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 , 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  claim 1 , wherein the  E. coli  contains an overexpression of at least 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 , wherein at least one gene is expressed by a strong promoter. 
     
     
         9 - 34 . 
     
     
         35 . The method of  claim 1 , 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, F110, motA, htpx, pgaC, ygdD, hemr, and ycls, or derivative thereof. 
     
     
         36 . (canceled) 
     
     
         37 . The method of  claim 1 , wherein the P450 enzyme has a deletion of part or all of its native N-terminal transmembrane domain. 
     
     
         38 - 53 . (canceled) 
     
     
         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 , 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 - 104 . (canceled) 
     
     
         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 - 131 . 
     
     
         132 . The host cell of  claim 105 , 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 . (canceled) 
     
     
         134 . The host cell of  claim 105 , wherein the P450 enzyme has a deletion of part or all of its native N-terminal transmembrane domain. 
     
     
         135 - 146 . (canceled) 
     
     
         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 - 162 . (canceled) 
     
     
         163 . A polynucleotide encoding the enzyme of  claim 147 .