205, 226, 236, 252, 255, 285, 290, 328, 353, 464, and 710.
  • an isolated or recombinant polypeptide that includes the amino acid sequence set forth in SEQ ID NO:12 is provided.
  • SEQ ID N0:12 provides an amino acid sequence of a P450 BM-3 variant. This variant is also designated 23-1D.
  • the polypeptide may contain up to 75, 50, 25, or 10 conservative amino acid substitutions excluding residues 47, 78, 82, 94, 142, 175, 184, 205, 226, 236, 252, 255, 290, 328, 353, 464, 645, and 710.
  • an isolated or recombinant polypeptide that includes the amino acid sequence set forth in SEQ ID NO: 13, is provided.
  • SEQ ID NO: 13 provides an amino acid sequence of a P450 BM-3 variant. This variant is also designated 21-4G.
  • the polypeptide may contain up to 75, 50, 25, or 10 conservative amino acid substitutions excluding residues 47, 78, 82, 94, 142, 175, 184, 205, 226, 236, 252, 255, 290, 328, 353, 464, 631, and 710.
  • isolated or recombinant polypeptides of the invention include: (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 7; (b) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 7; (c) a polypeptide comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, or 98% sequence identity to the amino acid sequence set forth in SEQ ID NO:7 excluding amino acid residues 47, 78, 82, 94, 142, 175, 184, 205, 226, 236, 252, 255, 290, 328, 353, 464, and 710 of SEQ ID NO: 7; or (d) a polypeptide comprising an amino acid sequence that can be optimally aligned with the sequence of SEQ ID NO:7 to generate a similarity score of at least 1830, using the BLOSUM62 matrix, a gap existence penalty of 11, and a gap extension penalty of 1, excluding amino acid residues 47,
  • isolated or recombinant polypeptides of the invention include: (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8; (b) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 8; or (c) a polypeptide comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, or 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8 excluding amino acid residues 47, 78, 82, 94, 142, 175, 184, 205, 226, 236, 252, 255, 290, 328, 353, 464, and 710 of SEQ ID NO: 8.
  • isolated or recombinant polypeptides of the invention include: (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 9; (b) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 9; or (c) a polypeptide comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, or 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9 excluding amino acid residues 47, 78, 82, 94, 142, 175, 184, 205, 226, 236, 252, 255, 290, 328, 353, 464, and 710 of SEQ ID NO:9.
  • isolated or recombinant polypeptides of the invention include: (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 10; (b) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 10; or (c) a polypeptide comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, or 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10 excluding amino acid residues 47, 78, 82, 94, 142, 175, 184, 205, 226, 236, 252, 255, 290, 328, 353, 464, and 710 of SEQ ID NO:10.
  • isolated or recombinant polypeptides of the invention include: (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 11; (b) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 11; or (c) a polypeptide comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, or 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11 excluding amino acid residues 47, 78, 82, 94, 142, 175, 184, 205, 226, 236, 252, 255, 285, 290, 328, 353, 464, and 710 of SEQ ID NO: 11.
  • isolated or recombinant polypeptides of the invention include: (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 12; (b) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 12; or (c) a polypeptide comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, or 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12 excluding amino acid residues 47, 78, 82, 94, 142, 175, 184, 205, 226, 236, 252, 255, 290, 328, 353, 464, 645, and 710 of SEQ ID NO: 12.
  • isolated or recombinant polypeptides of the invention include: (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 13; (b) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 13; or (c) a polypeptide comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, or 98% sequence identity to the amino acid sequence set forth in SEQ ID NO:13 excluding amino acid residues 47, 78, 82, 94, 142, 175, 184, 205, 226, 236, 252, 255, 290, 328, 353, 464, 631, and 710 of SEQ ID NO:13.
  • sequence identity herein means the extent to which two nucleotide or amino acid sequences are invariant.
  • Sequence alignment means the process of lining up two or more sequences to achieve maximal levels of identity (and, in the case of amino acid sequences, conservation) for the purpose of assessing the degree of similarity. Numerous methods for aligning sequences and assessing similarity/identity are known in the art such as, for example, the Cluster Method, wherein similarity is based on the MEGALIGN algorithm, as well as BLASTN, BLASTP, and FASTA (Lipman and Pearson, 1985; Pearson and Lipman, 1988) . When using all of these programs, the preferred settings are those that results in the highest sequence similarity.
  • ClustalW analysis version W 1.8 available from European Bioinformatics Institute, Cambridge, UK
  • Two sequences are "optimally aligned” when they are aligned for similarity scoring using a defined amino acid substitution matrix (e.g., BLOSUM62), gap existence penalty and gap extension penalty so as to arrive at the highest score possible for that pair of sequences.
  • Amino acid substitution matrices and their use in quantifying the similarity between two sequences are well-known in the art and described, e.g., in Dayhoff et al . (1978) "A model of evolutionary change in proteins” in "Atlas of Protein Sequence and Structure," Vol. 5, Suppl. 3 (ed. M. O. Dayhoff), pp. 345-352. Natl. Biomed. Res. Found., Washington, D. C.
  • the BLOSUM62 matrix (FIG. 10) is often used as a default scoring substitution ULCiUJ-J-X m sequence alignment protocols such as Gapped BLAST 2.0.
  • the gap existence penalty is imposed for the introduction of a single amino acid gap in one of the aligned sequences, and the gap extension penalty is imposed for each additional empty amino acid position inserted into an already opened gap.
  • the alignment is defined by the amino acids positions of each sequence at which the alignment begins and ends, and optionally by the insertion of a gap or multiple gaps in one or both sequences so as to arrive at the highest possible score.
  • Optimal alignments can be prepared using, e.g., PSI-BLAST, available through the NCBl website and described by Altschul et al . (1997) Nucl. Acids Res. 25:3389-3402.
  • an amino acid residue “corresponds to” the position in the reference sequence with which the residue is paired in the alignment.
  • the "position” is denoted by a number that sequentially identifies each amino acid in the reference sequence based on its position relative to the N-terminus . For example, in SEQ ID N0:l, position 1 is T, position 2 is I, position 3 is K, etc.
  • a residue in the test sequence that aligns with the K at position 3 is said to "correspond to position 3" of SEQ ID NO:1.
  • the amino acid residue number in a test sequence as determined by simply counting from the N- terminal will not necessarily be the same as the number of its corresponding position in the reference sequence.
  • the amino acid residue number in a test sequence as determined by simply counting from the N- terminal will not necessarily be the same as the number of its corresponding position in the reference sequence.
  • that insertion will not correspond to any amino acid position in the reference sequence.
  • isolated nucleic acid molecules are provided.
  • the invention provides a novel family of isolated or recombinant polynucleotides referred to herein as "P450 polynucleotides" or "P450 nucleic acid molecules.”
  • P450 polynucleotide sequences are characterized by the ability to encode a P450 polypeptide.
  • the invention includes any nucleotide sequence that encodes any of the novel P450 polypeptides described herein.
  • a P450 polynucleotide that encodes a P450 variant polypeptide with activity on alkanes.
  • polynucleotide “nucleotide sequence,” and “nucleic acid molecule” are used to refer to a polymer of nucleotides (A, C, T, U, G, etc. or naturally occurring or artificial nucleotide analogues), e.g., DNA or RNA, or a representation thereof, e.g., a character string, etc., depending on the relevant context.
  • a given polynucleotide or complementary polynucleotide can be determined from any specified nucleotide sequence.
  • the P450 polynucleotides comprise recombinant or isolated forms of naturally occurring nucleic acids isolated from an organism, e.g., a bacterial strain.
  • Exemplary P450 polynucleotides include those that encode the wild-type polypeptides set forth in SEQ ID NO: 1, 2, 3, 4, 5, or 6.
  • P450 polynucleotides are produced by diversifying, e.g., recombining and/or mutating one or more naturally occurring, isolated, or recombinant P450 polynucleotides.
  • P450 polynucleotides encoding P450 polypeptides with superior functional attributes, e.g., increased catalytic function, increased stability, or higher expression level, than a P450 polynucleotide used as a substrate or parent in the diversification process.
  • Exemplary polynucleotides include those that encode the P450 variant polypeptides set forth in SEQ ID NO: 7, 8, 9, 10, 11, 12, or 13.
  • the polynucleotides of the invention have a variety of uses in, for example recombinant production (i.e., expression) of the P450 polypeptides of the invention and as substrates for further diversity generation, e.g., recombination reactions or mutation reactions to produce new and/or improved P450 homologues, and the like.
  • recombinant production i.e., expression
  • substrates for further diversity generation e.g., recombination reactions or mutation reactions to produce new and/or improved P450 homologues, and the like.
  • P450 polynucleotides that do not encode active enzymes can be valuable sources of parental polynucleotides for use in diversification procedures to arrive at P450 polynucleotide variants, or non-P450 polynucleotides, with desirable functional properties (e.g., high k cat or k cat /K m , low K m , high stability towards heat or other environmental factors, high transcription or translation rates, resistance to proteolytic cleavage, etc.).
  • desirable functional properties e.g., high k cat or k cat /K m , low K m , high stability towards heat or other environmental factors, high transcription or translation rates, resistance to proteolytic cleavage, etc.
  • P450 polynucleotides including nucleotide sequences that encode P450 polypeptides and variants thereof, fragments of P450 polypeptides, related fusion proteins, or functional equivalents thereof, are used in recombinant DNA molecules that direct the expression of the P450 polypeptides in appropriate host cells, such as bacterial cells. Due to the inherent degeneracy of the genetic code, other nucleic acid sequences which encode substantially the same or a functionally equivalent amino acid sequence can also be used to clone and express the P450 polynucleotides.
  • host cell includes any cell type which is susceptible to transformation with a nucleic acid construct.
  • transformation means the introduction of a foreign (i.e., extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence.
  • the introduced gene or sequence may include regulatory or control sequences, such as start, stop, promoter, signal, secretion, or other sequences used by the genetic machinery of the cell.
  • a host cell that receives and expresses introduced DNA or RNA has been "transformed” and is a "transformant” or a “clone.”
  • the DNA or RNA introduced to a host cell can come from any source, including cells of the same genus or species as the host cell, or cells of a different genus or species .
  • Codons can be substituted to reflect the preferred codon usage of the host, a process sometimes called "codon optimization” or "controlling for species codon bias.”
  • Optimized coding sequences containing codons preferred by a particular prokaryotic or eukaryotic host can be prepared, for example, to increase the rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced from a non-optimized sequence.
  • Translation stop codons can also be modified to reflect host preference. For example, preferred stop codons for S.
  • nucleic acid molecules of the invention include: (a) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID N0:7, 8, 9, 10, 11, 12, or 13; (b) a nucleic acid molecule which encodes a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:7, 8, 9, 10, 11, 12, or 13; (c) a nucleic acid molecule which encodes a polypeptide comprising residues 1 to about 455 of the amino acid sequence set forth in SEQ ID NO: 7 or 11; (d) a nucleic acid molecule which encodes a polypeptide comprising residues about 456 to about 1088 of the amino acid sequence set forth in SEQ ID N0:7, 8, 9, 10, 12, or 13; (e) a nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO: 15; (f) a nucleic acid molecule consisting of the nucle
  • nucleic acid molecules of the invention include: (a) a nucleic acid molecule which encodes a polypeptide comprising residues 1 to about 455 of the amino acid sequence set forth in SEQ ID NO: 2 or 3 with the following amino acid residues: 48 and 206 are cysteine (C); 79 and 330 are phenylalanine (F); 83, 143, and 257 are serine (S); 95 and 176 are isoleucine (I); 185, 292, and 355 are valine (V); 227 is arginine (R) ; 238 is glutamine (Q) ; and 254 is glycine (G) ;
  • nucleic acid molecule which encodes a polypeptide consisting of residues 1 to about 455 of the amino acid sequence set forth in SEQ ID NO: 2 or 3 with the following amino acid residues: 48 and 206 are cysteine (C); 79 and 330 are phenylalanine (F); 83, 143, and 257 are serine (S); 95 and 176 are isoleucine (I); 185, 292, and 355 are valine (V); 227 is arginine (R) ; 238 is glutamine (Q) ; and 254 is glycine (G) ;
  • nucleic acid molecule which encodes a polypeptide comprising residues 1 to about 455 of the amino acid sequence set forth in SEQ ID NO: 4 with the following amino acid residues: 49 and 207 are cysteine (C); 80 and 331 are phenylalanine (F); 84, 144, and 258 are serine (S); 96 and 177 are isoleucine (I); 186, 293, and 357 are valine (V); 228 is arginine (R) ; 239 is glutamine (Q) ; and 255 is glycine (G) ;
  • nucleic acid molecule which encodes a polypeptide consisting of residues 1 to about 455 of the amino acid sequence set forth in SEQ ID NO: 4 with the following amino acid residues: 49 and 207 are cysteine (C); 80 and 331 are phenylalanine (F); 84, 144, and 258 are serine (S); 96 and 177 are isoleucine (I) ; 186, 293, and 357 are valine (V) ; 228 is arginine (R) ; 239 is glutamine (Q) ; and 255 is glycine (G) ;
  • nucleic acid molecule which encodes a polypeptide comprising residues 1 to about 455 of the amino acid sequence set forth in SEQ ID NO: 5 with the following amino acid residues: 54 and 212 are cysteine (C); 85 and 336 are phenylalanine (F); 89, 149, and 262 are serine (S); 101 and 182 are isoleucine (I); 191, 297, and 361 are valine (V); 232 is arginine (R) ; 243 is glutamine (Q) ; and 259 is glycine (G) ;
  • nucleic acid molecule which encodes a polypeptide consisting of residues 1 to about 455 of the amino acid sequence set forth in SEQ ID NO: 5 with the following amino acid residues: 54 and 212 are cysteine (C); 85 and 336 are phenylalanine (F); 89, 149, and 262 are serine (S); 101 and 182 are isoleucine (I); 191, 297, and 361 are valine (V); 232 is arginine (R) ; 243 is glutamine (Q) ; and 259 is glycine (G) ;
  • nucleic acid molecules of the invention include: (a) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 with the following amino acid residues: 48 and 206 are cysteine (C) ; 79 and 330 are phenylalanine (F) ; 83, 143, and 257 are serine (S); 95 and 176 are isoleucine (I); 185, 292, and 355 are valine (V); 227, 287, and 656 are arginine (R) ; 238 is glutamine (Q) ; 254 is glycine (G) ; 475 is glycine (G) , arginine (R) , tyrosine (Y) , or threonine (T) ; 641 is asparagine (N) ; 721 is threonine (T) ; and 980 is leucine; (b) a nucleic acid molecule which encodes
  • (S); 96 and 177 are isoleucine (I); 186, 293, and 357 are valine (V); 228, 288, and 659 are arginine (R); 239 is glutamine (Q) ; and 255 is glycine (G) ; 477 is glycine (G) , arginine (R) , tyrosine (Y) , or threonine (T) ; 644 is asparagine (N) ; 724 is threonine (T) ; and 983 is leucine; (g) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 5 with the following amino acid residues: 54 and 212 are cysteine (C); 85 and 336 are phenylalanine (F); 89, 149, and 262 are serine
  • (S); 101 and 182 are isoleucine (I); 191, 297, and 361 are valine (V); 232, 292, and 656 are arginine (R); 243 is glutamine (Q) ; and 259 is glycine (G) ; 472 is glycine (G.) , arginine (R) , tyrosine (Y) , or threonine (T) ; 640 is asparagine (N) ; 723 is threonine (T) ; and 985 is leucine; (h) a nucleic acid molecule which encodes a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 5 with the following amino acid residues: 54 and 212 are cysteine (C); 85 and 336 are phenylalanine (F); 89, 149, and 262 are serine
  • (S); 101 and 182 are isoleucine (I); 191, 297, and 361 are valine (V); 232, 292, and 656 are arginine (R); 243 is glutamine (Q) ; and 259 is glycine (G) ; 472 is glycine (G) , arginine (R) , tyrosine (Y) , or threonine (T) ; 640 is asparagine (N) ; 723 is threonine (T) ; and 985 is leucine; (i) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 6 with the following amino acid residues: 51 and 208 are cysteine (C); 82 and 337 are phenylalanine (F) ; 86, 146, and 262 are serine
  • (S); 98 and 179 are isoleucine (I); 188, 296, and 363 are valine (V); 231, 291, and 664 are arginine (R); 243 is glutamine (Q) ; and 258 is glycine (G) ; 480 is glycine (G) , arginine (R) , tyrosine (Y) , or threonine (T) ; 648 is asparagine (N) ; 733 is threonine (T) ; and 997 is leucine; and (j) a nucleic acid molecule which encodes a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 6 with the following amino acid residues: 51 and 208 are cysteine (C); 82 and 337 are phenylalanine (F); 86, 146, and 262 are serine (S); 98 and 179 are isoleucine (I); 188, 296, and 363 are valine
  • nucleotide sequences encoding modified P450 polypeptides of the invention may be produced, some of which bear substantial identity to the nucleic acid sequences explicitly disclosed herein.
  • codons AGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acid arginine.
  • the codon can be altered to any of the corresponding codons described above without altering the encoded polypeptide. It is understood that U in an RNA sequence corresponds to T in a DNA sequence.
  • silent variations are one species of “conservatively modified variations.”
  • AUG which is ordinarily the only codon for methionine
  • each silent variation of a nucleic acid which encodes a polypeptide is implicit in any described sequence.
  • the invention provides each and every possible variation of nucleic acid sequence encoding a polypeptide of the invention that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the nucleic acid sequence encoding a P450 homologue polypeptide of the invention.
  • the invention includes any polypeptide encoded by a modified P450 polynucleotide derived by mutation, recursive sequence recombination, and/or diversification of the polynucleotide sequences described herein.
  • a P450 polypeptide such as a heme domain and/or a reductase domain, is modified by single or multiple amino acid substitutions, a deletion, an insertion, or a combination of one or more of these types of modifications.
  • Substitutions can be conservative or non-conservative, can alter function or not, and can add new function. Insertions and deletions can be substantial, such as the case of a truncation of a substantial fragment of the sequence, or in the fusion of additional sequence, either internally or at N or C terminal .
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs .
  • the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • An "isolated" nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.
  • isolated includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated.
  • an "isolated” ' nucleic acid is free of sequences which naturally- flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • a nucleic acid molecule of the present invention e.g., a nucleic acid molecule that encodes a polypeptide set forth in any of SEQ NOs: 1-13, or having the nucleotide sequence of set forth in any of SEQ ID NOs: 15-18, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein.
  • a nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of a nucleotide sequence encoding a polypeptide set forth in any of SEQ NOs: 1-13, or having the nucleotide sequence of set forth in any of SEQ ID NOs: 15-18.
  • an isolated nucleic acid molecule of the present invention comprises a nucleotide sequence which is at least about 50%, 54%, 55%, 60%, 62%, 65%, 70%, 75%, 78%, 80%, 85%, 86%, 90%, 95%, 97%, 98% or more homologous to the nucleotide sequence encoding a polypeptide set forth in any of SEQ NOs: 1-13, or having the nucleotide sequence set forth in any of SEQ ID NOs: 15-18, or a portion of any of these nucleotide sequences.
  • DNA sequence polymorphisms that lead to changes in the amino acid sequences of the proteins may exist within a population.
  • Such genetic polymorphisms may exist among individuals within a population due to natural allelic variation.
  • Such natural allelic variations include both functional and non-functional proteins and can typically result in 1-5% variance in the nucleotide sequence of a gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in genes that are the result of natural allelic variation and that do not alter the functional activity of a protein are intended to be within the scope of the invention.
  • an isolated nucleic acid molecule of the invention hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence encoding a polypeptide set forth in any of SEQ NOs: 1-13, or having the nucleotide sequence set forth in any of SEQ ID NOs: 15-18.
  • the nucleic acid is at least 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 nucleotides in length.
  • Nucleic acid molecules are "hybridizable" to each other when at least one strand of one polynucleotide can anneal to another polynucleotide under defined stringency conditions .
  • Stringency of hybridization is determined, e.g., by (a) the temperature at which hybridization and/or washing is performed, and (b) the ionic strength and polarity (e.g., formamide) of the hybridization and washing solutions, as well as other parameters.
  • Hybridization requires that the two polynucleotides contain substantially complementary sequences; depending on the stringency of hybridization, however, mismatches may be tolerated.
  • hybridization of two sequences at high stringency (such as, for example, in an aqueous solution of 0.5 X SSC at 65°C) requires that the sequences exhibit some high degree of complementarity over their entire sequence.
  • Conditions of intermediate stringency such as, for example, an aqueous solution of 2 X SSC at 65 0 C
  • low stringency such as, for example, an aqueous solution of 2 X SSC at 55°C
  • Nucleic acid molecules that hybridize include those which anneal under suitable stringency conditions and which encode polypeptides or enzymes having the same function, such as the ability to catalyze the conversion of an alkane (e.g., methane) to an alcohol (e.g., methanol), of the invention.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 30%, 40%, 50%, or 60% homologous to each other typically remain hybridized to each other.
  • the conditions are such that sequences at least about 70%, more preferably at least about 80%, even more preferably at least about 85% or 90% homologous to each other typically remain hybridized to each other.
  • an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to a nucleic acid sequence encoding a polypeptide set forth in any of SEQ NOs: 1-13, or having the nucleotide sequence set forth in any of SEQ ID NOs: 15-18, corresponds to a naturally- occurring nucleic acid molecule.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • substitutions can be made in, for example, in the sequence encoding a polypeptide set forth in any of SEQ NOs: 7-13, or having the nucleotide sequence set forth in any of SEQ ID NOs: 15-18, without altering the ability of the enzyme to catalyze the oxidation of an alkane to an alcohol.
  • a "non-essential" amino acid residue is a residue that can be altered from the parent sequence without altering the biological activity of the resulting polypeptide, e.g., catalyzing the conversion of methane to methanol .
  • Also contemplated are those situations where it is desirable to alter the activity of a parent polypeptide such that the polypeptide has new or increased activity on a particular substrate.
  • polypeptides set forth in Table 4 describe specific amino acid substitutions that contribute to the alteration of the activity of a parent polypeptide.
  • SEQ ID NOs: 1-6 are P450 wild-type polypeptides.
  • These polypeptides may also be referred to as "parent" amino acid sequences because substitutions made to their amino acid sequence give rise to modified P450 polypeptides that can have altered or modified activity as compared to the parent sequence (see Table 4).
  • SEQ ID NO: 1 provided the parent sequence for the modified P450 polypeptide set forth in SEQ ID NO: 7.
  • SEQ ID NO: 7 includes amino acid substitutions that impart activities to the SEQ ID NO: 7 polypeptide not measurable in the parent polyprptide.
  • the nucleic acid molecule encoding the amino acid sequence of SEQ ID N0:l provides a "parent" nucleic acid molecule from which mutations can be made to obtain a nucleic acid molecule that encodes a modified polypeptide that includes amino acid substitutions. In general, these mutations provide non- conservative amino acid substitutions at indicated position (s) in a designated sequence.
  • a modified polypeptide can constitute a "parent" polypeptide from which additional substitutions can be made.
  • a parent polypeptide, and a nucleic acid molecule that encodes a parent polypeptide includes modified polypeptides and not just "wild-type" sequences.
  • the polypeptide of SEQ ID NO: 7 is a modified polypeptide with respect to SEQ ID N0;l (i.e., the "parent” polypeptide) .
  • the polypeptide of SEQ ID NO: 8 is a modified polypeptide with respect to SEQ ID NO: 7.
  • SEQ ID NO: 7 is the parent sequence of SEQ ID NO: 8.
  • the nucleic acid sequence encoding the polypeptide of SEQ ID NO: 7 provides the parent sequence from which mutations can be made to obtain a nucleic acid sequence that encodes the polypeptide of SEQ ID NO: 8.
  • an isolated nucleic acid molecule encoding a polypeptide homologous to the polypeptides if of SEQ ID NOs: 1-13 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence encoding the particular polypeptide, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into the nucleic acid sequence by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. In contrast to those positions where it may be desirable to make a non-conservative amino acid substitutions (see above) , in some positions it is preferable to make conservative amino acid substitutions.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine) , acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine) , nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) , beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,
  • Mutational methods of generating diversity include, for example, site-directed mutagenesis (Ling et al . (1997) "Approaches to DNA mutagenesis: an overview” Anal Biochem. 254(2): 157-178; Dale et al . (1996) “Oligonucleotide-directed random mutagenesis using the phosphorothioate method” Methods MoI. Biol. 57:369-374; Smith (1985) "In vitro mutagenesis” Ann. Rev. Genet.
  • constructs comprising one or more of the nucleic acid sequences as broadly described above.
  • the constructs comprise a vector, such as, a plasmid, a cosmid, a phage, a virus, a bacterial artificial chromosome (BAC) , a yeast artificial chromosome (YAC) , or the like, into which a nucleic acid sequence of the invention has been inserted, in a forward or reverse orientation.
  • the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available.
  • vectors that include a nucleic acid molecule of the invention are provided.
  • host cells transfected with a nucleic acid molecule of the invention, or a vector that includes a nucleic acid molecule of the invention are provided.
  • Host cells include eucaryotic cells such as yeast cells, insect cells, or animal cells.
  • Host cells also include procaryotic cells such as bacterial cells.
  • methods for producing a cell that converts an alkane to alcohol generally include: (a) transforming a cell with an isolated nucleic acid molecule encoding a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 7, 8, 9, 10, 11, 12, or 13; (b) transforming a cell with an isolated nucleic acid molecule encoding a polypeptide of the invention; or (c) transforming a cell with an isolated nucleic acid molecule of the invention.
  • methods for selecting a cell that converts an alkane to an alcohol generally include: (a) providing a cell containing a nucleic acid construct that includes a nucleotide sequence that encodes a modified cytochrome P450 polypeptide, the nucleotide sequence selected from: (i) a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 7, 8, 9, 10, 11, 12, or 13; (ii) a nucleic acid molecule encoding a polypeptide of the invention; or (iii) a nucleic acid molecule of the invention.
  • the methods further include (b) culturing the cell in the presence of a suitable alkane and under conditions where the modified cytochrome P450 is expressed at an effective level; and (c) detecting the production of an alcohol.
  • methods for producing an alcohol include: (a) providing a cell containing a nucleic acid construct comprising a nucleotide sequence that encodes a modified cytochrome P450 polypeptide, the nucleotide sequence selected from: (i) a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 7, 8, 9, 10, 11, 12, or 13; (ii) a nucleic acid molecule encoding a polypeptide of the invention; or (iii) a nucleic acid molecule of the invention.
  • the methods further include (b) culturing the cell in the presence of a suitable alkane and under conditions where the modified cytochrome P450 is expressed at an effective level; and (c) producing an alcohol by hydroxylation of the suitable alkane such as methane (CH 4 ) , ethane (C 2 He) , propane (C 3 H 8 ) , butane (C 4 Hi 0 ) , pentane (C 5 Hi 2 ) , hexane (C 6 H 14 ) , heptane (C7H16) , octane (C 8 Hi 8 ) r nonane (C 9 H 2 o) , decane (C10H22) / undecane (C11H2 4 ) , and dodecane (C12H26) •
  • the invention can include methanol, ethanol, propanol, butanol, pentanol, hexanol, hept
  • RNA polymerase mediated techniques e.g., NASBA
  • RNA can be converted into a double stranded DNA suitable for restriction digestion, PCR expansion and sequencing using reverse transcriptase and a polymerase. See, e.g., Ausubel, Sambrook and Berger, all supra.
  • engineered host cells that are transduced (transformed or transfected) with a vector provided herein (e.g., a cloning vector or an expression vector), as well as the production of polypeptides of the invention by recombinant techniques.
  • the vector may be, for example, a plasmid, a viral particle, a phage, etc.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying the P450 homologue gene.
  • vector means the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence.
  • a DNA or RNA sequence e.g. a foreign gene
  • Vectors typically comprise the DNA of a transmissible agent, into which foreign DNA encoding a protein is inserted by restriction enzyme technology.
  • a common type of vector is a "plasmid", which generally is a self-contained molecule of double-stranded DNA that can readily accept additional (foreign) DNA and which can readily introduced into a suitable host cell.
  • plasmid a vector that can readily accept additional (foreign) DNA and which can readily introduced into a suitable host cell.
  • plasmid plasmid and fungal vectors
  • Non-limiting examples include pKK plasmids (Clonetech) , pUC plasmids, pET plasmids (Novagen, Inc., Madison, Wis .
  • Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g., antibiotic resistance, and one or more expression cassettes.
  • the terms "express” and "expression” mean allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence.
  • a DNA sequence is expressed in or by a cell to form an "expression product" such as a protein.
  • the expression product itself e.g. the resulting protein, may also be said to be “expressed” by the cell.
  • a polynucleotide or polypeptide is expressed recombinantly, for example, when it is expressed or produced in a foreign host cell under the control of a foreign or native promoter, or in a native host cell under the control of a foreign promoter.
  • Polynucleotides provided herein can be incorporated into any one of a variety of expression vectors suitable for expressing a polypeptide.
  • Suitable vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, pseudorabies, adenovirus, adeno- associated viruses, retroviruses and many others. Any vector that transduces genetic material into a cell, and, if replication is desired, which is replicable and viable in the relevant host can be used.
  • Vectors can be employed to transform an appropriate host to permit the host to express an inventive protein or polypeptide.
  • appropriate expression hosts include: bacterial cells, such as E. coli, B. subtilis, Streptomyces, and Salmonella typhimurium; fungal cells, such as Saccharomyces cerevisiae, Pichia pastoris, and Neurospora crassa; insect cells such as Drosophila and Spodoptera frugiperda; mammalian cells such as CHO, COS, BHK, HEK 293 br Bowes melanoma; or plant cells or explants, etc.
  • bacterial cells such as E. coli, B. subtilis, Streptomyces, and Salmonella typhimurium
  • fungal cells such as Saccharomyces cerevisiae, Pichia pastoris, and Neurospora crassa
  • insect cells such as Drosophila and Spodoptera frugiperda
  • mammalian cells such as CHO, COS,
  • vectors which direct high level expression of fusion proteins that are readily purified can be desirable.
  • Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene) , in which the P450polypeptide coding sequence may be ligated into the vector in-frame with sequences for the amino-terminal Met and the subsequent 7 residues of beta-galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke & Schuster (1989) J. Biol. Chem. 254: 5503-5509); pET vectors (Novagen, Madison Wis.); and the like.
  • yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH may be used for production of the P450 polypeptides of the invention.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH.
  • a catalyst for the selective oxidation under mild conditions of gaseous alkanes into easily transported alcohols allows for the economical exploitation of both local, small natural gas resources and vast, remote reserves.
  • Prior to the present studies the selective conversion of ethane and methane mainly to their corresponding alcohols was not demonstrated.
  • Provided herein are enzyme-based catalysts that convert ethane to ethanol and methane to methanol with high selectivity and productivity.
  • the enzymes are function at ambient temperature and pressure, use dioxygen from the air as the oxidant, and produce little or no hazardous wastes.
  • Biological systems have evolved efficient and productive metalloenzymes that convert alkanes into alcohols using dioxygen from the air.
  • Methane monooxygenases catalyze the conversion of methane to methanol and thereby enable methanotrophic bacteria to use methane as a source of carbon and energy.
  • Microorganisms that grow on larger gaseous alkanes ethane, propane, butane
  • MMOs Methane monooxygenases
  • the relatively well-studied MMOs have long been a source of inspiration for designers of chemical catalysts .
  • these structurally complex enzymes have never been functionally expressed in a heterologous organism suitable for bioconversion and process optimization and therefore have proven to be of little practical use themselves for production of alcohols.
  • An exemplary hydroxylase includes cytochrome P450 BM-3 (CYP102A1) which is the third P450 identified in the bacterium Bacillus megaterium.
  • P450 BM-3 is a soluble, catalytically self-sufficient fatty acid monoxygenase isolated from Bacillus megaterium. It has a multi-domain structure, composed of three domains: one FAD, one FMN and one heme domain, fused on the same 119 kDa polypeptide chain of 1048 residues.
  • P450 BM-3 has been classified as a class II P450 enzyme, typical of microsomal eukaryotic P450s: it shares 30% sequence identity with microsomal fatty acid ⁇ -hydroxylase, 35% sequence identity with microsomal NADPH:P450 reductase, and only 20% homology with other bacterial P450s.
  • Homologues of P450 BM-3 (CYP102A1) have been recognized in other bacteria, including two characterized systems in Bacillus subtilis: CYP102A2 and CYP102A3.
  • Substrate binding must initiate electron transfer from the reductase domain to the heme domain during catalysis. Once electron transfer occurs, the active iron-oxo species must be capable of breaking the high energy C-H bond in ethane (101.1 kcal it ⁇ ol-1 vs. 95-99 kcal mol-1 for the secondary C-H bonds of the fatty acid substrates of wild type BM-3) . Finally, the singly-hydroxylated product must be released from the active site before further oxidation can occur.
  • the BM-3 active site consists of a hydrophobic channel located directly above the heme. Guided by the high- resolution crystal structure of P450 BM-3 with bound palmitoylglycine substrate eleven amino acid residues in this channel that lie within five A of the terminal eight carbons of the bound substrate were chosen for mutagenesis. These residues were targeted because they are likely to contact small alkanes in the active site during catalysis. Saturation mutagenesis libraries constructed for each of these residues were screened for mutants showing improved activity towards dimethyl ether.
  • P450 BM-3 mutant 53-5H also hydroxylates ethane to generate ethanol (see Table 1) .
  • 53-5H contains three active-site mutations, A78F, A82S, and A328F, all of which replace alanine with a larger side chain which reduce the volume of the active site and position small alkanes above the heme during catalysis.
  • 53-5H exhibits the highest regioselectivity (89% 2- octanol) and enantioselectivity (65% S-2-octanol) towards octane yet encountered in any BM-3 variant (see Table 1), further evidence of tighter substrate binding in the engineered active site.
  • Ethane hydroxylation by 53-5H was measured after 12- hour incubations of the enzyme in ethane-saturated buffer containing an ethanol-free NADPH regeneration system (commercial NADPH contains 2-3% ethanol) . While the reaction is uncoupled to NADPH oxidation in the presence of ethane (660 min "1 , see Table 2), 53-5H nevertheless consistently produces at least 50 equivalents of ethanol independent of the starting concentration of enzyme (see Figures 2 and 3) . Overoxidation was tested by supplying ethanol as a substrate and monitoring ethanol depletion by gas chromatography and the production of acetaldehyde and acetic acid by 13 C NMR using 13 C-labeled ethanol. No (i.e.
  • a further round of directed evolution was performed to increase the ethane activity of 53-5H, this time targeting mutations to the reductase domain, including the polypeptide linker that connects it to the heme domain.
  • the amino acid substitution E464G in the linker region increases total turnover in selected BM-3 mutants. Alone this mutation does not enhance the production of ethanol by 53-5H, but further improvement was found upon screening a library of 53-5H containing this mutation and random mutations in the reductase domain for high activity towards dimethyl ether and accompanied by reduced NADPH consumption rates in the absence of substrate.
  • the increased productivity likely reflects a prolonged catalyst lifetime, achieved by reducing non-productive cofactor oxidation, which inactivates the protein by forming various reactive species .
  • Amino acid residue 1710 by comparison to the crystal structure of the homologous rat P450 reductase, is located near the FAD cofactor .
  • variant P450s that have been adapted to hydroxylate ever smaller substrates .
  • the wildtype cytochrome P450 BM-3 enzyme is inactive on propane. Evolving it to become an octane hydroxylase, however, generated a small amount of activity towards propane. Similarly, increasing the newly acquired propane activity produced measurable activity towards ethane.
  • Directed evolution has generated a biocatalyst with activity towards ethane.
  • additional rounds of directed evolution has generated a BM-3 variant with the ability to convert methane to methanol. This enzyme provides the foundation for a novel biocatalytic route to methanol production from methane.
  • Restriction enzymes were purchased from Roche (Indianapolis, IN) or New England Biolabs (Beverly, MA), Taq DNA polymerase from Roche (Indianapolis, IN) , Pfu turbo DNA polymerase from Stratagene (La Jolla, CA) , and T4 DNA ligase from Invitrogen (Carlsbad, CA) .
  • the primers in the forward direction for each library were: 74NNN-for (5'-GTCAANNNCTTAAATTTGCACG-S') (SEQ ID NO:19) 75NNN-for (5'-GTCAAGCGNNNAAATTTGCACG-S') (SEQ ID NO:20) 78NNN-for (5'-GCTTAAATTTNNNCGTGATTTTGCAGG-S') (SEQ ID NO:21) 81NNN-for ( ⁇ '-CGTGATNNNGCAGGAGAC-S') (SEQ ID NO: 22)
  • 87F/I/V/L 88T for (5' GAGACGGGTTANTYACAAGCTGGAC) (SEQ ID NO: 37) and 87F/I/V/L 88C for ( ⁇ 'GAGACGGGTTANTYTGTAGCTGGAC) (SEQ ID NO: 37) and 87F/I/V/L 88C for ( ⁇ 'GAGACGGGTTANTYTGTAGCTGGAC) (SEQ ID NO: 37) and 87F/I/V/L 88C for ( ⁇ 'GAGACGGGTTANTYTGTAGCTGGAC) (SEQ ID NO: 37) and 87F/I/V/L 88C for ( ⁇ 'GAGACGGGTTANTYTGTAGCTGGAC) (SEQ ID NO: 37) and 87F/I/V/L 88C for ( ⁇ 'GAGACGGGTTANTYTGTAGCTGGAC) (SEQ ID NO: 37) and 87F/I/V/L 88C for ( ⁇ 'GAGACGGGTTANT
  • Nucleic acid mutations compared to wild-type P450 BM-3 include C142T, T234C, G235T, A237T, G247A, C248G, A249C,
  • Amino acid mutations compared to wild-type P450 BM-3 include R47C, V78F,
  • the amplified DNA was restriction digested using Sacl and EcoRI and then ligated with T4 ligase into the pCWori plasmid containing the gene for 53-5H with its reductase portion removed with Sacl and EcoRI.
  • a small library ( ⁇ 100 mutants) from each PCR condition was tested for mutation rate.
  • the 100 ⁇ M MnC12 condition produced the optimal mutation rate of 1-2 mutations per gene, and a larger library containing 2610 members from this condition was picked and screened.
  • Nucleic acid sequences of resulting ethane hydroxylating mutant 35-E11 include C142T, T234C, G235T, A237T, G247A, C248G, A249C, A284T, C324T, C427T, C527T, C554T, T617G, C681G, T711G, A758G, C766A, C872T, G985T, C986T, C1060G, A1119G, A1394G, T2132C, and A2313G.
  • Control reactions were performed by repeating these steps without substrate and with inactivated protein, to correct for background levels of ethanol .
  • Reaction buffer saturated with ethane and oxygen did not contain ethanol concentrations above this background.
  • Reactions with ethane and corresponding control reactions without ethane or with inactivated protein were carried out in triplicate.
  • the background ethanol concentration of around 1 ⁇ M (1.2 ⁇ 0.1 ⁇ M for the control reaction without ethane or 0.8 + 0.1 uM for the control reaction with inactivated P450)
  • the reactions yielded final ethanol concentrations corresponding to total turnover numbers of 50 and 250 for 53-5H and 35-Ell, respectively (see e.g., Figures 2 and 3) .
  • NADPH oxidation rates were measured over 20 s at 25 0 C using a BioSpec-1601 UV-Vis spectrophotometer (Shimadzu, Columbia, MD) and 1 cm pathlength cuvettes.
  • a 1 mL reaction mixture contained 100 nM P450, 166 ⁇ M NADPH and either methane-, ethane- or propane-saturated potassium phosphate buffer (0.1 M, pH 8.0) or 4 mM octane and 1% ethanol in potassium phosphate buffer (0.1M, pH 8.0) .
  • the reaction was initiated by the addition of NADPH and the decrease in absorption at 340 nm was monitored. NADPH consumption rates were also measured without substrate (see Table 2) .
  • Table 2 provides NADPH oxidation rates of 53-5H and 35-Ell in presence and absence of substrate. The coupling efficiency is estimated from product formation rate / NADPH oxidation rate. In general, NADPH oxidation rates cannot be measured on the same time scale as ethanol formation rates. [00207] Table 2
  • the engineered cytochromes P450 mutants described throughout the present disclosure were acquired by accumulating point mutations in directed evolution experiments .
  • An alternative method for making libraries for directed evolution to obtain P450s with new or altered properties is recombination, or chimeragenesis, in which portions of homologous P450s are swapped to form functional chimeras.
  • Recombining equivalent segments of homologous proteins generates variants in which every amino acid substitution has already proven to be successful in one of the parents. Therefore, the amino acid mutations made in this way are less disruptive, on average, than random mutations.
  • a structure-based algorithm, such as SCHEMA identifies fragments of proteins that can be recombined to minimize disruptive interactions that would prevent the protein from folding into its active -form.
  • SCHEMA has been used to design chimeras of P450 BM-3 and its homolog CYP102A2, sharing 63% amino acid sequence identity.
  • Fourteen of the seventeen constructed hybrid proteins were able to fold correctly and incorporate the heme cofactor, as determined by CO difference spectra.
  • Half of the chimeras had altered substrate specificities, while three mutants acquired activities towards a new substrate not hydroxylated by either parent.
  • a large library of chimeras made by SCHEMA-guided recombination of BM- 3 with its homologs CYP102A2 and CYP102A3 contains more than 3000 new properly folded variants of BM-3.
  • cytochrome P450 BM-3 that catalyze the direct conversion of methane to methanol.
  • the reaction uses dioxygen from the air and the reduced form of the biological cofactor nicotinamide adenine dinucleotide phosphate (NADPH) .
  • NADPH biological cofactor nicotinamide adenine dinucleotide phosphate
  • the stoichiometry of the reactions catalyzed by the BM-3 variant is CH 4 + O 2 + NADPH + H + ⁇ CH 3 OH + H 2 O + NADP + .
  • a "variant" cytochrome P450 contains one or more amino acid substitutions, insertions or deletions relative to its parent cytochrome P450.
  • the term “variant” is often used interchangeably with "mutant”.
  • a cytochrome P450 variant is suitably prepared on the basis of a parent cytochrome P450 having one of the sequences shown, for example, in SEQ ID NO: 1, 2 or 3.
  • the parent cytochromes P450 shown in SEQ NO: 2 and 3 are obtainable from Bacillus subtilis strain IAl.15 These cytochromes P450 are known as CYP102A2 and CYP102A3, respectively.
  • the cytochrome P450 of SEQ ID NO: 1 is known as the cytochrome P450 BM-3, from Bacillus megaterium. It is also known in the CYP nomenclature as CYP102A1.
  • the parent cytochromes P450 shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 are obtainable from Bacillus cereus (CYP102A4), Ralstonia metallidurans (CYP102E1) and Bradyrhizobium japonicum (CYP102A6) , respectively.
  • CYP102A4 Bacillus cereus
  • CYP102E1 Ralstonia metallidurans
  • CYP102A6 Bradyrhizobium japonicum
  • Figure 6A - 6F the % identity between the parent sequences and CYP102A1 are reported from alignments made with standard in the art software. As shown in Figure 6A - 6F, the identity in the heme domain is often higher than reported because of large differences in the link
  • cytochromes P450 are closely related to one another and show a high degree of sequence identity.
  • the sequences can be aligned based on the sequence homology, and that alignment is shown in Figure 6A - 6F.
  • This alignment defines "equivalent positions" in the sequences.
  • An equivalent position denotes a position which, on the basis of the alignment of the sequence of the parent cytochrome P450 in question with the "reference" cytochrome P450 amino acid sequence in question (e.g. SEQ ID NO: 1) so as to achieve juxtapositioning of amino acid residues which are common to both, corresponds most closely to a particular position in the reference sequence in question. This process can cause gaps or insertions to appear in the sequences.
  • equivalent positions are shown lined up vertically with one another. For example, position 87 in SEQ ID NO: 1 is equivalent to position 88 in SEQ ID NO: 2 and 3 and position 89 in SEQ ID NO: 4.
  • TTN total turnover number
  • P cofactor
  • Coupling means the ratio, in percent, of product formed to cofactor (NAD(P)H) consumed during the enzyme-catalyzed reaction. If 2 moles of cofactor are consumed for every mole of product made, then the coupling for the reaction is 50%. Coupling is also an important figure of merit because it allows one to assess how much cofactor is required to produce a given amount of product.
  • the BM-3-based catalyst functions at ambient temperature and pressure. Unlike methane monooxygenase enzymes, this BM-3 based biocatalyst can be expressed in host organisms that do not consume the methanol as it is produced. Thus it may be incorporated into a whole-cell biocatalysis system that can accumulate methanol in the presence of methane and air.
  • Methane hydroxylase variants were generated by an Evolutionary' strategy in which mutations were accumulated in multiple generations of random mutagenesis and screening, in essence adapting the enzyme to exhibit higher turnover on smaller and smaller alkanes. In the first generations, the activity of BM-3 towards octane was increased and a small but appreciable activity on propane was acquired.
  • the ethane-hydroxylating P450 BM-3 variant 35-E11 was obtained by improving the electron transfer in the triple active site variant 53-5H as described. Briefly, the active site mutations in 53-5H (V78F, A82S, and A328F) were obtained by screening a library containing all possible combinations of single-site mutations of a BM-3 variant previously evolved for high activity towards propane.
  • the 15 mutations in 53-5H (12 are not in the active site) are located in the heme domain: R47C, V78F, A82S, K94I, P142S, T175I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V, A328F, L353V. While not bound to a particular mechanism of action, it is believed that the active site mutations serve to decrease the size of the active site so that ethane is bound and hydroxylated. Mutant 53-5H has only limited activity towards ethane, producing only about 50 total turnovers before it is inactivated.
  • Variant 35-E11 was obtained by modifying the reductase domain of 53-5H to more efficiently transfer electrons to the active site during catalysis.
  • This variant contains two mutations, one in the linker region connecting the two domains (E464G) and one in the reductase domain (I710T) , that improve coupling and increase the total turnovers on ethane to more than 250.
  • the 35-Ell variant was tested for the ability to convert methane to methanol using the method of Anthon and Barrett (Agric. Food Chem., 52:3749 (2004)). The method was optimized so that it could detect methanol concentrations in water as low as 10 ⁇ M.
  • 35-Ell variant was identified as producing methanol from methane.
  • Libraries of mutants of 35-Ell were screened for phenotypes indicative of either increased product formation on small gaseous substrates slightly larger than methane. Additionally, they were screened for increased coupling of the consumption of NADPH reducing equivalents to product formation. Product formation was determined by measuring total amount of dimethyl ether hydroxylation while increased coupling was determined primarily by decreased background NADPH consumption rates (measured spectrophotometrically at 340 nm in the absence of substrate) .
  • Four libraries of mutants of 35-Ell were constructed.
  • Two saturation mutagenesis libraries were prepared in 35-Ell, in which all possible amino acid substitutions were introduced at the positions of two non-heme domain mutations, E464G and I710T. Additionally, two random mutagenesis libraries of 35-Ell were prepared by error-prone PCR such that each library contained an average of 1-2 amino acid mutations. One of these random mutagenesis libraries contained only mutations in the heme domain (residues 1 to about 455) while the other library contained mutations only in the linker between the two domains and the reductase domain (residues about 470 to 1048) . These four libraries were screened as described above, and several improved variants (described below) were isolated, sequenced, and purified for further analysis.
  • an NADPH regeneration system consisting of NADP+, isocitrate, and isocitric dehydrogenase was added through a syringe. The reaction was allowed to stir 12-15 hours. Methanol production was measured using a coupled enzyme assay.
  • mutants None of these mutants exhibited higher activity towards the screening substrate DME, but all of them had a 30- 40% lower NADPH background consumption rate in the screen, indicating that the roughly equivalent production of oxidized DME they produced compared to the parent 35-E11 required less NADPH.
  • This increase in coupling on DME translated to an increase in TTN of methane (from 8 to 11-13) .
  • mutant 20-D3 was identified for its increased activity towards DME. Even though the background consumption rate of NADPH was increased by an equivalent amount in the screen, more of these reducing equivalents are transferred to the heme domain of this mutant in the presence of substrate, leading to an increase in methanol formation (from 8 to 10 TTN) .
  • This mutation, H285R is located on the surface of the heme domain, near the putative interface between the heme and reductase domains.
  • two mutants, 23-1D and 21-4G were identified for their decreased background NADPH consumption rates, leading to an increase in methanol formation.
  • the mutations in these variants, Q645R/P968L and D631N, respectively, are located outside of the electron transfer pathway. Their presence, however, gives 35-E11 increased TTN on methane (from 8 to 10-11) .
  • the methane hydroxylation activity reported here is not sufficiently high for a practical process for converting methane to methanol. TTN must be increased, and coupling of methanol production to consumption of the cofactor must be increased, or the enzyme will use too must However, the methods of making mutations and screening the libraries described here can be used to further improve the activity, TTN and coupling.
  • these P450 enzymes can be subjected to rounds of directed evolution using the techniques and screens described herein to obtain and/or increase methane hydroxylation activity.
  • Less closely-related cytochromes P450 with similar domain architecture to BM-3 have also been reported in the literature, including P450s from the unrelated ⁇ -proteobacterium Ralstonia metallidurans (CYP102E1) and the gram negative bacterium Bradyrhizobium japonicum (CYP102A6) , and could potentially be similarly engineered for methane oxidation (see Figures 6A - 6F and Table 4 for corresponding mutations in these two P450s) .
  • the alkane content of natural gas generally comprises about 80% methane and 20% other small alkanes, primarily ethane and propane.
  • Rhodococcus Rhodococcus
  • engineered laboratory strains of E. coli can grow using small alcohols as their sole carbon source.
  • Expression of optimized BM-3 based biocatalysts in one of these bacterial strains will allow the bacteria to use the alkane impurities in natural gas as their sole source of carbon and energy (and cofactor regeneration) while converting the methane into methanol.
  • Expression of our BM-3 enzymes in any of these bacteria can be done by standard in the art techniques using muli-host plasmids and shuttle vectors .
  • Methane generated by the decomposition of biomass in landfills and agricultural waste does not contain other alkanes.
  • an external carbon and energy source such as glucose must be provided to the host cells to regenerate NADPH.
  • Glucose or similar compounds can be added directly to these whole cell reactions or generated from biomass present in the methane-producing waste.
  • the amount of cofactor a host organism generates from these carbon sources can also be increased by streamlining the cell strain for methane production. Expression of optimized BM-3 enzymes in these bacteria can be done by standard in the art techniques using strain specific plasmids. Modification of E.
  • coli strains to optimize NADPH availability can also be performed using standard in the art metabolic engineering techniques such as chromosomal insertion of phage-delivered DNA and recombination of homologous gene fragments from plasmids.
  • methylotrophs i.e. methanol- metabolizing yeast (e.g. strains of Pichia) and bacterial cell strains (e.g. strains of Methylobacterium)
  • BM-3 biocatalysts can serve as hosts for BM-3 biocatalysts.
  • the engineered BM-3 enzymes supply all of the methanol the hosts need to grow and live while generating excess methanol for production.
  • a Process for Bioconversion of Methane to Methanol Using Cytochromes P450 Provided herein are novel cytochrome P450-based variants that are capable of ethane and/or methane hydroxylation . Accordingly, processes that use such catalysts for the bioconversion of ethane into ethanol or methane into methanol can be performed in procedures described below. However, it is understood that the following procedures are exemplary only. The novel enzymes of the invention allow those skilled in the art of bioconversion to modify the exemplary procedures to suit their particular needs.
  • cell mass is generated using a carbon source such as glucose and nitrogen and other nutrients supplied to the cells.
  • a carbon source such as glucose and nitrogen and other nutrients supplied to the cells.
  • expression of the P450 biocatalyst is induced using strategies well known in the art.
  • nitrogen is removed from the medium to prevent further growth of the cell, ensuring that any additional carbon source supplied to the non-growing cells is converted into reducing equivalents for methanol production.
  • Methane and oxygen are then supplied to the culture at saturating levels assisted by mixing with either propeller- type mixing or air-lift mixing.
  • the process can be constructed as either a batch or continuous process.
  • Catalyst expression will be induced by the addition of 1 mM IPTG and 0.5 mM ⁇ -amino levulinic acid.
  • the culture will be centrifuged to collect the cells containing biocatalyst. After removal of the supernatant, the cells will be resuspended and washed in a nitrogen free minimal medium to remove traces of the terrific broth.
  • the cells in nitrogen free minimal medium will then be added to a fermentor and fed 0.5% glucose. During the reaction, additional glucose will be added to maintain this level. In the fermentor, oxygen will be added to the culture at saturating levels and will be monitored and added during the reaction as needed to maintain these levels. To initiate the reaction, methane will be bubbled through the culture. The lack of nitrogen in the culture forces the cells into a non- growing state, allowing most of the glucose aerobically consumed to be converted into NADPH for the biocatalyst .39 An E. coli cell functioning as an aerobe also has a limited ability to metabolize alcohols, including the methanol product.40 Methanol will be measured in this culture as described for the in vitro reactions in this work.
  • the present data demonstrates that methane hydroxylation activity can be achieved through modification of various cytochrome P450 enzymes by directed evolution. Since this enzyme can be expressed in heterologous hosts that do not metabolize methanol as it is produced, the invention of this biocatalyst also makes the production of methanol using a whole cell process possible.
  • a 470 ⁇ L reaction mixture containing 5 ⁇ M purified BM-3 variant in 0.1 M potassium phosphate buffer (pH 8.0) was first added to the vial.
  • the vial was immediately topped with a septum, pressurized with 30 psi of methane and shaken at 4 0 C for 1 hour.
  • 30 uL of an NADPH regeneration system (1.66 mM NADP+, 167 U/mL isocitric dehydrogenase, 416 mM isocitrate) was added to initiate the reaction. For determination of total turnover, the reaction was allowed to proceed for 20 hours at room temperature.
  • the amount of methanol product formed during the 20 hr methane reaction was quantified colorimetrically with the use of alcohol oxidase and Purpald. 190 ⁇ L of the reaction mixture is transferred onto a single cell of a 96 well plate. 10 ⁇ L of 0.05U/ ⁇ L solution of alcohol oxidase (from Pichia pastoris, purchased from Sigma-Aldrich) was added to cell and allowed to incubate at room temperature for 5 minutes. During this time, the alcohol oxidase converts the methanol into formaldehyde. Purpald (168 mM in 2 M NAOH) was then added to form a purple product with the formaldehyde in solution.
  • the purple color was read approximately 1 hour later at 550 nm using a Spectramax Plus microtiter plate reader.
  • concentration of methanol was determined based on the measure absorbance in comparison to a methanol calibration curve.
  • the methanol calibration curve was generated with several standards. Each standard contained 100 ⁇ L of a methanol solution (10, 20, 40, 60, 100 ⁇ M) and 90 ⁇ L of the methane reaction mixture described above, without the methane pressurization .
  • Table 3 provides amino acid substitution, screening and activity data for second-generation 35-E11 variants exhibiting methane hydroxylation activity.
  • the relative dimethyl ether (DME) activity was measured, 1 hour after treatment with Purpald, as the average net and absorbance at 550 nm from 8 microtitre plate DME reactions of the mutant compared to 8 equivalent reactions of 35-E11. All values were normalized by measuring the P450 concentration in each well with CO binding. "Asteriks" indicate criteria used to select mutants from libraries for further characterization.
  • the oxidation rate is measured as absorbance change at 340 nm over 1 minute upon the addition of NADPH to 8 microtitre plate wells containing P450-containing cell lysate and compared to 8 equivalent reactions of 35-E11. All values were normalized by measuring the P450 concentration in each well with CO binding.
  • the total turnover number (TTN) was measured as the absorbance at 550 nm of methane reactions with each mutant after treatment with alcohol oxidase and Purpald. [00239] Table 3
  • Table 4 provides an extensive list of amino acid substitutions in various parental P450 sequences (e.g., SEQ ID NO: 1, 2, 3, 4, 5, and 6) .
  • cytochrome P450 BM-3 is a soluble protein that contains an hydroxylase domain and a reductase domain on a single polypeptide. Also as previously noted, cytochrome P450 BM-3 variants are provided that exhibit efficient alkane hydroxylase activity. One of these variants, 9-lOA, was further modified at eleven active site residues that are in close contact to the substrate and screened for activity. Beneficial mutations were recombined, generating variants that catalyze the hydroxylation of linear alkanes with varying regioselectivities .
  • Mutant 9-10A exhibits high activity towards alkanes as small as propane.
  • the alkane hydroxylation properties of this mutant are detailed in Tables 5 and 6.
  • Octane reactions contained 10OnM P450, 500 uM NADPH, and 4 mM octane in 1% ethanol and potassium phosphate buffer.
  • Propane reactions contained 1 uM P450, 500 uM NADPH, and propane-saturated potassium phosphate buffer. Coupling was determined by ratio of product formation rate to NADPH consumption rate. Total turnover numbers were determined as nmol product/nmol enzyme.
  • Octane reactions contained 10-25 nM P450, 500 uM NADPH, and 4 mM octane in 1% ethanol and potassium phosphate buffer.
  • Propane reactions contained 10-25 nM protein, potassium phosphate buffer saturated with propane, and an NADPH regeneration system containing 100 uM NADP+, 2 U/mL isocitrate dehydrogenase, and 10 mM isocitrate. [00249] Table 6
  • 9-lOA hydroxylates octane into a mixture of octanols and ketones.
  • the distribution of these products is different from the distribution produced by wild type BM-3.
  • 9-lOA contains 14 mutations in the heme domain, only one of which, V78A, is located in the active site.
  • This data indicates that active site changes, especially multiple changes, may alter product distributions and increase activity.
  • two other active-site mutations, A82L and A328V shifted the regioselectivity of alkane hydroxylation predominantly to a single, subterminal ( ⁇ -1) position. Based on these results, active site variants were generated in order to obtain enzymes with altered regioselectivity of hydroxylation and altered substrate specificities .
  • Screening libraries by spectroscopically monitoring NADPH consumption rates in the presence of the alkane often selects for mutants with NADPH oxidation rates highly uncoupled to product formation.
  • This can be overcome through the use of surrogate substrates that mimic desired substrates, yet yield species when hydroxylated that are easily detected either directly or with the addition of a dye.
  • the screen for the high-throughput identification of mutant BM-3 enzymes with activity towards propane uses dimethyl ether (DME) as a propane surrogate substrate. Upon hydroxylation, it decomposes into methanol and dye-sensitive formaldehyde.
  • DME dimethyl ether
  • HME hexyl methyl ether
  • HME hexyl methyl ether
  • Formaldehyde is approximately 42 times more sensitive to Purpald than hexanal (see Figure 26) . This disproportionate response makes HME, in contrast to the symmetric ether DME, a regio-selective screen for terminal alkane hydroxylation.
  • the substrate free structure displays an open access channel with 17 to 21 ordered water molecules.
  • Substrate recognition serves as a conformational trigger to close the channel, which dehydrates the active site, increases the redox potential, and allows dioxygen to bind to the heme.
  • saturation mutagenesis libraries were made at each of these 11 positions. The libraries were screened for activity towards dimethyl ether (DME) and hexylmethyl ether (HME) . [00253] Twenty-one single active site mutants with increased activity towards either of these substrates were isolated.
  • Table 8 provides a listing of mutations, product distribution, rates and total turnover numbers of octane hydroxylation reactions catalyzed by BM-3 active site mutants.
  • Mutations are relative to variant 9-10A (described above) . Wild-type has a valine at position 78. The product distribution determined as ratio of a specific alcohol product to the total amount of alcohol products (given in %) . The formation of ketones was also observed but is less than 5% of the total amount of product. The initial rates of product formation were measured by GC over 60 s as nmol total products /min/nmol protein.
  • the HME screening procedure was used to identify BM- 3 variants with a regioselectivity shifted towards the terminal methyl carbon of the alkane chain.
  • mutants with high total turnover of HME in the screen produced more 1-octanol than compared to 1-12G.
  • the total amount of 1- octanol formed correlates well with the amount of HME converted.
  • mutant 77-9H is not improved relative to 1-12G when considering rates and total turnovers on octane, but this mutant converts octane to 52% 1-octanol, making it highly active towards HME in the screen.
  • the proportion of 1-octanol in the product mixture varies from three to 51%.
  • Single active site mutants that showed significantly different regioselectivities were recombined.
  • all the identified variants are selective for the terminal methyl or penultimate subterminal methylene group.
  • Screening the recombination library e.g. for hydroxylation of the third or fourth carbon of a linear alkane chain may identify variants that exhibit this specific regioselectivity.
  • the recombination library contains approximately 9,000 different mutants of P450 BM-3, each of which contains a characteristic active site. More than 70% of these were active towards DME or HME. Data provided herein indicate that mutations in the active site can yield variants that hydroxylate complex substrates such as 2-cyclopentyl- benzoxazole with high regio- and enantioselectivity, yielding potentially valuable intermediates for chemical synthesis (see example below) .
  • P450 BM-3 and homologs thereof are soluble fusion proteins consisting of a catalytic heme-domain and an FAD- and FMN- containing NADPH reductase. They require dioxygen and an NADPH cofactor to catalyze substrate hydroxylation.
  • the heme domain can utilize hydrogen peroxide via the peroxide "shunt" pathway for catalysis. While this 'peroxygenase ' activity is low in CYP102A1, it is enhanced by the amino acid substitution F87A. A similar effect has been shown for the equivalent F88A mutation in CYP102A2.
  • SCHEMA was used to design chimeras of P450 BM-3 and its homolog CYP102A2 sharing 63% amino acid sequence identity. Fourteen of the seventeen constructed hybrid proteins were able to fold correctly and incorporate the heme cofactor, as determined by CO difference spectra. The folded chimeras exhibited peroxygenase activity. A large library of chimeras made by SCHEMA-guided recombination of BM-3 with its hor ⁇ ologs CYP102A2 and CYP102A3 contains more than 3000 new properly folded variants of BM-3.
  • mutant 77-9H shows a similar trend, with selectivity for specific subterminal positions (see Table 10) .
  • Residues that contact the terminal eight carbons of the substrate were altered based on the crystal structure with bound palmitoylglycine. Amino acid residues outside this region have not been modified, particularly those interacting with the carboxyl moiety.
  • Table 10 shows product distributions for hydroxylation of fatty acids catalyzed by cytochrome P450 BM-3 77-9H. Product distribution was determined as ratio of a specific hydroxylation product to the total amount of hydroxylation products (given in %) .
  • Table 12 shows product distributions for fatty acid hydroxylation catalyzed by cytochrome P450 BM-3 variants wt F87A and 9-10A F87A. [00271] Table 12
  • the background NADPH oxidation rate (without substrate) rate was 220/min.
  • the coupling efficiency is the ratio of product formation rate to NADPH oxidation rate.
  • Wildtype P450 BM-3 tightly regulates electron transfer from the cofactor (NADPH) to the heme.
  • NADPH cofactor
  • a weakly-bound water molecule acts as the sixth, axial ligand of the heme iron.
  • Substrate replaces this water molecule, perturbing the spin-state equilibrium of the heme iron in favor of the high-spin form and also increases the heme iron reduction potential by approximately 130 mV.
  • Linear alkanes are challenging to hydroxylate selectively because they contain no functional groups that help fix their orientation for reaction with the active heme iron-oxo species of cytochrome P450.
  • Model studies with metalloporphyrins show that, absent substrate binding effects, the regioselectivity of n-alkane hydroxylation is determined by the relative bond dissociation energies.
  • Engineering a P450 for terminal hydroxylase activity must therefore override the inherent specificity of the catalytic species for the methylene groups so that only the terminal methyl carbon is activated.
  • Wildtype cytochrome P450 BM-3 hydroxylates its preferred substrates, medium-chain fatty acids, at subterminal positions. The terminal carbon is not hydroxylated.
  • By screening saturation mutagenesis libraries at 11 active site positions and recombining beneficial mutations a set of P450 BM-3 mutants that exhibit a range of regioselectivities for fatty acid and alkane hydroxylation are provided herein.
  • One mutant, 77-9H hydroxylates octane at the terminal position with 51% selectivity, but exhibits this selectivity only for octane.
  • the active site of 77-9H is therefore not restricted near the activated oxygen to prevent subterminal hydroxylation.
  • the specificity for the terminal methyl group of octane probably reflects specific interactions between active site residues and octane methylene groups and/or the methyl group at the unreacted end.
  • the hexyl methyl ether substrate used for screening terminal hydroxylation activity has the same chain length as octane.
  • the mutations that enable terminal hydroxylation of HME likely act in a similar fashion to promote terminal hydroxylation of octane.
  • Recombining active site mutations for linear, terminal alkene epoxidation The target reaction was the selective epoxidation of linear, terminal alkenes. The same saturation mutagenesis libraries were screened to identify single active site mutants that exhibit this activity.
  • the work presented herein demonstrates that active site mutants are well suited for the preparation of chiral intermediates for chemical synthesis.
  • the hydroxylated product can be used to as an intermediate in the synthesis of, for example, Carbovir, a carboxylic nucleoside potentially active against human HIV.
  • the mutant with the highest activity on propane, 53- 5H was shown to catalyze thousands of turnovers of propane to propanol at a rate of 370 min "1 .
  • This mutant also hydroxylates ethane to generate ethanol 53-5H contains three active-site mutations, A78F, A82S, and A328F, all of which replace alanine with a larger side chain and presumably reduce the volume of the active site and position small alkanes above the heme during catalysis.
  • 53-5H exhibits the highest regioselectivity (89% 2-octanol) and enantioselectivity (65% S ⁇ 2-octanol) towards octane of any BM-3 variant identified herein. This information further indicates that tighter substrate binding in the engineered active site occurs in the modified enzymes.
  • the dehalogenation reaction is based on the hydroxylation of the carbon atom carrying the halogen by a spontaneous (base catalyzed) alpha-elimination reaction which leads to the formation of an aldehyde or ketone depending on the starting haloalkane.
  • the expected reaction mechanism for the dehalogenation of a general alpha-haloalkane is described by the scheme:
  • chloromethane was chosen as substrate for the dehalogenation reaction.
  • chloromethane (CM) has the advantage to be moderately soluble in water (0.5g/100 ml) and to lead to formaldehyde as end product of the hydroxylation/alpha-elimination reaction. Formaldehyde can be then readily detected by colorimetric reaction with Purpald as described previously.
  • NADPH consumption 1 " 1 19.9 36.3 12.5 n.d. 16.2 n.d. product formation 1 " 1 - 4.3 3.0 7.0 1.1 n.d. coupling (%) - 11.8 24.2 8.0 6.8 n.d.
  • halomethane is extendable to other halomethanes such as bromo- and iodomethane as well as longer haloalkanes (e.g., haloethane, 1- and 2-halopropane) .
  • haloalkanes e.g., haloethane, 1- and 2-halopropane
  • the resulting haloalcohols are reactive intermediates which can form the corresponding carbonyl compounds (aldehyde, ketones) or epoxides depending on the regioselectivity of the hydroxylation reaction and the nature of starting compounds ( Figure 30) . These reactions are useful for bioremediation as well as for synthetic purposes.

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Abstract

Cette invention concerne des hydroxylases modifiées. Elle concerne également des cellules exprimant de telles hydroxylases modifiées, et des méthodes de production d'alcanes hydroxylés consistant à placer un substrat approprié au contact desdites cellules.
PCT/US2006/011273 2005-03-28 2006-03-28 Oxydation d'alcanes par des hydroxylases modifiees WO2006105082A2 (fr)

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US14/270,268 US9074178B2 (en) 2005-03-28 2014-05-05 Alkane oxidation by modified hydroxylases
US14/788,365 US9404096B2 (en) 2005-03-28 2015-06-30 Alkane oxidation by modified hydroxylases
US15/224,900 US9963720B2 (en) 2005-03-28 2016-08-01 Alkane oxidation by modified hydroxylases
US15/942,001 US10648006B2 (en) 2005-03-28 2018-03-30 Alkane oxidation by modified hydroxylases
US16/872,275 US11214817B2 (en) 2005-03-28 2020-05-11 Alkane oxidation by modified hydroxylases
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EP2121907A2 (fr) * 2007-02-08 2009-11-25 The California Institute of Technology Oxydation d'alcane par des hydroxylases modifiées
US20090312196A1 (en) * 2008-06-13 2009-12-17 Codexis, Inc. Method of synthesizing polynucleotide variants
US20100093560A1 (en) * 2008-06-13 2010-04-15 Codexis, Inc. Combined automated parallel synthesis of polynucleotide variants
JP2010539967A (ja) * 2007-10-08 2010-12-24 アイシス イノベーション リミテッド 変異酵素
US7863030B2 (en) 2003-06-17 2011-01-04 The California Institute Of Technology Regio- and enantioselective alkane hydroxylation with modified cytochrome P450
US8026085B2 (en) 2006-08-04 2011-09-27 California Institute Of Technology Methods and systems for selective fluorination of organic molecules
EP2426198A1 (fr) * 2010-09-03 2012-03-07 B.R.A.I.N. Biotechnology Research And Information Network AG Variantes de cytochrome P450 monooxygénase
US8252559B2 (en) 2006-08-04 2012-08-28 The California Institute Of Technology Methods and systems for selective fluorination of organic molecules
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GB9914373D0 (en) * 1999-06-18 1999-08-18 Isis Innovation Process for oxidising aromatic compounds
US7524664B2 (en) * 2003-06-17 2009-04-28 California Institute Of Technology Regio- and enantioselective alkane hydroxylation with modified cytochrome P450

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US8252559B2 (en) 2006-08-04 2012-08-28 The California Institute Of Technology Methods and systems for selective fluorination of organic molecules
US8026085B2 (en) 2006-08-04 2011-09-27 California Institute Of Technology Methods and systems for selective fluorination of organic molecules
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US8802401B2 (en) 2007-06-18 2014-08-12 The California Institute Of Technology Methods and compositions for preparation of selectively protected carbohydrates
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JP2010539967A (ja) * 2007-10-08 2010-12-24 アイシス イノベーション リミテッド 変異酵素
JP2016000037A (ja) * 2007-10-08 2016-01-07 アイシス イノベーション リミテッドＩｓｉｓ Ｉｎｎｏｖａｔｉｏｎ Ｌｉｍｉｔｅｄ 変異酵素
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US8383346B2 (en) * 2008-06-13 2013-02-26 Codexis, Inc. Combined automated parallel synthesis of polynucleotide variants
US20150024971A1 (en) * 2008-06-13 2015-01-22 Codexis, Inc. Combined automated parallel synthesis of polynucleotide variants
US20090312196A1 (en) * 2008-06-13 2009-12-17 Codexis, Inc. Method of synthesizing polynucleotide variants
US20100093560A1 (en) * 2008-06-13 2010-04-15 Codexis, Inc. Combined automated parallel synthesis of polynucleotide variants
US20130143767A1 (en) * 2008-06-13 2013-06-06 Codexis, Inc. Combined automated parallel synthesis of polynucleotide variants
EP2451951B1 (fr) * 2009-06-11 2016-04-13 Codexis, Inc. Synthèse en parallèle automatisée combinée de variants polynucléotidique
EP3026113A3 (fr) * 2009-06-11 2016-07-27 Codexis, Inc. Synthèse parallèle automatisée combinée de variants polynucléotidiques
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US9322007B2 (en) 2011-07-22 2016-04-26 The California Institute Of Technology Stable fungal Cel6 enzyme variants

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