Increasing productivity of e. coli host cells that functionally express p450 enzymes

Abstract

The present invention relates to the production of chemical species in bacterial host cells. Particularly, the present invention provides for the production of chemical species in Escherichia coli (E. coli) host cells that functionally express engineered P450 enzymes.

1 . A method for biosynthesis of one or more chemical species in  E. coli,  comprising:
 expressing one or more biosynthetic pathways in  E. coli,  the one or more biosynthetic pathways comprising at least one membrane-anchored P450 enzyme having a transmembrane domain derived from an  E. coli  inner membrane cytoplasmic C-terminus protein, and   culturing the  E. coli  to produce the one or more chemical species from the biosynthetic pathway(s).   
     
     
         2 . The method of  claim 1 , wherein the  E. coli  does not exhibit a substantially stressed phenotype during the culturing. 
     
     
         3 . The method of  claim 1  or  2 , wherein the  E. coli  expresses at least two, at least three, or at least four recombinant enzymes. 
     
     
         4 . The method of  claim 3 , wherein the biosynthetic pathway(s) produce a secondary metabolite through the overexpression of at least two foreign genes. 
     
     
         5 . The method of  claim 4 , wherein the biosynthetic pathway(s) produce a secondary metabolite through the overexpression of at least three foreign genes, at least four foreign genes, or at least five foreign genes. 
     
     
         6 . The method of any one of  claims 1  to  5 , wherein the  E. coli  contains an overexpression of at leak two  E. coli  genes. 
     
     
         7 . The method of  claim 6 , wherein the  E. coli  overexpresses at least one gene in the MEP pathway. 
     
     
         8 . The method of  claim 6  or  7 , wherein at least one gene is expressed by a strong promoter. 
     
     
         9 . The method of any one of  claims 6  to  8 , wherein at least one gene is expressed from a plasmid. 
     
     
         10 . The method of any one of  claims 6  to  9 , wherein at least one gene is chromosomally integrated. 
     
     
         11 . The method of any one of  claims 1  to  10 , wherein at least one P450 enzyme is not strongly expressed. 
     
     
         12 . The method of any one of  claims 1  to  11 , wherein the  E. coli  expresses at least two P450 enzymes, which are optionally derived from plant P450 enzymes. 
     
     
         13 . The method of  claim 12 , wherein the  E. coli  expresses a membrane-anchored P450 selected from CiVO, HmPO, LsGAO, BsGAO, NtEAO, SrKO, SrKAH, AtKAEI, ZzHO, CpVO, MsL6OH, NtVO, StVO, AtKO, Ci2VO, AaAO, and Taxus 5-alpha hydroxylase, or derivative thereof. 
     
     
         14 . The method of any one of  claims 1  to  13 , wherein the biosynthetic pathway produces a secondary metabolite selected from a terpenoid, alkaloid, cannabinoid, steroid, saponin, glycoside, stilbenoid, polyphenol, antibiotic, polyketide, fatty acid, or non-ribosomal peptide. 
     
     
         15 . The method of  claim 14 , wherein the biosynthetic pathway produces a terpenoid selected from a monoterpenoid, a sesquiterpenoid, diterpenoid, a sesterpenoid, or a triterpenoid. 
     
     
         16 . The method of  claim 15 , wherein the biosynthetic pathway involves overexpression of a geranyl diphosphate synthase (GPS), a gernanylgeranyl diphosphate synthase (GGPS), a farnsesyl diphosphate synthase (FPS), or a farnesyl geranyl diphosphate synthase (FGPPS). 
     
     
         17 . The method of any one of  claims 14  to  16 , wherein the biosynthetic pathway(s) produce at least one terpenoid selected from alpha-sinensal, beta-Thujone, Camphor, Carveol, Carvone, Cineole, Citral, Citronellal, Cubebol, Geraniol, Limonene, Menthol, Menthone, Myrcene, Nootkatone, Nootkatoi, Patchouli, Piperitone, Sabinene, Steviol, Steviol glycoside, Taxadiene, Thymol, and Valencene, or derivative thereof. 
     
     
         18 . The method of  claim 17 , wherein the biosynthetic pathway(s) produce steviol or steviol glycoside. 
     
     
         19 . The method of  claim 18 , wherein the biosynthetic pathway comprises a geranylgeranyl pyrophosphate synthase (GPPS), a copalyl diphosphate synthase (CPPS), and a kaurene synthase (KS), as well as a kaurene oxidase (KO) and a kaureneoic acid hydroxylase (KAH) having the transmembrane domain derived from an  E. coli  gene. 
     
     
         20 . The method of  claim 18  or  19 , wherein the biosynthetic pathway further comprises one or more uridine diphosphate dependent glycosyltransferase enzymes (UGT). 
     
     
         21 . The method of  claim 17 , wherein the biosynthetic pathway produces Valencene and/or Nootkatone. 
     
     
         22 . The method of  claim 21 , wherein the biosynthetic pathway comprises a farnesyl pyrophosphate synthase, a Valencene Synthase, and a Valencene Oxidase. 
     
     
         23 . The method of any one of  claims 1  to  22 , wherein IbpA is not overexpressed during the culturing. 
     
     
         24 . The method of any one of  claims 1  to  23 , wherein the culturing is conducted at 30° C. or greater. 
     
     
         25 . The method of  claim 24 , wherein the culturing is conducted at 32° C. or greater. 
     
     
         26 . The method of  claim 24 , wherein the culturing is conducted at 34° C. or greater. 
     
     
         27 . The method of any one of  claims 1  to  26 , wherein the size of the culture is at least 100 L. 
     
     
         28 . The method of  claim 27 , wherein the size of the culture is at least 1000 L. 
     
     
         29 . The method of  claim 28 , wherein the culturing is conducted in batch culture. 
     
     
         30 . The method of  claim 28 , wherein the culturing is conducted in continuous culture or semi-continuous culture. 
     
     
         31 . The method of  claims 23  to  30 , wherein the  E. coli  expresses one or more CPR enzymes as a translational fusion or operon with the P450 enzymes. 
     
     
         32 . The method of any one of  claims 1  to  31 , wherein the P450 and the CPR are expressed separately, and the level of expression of the P450 enzyme and the CPR are approximately 2:1 to 1:2. 
     
     
         33 . The method of any one of  claims 1  to  32 , wherein the cell expresses a single CPR protein. 
     
     
         34 . The method of any one of  claims 1  to  33 , wherein at least one CPR partner comprises a membrane-anchor having a single pass transmembrane domain derived from an  E. coli  gene. 
     
     
         35 . The method of any one of  claims 1  to  34 , wherein at least one membrane anchor is a single pass transmembrane domain derived from an  E. coli  gene selected from waaA, vpfN, yhcB, yhbM, yhhm, zipA, ycgG, djlA, sohB, lpxK, F11O, motA, htpx, pgaC, ygdD, hemr, and ycls, or derivative thereof. 
     
     
         36 . The method of  claim 35 , wherein at least one membrane anchor is a single pass transmembrane domain derived from yhcB or zipA, or a derivative thereof. 
     
     
         37 . The method of any one of  claims 1  to  36 , wherein the P450 enzyme has a deletion of part or all of its native N-terminal transmembrane domain. 
     
     
         38 . The method of  claim 37 , wherein the P450 enzyme has an N-terminal truncation of from about 10 to about 50 amino acids with respect to the wildtype enzyme. 
     
     
         39 . The method of  claim 37 , wherein the P450 enzyme has a N-terminal truncation of from about 15 to about 45 amino acids with respect to the wildtype enzyme. 
     
     
         40 . The method of  claim 37 , wherein the P450 enzyme has an N-terminal truncation of from about 20 to about 40 amino acids with respect to the wildtype enzyme. 
     
     
         41 . The method of  claim 37 , wherein the P450 enzyme has an N-terminal truncation of from about 20 to about 35 amino acids with respect to the wildtype enzyme. 
     
     
         42 . The method of  claim 37 , wherein the P450 enzyme has an N-terminal truncation of about 29 or 30 amino acids with respect to the wildtype enzyme. 
     
     
         43 . The method of any one of  claims 1  to  42 , wherein the membrane anchor is from about 8 to about 75 amino acids in length. 
     
     
         44 . The method of  claim 43 , wherein the membrane anchor is from about 15 to about 50 amino acids in length. 
     
     
         45 . The method of  claim 43 , wherein the membrane anchor is from about 20 to about 40 amino acids in length. 
     
     
         46 . The method of  claim 43 , wherein the membrane anchor is from about 20 to about 30 amino acids in length. 
     
     
         47 . The method of  claim 43 , wherein at least one membrane anchor is selected from:
 about the N-terminal 20 to 22 amino acids of yhcB,   about the N-terminal 19 to 21 amino acids of yhhM,   about the N-terminal 24 to 26 amino acids of zipA,   about the N-terminal 21 to 23 amino acids of ypfN,   about the N-terminal 27 to 29 amino acids of SohB, and   about the N-terminal 20-22 amino acids of waaA, or derivative thereof.   
     
     
         48 . The method of any one of  claims 1  to  47 , wherein the membrane anchor has from 1 to about 8 deletions, insertions, or substitutions relative to the wildtype  E. coli  sequence. 
     
     
         49 . The method of any one of  claims 37  to  48 , wherein the length of the truncation and selection of anchor sequence is by testing ibpA expression in cultures. 
     
     
         50 . The method of any one of  claims 1  to  49 , further comprising recovering the chemical species from the culture. 
     
     
         51 . The method of any one of  claims 1  to  49 , wherein the culturing produces at least 25 mg/L of the chemical species. 
     
     
         52 . The method of  claim 51 , wherein the culturing produces at least 50 mg/L of the chemical species or at least 100 mg/L of the chemical species. 
     
     
         53 . The method of any one of  claims 1  to  52 , further comprising, incorporating the chemical species into a product. 
     
     
         54 . A method for producing a product comprising one or more terpenoid compounds, comprising:
 expressing a terpenoid biosynthetic pathway in  E. coli,  the biosynthetic pathway comprising at least one membrane-anchored P450 enzyme having a transmembrane domain derived from an  E. coli  inner membrane cytoplasmic C-terminus protein; and   culturing the  E. coli  to produce the one or more terpenoids from the biosynthetic pathway;   recovering the terpenoid(s) from the culture; and   incorporating the terpenoid into a product.   
     
     
         55 . The method of  claim 54 , wherein the  E. coli  does not exhibit a substantially stressed phenotype during the culturing. 
     
     
         56 . The method of  claim 54  or  55 , wherein the  E. coli  expresses at least two, at least three, or at least four recombinant enzymes. 
     
     
         57 . The method of  claim 56 , wherein the terpenoid biosynthetic pathway comprises the overexpression of at least two foreign genes. 
     
     
         58 . The method of  claim 57 , wherein the terpenoid biosynthetic pathway comprises the overexpression of at least three foreign genes, at least four foreign genes, or at least five foreign genes. 
     
     
         59 . The method of any one of  claims 54  to  58 , wherein the  E. coli  contains an overexpression of at least two  E. coli  genes. 
     
     
         60 . The method of  claim 59 , wherein the  E. coli  overexpresses at least one gene in the MEP pathway. 
     
     
         61 . The method of  claim 59  or  60 , wherein at least one gene is expressed by a strong promoter. 
     
     
         62 . The method of any one of  claims 59  to  61 , wherein at least one gene is expressed from a plasmid. 
     
     
         63 . The method of any one of  claims 59  to  62 , wherein at least one gene is chromosomally integrated. 
     
     
         64 . The method of any one of  claims 54  to  63 , wherein at least one P450 enzyme is not strongly expressed. 
     
     
         65 . The method of any one of  claims 54  to  64 , wherein the  E. coli  expresses at least two P450 enzymes, which are optionally derived from plant P450 enzymes. 
     
     
         66 . The method of  claim 65 , wherein the  E. coli  expresses a membrane-anchored P450 selected from CiVO, HmPO, LsGAO, BsGAO, NtEAO, SrKO, SrKAH, AtKAH, ZzHO, CpVO, MsL6OH, NtVO, StVO, AtKO, Ci2VO, AaAO, and Taxus 5-alpha hydroxylase, or derivative thereof. 
     
     
         67 . The method of any one of  claims 54  to  66 , wherein the biosynthetic pathway produces a terpenoid selected from a monoterpenoid, a sesquiterpenoid, diterpenoid, a sesterpenoid, or a triterpenoid. 
     
     
         68 . The method of  claim 67 , wherein the biosynthetic pathway involves overexpression of a geranyl diphosphate synthase (GPS), a gernanylgeranyl diphosphate synthase (GGPS), a farnsesyl diphosphate synthase (FPS), or a famesyl geranyl diphosphate synthase (FGPPS). 
     
     
         69 . The method of any one of  claims 54  to  68 , wherein the biosynthetic pathway produces at least one terpenoid selected from alpha-sinensal, beta-Thujone, Camphor, Carveol, Carvone, Cineole, Citral, Citronellal, Cubebol, Geraniol, Limonene, Menthol, Menthone, Myrcene, Nootkatone, Nootkatol, Patchouli, Piperitone, Sabinene, Steviol, Steviol glycoside, Taxadiene, Thymol, and Valencene, or derivative thereof. 
     
     
         70 . The method of  claim 69 , wherein the biosynthetic pathway(s) produce steviol or steviol glycoside. 
     
     
         71 . The method of  claim 70 , wherein the biosynthetic pathway comprises a geranylgeranyl pyrophosphate synthase (GPPS), a copalyl diphosphate synthase (CPPS), and a kaurene synthase (KS), as well as a kaurene oxidase (KO) and a kaureneoic acid hydroxylase (KAH) having the transmembrane domain derived from an  E. coli  gene. 
     
     
         72 . The method of  claim 71 , wherein the biosynthetic pathway further comprises one or more uridine diphosphate dependent glycosyltransferase enzymes (UGT). 
     
     
         73 . The method of  claim 69 , wherein the biosynthetic pathway produces Valencene and/or Nootkatone. 
     
     
         74 . The method of  claim 73 , wherein the biosynthetic pathway comprises a farnesyl pyrophosphate synthase, a Valencene Synthase, and a Valencene Oxidase. 
     
     
         75 . The method of any one of  claims 54  to  74 , wherein IbpA is not overexpressed during the culturing. 
     
     
         76 . The method of any one of  claims 54  to  75 , wherein the culturing is conducted at 30° C. or greater. 
     
     
         77 . The method of  claim 76 , wherein the culturing is conducted at 32° C. or greater. 
     
     
         78 . The method of  claim 76 , wherein the culturing is conducted at 34° C. or greater. 
     
     
         79 . The method of any one of  claims 54  to  78 , wherein the size of the culture is at least 100 L. 
     
     
         80 . The method of  claim 79 , wherein the size of the culture is at least 1000 L. 
     
     
         81 . The method of  claim 79  or  80 , wherein the culturing is conducted in batch culture. 
     
     
         82 . The method of any one of  claims 76  to  81 , wherein the culturing is conducted in continuous culture or semi-continuous culture. 
     
     
         83 . The method of  claims 76  to  82 , wherein the  E. coli  expresses one or more CPR enzymes as a translational fusion or operon with the P450 enzymes. 
     
     
         84 . The method of any one of  claims 54  to  83 , wherein the P450 and the CPR are expressed separately, and the level of expression of the P450 enzyme and the CPR are approximately 2:1 to 1:2. 
     
     
         85 . The method of any one of  claims 54  to  84 , wherein the cell expresses a single CPR protein. 
     
     
         86 . The method of any one of  claims 54  to  85 , wherein at least one CPR partner comprises a membrane-anchor having a single pass transmembrane domain derived from an  E. coli  gene. 
     
     
         87 . The method of any one of  claims 54  to  86 , wherein at least one membrane anchor is a single pass transmembrane domain derived from an  E. coli  gene selected from waaA, ypfN, yhcB, yhbM, yhhm, zipA, ycgG, djlA, sohB, lpxK, F11O, motA, htpx, pgaC, ygdD, hemr, and ycls, or derivative thereof. 
     
     
         88 . The method of  claim 87 , wherein at least one membrane anchor is a single pass transmembrane domain derived from yhcB or zipA, or derivative thereof. 
     
     
         89 . The method of any one of  claims 54  to  88 , wherein the P450 enzyme has a deletion of part or all of its native N-terminal transmembrane domain. 
     
     
         90 . The method of  claim 89 , wherein the P450 enzyme has an N-terminal truncation of from about 10 to about 50 amino acids with respect to the wildtype enzyme. 
     
     
         91 . The method of  claim 89 , wherein the P450 enzyme has a N-terminal truncation of from about 15 to about 45 amino acids with respect to the wildtype enzyme. 
     
     
         92 . The method of  claim 89 , wherein the P450 enzyme has an N-terminal truncation of from about 20 to about 40 amino acids with respect to the wildtype enzyme. 
     
     
         93 . The method of  claim 89 , wherein the P450 enzyme has an N-terminal truncation of from about 20 to about 35 amino acids with respect to the wildtype enzyme. 
     
     
         94 . The method of  claim 89 , wherein the 1450 enzyme has an N-terminal truncation of about 29 or 30 amino acids with respect to the wildtype enzyme. 
     
     
         95 . The method of any one of  claims 54  to  94 , wherein the membrane anchor is from about 8 to about 75 amino acids in length. 
     
     
         96 . The method of  claim 95 , wherein the membrane anchor is from about 15 to about 50 amino acids in length. 
     
     
         97 . The method of  claim 95 , wherein the membrane anchor is from about 20 to about 40 amino acids in length. 
     
     
         98 . The method of  claim 95 , wherein the membrane anchor is from about 20 to about 30 amino acids in length. 
     
     
         99 . The method of  claim 95 , wherein at least one membrane anchor is selected from:
 about the N-terminal 20 to 22 amino acids of yhcB,   about the N-terminal 19 to 21 amino acids of yhhM,   about the N-terminal 24 to 26 amino acids of zipA,   about the N-terminal 21 to 23 amino acids of ypfN,   about the N-terminal 27 to 29 amino acids of SohB, and   about the N-terminal 20-22 amino acids of waaA, or derivative thereof.   
     
     
         100 . The method of any one of  claims 54  to  99 , wherein the membrane anchor has from 1 to about 8 deletions, insertions, or substitutions relative to the wildtype  E. coli  sequence. 
     
     
         101 . The method of  claim 100 , wherein the length of the truncation and selection of anchor sequence is by testing ibpA expression in cultures. 
     
     
         102 . The method of any one of  claims 54  to  101 , further comprising recovering the chemical species from the culture. 
     
     
         103 . The method of  claim 102 , wherein the culturing produces at least 25 mg/L of the chemical species. 
     
     
         104 . The method of  claim 102 , wherein the culturing produces at least 50 mg/L of the chemical species or at least 100 mg/L of the chemical species. 
     
     
         105 . An  E. coli  host cell expressing one or more recombinant biosynthetic pathways, where the biosynthetic pathways comprise at least one membrane-anchored P450 protein having a transmembrane domain derived from an  E. coli  inner membrane cytoplasmic C-terminus protein. 
     
     
         106 . The host cell of  claim 105 , wherein the  E. coli  does not exhibit a substantially stressed phenotype during culturing. 
     
     
         107 . The host cell of  claim 105  or  106 , wherein the  E. coli  expresses at least two, at least three, or at least four recombinant enzymes. 
     
     
         108 . The host cell of  claim 107 , wherein the biosynthetic pathway(s) produce a secondary metabolite through the overexpression of at least two foreign genes. 
     
     
         109 . The host cell of  claim 108 , wherein the biosynthetic pathway(s) produce a secondary metabolite through the overexpression of at least three foreign genes, at least four foreign genes, or at least five foreign genes. 
     
     
         110 . The host cell of any one of  claims 105  to  109 , wherein the  E. coli  contains an overexpression of at least two  E. coli  genes. 
     
     
         111 . The host cell of  claim 110 , wherein the  E. coli  overexpresses at least one gene in the MFP pathway. 
     
     
         112 . The host cell of  claim 110  or  111 , wherein at least one gene is expressed by a strong promoter. 
     
     
         113 . The host cell of any one of  claims 110  to  112 , wherein at least one gene is expressed from a plasmid. 
     
     
         114 . The host cell of any one of  claims 110  to  113 , wherein at least one gene is chromosomally integrated. 
     
     
         115 . The host cell of any one of  claims 105  to  114 , wherein at least one P450 enzyme is not strongly expressed. 
     
     
         116 . The host cell of any one of  claims 105  to  114 , wherein the  E. coli  expresses at least two P450 enzymes, which are optionally derived from plant P450 enzymes. 
     
     
         117 . The host cell of  claim 116 , wherein the  E. coli  expresses a membrane-anchored P450 selected from CiVO, HmPO, LsGAO, BsGAO, NtEAO, SrKO, SrKAH, AtKAH, ZzHO, CpVO, MsL6OH, NtVO, StVO, AtKO, Ci2VO, AaAO, and Taxus 5-alpha hydroxylase, or derivative thereof. 
     
     
         118 . The host cell of any one of  claims 105  to  117 , wherein the biosynthetic pathway produces a secondary metabolite selected from a terpenoid, alkaloid, cannabinoid, steroid, saponin, glycoside, stilbenoid, polyphenol, antibiotic, polyketide, fatty acid, or non-ribosomal peptide. 
     
     
         119 . The host cell of  claim 118 , wherein the biosynthetic pathway produces a terpenoid selected from a monoterpenoid, a sesquiterpenoid, diterpenoid, a sesterpenoid, or a triterpenoid. 
     
     
         120 . The host cell of  claim 119 , wherein the biosynthetic pathway involves overexpression of a geranyl diphosphate synthase (GPS), a gernanylgeranyl diphosphate synthase (GGPS), a farnsesyl diphosphate synthase (FPS), or a farnesyl geranyl diphosphate synthase (FGPPS). 
     
     
         121 . The host cell of any one of  claims 118  to  120 , wherein the biosynthetic pathway(s) produce at least one terpenoid selected from alpha-sinensal, beta-Thujone, Camphor, Carveol, Carvone, Cineole, Citral, Citronellal, Cubebol, Geraniol, Limonene, Menthol, Menthone, Myrcene, Nootkatone, Nootkatol, Patchouli, Piperitone, Sabinene, Steviol, Steviol glycoside, Taxadiene, Thymol, and Valencene, or derivative thereof. 
     
     
         122 . The host cell of  claim 121 , wherein the biosynthetic pathway(s) produce steviol or steviol glycoside. 
     
     
         123 . The host cell of  claim 122 , wherein the biosynthetic pathway comprises a geranylgeranyl pyrophosphate synthase (GPPS), a copalyl diphosphate synthase (CPPS), and a kaurene synthase (KS), as well as a kaurene oxidase (KO) and a kaureneoic acid hydroxylase (KAH) having the transmembrane domain derived from an  E. coli  gene. 
     
     
         124 . The host cell of  claim 122  or  123 , wherein the biosynthetic pathway further comprises one or more uridine diphosphate dependent glycosyltransferase enzymes (UGT). 
     
     
         125 . The host cell of  claim 121 , wherein the biosynthetic pathway produces Valencene and/or Nootkatone. 
     
     
         126 . The host cell of  claim 125 , wherein the biosynthetic pathway comprises a farnesyl pyrophosphate synthase, a Valencene Synthase, and a Valencene Oxidase. 
     
     
         127 . The host cell of any one of  claims 105  to  126 , wherein IbpA is not overexpressed during culturing. 
     
     
         128 . The host cell of  claims 105  to  127 , wherein the  E. coli  expresses one or more CPR enzymes as a translational fusion or operon with the P450 enzymes. 
     
     
         129 . The host cell of any one of  claims 105  to  128 , wherein the P450 and the CPR are expressed separately, and the level of expression of the P450 enzyme and the CPR are approximately 2:1 to 1:2. 
     
     
         130 . The host cell of any one of  claims 105  to  129 , wherein the cell expresses a single CPR protein. 
     
     
         131 . The host cell of any one of  claims 105  to  130 , wherein at least one CPR partner comprises a membrane-anchor having a single pass transmembrane domain derived from an  E. coli  gene. 
     
     
         132 . The host cell of any one of  claims 105  to  131 , wherein at least one membrane anchor is a single pass transmembrane domain derived from an  E. coli  gene selected from waaA, ypfN, yhcB, yhbM, yhhm, zipA, ycgG, djlA, sohB, lpxK, F11O, motA, htpx, pgaC, ygdD, hemr, and ycls, or derivative thereof. 
     
     
         133 . The host cell of  claim 132 , wherein at least one membrane anchor is a single pass transmembrane domain derived from yhcB or zipA, or derivative thereof. 
     
     
         134 . The host cell of any one of  claims 105  to  133 , wherein the P450 enzyme has a deletion of part or all of its native N-terminal transmembrane domain. 
     
     
         135 . The host cell of  claim 134 , wherein the P450 enzyme has an N-terminal truncation of from about 10 to about 50 amino acids with respect to the wildtype enzyme. 
     
     
         136 . The host cell of  claim 134 , wherein the P450 enzyme has a N-terminal truncation of from about 15 to about 45 amino acids with respect to the wildtype enzyme. 
     
     
         137 . The host cell of  claim 134 , wherein the P450 enzyme has an N-terminal truncation of from about 20 to about 40 amino acids with respect to the wildtype enzyme. 
     
     
         138 . The host cell of  claim 134 , wherein the P450 enzyme has an N-terminal truncation of from about 20 to about 35 amino acids with respect to the wildtype enzyme. 
     
     
         139 . The host cell of  claim 134 , wherein the P450 enzyme has an N-terminal truncation of about 29 or 30 amino acids with respect to the wildtype enzyme. 
     
     
         140 . The host cell of any one of  claims 105  to  139 , wherein the membrane anchor is from about 8 to about 75 amino acids in length. 
     
     
         141 . The host cell of  claim 140 , wherein the membrane anchor is from about 15 to about 50 amino acids in length. 
     
     
         142 . The host cell of  claim 140 , wherein the membrane anchor is from about 20 to about 40 amino acids in length. 
     
     
         143 . The host cell of  claim 140 , wherein the membrane anchor is from about 20 to about 30 amino acids in length. 
     
     
         144 . The host cell of  claim 140 , wherein at least one membrane anchor is selected from:
 about the N-terminal 20 to 22 amino acids of yhcB,   about the N-terminal 19 to 21 amino acids of yhhM,   about the N-terminal 24 to 26 amino acids of zipA,   about the N-terminal 21 to 23 amino acids of ypfN,   about the N-terminal 27 to 29 amino acids of SohB, and   about the N-terminal 20-22 amino acids of waaA, or derivatives thereof.   
     
     
         145 . The host cell of any one of  claims 105  to  144 , wherein the membrane anchor has from 1 to about 8 deletions, insertions, or substitutions relative to the wildtype  E. coli  sequence. 
     
     
         146 . The host cell of  claim 145 , wherein the length of the truncation and selection of anchor sequence is by testing ibpA expression in cultures. 
     
     
         147 . A plant P450 enzyme comprising an N-terminal truncation and a single-pass transmembrane region derived from an  E. coli  inner membrane cytoplasmic C-terminus protein. 
     
     
         148 . The enzyme of  claim 147 , wherein the membrane-anchored P450 is selected from CiVO, HmPO, LsGAO, BsGAO, NtEAO, SrKO, SrKAH, AtKAH, ZzHO, CpVO, MsL6OH, NtVO, StVO, AtKO, Ci2VO, AaAO, and Taxus 5-alpha hydroxylase, or derivative thereof. 
     
     
         149 . The enzyme of  claim 148 , wherein at least one membrane anchor is a single pass transmembrane domain derived from an  E. coli  gene selected from waaA, ypfN, yhcB, yhbM, yhhm, zipA, ycgG, djlA, sohB, lpxK, F11O, motA, htpx, pgaC, ygdD, hemr, and ycls, or derivative thereof. 
     
     
         150 . The enzyme of  claim 149 , wherein at least one membrane anchor is a single pass transmembrane domain derived from yhcB or zipA, or derivative thereof. 
     
     
         151 . The enzyme of any one of  claims 147  to  150 , wherein the P450 enzyme has a deletion of part or all of its native N-terminal transmembrane domain. 
     
     
         152 . The enzyme of  claim 151 , wherein the P450 enzyme has an N-terminal truncation of from about 10 to about 50 amino acids with respect to the wildtype enzyme. 
     
     
         153 . The enzyme of  claim 151 , wherein the P450 enzyme has a N-terminal truncation of from about 15 to about 45 amino acids with respect to the wildtype enzyme. 
     
     
         154 . The enzyme of  claim 151 , wherein the P450 enzyme has an N-terminal truncation of from about 20 to about 40 amino acids with respect to the wildtype enzyme. 
     
     
         155 . The enzyme of  claim 151 , wherein the P450 enzyme has an N-terminal truncation of from about 20 to about 35 amino acids with respect to the wildtype enzyme. 
     
     
         156 . The enzyme of  claim 151 , wherein the P450 enzyme has an N-terminal truncation of about 29 or 30 amino acids with respect to the wildtype enzyme. 
     
     
         157 . The enzyme of any one of  claims 147  to  156 , wherein the membrane anchor is from about 8 to about 75 amino acids in length. 
     
     
         158 . The enzyme of  claim 157 , wherein the membrane anchor is from about 15 to about 50 amino acids in length. 
     
     
         159 . The enzyme of  claim 157 , wherein the membrane anchor is from about 20 to about 40 amino acids in length. 
     
     
         160 . The enzyme of  claim 157 , wherein the membrane anchor is from about 20 to about 30 amino acids in length. 
     
     
         161 . The enzyme of  claim 157 , wherein at least one membrane anchor is selected from:
 about the N-terminal 20 to 22 amino acids of yhcB,   about the N-terminal 19 to 21 amino acids of yhhM,   about the N-terminal 24 to 26 amino acids of zipA,   about the N-terminal 21 to 23 amino acids of ypfN,   about the N-terminal 27 to 29 amino acids of SohB, and   about the N-terminal 20-22 amino acids of waaA, or derivative thereof.   
     
     
         162 . The enzyme of any one of  claims 147  to  161 , wherein the membrane anchor has from 1 to about 8 deletions, insertions, or substitutions relative to the wildtype  E. coli  sequence. 
     
     
         163 . A polynucleotide encoding the enzyme of any one of  claims 147  to  162 .