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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 Escherichia coli , said method comprising:
expressing one or more recombinant biosynthetic pathways that produce one or more chemical species in Escherichia coli , the one or more biosynthetic pathways comprising at least one membrane-anchored P450 enzyme, wherein at least a portion of the transmembrane region of the P450 enzyme is substituted with a transmembrane domain derived from an Escherichia coli inner membrane cytoplasmic C-terminus protein, and
culturing the Escherichia coli to produce the one or more chemical species from the one or more biosynthetic pathways.
2. The method of claim 1 , wherein the Escherichia coli overexpresses at least one gene in the MEP pathway.
3. The method of claim 1 , wherein the Escherichia coli expresses at least two membrane-anchored P450 enzymes, wherein at least a portion of the transmembrane region of at least one of the P450 enzymes is substituted with a transmembrane domain derived from an Escherichia coli inner membrane cytoplasmic C-terminus protein, wherein the P450 enzymes are optionally derived from plant P450 enzymes.
4. The method of claim 1 , wherein the at least one membrane-anchored P450 enzyme, is CiVO, HmPO, LsGAO, BsGAO, NtEAO, SrKO, SrKAH, AtKAH, ZzHO, CpVO, MsL60H, NtVO, StVO, AtKO, Ci2VO, AaAO, or Taxus 5-alpha hydroxylase.
5. The method of claim 1 , wherein the chemical species is a terpenoid.
6. The method of claim 5 , wherein the terpenoid is selected from the group consisting of a monoterpenoid, a sesquiterpenoid, diterpenoid, a sesterpenoid, and a triterpenoid.
7. The method of claim 6 , 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).
8. The method of claim 5 , wherein the terpenoid is selected from the group consisting of 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.
9. The method of claim 1 , wherein IbpA is not overexpressed during the culturing.
10. The method of claim 1 , wherein the Escherichia coli expresses one or more CPR enzymes as a translational fusion or operon with the P450 enzymes; or 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.
11. The method of claim 10 , wherein the cell expresses a single CPR protein.
12. The method of claim 10 , wherein at least one CPR partner comprises a membrane-anchor having a single pass transmembrane domain derived from an Escherichia coli protein.
13. The method of claim 1 , wherein the transmembrane domain derived from an Escherichia coli inner membrane cytoplasmic C-terminus protein is a single pass transmembrane domain encoded by an Escherichia coli gene selected from the group consisting of waaA, ypfN, yhcB, yhbM, yhhm, zipA, ycgG, djlA, sohB, IpxK, F110, motA, htpx, pgaC, ygdD, hemr, and ycls, or derivative thereof.
14. The method of claim 13 , wherein the single pass transmembrane domain is derived from yhcB or zipA gene, or a derivative thereof.
15. The method of claim 1 , wherein the transmembrane domain derived from an Escherichia coli inner membrane cytoplasmic C-terminus protein is from 8 to 75 amino acids in length.
16. The method of claim 15 , wherein at least one membrane anchor is selected from:
the N-terminal 20 to 22 amino acids of yhcB (SEQ ID NO: 18),
the N-terminal 19 to 21 amino acids of yhhM (SEQ ID NO: 20),
the N-terminal 24 to 26 amino acids of zipA (SEQ ID NO: 21),
the N-terminal 21 to 23 amino acids of ypfN (SEQ ID NO: 17),
the N-terminal 27 to 29 amino acids of SohB (SEQ ID NO: 24), and
the N-terminal 20-22 amino acids of waaA (SEQ ID NO: 16), or derivative thereof.
17. The method of claim 1 , further comprising recovering the chemical species from the culture.
18. An Escherichia coli host cell expressing one or more recombinant biosynthetic pathways that produce one or more chemical species, where the biosynthetic pathways comprise at least one membrane-anchored P450 protein, wherein at least a portion of the transmembrane region of the P450 enzyme is substituted with a transmembrane domain derived from an Escherichia coli inner membrane cytoplasmic C-terminus protein.
19. A plant P450 enzyme comprising an N-terminal truncation and a single-pass transmembrane region derived from an Escherichia coli inner membrane cytoplasmic C-terminus protein.
20. A polynucleotide encoding the enzyme of claim 19 .