US20230323314A1

US20230323314A1 - Increasing productivity of e. coli host cells that functionally express p450 enzymes

Info

Publication number: US20230323314A1
Application number: US18/161,938
Authority: US
Inventors: Jason Donald; Christopher Pirie; Liwei Li; Huey-Ming MAK; Srishti TIBREWALA; Ajikumar PARAYIL KUMARAN
Original assignee: Manus Bio Inc
Current assignee: Manus Bio Inc
Priority date: 2015-08-21
Filing date: 2023-01-31
Publication date: 2023-10-12
Also published as: EP3337893A4; WO2017034942A1; US10774314B2; EP3337893A1; ES2959560T3; US20180251738A1; EP3337893B1; US20200377865A1

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.

Description

FIELD OF THE INVENTION

The present invention relates to production of chemical species through oxidative chemistry in bacterial host cells. Particularly, the present invention provides P450 enzymes engineered for functional expression in bacterial host cells such as E. coli.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The instant application contains a sequence listing, which has been submitted in XML format via EFS-Web. The contents of the XML copy named “MAN-006C2_Sequence Listing”, which was created on Jan. 30, 2023 and is 84,000 bytes in size, the contents of which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

E. coli is widely used for production of chemicals by the recombinant expression of biosynthetic pathways, which can involve overexpression of several native and/or foreign genes. However, where the biosynthetic pathway involves recombinant expression of one or more cytochrome P450 enzymes (e.g., to perform oxidative chemistry), other host organisms such as yeast are generally preferred. See Chang & Keasling, Nat. Chem. Bio. 2006. The perceived limitations of the bacterial system for oxidative chemistry include the absence of electron transfer machinery and P450-reductases (CPRs), and translational incompatibility of the membrane signal modules of P450 enzymes due to the lack of an endoplasmic reticulum. Thus, it remains a commonly held belief in the scientific community that E. coli is a generally unsuitable host for P450 expression and expression of biosynthetic pathways incorporating the same. See Tippman et al., Biotech. Journal (2013); Thodey et al., Nat. Chem. Bio. (2014).
It is an object of the invention to improve productivity of P450 enzymes in bacterial platforms such as E. coli, to thereby expand the utility of these platforms for P450 chemistry.

SUMMARY OF THE INVENTION

In various aspects, the invention provides P450 enzymes engineered for functional expression in bacterial cells (e.g., E. coli), and polynucleotides encoding the same. The invention further provides bacterial host cells expressing the engineered P450 enzymes, and methods for producing chemical species through recombinant expression of biosynthetic pathways involving P450 enzymes.
The engineered P450 enzymes described herein have a deletion of all or part of the wild type P450 N-terminal transmembrane region, and the addition of a transmembrane domain derived from an E. coli inner membrane, cytoplasmic C-terminus protein. It is believed that the transmembrane domain acts to anchor the P450 in the E. coli inner membrane. In various embodiments, the transmembrane domain is a single-pass transmembrane domain.
In various embodiments, the transmembrane domain (or “N-terminal anchor”) is 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. These genes were identified as inner membrane, cytoplasmic C-terminus proteins through bioinformatic prediction as well as experimental validation. The invention may employ an N-terminal anchor sequence that is a derivative of the E. coli wild-type transmembrane domain, that is, having one or more mutations with respect to the wild-type sequence.
In some aspects, the invention provides methods for the production of chemical species by expressing in E. coli cells one or more biosynthetic pathways including at least one membrane-anchored P450 (CYP) enzyme, and culturing the E. coli cells to produce the chemical species. At least one membrane-anchored P450 enzyme contains a transmembrane domain derived from an E. coli inner membrane, cytoplasmic C-terminus protein. As demonstrated herein, previous methods for expressing P450 proteins in E. coli can result in a substantial stress response, which limits productivity of the host cell. E. coli cells expressing the engineered P450 enzymes described herein do not exhibit a substantially stressed phenotype in some embodiments, thereby improving pathway productivity.
The invention in various aspects is applicable to various P450 enzymes, including plant-derived P450 enzymes, which can be further engineered for productivity in a bacterial host cell system. These engineered P450 enzymes can be used in the production of a variety of chemical species through recombinant pathway expression, including but not limited to production of terpenoid compounds. Terpenoids represent a diverse class of molecules that provide beneficial health and nutritional attributes, as well as numerous other commercial applications. For example, terpenoids find use in the food and beverage industries as well as the perfume, cosmetic and health care industries.
In various embodiments, the E. coli cell is used for the production of chemicals by the recombinant expression of biosynthetic pathways, which can involve overexpression of several native and/or foreign genes. Often, expression of several foreign genes in E. coli and/or overexpression of native E. coli genes can induce a substantial stress response, which limits productivity. Conventional expression of P450 enzymes in E. coli, together with cytochrome P450 reductase (CPR) partners to regenerate the cofactor, can substantially add to this stress response, as exhibited for example by overexpression of IbpA, a protein that is overexpressed in E. coli under conditions of high protein aggregation and stress. It is critical that the P450 enzyme expression induce as little cell stress as possible to avoid limits on pathway productivity. Accordingly, the invention helps minimize cellular stress in a host E. coli cell to increase productivity of the host cell for production of chemical species.
Other aspects and embodiments of the invention will be apparent from the following detailed disclosure.

DESCRIPTION OF THE FIGURES

FIG. 1 illustrates N-terminal anchors for expressing P450 proteins in E. coli, including the previous designs based on truncation of the P450 transmembrane helix with the addition of an 8-amino acid peptide (8RP), and the use of single-pass and multi-pass transmembrane helices from E. coli proteins as described herein.

FIG. 2 shows a computational prediction of signal peptides and/or transmembrane helices using the Phobius predictive tool. SrKO and AtKAH are predicted to have an N-terminal transmembrane region. Bovine P450-C17 N-terminus is predicted as a signal peptide.

FIG. 3 shows the total terpenoid flux and oxygenated terpenoid formation upon expression in E. coli of Valencene Oxidase (VO) enzymes truncated at residue 30 and having various E. coli anchors. The E. coli cells express a valencene synthesis pathway, producing the valencene substrate. Results with control VO enzymes having the 8rp signal peptide with truncation of 20 or 30 residues are also shown. Enzymes include a translationally coupled CPR.

FIG. 4 shows the total terpenoid flux and oxygenated terpenoid formation upon expression in E. coli of VO enzymes truncated at residue 30 with candidate E. coli anchors (sequences from yhcB, yhhM, zipA, ypfN, sohB, and waaA).

FIG. 5 shows the total terpenoid flux and oxygenated terpenoid formation upon expression of VO enzymes from a p10 expression plasmid or a p5 expression plasmid. While the 8RP anchor shows markedly decreased productivity at the higher expression level provided by a p10 plasmid, the higher expression level has little impact on productivity with the VO enzymes engineered with the E. coli anchor sequences.

FIG. 6 shows the total terpenoid flux and oxygenated terpenoid titers upon expression of the VO enzymes in a valencene-producing E. coli strain with a cytochrome P450 reductase (CPR) partner. The VO enzymes were expressed with the CPR either as separate proteins from the same operon or were translationally coupled.

FIG. 7 shows the level of VO protein expression with candidate N-terminal E. coli anchors (i.e., yhcB, yhhm, zipA, and ypfN), in comparison to t20-8RP. All four of the native E. coli anchors show lower total VO expression compared to 8RP, which results in a significantly lower relative VO/CPR ratio.

FIG. 8 shows the cellular stress response to VO expression with different N-terminal E. coli anchors, by assessing known E. coli stress response proteins. The IbpA protein, which is overexpressed in E. coli under conditions of high protein aggregation, was highly expressed in response to t20-8RP expression, but not with the native E. coli anchors.

FIG. 9 is a diagram showing the optimization of truncation length and anchor length of yhcB anchored VO enzymes.

FIG. 10 shows the total terpenoid flux and oxygenated terpenoid production with various truncated and yhcB anchored VO enzymes. Truncations varied from 28 to 32 amino acids of the Valencene Oxidase, and the anchored VO included from 20 to 24 amino acids of the yhcB N-terminus.

FIG. 11 shows the E(z) score of AtKAH enzymes truncated at residue 26 with various E. coli anchors. Wild-type (wt) is the score in the wild-type E. coli enzyme, nXX is the number of residues taken from the protein for a swap with the E. coli anchor, and the name of the source protein. Asterisks show truncations not tested with VO. E(z) score estimates the suitability of a protein sequence for insertion into a cell membrane based on statistics from solved transmembrane crystal structures.

FIG. 12A shows kaurenoic acid formation upon expression in kaurene-producing strains of SrKO enzymes engineered by truncation at residue 30, and with addition of E. coli anchors. The enzymes were expressed from a p5 plasmid along with a cytochrome P450 reductase (CPR) in the same operon. Kaurenoic acid formation is shown at 30° C. and 34° C. FIG. 12B shows the kaurenoic acid formation/OD of the E. coli strains.

FIG. 13 shows a detailed proteomic analysis of SrKO expressing cells. The relative abundance of various pathway and stress response proteins is assessed. SrKO is significantly over expressed when paired with non-native anchors (E. coli or 8rp) although the increased expression is dampened at higher temperatures (34° C. vs. 30° C.). IbpA stress response is also significantly increased at the higher temperature.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides for improved functional expression of P450 enzymes in bacterial host cells, and in particular, bacterial cells that do not naturally possess P450 enzymes, such as E. coli. While these bacterial platforms are widely used for production of a wide variety of chemicals, they are generally considered insufficient when P450 chemistry is required. The perceived limitations of bacterial systems such as E. coli for oxidative chemistry include the absence of electron transfer machinery and P450-reductases (CPRs), and translational incompatibility of the membrane signal modules of P450 enzymes due to the lack of an endoplasmic reticulum.
Basic P450 expression in E. coli has been obtained by co-expression of the P450 with a CPR. The P450 enzyme contained a truncation of at least part of the native P450 transmembrane region, which was replaced with the 8 amino acid tag MALLLAVF (which is derived from bovine P45017a) at the N-terminus. The present invention demonstrates that this tag is far from optimal, and results in a substantial cell stress response.
The invention provides P450 enzymes engineered for functional expression in bacterial platforms such as E. coli. The P450 enzymes comprise an N-terminal membrane anchor sequence derived from a native E. coli inner membrane protein having a cytoplasmic C-terminus. The N-terminal membrane anchor sequence replaces some or the entire native P450 N-terminal transmembrane region, where present in the wild-type enzyme. Expression of the engineered P450 enzymes in bacteria induces less cell stress than previous attempts to functionally express P450 enzymes in E. coli, for example. The invention allows for increases in biosynthetic productivity in bacterial host platforms, due in-part, to substantial improvements in P450 efficiency and to minimizing the cell stress response.
In one aspect, the present invention relates to a P450 enzyme having a transmembrane domain derived from an E. coli inner membrane protein having a cytoplasmic C-terminus. The E. coli transmembrane domain (or derivative thereof) replaces part or the entire native P450 N-terminal transmembrane region. In some embodiments, the transmembrane domain is a single-pass transmembrane domain, or in other embodiments, is a multi-pass (e.g., 2, 3, or more transmembrane helices) transmembrane domain.
The P450 enzyme may be derived from any source, including plants, animals, or microbes. The P450 enzyme may be a CYP70, CYP71, CYP73, CYP76 (e.g., CYP76F), CYP82 or CYP92 family P450. The P450 may be an enzyme disclosed in US 8,722,363, which is hereby incorporated by reference. In some embodiments, the P450 is a plant P450 enzyme. Plant cytochrome P450s are involved in a wide range of biosynthetic reactions, leading to various fatty acid conjugates, plant hormones, defensive compounds, or medically and commercially important compounds, including terpenoids. Terpenoids represent the largest class of characterized natural plant compounds and are often substrates for plant P450 enzymes. In some embodiments, the P450 is derived from a species selected from Zingiber sp., Barnadesia sp., Hyoscyamus sp., Latuca sp., Nicotiana sp., Citrus sp., Artemesia sp., Arabidopsis sp, Stevia sp., Bacillus sp., Pleurotus sp., Cichorium sp., Helianthus sp., and Physcomitrella sp., Taxus sp., Rosa sp., Cymbopogon sp., Humulus sp., Pogostemon sp., and Cannabis sp. Wild-type P450 enzyme sequences are known and publically available, and/or can be obtained by genetic analysis of select plants based on well-known P450 motifs. See, for example, Saxena A. et al., Identification of cytochrome P450 heme motif in plants proteome, Plant Omics (2013); Chapple C., Molecular-genetic analysis of plant cytochrome p450-dependent monooxygenases, Annual Review of Plant Physiology and Plant Molecular Biology Vol. 49:311-343 (1998).
Table 1 provides a list of exemplary P450 enzymes that may be engineered in accordance with the invention:

TABLE 1

Species	Name	Native Substrate	Native Reaction Product
Zingiber zerumbet	zzHO	a-humulene	8-hydroxy-a-humulene
Barnadesia spinosa	BsGAO	germacrene A	germacra-1(10),4,11(13)-trien-12-ol
Hyoscyamus muticus	HmPO	premnaspirodiene	solavetivol
Latuca spicata	LsGAO	germacrene A	germacra-1(10),4,11(13)-trien-12-ol
Nicotiana tabacum	NtEAO	5-epi-aristolochene	capsidiol
Citrus x paradisi	CpVO	valencene	nootkatol
Artemesia annua	AaAO	amorphadiene	artemisinic acid
Arabidopsis thaliana	AtKO	kaurene	kaurenoic acid
Stevia rebaudiana	SrKO	kaurene	kaurenoic acid
Physcomitrella patens	PpKO	kaurene	kaurenoic acid
Bacillus megaterium	BmVO	fatty acids	hydroxylated FAs
Pleurotus sapidus	PsVO	valencene	nootkatone
Pleurotus ostreatus	PoLO	unknown	unknown
Cichorium intybus	CiVO	valencene	nootkatone
Helianthus annuus	HaGAO	germacrene A	germacrene A acid

Thus, the engineered P450 enzyme may be based on wild-type sequences of ZzHO (SEQ ID NO: 1), BsGAO (SEQ ID NO: 2), HmPO (SEQ ID NO: 3), LsGAO (SEQ ID NO: 4), NtEAO (SEQ ID NO: 5), CpVO (SEQ ID NO: 6), AaAO (SEQ ID NO: 7), AtKO (SEQ ID NO: 8), SrKO (SEQ ID NO: 9), PpKO (SEQ ID NO:10), BmVO (SEQ ID NO: 11), PsVO (SEQ ID NO:12), PoLO (SEQ ID NO: 13), CiVO (SEQ ID NO: 14), or HaGAO (SEQ ID NO: 15).
Additional P450 enzymes that can be engineered in accordance with the invention include limonene-6-hydroxylase (AAQ18706.1, AAD44150.1), (-)-limonene-3-hydroxylase (EF426464, AY622319), kaurenoic acid 13-hydroxylase (EU722415.1), carotenoid cleavage dioxygenase (ABY60886.1, BAJ05401.1), beta-carotene hydroxylase (AAA64983.1), amorpha-4,11-diene monoxygenase (DQ315671), taxadiene 5-alpha hydroxylase (AY289209.2), 5-alpha-taxadienol-10-beta-hydroxylase (AF318211.1), taxoid 10-beta hydroxylase (AY563635.1), taxane 13-alpha-hydroxylase (AY056019.1), taxane 14b-hydroxylase (AY188177.1), taxoid 7-beta-hydroxylase (AY307951.1). The amino acid and encoding nucleotide sequences of these enzymes are hereby incorporated by reference. Derivatives of these P450s may be constructed in accordance with this disclosure.
The particular P450 enzyme scaffold can be selected based on the desired substrate specificity, which may be its natural substrate, or a non-natural substrate similar to the natural substrate, or otherwise determined experimentally. P450′s can have varying substrate specificities, and thus can be engineered for chemistry on non-natural substrates. See, for example, Wu et al., Expansion of substrate specificity of cytochrome P450 2A6 by random and site-directed mutagenesis, J. Biol. Chem. 280(49): 41090-100 (2005). Exemplary substrates for P450 chemistry include various secondary metabolites such as, without limitation, terpenoids, alkaloids, cannabinoids, steroids, saponins, glycosides, stilbenoids, polyphenols, antibiotics, polyketides, fatty acids, and non-ribosomal peptides. Exemplary products that may be produced through P450 chemistry include, without limitation, lutein, tocopherol, abietic acid, mogroside, forskolin, amyrin, lupeol, butyrospermol, quillic acid, triterpenoid saponins, oleanic acid, betulinic acid, boswellic acid, gymnemic acid, banaba/corosolic acid, cissus keto-steroid, curcurbitane triterpenoid, santalol, marrubiin, montbretin A, tropolone, sclareol, pseudolaric acid, grindelic acid, kauralexin, viteagnusin, diterpenoid epoxide triptolide, quinone triterpene celastrol, gibberellic acid, pseudolaric acid, carveol, carvone, nootkatol, nootkatone, piperitone, steviol, perillaldehyde, tagetone, verbenone, menthol, thymol, 3-oxo-alpha-lonone, zeanthin, artemisinin, taxol, gingkolide, gossypol, pseudoterosin, crotophorbolone, englerin, psiguadial, stemodinone, maritimol, cyclopamine, veratramine, aplyviolene, macfarlandin E, betulinic acid, oleanolic acid, ursoloic acid, dolichol, lupeol, euphol, cassaic acid, erthroxydiol, trisporic acid, podocarpic acid, retene, dehydroleucodine, phorbol, cafestol, kahweol, tetrahydrocannabinol, androstenol, tanshinone IIA or IIB or VI, cryptotanshinone, 15,16-dihydrotanshinone, trijuganone A or B, dihydrotanshinone I, miltirone, ferruginol, hydrotanshinone IIA, and 1,2-dihydrocrytotanshinone.
Exemplary terpenoid products that may be produced in accordance with the invention are described in US 8,927,241, which is hereby incorporated by reference, and include: alpha-sinensal, beta-Thujone, Camphor, Carveol, Carvone, Cineole, Citral, Citronellal, Cubebol, Geraniol, Limonene, Menthol, Menthone, Nootkatone, Nootkatol, Patchouli, Piperitone, Sabinene, Steviol, Steviol glycoside, Taxadiene, and Thymol.
In various embodiments, the engineered P450 enzyme comprises an amino acid sequence that has at least about 30% sequence identity, at least about 40% sequence identity, or at least about 50% sequence identity to any one of SEQ ID NOS: 1-15, or other P450 enzyme described herein. While the P450 need not display high sequence identity to these exemplary P450 enzymes in some embodiments, the P450 exhibits well-known P450 motifs and/or secondary structure. Generally, P450 sequence identity is determined by alignment of the full amino acid sequences, except for the N-terminal transmembrane regions (e.g., the alignment does not include about the first 30 amino acids of the wild-type sequence). In some embodiments, the engineered P450 enzyme comprises an amino acid sequence that has at least about 60% identity, at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, or at least about 98% identity to any one of SEQ ID NOS: 48 to 60. SEQ ID NOS: 48 to 60 show P450 enzymes without the predicted transmembrane region (txx is the length of the N-terminal truncation): t22ZzHO (SEQ ID NO:48), t20BsGAO (SEQ ID NO:49), t16HmPO (SEQ ID NO:50), t19LsGAO (SEQ ID NO:51), t16NtEAO (SEQ ID NO:52), t26CpVO (SEQ ID NO:53), t23AaAO (SEQ ID NO:54), t21AtKO (SEQ ID NO:55), t30SrKO (SEQ ID NO:56), t52PpKO (SEQ ID NO:57), t15PsVO (SEQ ID NO:58), t20CiVO (SEQ ID NO:59), t20HaGAO (SEQ ID NO:60).
The similarity of nucleotide and amino acid sequences, i.e. the percentage of sequence identity, can be determined via sequence alignments. Such alignments can be carried out with several art-known algorithms, such as with the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), with hmmalign (HMMER package, http://hmmer.wustl.edu/) or with the CLUSTAL algorithm (Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-80). The grade of sequence identity (sequence matching) may be calculated using e.g. BLAST, BLAT or BlastZ (or BlastX). A similar algorithm is incorporated into the BLASTN and BLASTP programs of Altschul et al (1990) J. Mol. Biol. 215: 403-410. BLAST polynucleotide searches can be performed with the BLASTN program, score = 100, word length = 12.
BLAST protein searches may be performed with the BLASTP program, score = 50, word length = 3. To obtain gapped alignments for comparative purposes, Gapped BLAST is utilized as described in Altschul et al (1997) Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs are used. Sequence matching analysis may be supplemented by established homology mapping techniques like Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1:154-162) or Markov random fields.
In various embodiments, the engineered P450 enzyme may comprise an amino acid sequence having one or more amino acid mutations relative to the wild-type sequence, not including the modifications to the N-terminal transmembrane region (e.g., about the first 18 to 30 amino acids). For example, the P450 enzyme may comprise an amino acid sequence having from 1 to about 50, or from 1 to about 40, or from 1 to about 30, or from 1 to about 25, or from 1 to about 20, or from 1 to about 15, or from 1 to about 10, or from 1 to about 5 mutations relative to the wild-type sequence (e.g., any one of SEQ ID NOS: 48 to 60). In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations. In some embodiments, the amino acid mutations are amino acid substitutions, and may include conservative and/or non-conservative substitutions.
“Conservative substitutions” may be made, for instance, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the amino acid residues involved. The 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups:

(1) hydrophobic: Met, Ala, Val, Leu, Ile;
(2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gin;
(3) acidic: Asp, Glu;
(4) basic: His, Lys, Arg;
(5) residues that influence chain orientation: Gly, Pro; and
(6) aromatic: Trp, Tyr, Phe.

As used herein, “conservative substitutions” are defined as exchanges of an amino acid by another amino acid listed within the same group of the six standard amino acid groups shown above. For example, the exchange of Asp by Glu retains one negative charge in the so modified polypeptide. In addition, glycine and proline may be substituted for one another based on their ability to disrupt a-helices. Some preferred conservative substitutions within the above six groups are exchanges within the following sub-groups: (i) Ala, Val, Leu and Ile; (ii) Ser and Thr; (ii) Asn and Gin; (iv) Lys and Arg; and (v) Tyr and Phe.
As used herein, “non-conservative substitutions” are defined as exchanges of an amino acid by another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above.
In various embodiments, the engineered P450 enzyme has a deletion or truncation of part or all of it native transmembrane domain. Generally, the deletion or truncation is about the first 15 to 30 amino acids, and the desired length can be determined based on the present disclosure and using predictive tools known in the art (e.g., PHOBIUS, http://phobius.sbc.su.se/). See Lukas K, et al., A Combined Transmembrane Topology and Signal Peptide Prediction Method, Journal of Molecular Biology, 338(5):1027-1036 (2004); Lukas K, et al., An HMM posterior decoder for sequence feature prediction that includes homology information, Bioinformatics, 21 (Suppl 1):i251-i257 (2005); Lukas K, et al., Advantages of combined transmembrane topology and signal peptide prediction--the Phobius web server, Nucleic Acids Res., 35:W429-32 (2007).
In various embodiments, the engineered P450 enzyme may have an N-terminal truncation of from about 10 to about 55 amino acids, or from about 15 to about 45 amino acids, or from about 15 to about 40 amino acids, or from about 15 to about 35 amino acids with respect to the wild-type enzyme. In various embodiments, the engineered P450 enzyme may have an N-terminal truncation of about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23 about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, or about 40 amino acids with respect to the wild-type enzyme.
The wild-type transmembrane region is replaced with a membrane anchor sequence derived from an E. coli protein. The E. coli protein is an inner membrane protein with its C-terminus in the cytoplasm. The membrane anchor derived from E. coli may be a single-pass transmembrane domain or a multiple-pass transmembrane domain. In some embodiments, the membrane anchor is a single-pass transmembrane domain. Exemplary single-pass transmembrane domains derived from E. coli include, but are not limited to, N-terminal domains from the following genes: waaA (SEQ ID NO: 16), ypfN (SEQ ID NO: 17), yhcB (SEQ ID NO: 18), yhbM (SEQ ID NO: 19), yhhm (SEQ ID NO: 20), zipA (SEQ ID NO: 21), ycgG (SEQ ID NO: 22), djlA (SEQ ID NO: 23), sohB (SEQ ID NO: 24), lpxK (SEQ ID NO: 25), F11O (SEQ ID NO: 26), motA (SEQ ID NO: 27), htpx (SEQ ID NO: 28), pgaC (SEQ ID NO: 29), ygdD (SEQ ID NO: 30), hemr (SEQ ID NO: 31), and ycls (SEQ ID NO: 32). In an embodiment, the transmembrane domain is derived from yhcB, yhhm, zipA, sohB, and waaA. The transmembrane regions can likewise be determined by predictive tools known in the art (including PHOBIUS).
In various embodiments, the membrane anchor sequence is from about 8 to about 75 amino acids in length. For example, the membrane anchor may be from about 15 to about 50, or from about 15 to about 40, or from about 15 to about 30, or from about 20 to about 40, or from about 20 to about 30 amino acids in length. In various embodiments, the membrane anchor is about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, or about 75 amino acids in length.
In an embodiment, the transmembrane domain is a yhcB transmembrane domain or a derivative thereof. For example, the transmembrane domain may include the N-terminal 15 to 30 amino acids of yhcB, such as the N-terminal 20 to 22 amino acids of yhcB. For example, the transmembrane domain may include the N-terminal 20, 21, or 22 amino acids of yhcB. The transmembrane domain may have one or more amino acid mutations relative to the wild-type yhcB domain (SEQ ID NO: 18). In some embodiments, the transmembrane domain may have from about 1 to about 8, or from about 1 to about 7, or from about 1 to about 6, or from about 1 to about 5, or from 1 to about 3 mutations relative to the wild-type yhcB sequence. The one or more amino acid mutations may be independently selected from substitutions, insertions, or deletions. In some embodiments, the amino acid mutations are amino acid substitutions. In some embodiments, mutations are selected based on their predicted score as a transmembrane region, using known predictive tools.
In an embodiment, the transmembrane domain is a yhhM transmembrane domain or derivative thereof. For example, the transmembrane domain may include the N-terminal 15 to 30, or 19 to 21, amino acids of yhhM. For example, the transmembrane domain may include the N-terminal 19, 20, or 21 amino acids of yhhM. The transmembrane domain may have one or more amino acid mutations relative to the wild type yhhM domain (SEQ ID NO: 20). In some embodiments, the transmembrane domain may have from about 1 to about 8, or from about 1 to about 7, or from about 1 to about 6, or from about 1 to about 5, or from 1 to about 3 mutations relative to the wild-type yhhM sequence. The one or more amino acid mutations may be independently selected from substitutions, insertions, or deletions. In some embodiments, the amino acid mutations are amino acid substitutions. In some embodiments, mutations are selected based on their predicted score as a transmembrane region, using known predictive tools.
In an embodiment, the transmembrane domain is a zipA transmembrane domain or derivative thereof. In such an embodiment, the transmembrane domain may include the N-terminal 15 to 30, or 24 to 26, amino acids of zipA. For example, the transmembrane domain may include the N-terminal 24, 25, or 26 amino acids of zipA. The transmembrane domain may have one or more amino acid mutations relative to the wild type zipA domain (SEQ ID NO: 21). In some embodiments, the transmembrane domain may have from about 1 to about 8, or from about 1 to about 7, or from about 1 to about 6, or from about 1 to about 5, or from 1 to about 3 mutations relative to the wild-type zipA sequence. The one or more amino acid mutations may be independently selected from substitutions, insertions, or deletions. In some embodiments, the amino acid mutations are amino acid substitutions. In some embodiments, mutations are selected based on their predicted score as a transmembrane region, using known predictive tools.
In an embodiment, the transmembrane domain is a ypfN transmembrane domain or derivative thereof. In such an embodiment, the transmembrane domain may include the N-terminal 15 to 30, or 21 to 23, amino acids of ypfN. For example, the transmembrane domain may include the N-terminal 21, 22, or 23 amino acids of ypfN. The transmembrane domain may have one or more amino acid mutations relative to the wild-type ypfN domain (SEQ ID NO: 17). In some embodiments, the transmembrane domain may have from about 1 to about 8, or from about 1 to about 7, or from about 1 to about 6, or from about 1 to about 5, or from 1 to about 3 mutations relative to the wild-type ypfN sequence. The one or more amino acid mutations may be independently selected from substitutions, insertions, or deletions. In some embodiments, the amino acid mutations are amino acid substitutions. In some embodiments, mutations are selected based on their predicted score as a transmembrane region, using known predictive tools.
In an embodiment, the transmembrane domain is a sohB transmembrane domain or derivative. In such an embodiment, the transmembrane domain may include the N-terminal 20 to 35, or 27 to 29, amino acids, of sohB. For example, the transmembrane domain may include the N-terminal 27, 28, or 29 amino acids of sohB. The transmembrane domain may have one or more amino acid mutations relative to the wild-type sohB domain (SEQ ID NO: 24). In some embodiments, the transmembrane domain may have from about 1 to about 8, or from about 1 to about 7, or from about 1 to about 6, or from about 1 to about 5, or from 1 to about 3 mutations relative to the wild-type sohB sequence. The one or more amino acid mutations may be independently selected from substitutions, insertions, or deletions. In some embodiments, the amino acid mutations are amino acid substitutions. In some embodiments, mutations are selected based on their predicted score as a transmembrane region, using known predictive tools.
In an embodiment, the transmembrane domain is a waaA transmembrane domain or derivative thereof. In such an embodiment, the transmembrane domain may include the N-terminal 15 to 30, or 20 to 22, amino acids of waaA. For example, the transmembrane domain may include the N-terminal 20, 21, or 22 amino acids of waaA. The transmembrane domain may have one or more amino acid mutations relative to the wild-type waaA domain (SEQ ID NO: 16). In some embodiments, the transmembrane domain may have from about 1 to about 8, or from about 1 to about 7, or from about 1 to about 6, or from about 1 to about 5, or from 1 to about 3 mutations relative to the wild-type waaA sequence. The one or more amino acid mutations may be independently selected from substitutions, insertions, or deletions. In some embodiments, the amino acid mutations are amino acid substitutions. In some embodiments, mutations are selected based on their predicted score as a transmembrane region, using known predictive tools.
In still other embodiments, the transmembrane domain is a transmembrane domain of yhbM (SEQ ID NO: 19), ycgG (SEQ ID NO: 22), djlA (SEQ ID NO: 23), lpxK (SEQ ID NO: 25), F110 (SEQ ID NO: 26), motA (SEQ ID NO: 27), htpx (SEQ ID NO: 28), pgaC (SEQ ID NO: 29), ygdD (SEQ ID NO: 30), hemr (SEQ ID NO: 31), or ycls (SEQ ID NO: 32), or a derivative thereof. The derivative may have one or more amino acid mutations relative to the wild-type E. coli sequence. In some embodiments, the transmembrane domain may have from about 1 to about 10, or from about 1 to about 8, or from about 1 to about 5, or from about 1 to about 3 mutations relative to the wild-type E. coli sequence. In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, and deletions. In some embodiments, the amino acid mutations are amino acid substitutions. In some embodiments, mutations are selected based on their predicted score as a transmembrane region, using known predictive tools.
In other aspects, the invention provides polynucleotides comprising a nucleotide sequence encoding an engineered P450 enzyme described above. The polynucleotide may be codon optimized for expression in E. coli in some embodiments. In another example, the polynucleotide may comprise a nucleotide sequence encoding at least one engineered P450 enzyme with one or more cytochrome P450 reductase (CPR) enzymes described herein as a translational fusion or operon. Such polynucleotides may further comprise, in addition to sequences encoding the engineered P450 enzyme, one or more expression control elements. For example, the polynucleotide may comprise one or more promoters or transcriptional enhancers, ribosomal binding sites, transcription termination signals, as expression control elements. The polynucleotide may be inserted within any suitable vector, including an expression vector, and which may be contained within any suitable host cell for expression. The polynucleotide may be designed for introduction and/or protein expression in any suitable host cell, including bacterial cells such as E. coli cells.
In other aspects, the invention provides E. coli host cells expressing the engineered P450 enzyme, either integrated into the genome, or extrachromosomally (e.g., on a plasmid). In some embodiments, the P450 enzyme is expressed by a strong promoter, such as T7, T5, T3, or Trc, or a promoter having promoter strength in E. coli equal to or more than T7, T5, T3, or Trc. The promoter may be a strong constitutive E. coli promoter or a coliphage promoter, or a variant thereof. Deuschle et al., Promoters of Escherichia coli: a hierarchy of in vivo strength indicates alternate structures, EMBO J. 5(11): 2987-2994 (1986); http://parts.igem.org/Promoters/Catalog/Ecoli/Constitutive. When expressed from a plasmid, the plasmid may be a low or high copy number plasmid (e.g., p5, p10, p20). In another embodiment, the P450 gene is chromosomally integrated into the genome of the host E. coli cell, which may further include tandem repeats of the gene to increase the expression level. See US 20110236927, which is hereby incorporated by reference in its entirety.
The E. coli cells may be fed the desired P450 substrate for chemical transformation, or biochemical pathways may be expressed in the host cell to generate the substrate in vivo.
In some embodiments the bacterial host expresses one or more recombinant biosynthetic pathways. For example, an E. coli host cell may express one or more recombinant biosynthetic pathways that include at least 1, at least 2, at least 3, at least 4, or at least 5 recombinant enzymes. The biosynthetic pathways may produce a secondary metabolite through the overexpression of at least 1, at least 2, at least 3, at least 4, or at least 5 foreign genes. In these or other embodiments, the E. coli host cell may overexpress at least 1, at least 2, at least 3, at least 4, or at least 5 E. coli genes. Overexpression of several E. coli genes and/or foreign genes can produce substantial cell stress responses. Where these pathways include one or more P450 enzymes, these stress responses can be substantially higher. For example, as shown herein, P450 enzymes can induce substantial overexpression of the IbpA protein, a protein that is overexpressed under conditions of protein aggregation and cell stress. For example, overexpression of native E. coli genes as well as foreign genes can result in conditions of protein aggregation that induce a cell stress response (e.g., as observed by overexpression of IbpA).
In various embodiments, the invention results in reduced cell stress such that the E. coli cell does not exhibit a substantially stressed phenotype during culturing, or the cell stress is minimized. Cell stress may be assessed by measuring the expression of various cell stress proteins including, but not limited to, IbpA, DnaK, GrpE, and GroL. In some embodiments, methods of the invention do not result in overexpression of the cell stress protein IbpA. In this context, overexpression refers to at least two times the IbpA expression level of the parent strain. In some embodiments, the engineering of the P450 enzyme of the invention may be guided by testing IbpA expression in cultures. For example, a determination of the length of the truncation of the P450 enzyme and/or the anchor size and/or sequence may be guided by IbpA expression levels in the culture.
In some embodiments, at least one foreign gene is expressed by a strong promoter, such as T7, T5, T3, or Trc, or a promoter having promoter strength in E. coli equal to or more than T7, T5, T3, or Trc. The promoter may be a strong constitutive E. coli promoter or a coliphage promoter, or a variant thereof. Deuschle et al., Promoters of Escherichia coli: a hierarchy of in vivo strength indicates alternate structures, EMBO J. 5(11): 2987-2994 (1986); http://parts.igem.org/Promoters/Catalog/Ecoli/Constitutive. In an embodiment, the genes are expressed from a plasmid, which may be a low or high copy number plasmid (e.g., p5, p10, p20). In another embodiment, the genes are chromosomally integrated into the genome of the host E. coli cell, which may further include tandem repeats of the gene to increase the expression level. See US 20110236927, which is hereby incorporated by reference in its entirety.
In some embodiments, the E. coli produces a compound from isopentyl pyrophosphate (IPP) and/or dimethylallyl pyrophosphate DMAPP, such as a terpene or terpenoid compound. In an exemplary embodiment, the E. coli cell may overexpress at least one gene in the MEP pathway, which is endogenous to E. coli. The MEP (2-C-methyl-D-erythritol 4-phosphate) pathway, also called the MEP/DOXP (2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate) pathway or the non-mevalonate pathway or the mevalonic acid-independent pathway refers to the pathway that converts glyceraldehyde-3-phosphate and pyruvate to IPP and DMAPP. In the MEP pathway, pyruvate and D-glyceraldehyde-3-phosphate are converted via a series of reactions to IPP and DMAPP. The pathway typically involves action of the following enzymes: 1-deoxy-D-xylulose-5-phosphate synthase (Dxs), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (IspC), 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (IspD), 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE), 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF), 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (IspG), and isopentenyl diphosphate isomerase (IspH). The MEP pathway, and the genes and enzymes that make up the MEP pathway, are described in US 8,512,988, which is hereby incorporated by reference in its entirety. For example, genes that make up the MEP pathway include dxs, ispC, ispD, ispE, ispF, ispG, ispH, idi, ispA, and ispB. In some embodiments, one or more terpenoid compounds are produced at least in part by metabolic flux through an MEP pathway. In an embodiment, the E. coli host cell may express at least one additional copy of a dxs and/or idi gene. In some embodiments, the E. coli host as at least one additional copy of dxs, ispD, ispF, and/or idi gene, so as to overexpress these gene products.
In various embodiments, the E. coli cell expresses one or more biosynthetic pathways that include at least one membrane-anchored engineered P450 enzyme as described herein. In some embodiments, the P450 enzyme is not strongly expressed, but the more efficient membrane anchoring in accordance with the invention allows for sufficient activity without stronger expression. In various embodiments, the E. coli cell expresses at least 1, at least 2, at least 3, at least 4, or at least 5 P450 enzymes, which may operate in serial fashion in a biosynthetic pathway.
In some embodiments, the P450 enzyme is expressed from a strong (e.g., constitutive or inducible) E. coli or coliphage promoter, or variant thereof (e.g., Trc, T7, T5, or T3, or variant thereof). While overexpression of P450 enzymes can induce significant cell stress, the membrane anchoring system in accordance with the invention renders the P450-membrane association more productive, with less protein misfolding and/or aggregation, which would otherwise induce cell stress.
In various embodiments, the E. coli host cell expresses the engineered P450 enzyme alongside one or more cytochrome P450 reductase (CPR) partner that regenerates the P450 enzyme. As used herein, the term “cytochrome P450 reductase partner” or “CPR partner” refers to a cytochrome P450 reductase capable of regenerating the cofactor component of the cytochrome P450 oxidase of interest for oxidative chemistry. The CPR may be a natural CPR partner for the P450 enzyme, and in other embodiments, the CPR partner is not the natural CPR partner for the P450 enzyme. In nature, cytochrome P450 reductase is a membrane protein generally found in the endoplasmic reticulum. It catalyzes pyridine nucleotide dehydration and electron transfer to membrane bound cytochrome P450s. CPRs may be derived from any species that naturally employs P450 biochemistry, including: Zingiber sp., Barnadesia sp., Hyoscyamus sp., Latuca sp., Nicotiana sp., Citrus sp., Artemesia sp., Arabidopsis sp, Stevia sp., Bacillus sp., Pleurotus sp., Cichorium sp., Helianthus sp., and Physcomitrella sp., Taxus sp., Rosa sp., Cymbopogon sp., Humulus sp., Pogostemon sp., and Cannabis sp. Exemplary CPRs include those from Stevia rebaudiana (e.g., SEQ ID NO: 33, 40, 41, and 42), Arabidopsis thaliana (SEQ ID NO: 34, 37, 38, and 39), Taxus cuspidata (SEQ ID NO: 35), Atemisia annua (SEQ ID NO:36), and Pelargonium graveolans (SEQ ID NO: 43). In various embodiments, the wild-type CPR or derivative thereof is expressed separately from the P450 enzymes (e.g., from the same or different operon), or in some embodiments as a translational fusion with the P450 enzyme. Generally, CPR derivatives comprise amino acid sequences having at least 70%, or at least 80%, or at least 90%, or at least 95% identity to the wild-type sequence (e.g., SEQ ID NOS: 33-43), and which can be employed in the various embodiments.
In an embodiment, the CPR may be expressed as a translational fusion protein with an engineered P450 enzyme. The CPR may be fused to the P450 enzyme through a linker. Exemplary linker sequences can be predominantly serine, glycine, and/or alanine, and may be from three to one hundred amino acids in various embodiments. Linker sequences include, for example, GSG, GSGGGGS (SEQ ID NO: 44), GSGEAAAK (SEQ ID NO: 45), GSGEAAAKEAAAK (SEQ ID NO: 46), and GSGMGSSSN (SEQ ID NO: 47).
In some embodiments, the invention allows for better control of P450 enzyme efficiency, by allowing for efficient ratios of expression of P450 enzymes in relation to the CPR partner (when expressed separately). In some embodiments, the ratio of the expression levels of the P450 enzyme(s) and the CPR partners may range from about 5:1 to about 1:5, for example, about 5:1, or about 4:1, or about 3:1, or about 2:1, or about 1:1, or about 1:2, or about 1:3, or about 1:4, or about 1:5. For example, the ratio of the expression levels of the P450 enzyme(s) and the CPR partner may be from about 2:1 to about 1:2. In various embodiments, the CPR may also be modified to include at least one membrane anchor sequence derived from an E. coli protein as described herein. In an embodiment, the E. coli cell expresses a single CPR protein, and optionally expresses more than one P450 enzyme.
In various embodiments, the E. coli host cell expresses a biosynthetic pathway that produces a secondary metabolite selected from a terpenoid, alkaloid, cannabinoid, steroid, saponin, glycoside, stilbenoid, polyphenol, antibiotic, polyketide, fatty acid, or non-ribosomal peptide. In certain embodiments, the E. coli cell produces one or more terpenoid compounds. A terpenoid, also referred to as an isoprenoid, is an organic chemical derived from a five-carbon isoprene unit (C5). Several non-limiting examples of terpenoids, classified based on the number of isoprene units that they contain, include: hemiterpenoids (1 isoprene unit), monoterpenoids (2 isoprene units), sesquiterpenoids (3 isoprene units), diterpenoids (4 isoprene units), sesterterpenoids (5 isoprene units), triterpenoids (6 isoprene units), tetraterpenoids (8 isoprene units), and polyterpenoids with a larger number of isoprene units. In an embodiment, the E. coli host cell produces a terpenoid selected from a monoterpenoid, a sesquiterpenoid, diterpenoid, a sesterpenoid, or a triterpenoid. Terpenoids represent a diverse class of molecules that provide numerous commercial applications, including in the food and beverage industries as well as the perfume, cosmetic and health care industries. By way of example, terpenoid compounds find use in perfumery (e.g. patchoulol), in the flavor industry (e.g., nootkatone), as sweeteners (e.g., steviol), or therapeutic agents (e.g., taxol) and many are conventionally extracted from plants. Nevertheless, terpenoid molecules are found in ppm levels in nature, and therefore require massive harvesting to obtain sufficient amounts for commercial applications.
Where the chemical species is a terpenoid, the host cell will generally contain a recombinant downstream pathway that produces the terpenoid from IPP and DMAPP precursors. Terpenes such as Monoterpenes (C10), Sesquiterpenes (C15) and Diterpenes (C20) are derived from the prenyl diphosphate substrates, geranyl diphosphate (GPP), farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) respectively through the action of a very large group of enzymes called the terpene (terpenoid) synthases. These enzymes are often referred to as terpene cyclases since the product of the reactions are cyclized to various monoterpene, sesquiterpene and diterpene carbon skeleton products. Many of the resulting carbon skeletons undergo subsequence oxygenation by cytochrome P450 hydrolysase enzymes to give rise to large families of derivatives. In various embodiments, the E. coli cell expresses a biosynthetic pathway involving the overexpression of a geranyl diphosphate synthase (GPS), a gernanylgeranyl diphosphate synthase (GGPS), a farnsesyl diphosphate synthase (FPS), or a farnesyl geranyl diphosphate synthase (FGPPS).
The product of the invention in some embodiments is one or more oxygenated terpenoids. As used herein, the term “oxygenated terpenoid” refers to a terpene scaffold having one or more oxygenation events, producing a corresponding alcohol, aldehyde, carboxylic acid and/or ketone. In some embodiments, the E. coli cell 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, either as a P450 oxygenated product or as a substrate for P450 chemistry.
In another embodiment, the E. coli cell produces Valencene and/or Nootkatone. In such an embodiment, the E. coli cell may express a biosynthetic pathway that further includes a farnesyl pyrophosphate synthase, a Valencene Synthase, and a Valencene Oxidase (the VO comprising the membrane anchor described herein). Farnesyl pyrophosphate synthases (FPPS) produce farnesyl pyrophosphates from iso-pentyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). An exemplary farnesyl pyrophosphate synthase is ERG20 of Saccharomyces cerevisiae (NCBI accession P08524) and E. coli ispA. Valencene synthase produces sesquiterpene scaffolds and are described in, for example, US 2012/0107893, US 2012/0246767, and US 7,273,735, which are hereby incorporated by reference in their entireties.
In an embodiment, the E. coli cell produces steviol or steviol glycoside (e.g., RebM). Steviol is produced from kaurene by the action of two P450 enzymes, kaurene oxidase (KO) and kaurenoic acid hydroxylase (KAH). After production of steviol, various steviol glycoside products may be produced through a series of glycosylation reactions, which can take place in vitro or in vivo. Pathways and enzymes for production of steviol and steviol glycosides are disclosed in US 2013/0171328, US 2012/0107893, WO 2012/075030, WO 2014/122328, which are hereby incorporated by reference in their entireties.
In various embodiments, the E. coli cell may express a biosynthetic pathway involving 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 a single pass transmembrane domain derived from an E. coli gene. In some embodiments, the biosynthetic pathway may further include or more uridine diphosphate dependent glycosyltransferase enzymes (UGT).
Other biosynthetic pathways for production of terpenoid compounds are disclosed in US 8,927,241, which is hereby incorporated by reference in its entirety.
In various embodiments, E. coli cell is cultured to produce the one or more chemical species. For example, the E. coli cell may be cultured to produce one or more terpenoid compounds.
While commercial biosynthesis in E. coli can be limited by the temperature at which overexpressed and/or foreign enzymes are stable, the substantial improvements in stability of the P450 enzymes described herein, may allow for cultures to be maintained at higher temperatures, resulting in higher yields and higher overall productivity. In some embodiments, the culturing is conducted at about 30° C. or greater, about 31° C. or greater, about 32° C. or greater, about 33° C. or greater, about 34° C. or greater, about 35° C. or greater, about 36° C. or greater, or about 37° C. or greater.
The host cells are further suitable for commercial production of chemical species, and therefor can be productive at commercial scale. In some embodiments, the size of the culture is at least about 100 L, at least about 200 L, at least about 500 L, at least about 1,000 L, or at least about 10,000 L. In an embodiment, the culturing may be conducted in batch culture, continuous culture, or semi-continuous culture.
In various embodiments, methods of the invention further include recovering the one or more chemical species such as one or more terpenoid compounds from the cell culture or from cell lysates. In some embodiments, the culture produces at least about 20 mg/L, at least about 25 mg/L, at least about 30 mg/L, at least about 35 mg/L, at least about 40 mg/L, at least about 45 mg/L, at least about 50 mg/L, at least about 55 mg/L, at least about 60 mg/L, at least about 65 mg/L, at least about 70 mg/L, at least about 75 mg/L, at least about 80 mg/L, at least about 85 mg/L, at least about 90 mg/L, at least about 95 mg/L, at least about 100 mg/L, at least about 150 mg/L, or at least about 200 mg/L of the chemical species.
In some embodiments involving the production of a terpenoid compound, the production of indole is used as a surrogate marker for terpenoid production, and/or the accumulation of indole in the culture is controlled to increase terpenoid production. For example, in various embodiments, accumulation of indole in the culture is controlled to below about 100 mg/L, or below about 75 mg/L, or below about 50 mg/L, or below about 25 mg/L, or below about 10 mg/L. The accumulation of indole can be controlled by balancing protein expression and activity using the multivariate modular approach as described in US 8,927,241 (which is hereby incorporated by reference), and/or is controlled by chemical means.
The oxidized product can be recovered by any suitable process, including partitioning the desired product into an organic phase. The production of the desired product can be determined and/or quantified, for example, by gas chromatography (e.g., GC-MS). The desired product can be produced in batch or continuous bioreactor systems. Production of product, recovery, and/or analysis of the product can be done as described in US 2012/0246767, which is hereby incorporated by reference in its entirety. For example, in some embodiments, particularly in relation to terpenoids, oxidized oil is extracted from aqueous reaction medium using an organic solvent, such as an alkane such as heptane, followed by fractional distillation. Terpenoid components of fractions may be measured quantitatively by GC/MS, followed by blending of fractions to generate a desired product profile.
In various embodiments, the recovered chemical species such as one or more terpenoid compounds are incorporated into a product (e.g., a consumer or industrial product). For example, the product may be a flavor product, a fragrance product, a sweetener, a cosmetic, a cleaning product, a detergent or soap, or a pest control product. In some embodiments, the oxygenated product recovered is nootkatol and/or nootkatone, and the product is a flavor product selected from a beverage, a chewing gum, a candy, or a flavor additive, or the product is an insect repellant. In some embodiments, the oxygenated product is steviol or a steviol glycoside, which is provided as a sweetener, or is incorporated into beverages or food products.
The invention further provides methods of making products such as foods, beverages, texturants (e.g., starches, fibers, gums, fats and fat mimetics, and emulsifiers), pharmaceutical products, tobacco products, nutraceutical products, oral hygiene products, and cosmetic products, by incorporating the chemical species produced herein. The higher yields of such species produced in embodiments of the invention can provide significant cost advantages as well as sustainability.

EXAMPLES

Example 1. Identification of Candidate E. Coli Genes With Membrane Anchor Sequences

FIG. 1 illustrates the N-terminal membrane anchor concept. Previously, P450 enzymes were expressed in E. coli by truncation of at least a portion of the P450 N-terminal transmembrane region, with the addition of an 8 amino acid peptide (MALLLAVF) derived from bovine P45017a. See, for example, Barnes HJ, et al., Expression and enzymatic activity of recombinant cytochrome P450 17a-hydroxylase in Escherichia coli, PNAS 88: 5597-5601 (1991); Ajikumar PK, et al., Isoprenoid pathway optimization for taxol precursor overproduction in Escherichia coli, Science 330(6000): 70-74 (2010). However, activity of these P450 constructs in E. coli was generally limiting, making eukaryotes such as yeast the preferred hosts for oxidative transformations involving P450 enzymes. In part, the lack of P450 productivity in E. coli could result from a cell stress response triggered by the non-native membrane anchoring. By engineering P450 proteins to contain an N-terminal anchor derived from an E. coli protein that is natively anchored in the inner membrane by its N-terminus (either by single pass or multi-pass transmembrane helices), this cell stress response could be minimized, and other benefits for the functional expression of P450 enzymes might be identified.
A proteomic analysis was carried out to identify candidate E. coli anchor sequences for functional expression in E. coli. Specifically, the EcoGene 3.0 software was used to identify E. coli inner membrane proteins that include a C-terminus in the cytoplasm. The Phobius predictive tool was used to assess candidate E. coli genes for the presence of signal peptides and/or transmembrane regions. The analysis identified 395 genes which were experimentally determined to be inner membrane proteins. In addition, 85 predicted inner membrane genes were identified. The prediction of transmembrane helices or signal peptides is illustrated in FIG. 2 for three known membrane-anchored P450 enzymes. Stevia rebaudiana Kaurene Oxidase (SrKO) and Arabidopsis thaliana kaurenoic acid 13-hydroxylase (AtKAH) were predicted to include transmembrane domains. The bovine P450-C17 enzyme was predicted to have a N-terminal signal peptide - 8rp.

Example 2. Construction and Functional Analysis of Engineered Valencene Oxidase (VO) Enzymes

Various N-terminal anchors based on single pass E. coli transmembrane regions were constructed and incorporated into Valencene Oxidase (VO) truncated at residue 30. The Valencene Oxidase is derived from Stevia rebaudiana Kaurene Oxidase (SrKO), which has been shown to be functional for oxidative transformation of Valencene to Nootkatone and Nootkatol, which are sesquiterpene compounds. Constructs were cloned into the p5Trc-tc-CPR plasmid which includes a translationally coupled cytochrome P450 reductase (CPR). As a control, the 8rp signal peptide was also incorporated into VO truncated at either residue 20 or residue 30 (8rp-t20 or 8rp-t30).
The engineered VO enzymes with candidate E. coli anchor sequences were expressed in host E. coli cells that produce valencene through a recombinant valencene synthesis pathway. The cells were incubated at 30° C. for 48 hours in 96 dispensing well plates with a dodecane overlayer. As shown in FIG. 3 , many of the E. coli anchored enzymes outperformed the 8rp-t20 and 8rp-t30 constructs in both total terpenoid flux and oxygenated product formation.
Lead VO constructs with E. coli anchor sequences were assessed. The constructs were expressed in host E. coli cells which were incubated at 30° C. for 48 hours in 24 dispensing well plates with a vegetable oil overlay. Consistent with previous results, the E. coli anchored enzymes outperformed the 8rp-t20 and 8rp-t30 constructs in both total terpenoid flux and oxygenated product formation (FIG. 4 ).
The effects of increased expression of engineered P450 constructs on E. coli cells were evaluated. Specifically, VO constructs with lead E. coli anchor sequences were cloned into a p10 plasmid and expressed in valencene-producing E. coli cells which were incubated at 30° C. for 48 hours in 24 dispensing well plates with a vegetable oil overlay. As shown in FIG. 5 , overexpression of 8rp anchored P450 enzymes (from the p10 plasmid) resulted in reduced activity including a three-fold loss in the formation of oxygenated terpenoids compared to a weaker expressed enzyme (from a p5 plasmid). In contrast, the P450 enzymes with E. coli anchors (particularly yhcB and ZipA) showed only a modest loss of oxygenated product when expressed from a p10 plasmid. Further, P450 enzymes with E. coli anchors expressed from a p10 plasmid produced as much product as the 8rp anchored enzyme expressed from a p5 plasmid. Accordingly, the E. coli anchored P450 enzymes allowed for high functional expression in E. coli cells.
Functional expression with the CPR partner (from Stevia) was also assessed. In particular, the CPR enzyme was expressed in E. coli cells with the P450 enzymes either in the same operon or translationally coupled from one plasmid. The cells were incubated at 30° C. for 48 hours in 24 dispensing well plates with a vegetable oil overlay. It was observed that membrane anchors based on native E. coli sequences (e.g., yhcB) provided increased oxygenated titer when translationally coupled to a CPR partner or when expressed separately (FIG. 6 ). In addition, cells with the E. coli anchors grew to a higher final OD and with less emulsion, suggesting that the cells are less stressed, despite overexpressing an upstream MEP pathway and downstream terpenoid biosynthesis (Nootkatone) pathway.
During translation initiation, N-terminal sequences can significantly affect the expression of a protein. As such, a comparison was conducted to examine VO expression and VO/CPR ratio with different N-terminal anchor regions. As shown in FIG. 7 , four anchors tested (yhcB, yhhm, zipA, and ypfN) showed lower total VO expression compared to the control 8rp anchor, allowing for a five-fold lower expression relative to CPR expression. This ratio is important for efficient P450 electron transfer without deleterious effects on coupling efficiency.
A proteomics approach was undertaken to assess cellular stress response to variable P450 expression. For example, IbpA, which is overexpressed in E. coli under conditions of high protein aggregation and stress, is strongly expressed with the 8rp anchored P450 enzyme, but not with the enzymes with E. coli anchors (FIG. 8 ).
The yhcB-anchored VO enzyme was further analyzed to determine the impact of anchor and P450 truncation length on overall productivity (FIG. 9 ). Variants with truncations in anchor and P450 sequences were generated using the GeneArt technology and subcloned into a p5T7-BCD18 screening plasmid. A list of the variants is provided in Table 2.

TABLE 2

Truncation Optimization
n20yhcB_t28VOR1	n22yhcB t31VOR1
n20yhcB_t29VOR1	n22yhcB_t32VOR1
n20yhcB_t30VOR1	n23yhcB_t28VOR1
n20yhcB_t31VOR1	n23yhcB_t29VOR1
n20yhcB_t32VOR1	n23yhcB_t30VOR1
n21yhcB_t28VOR1	n23yhcB_t31VOR1
n21yhcB_t29VORl	n23yhcB_t32VOR1
n21yhcB_t30VOR1	n24yhcB_t28VOR1
n21yhcB_t31VOR1	n24yhcB_t29VOR1
n21yhcB_t32VOR1	n24yhcB_t30VOR1
n22yhcB_t28VOR1	n24yhcB_t31VOR1
n22yhcB_t29VOR1	n24yhcB_t32VOR1

The variants were assayed for product formation in a MEP/FPPS/VS integrated E. coli strain. The E. coli cells were incubated at 30° C. for 48 hours in 96 dispensing well plates with a dodecane overlay. Products were subsequently extracted with ethyl acetate. As shown in FIG. 10 , modification of the anchor and P450 truncation lengths has only modest impact on overall productivity. For example, the n20yhcB-t29VO1 construct yielded an approximately 1.2-fold increase in total terpenoid flux and oxygenated products. These results suggest that the E. coli anchored P450 enzymes are relatively robust to changes in anchor and truncation length.

Example 3. Construction and Functional Analysis of Additional Engineered P450 Enzymes

Modified Arabidopsis thaliana kaurenoic acid 13- hydroxylase (AtKAH) and Stevia rebaudiana Kaurene Oxidase (SrKO) enzymes with E. coli anchor sequences were generated.
Engineered enzymes were produced with the AtKAH enzymes truncated at position 26. The N-terminus of the truncated AtKAH enzyme is shown below:
t25 AtKAH HVYGRAVVEQWR
t26 AtKAH VYGRAVVEQWR
The E(z) scoring system was used to guide truncation of the E. coli anchor sequence by similarity of scoring to the parent protein (FIG. 11 ). Eight combinations of E. coli anchored N-terminal segments and AtKAH truncation lengths were selected for in vivo screening.
E. coli membrane anchors (e.g., zipA, yhhm, and yhcB) were also incorporated into SrKO for production of kaurenoic acid in E. coli. The N-terminus of the truncated SrKO enzyme is shown below:
t29 SrVO EWYLKSYTSARR
t30 SrVO WYLKSYTSARR
The constructs were cloned into the p5Trc plasmid which include an operon expressed CPR. The E. coli cells were incubated at either 30° C. or 34° C. for 72 hours in 96 dispensing well plates. Similar to results obtained with the engineered VO enzymes, variant SrKO enzymes with E. coli anchor sequences also showed improved activity compared to enzymes with 8rp anchor sequences. Particularly, as shown in FIG. 12A, the zipA anchored P450 enzyme produced the highest kaurenoic acid titer (about 5 mg/L). In addition, all SrKO variants showed a drop in activity at 34° C. FIG. 12B shows that the final OD is higher in all variants except zipA when incubated at 34° C.
A proteomics analysis was undertaken to examine the relative abundance of various pathway and stress response proteins in cells expressing SrKO. Both soluble and insoluble cell fractions were analyzed by tryptic digest proteomics. As shown in FIG. 13 , SrKO enzymes with either E. coli anchors zipA and yhcB or control anchor 8rP all showed higher expressions than full-length SrKO at 30° C. and 34° C. Similar results were seen in the insoluble fractions, including higher expression of the upstream pathway enzyme dxs. At the higher temperature of 34° C., there was also an increase in the stress response gene IbpA in the insoluble fractions.
In summary, P450 enzymes with E. coli anchor sequences showed improvement in total terpenoid flux and oxygenated terpenoid formation, and provided higher catalytic efficiency and reduced stress response, as compared to 8rp anchored P450 enzymes. This N-terminal engineering approach is broadly applicable to E. coli cells having several overexpressed genes, including complete biosynthetic pathways, and including one or multiple P450 enzymes, where managing cell stress is crucial to obtaining commercial yields.

SEQUENCES

P450 Enzymes

>ZzHO [Zingiber zerumbet]

MEAISLFSPFFFITLFLGFFITLLIKRSSRSSVHKQQVLLASLPPSPPRL

PLIGNIHQLVGGNPHRILLQLARTHGPLICLRLGQVDQVVASSVEAVEEI

IKRHDLKFADRPRDLTFSRIFFYDGNAVVMTPYGGEWKQMRKIYAMELLN

SRRVKSFAAIREDVARKLTGEIAHKAFAQTPVINLSEMVMSMINAIVIRV

AFGDKCKQQAYFLHLVKEAMSYVSSFSVADMYPSLKFLDTLTGLKSKLEG

VHGKLDKVFDEIIAQRQAALAAEQAEEDLIIDVLLKLKDEGNQEFPITYT

SVKAIVMEIFLAGTETSSSVIDWVMSELIKNPKAMEKVQKEMREAMQGKT

KLEESDIPKFSYLNLVIKETLRLHPPGPLLFPRECRETCEVMGYRVPAGA

RLLINAFALSRDEKYWGSDAESFKPERFEGISVDFKGSNFEFMPFGAGRR

ICPGMTFGISSVEVALAHLLFHFDWQLPQGMKIEDLDMMEVSGMSATRRS

PLLVLAKLIIPLP (SEQ ID NO: 1)

>BsGAO [Barnadesia spinosa]

MELTLTTSLGLAVFVFILFKLLTGSKSTKNSLPEAWRLPIIGHMHHLVGT

LPHRGVTDMARKYGSLMHLQLGEVSTIVVSSPRWAKEVLTTYDITFANRP

ETLTGEIVAYHNTDIVLSPYGEYWRQLRKLCTLELLSAKKVKSFQSLREE

ECWNLVKEVRSSGSGSPVDLSESIFKLIATILSRAAFGKGIKDQREFTEI

VKEILRLTGGFDVADIFPSKKILHHLSGKRAKLTNIHNKLDSLINNIVSE

HPGSRTSSSQESLLDVLLRLKDSAELPLTSDNVKAVILDMFGAGTDTSSA

TIEWAISELIRCPRAMEKVQTELRQALNGKERIQEEDIQELSYLKLVIKE

TLRLHPPLPLVMPRECREPCVLAGYEIPTKTKLIVNVFAINRDPEYWKDA

ETFMPERFENSPINIMGSEYEYLPFGAGRRMCPGAALGLANVELPLAHIL

YYFNWKLPNGARLDELDMSECFGATVQRKSELLLVPTAYKTANNSA (SE

Q ID NO: 2)

>HmPO [Hyoscyamus muticus]

MQFFSLVSIFLFLSFLFLLRKWKNSNSQSKKLPPGPWKLPLLGSMLHMVG

GLPHHVLRDLAKKYGPLMHLQLGEVSAVVVTSPDMAKEVLKTHDIAFASR

PKLLAPEIVCYNRSDIAFCPYGDYWRQMRKICVLEVLSAKNVRSFSSIRR

DEVLRLVNFVRSSTSEPVNFTERLFLFTSSMTCRSAFGKVFKEQETFIQL

IKEVIGLAGGFDVADIFPSLKFLHVLTGMEGKIMKAHHKVDAIVEDVINE

HKKNLAMGKTNGALGGEDLIDVLLRLMNDGGLQFPITNDNIKAIIFDMFA

AGTETSSSTLVWAMVQMMRNPTILAKAQAEVREAFKGKETFDENDVEELK

YLKLVIKETLRLHPPVPLLVPRECREETEINGYTIPVKTKVMVNVWALGR

DPKYWDDADNFKPERFEQCSVDFIGNNFEYLPFGGGRRICPGISFGLANV

YLPLAQLLYHFDWKLPTGMEPKDLDLTELVGVTAARKSDLMLVATPYQPS

RE (SEQ ID NO: 3)

>LsGAO [Lactuca sativa]

MELSITTSIALATIVFFLYKLATRPKSTKKQLPEASRLPIIGHMHHLIGT

MPHRGVMDLARKHGSLMHLQLGEVSTIVVSSPKWAKEILTTYDITFANRP

ETLTGEIIAYHNTDIVLAPYGEYWRQLRKLCTLELLSVKKVKSFQSIREE

ECWNLVKEVKESGSGKPINLSESIFTMIATILSRAAFGKGIKDQREFTEI

VKEILRQTGGFDVADIFPSKKFLHHLSGKRARLTSIHKKLDNLINNIVAE

HHVSTSSKANETLLDVLLRLKDSAEFPLTADNVKAIILDMFGAGTDTSSA

TVEWAISELIRCPRAMEKVQAELRQALNGKEKIQEEDIQDLAYLNLVIRE

TLRLHPPLPLVMPRECREPVNLAGYEIANKTKLIVNVFAINRDPEYWKDA

EAFIPERFENNPNNIMGADYEYLPFGAGRRMCPGAALGLANVQLPLANIL

YHFNWKLPNGASHDQLDMTESFGATVQRKTELLLVPSF (SEQ ID NO:

4)

>NtEAO [Nicotiani tabacum]

MQFFSLVSIFLFLSFLFLLRKWKNSNSQSKKLPPGPWKIPILGSMLHMIG

GEPHHVLRDLAKKYGPLMHLQLGEISAVVVTSRDMAKEVLKTHDVVFASR

PKIVAMDIICYNQSDIAFSPYGDHWRQMRKICVMELLNAKNVRSFSSIRR

DEVVRLIDSIRSDSSSGELVNFTQRIIWFASSMTCRSAFGQVLKGQDIFA

KKIREVIGLAEGFDVVDIFPTYKFLHVLSGMKRKLLNAHLKVDAIVEDVI

NEHKKNLAAGKSNGALGGEDLIDVLLRLMNDTSLQFPITNDNIKAVIVDM

FAAGTETSSTTTVWAMAEMMKNPSVFTKAQAEVREAFRDKVSFDENDVEE

LKYLKLVIKETLRLHPPSPLLVPRECREDTDINGYTIPAKTKVMVNVWAL

GRDPKYWDDAESFKPERFEQCSVDFFGNNFEFLPFGGGRRICPGMSFGLA

NLYLPLAQLLYHFDWKLPTGIMPRDLDLTELSGITIARKGGLYLNATPYQ

PSRE (SEQ ID NO: 5)

>CpVO [Citrus x paradisi]

MELPLKSIALTIVIVTVLTWAWRVLNWVWLRPKKLEKFLRQQGLKGNSYR

LLFGDLKENSIELKEAKARPLSLDDDIAIRVNPFLHKLVNDYGKNSFMWF

GPTPRVNIMNPDQIKAIFTKINDFQKVNSIPLARLLIVGLATLEGEKWAK

HRKLINPAFHQEKLKLMLPAFYLSCIEIITKWEKQMSVEGSSELDVWPYL

ANLTSDVISRTAFGSSYEEGRRIFQLQAELAELTMQVFRSVHIPGWRFLP

TKRNRRMKEIDKEIRASLMGIIKNREKAMRAGEAANNDLLGILMETSFRE

IEEHGNNKNVGFSMNDVIEECKLFYFAGQETTSVLLNWTMVLLSKHQDWQ

ERARQEVLQVFGNNKPDYDGLNHLKIVQMILYEVLRLYPPVTVLSRAVFK

ETKLGNLTLPAGVQIGLPMILVHQDPELWGDDAVEFKPERFAEGISKAAK

NQVSYFPFALGPRICVGQNFALVEAKMATAMILQNYSFELSPSYVHAPTA

VPTLHPELGTQLILRKLWCKNN (SEQ IDNO: 6)

>AaAO [Artemesia annua]

MKSILKAMALSLTTSIALATILLFVYKFATRSKSTKKSLPEPWRLPIIGH

MHHLIGTTPHRGVRDLARKYGSLMHLQLGEVPTIVVSSPKWAKEILTTYD

ITFANRPETLTGEIVLYHNTDVVLAPYGEYWRQLRKICTLELLSVKKVKS

FQSLREEECWNLVQEIKASGSGRPVNLSENVFKLIATILSRAAFGKGIKD

QKELTEIVKEILRQTGGFDVADIFPSKKFLHHLSGKRARLTSLRKKIDNL

IDNLVAEHTVNTSSKTNETLLDVLLRLKDSAEFPLTSDNIKAIILDMFGA

GTDTSSSTIEWAISELIKCPKAMEKVQAELRKALNGKEKIHEEDIQELSY

LNMVIKETLRLHPPLPLVLPRECRQPVNLAGYNIPNKTKLIVNVFAINRD

PEYWKDAEAFIPERFENSSATVMGAEYEYLPFGAGRRMCPGAALGLANVQ

LPLANILYHFNWKLPNGVSYDQIDMTESSGATMQRKTELLLVPSF (SEQ

ID NO: 7)

>AtKO [Arabidopsis thaliana]

MAFFSMISILLGFVISSFIFIFFFKKLLSFSRKNMSEVSTLPSVPVVPGF

PVIGNLLQLKEKKPHKTFTRWSEIYGPIYSIKMGSSSLIVLNSTETAKEA

MVTRFSSISTRKLSNALTVLTCDKSMVATSDYDDFHKLVKRCLLNGLLGA

NAQKRKRHYRDALIENVSSKLHAHARDHPQEPVNFRAIFEHELFGVALKQ

AFGKDVESIYVKELGVTLSKDEIFKVLVHDMMEGAIDVDWRDFFPYLKWI

PNKSFEARIQQKHKRRLAVMNALIQDRLKQNGSESDDDCYLNFLMSEAKT

LTKEQIAILVWETIIETADTTLVTTEWAIYELAKHPSVQDRLCKEIQNVC

GGEKFKEEQLSQVPYLNGVFHETLRKYSPAPLVPIRYAHEDTQIGGYHVP

AGSEIAINIYGCNMDKKRWERPEDWWPERFLDDGKYETSDLHKTMAFGAG

KRVCAGALQASLMAGIAIGRLVQEFEWKLRDGEEENVDTYGLTSQKLYPL

MAIINPRRS (SEQ ID NO: 8)

>SrKO [Stevia rebaudiana]

MDAVTGLLTVPATAITIGGTAVALAVALIFWYLKSYTSARRSQSNHLPRV

PEVPGVPLLGNLLQLKEKKPYMTFTRWAATYGPIYSIKTGATSMVVVSSN

EIAKEALVTRFQSISTRNLSKALKVLTADKTMVAMSDYDDYHKTVKRHIL

TAVLGPNAQKKHRIHRDIMMDNISTQLHEFVKNNPEQEEVDLRKIFQSEL

FGLAMRQALGKDVESLYVEDLKITMNRDEIFQVLVVDPMMGAIDVDWRDF

FPYLKWVPNKKFENTIQQMYIRREAVMKSLIKEHKKRIASGEKLNSYIDY

LLSEAQTLTDQQLLMSLWEPIIESSDTTMVTTEWAMYELAKNPKLQDRLY

RDIKSVCGSEKITEEHLSQLPYITAIFHETLRRHSPVPIIPLRHVHEDTV

LGGYHVPAGTELAVNIYGCNMDKNVWENPEEWNPERFMKENETIDFQKTM

AFGGGKRVCAGSLQALLTASIGIGRMVQEFEWKLKDMTQEEVNTIGLTTQ

MLRPLRAIIKPRI (SEQ ID NO: 9)

>PpKO [Physcomitrella patens]

MAKHLATQLLQQWNEALKTMPPGFRTAGKILVWEELASNKVLITIALAWV

LLFVARTCLRNKKRLPPAIPGGLPVLGNLLQLTEKKPHRTFTAWSKEHGP

IFTIKVGSVPQAVVNNSEIAKEVLVTKFASISKRQMPMALRVLTRDKTMV

AMSDYGEEHRMLKKLVMTNLLGPTTQNKNRSLRDDALIGMIEGVLAELKA

SPTSPKVVNVRDYVQRSLFPFALQQVFGYIPDQVEVLELGTCVSTWDMFD

ALVVAPLSAVINVDWRDFFPALRWIPNRSVEDLVRTVDFKRNSIMKALIR

AQRMRLANLKEPPRCYADIALTEATHLTEKQLEMSLWEPIIESADTTLVT

SEWAMYEIAKNPDCQDRLYREIVSVAGTERMVTEDDLPNMPYLGAIIKET

LRKYTPVPLIPSRFVEEDITLGGYDIPKGYQILVNLFAIANDPAVWSNPE

KWDPERMLANKKVDMGFRDFSLMPFGAGKRMCAGITQAMFIIPMNVAALV

QHCEWRLSPQEISNINNKIEDVVYLTTHKLSPLSCEATPRISHRLP (SE

Q ID NO: 10)

>BmVO [Bacillus megaterium]

MTIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVT

RYLSSQRLIKEACDESRFDKNLSQALKFVRDFAGDGLATSWTHEKNWKKA

HNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVPEDMTRLT

LDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYD

ENKRQFQEDIKVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPL

DDENIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKAAEEAARVLV

DPVPSYKQVKQLKYVGMVLNEALRLWPTIPAFSLYAKEDTVLGGEYPLEK

GDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRA

CIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLTLKPEGFVVKAK

SKKIPLGGIPSPSTEQSAKKVRKKAENAHNTPLLVLYGSNMGTAEGTARD

LADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKQFVD

WLDQASADEVKGVRYSVFGCGDKNWATTYQKVPAFIDETLAAKGAENIAD

RGEADASDDFEGTYEEWREHMWSDVAAYFNLDIENSEDNKSTLSLQFVDS

AADMPLAKMHGAFSTNVVASKELQQPGSARSTRHLEIELPKEASYQEGDH

LGVIPRNYEGIVNRVTARFGLDASQQIRLEAEEEKLAHLPLAKTVSVEEL

LQYVELQDPVTRTQLRAMAAKTVCPPHKVELEALLEKQAYKEQVLAKRLT

MLELLEKYPACEMKFSEFIALLPSIRPRYYSISSSPRVDEKQASITVSVV

SGEAWSGYGEYKGIASNYLAELQEGDTITCFISTPQSEFTLPKDPETPLI

MVGPGTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEEL

ENAQSEGIITLHTAFSRMPNQPKTYVQHVMEQDGKKLIELLDQGAHFYIC

GDGSQMAPAVEATLMKSYADVHQVSEADARLWLQQLEEKGRYAKDVWAG

(SEQ IDNO: 11)

>PsVO [Pleurotus sapidus]

MRYGCAAVALFYLTAMGKLHPLAIIPDYKGSMAASVTIFNKRTNPLDISV

NQANDWPWRYAKTCVLSSDWALHEMIIHLNNTHLVEEAVIVAAQRKLSPS

HIVFRLLEPHWVVTLSLNALARSVLIPEVIVPIAGFSAPHIFQFIRESFT

NFDWKSLYVPADLESRGFPVDQLNSPKFHNYAYARDINDMWTTLKKFVSS

VLQDAQYYPDDASVAGDTQIQAWCDEMRSGMGAGMTNFPESITTVDDLVN

MVTMCIHIAAPQHTAVNYLQQYYQTFVSNKPSALFSPLPTSIAQLQKYTE

SDLMAALPLNAKRQWLLMAQIPYLLSMQVQEDENIVTYAANASTDKDPII

ASAGRQLAADLKKLAAVFLVNSAQLDDQNTPYDVLAPEQLANAIVI(SEQ

ID NO: 12)

>PoLO [Pleurotus ostreatus]

MAPTMSLSRSALKNVHLPYMVQHPEPTDCSTAMKHAAEGYDRARQMIAFL

FDILDYESSVPQKFTPEEKKEKYTWSHSDKFPPHLAIIPEDIDVPAYIIF

SIVRLVQTLSIMSGIQCNERLAPGPEQNTMEKLTKWNAERHKNQGWVKDM

FNEPNIGLRNDWYTDAVFAQQFFTGPNPTTITLASDTWMKAFTEEAASQG

KRDLISLFRSAPPNSFYVQDFSDFRARMGAKPDEELCATSDGGVTRYGCA

AVALFYLPPTGELHPLAIVPDYKGSMAASITLFNKRVDPSDASVDQANDW

PWRYAKTCVLSADWVLHEMIIHLNNTHLVQEAVIVAVQRTLPDSHIVFRL

LKPHWVVTLSLNAQARSVLIPEVIVPIAGFSELRIFQFVGHAFTNFDWKA

LYVPTDLEFRGFPLDRLDDDKFHNYAYAKDIKDMWMALRKFVSSVLKDGK

YYPDDSAVAADAQIQDWCDEMRSEKGAGMKKFPESISTLDDLIDMVTMCI

HIAAPQHTAVNYLQQYYQTFVPNKPSALFSPLPTLLSQLESYTESDLMAA

LPLGAKQEWLLMAQVPYLLSKEVEQDGNIVTYAGTASNNEDPIIAAAGKE

LSADLVILAGVFLKNSEKLDDQNTAYNVLAPDQLANAIVI (SEQ ID N

O: 13)

>CiVO [Cichorium intybus]

MEISIPTTLGLAVIIFIIFKLLTRTTSKKNLLPEPWRLPIIGHMHHLIGT

MPHRGVMELARKHGSLMHLQLGEVSTIVVSSPRWAKEVLTTYDITFANRP

ETLTGEIVAYHNTDIVLAPYGEYWRQLRKLCTLELLSNKKVKSFQSLREE

ECWNLVKDIRSTGQGSPINLSENIFKMIATILSRAAFGKGIKDQMKFTEL

VKEILRLTGGFDVADIFPSKKLLHHLSGKRAKLTNIHNKLDNLINNIIAE

HPGNRTSSSQETLLDVLLRLKESAEFPLTADNVKAVILDMFGAGTDTSSA

TIEWAISELIRCPRAMEKVQTELRQALNGKERIQEEDLQELNYLKLVIKE

TLRLHPPLPLVMPRECREPCVLGGYDIPSKTKLIVNVFAINRDPEYWKDA

ETFMPERFENSPITVMGSEYEYLPFGAGRRMCPGAALGLANVELPLAHIL

YFNWKLPNGKTFEDLDMTESFGATVQRKTELLLVPTDFQTLTAST (SEQ

ID NO: 14)

>HaGAO [Helianthus annuus]

MEVSLTTSIALATIVFFLYKLLTRPTSSKNRLPEPWRLPIIGHMHHLIGT

MPHRGVMDLARKYGSLMHLQLGEVSAIVVSSPKWAKEILTTYDIPFANRP

ETLTGEIIAYHNTDIVLAPYGEYWRQLRKLCTLELLSVKKVKSFQSLREE

ECWNLVQEIKASGSGTPFNLSEGIFKVIATVLSRAAFGKGIKDQKQFTEI

VKEILRETGGFDVADIFPSKKFLHHLSGKRGRLTSIHNKLDSLINNLVAE

HTVSKSSKVNETLLDVLLRLKNSEEFPLTADNVKAIILDMFGAGTDTSSA

TVEWAISELIRCPRAMEKVQAELRQALNGKERIKEEEIQDLPYLNLVIRE

TLRLHPPLPLVMPRECRQAMNLAGYDVANKTKLIVNVFAINRDPEYWKDA

ESFNPERFENSNTTIMGADYEYLPFGAGRRMCPGSALGLANVQLPLANIL

YYFKWKLPNGASHDQLDMTESFGATVQRKTELMLVPSF (SEQ ID NO:

15)

E. Coli Inner Membrane Proteins

>waaA [Escherichia coli]

MLELLYTALLYLIQPLIWIRLWVRGRKAPAYRKRWGERYGFYRHPLKPGG

IMLHSVSVGETLAAIPLVRALRHRYPDLPITVTTMTPTGSERVQSAFGKD

VQHVYLPYDLPDALNRFLNKVDPKLVLIMETELWPNLIAALHKRKIPLVI

ANARLSARSAAGYAKLGKFVRRLLRRITLIAAQNEEDGARFVALGAKNNQ

VTVTGSLKFDISVTPQLAAKAVTLRRQWAPHRPVWIATSTHEGEESVVIA

AHQALLQQFPNLLLILVPRHPERFPDAINLVRQAGLSYITRSSGEVPSTS

TQVVVGDTMGELMLLYGIADLAFVGGSLVERGGHNPLEAAAHAIPVLMGP

HTFNFKDICARLEQASGLITVTDATTLAKEVSSLLTDADYRSFYGRHAVE

VLYQNQGALQRLLQLLEPYLPPKTH (SEQ ID NO: 16)

>ypfN [Escherichia coli]
MDWLAKYWWILVIVFLVGVLLNVIKDLKRVDHKKFLANKPELPPHRDFND

KWDDDDDWPKKDQPKK(SEQ ID NO: 17)
>yhcB [Escherichia coli]

MTWEYALIGLVVGIIIGAVAMRFGNRKLRQQQALQYELEKNKAELDEYRE

ELVSHFARSAELLDTMAHDYRQLYQHMAKSSSSLLPELSAEANPFRNRLA

ESEASNDQAPVQMPRDYSEGASGLLRTGAKRD(SEQ ID NO: 18)

>yhbM [Escherichia coli]

MKPFLRWCFVATALTLAGCSNTSWRKSEVLAVPLQPTLQQEVILARMEQI

LASRALTDDERAQLLYERGVLYDSLGLRALARNDFSQALAIRPDMPEVFN

YLGIYLTQAGNFDAAYEAFDSVLELDPTYNYAHLNRGIALYYGGRDKLAQ

DDLLAFYQDDPNDPFRSLWLYLAEQKLDEKQAKEVLKQHFEKSDKEQWGW

NIVEFYLGNISEQTLMERLKADATDNTSLAEHLSETNFYLGKYYLSLGDL

DSATALFKLAVANNVHNFVEHRYALLELSLLGQDQDDLAESDQQ (SEQ

ID NO: 19)

>yhhm [Escherichia coli]

MSKPPLFFIVIIGLIVVAASFRFMQQRREKADNDMAPLQQKLVVVSNKRE

KPINDRRSRQQEVTPAGTSIRYEASFKPQSGGMEQTFRLDAQQYHALTVG

DKGTLSYKGTRFVSFVGEQ (SEQ ID NO:20)

>zipA [Escherichia coli]

MMQDLRLILIIVGAIAIIALLVHGFWTSRKERSSMFRDRPLKRMKSKRDD

DSYDEDVEDDEGVGEVRVHRVNHAPANAQEHEAARPSPQHQYQPPYASAQ

PRQPVQQPPEAQVPPQHAPHPAQPVQQPAYQPQPEQPLQQPVSPQVAPAP

QPVHSAPQPAQQAFQPAEPVAAPQPEPVAEPAPVMDKPKRKEAVIIMNVA

AHHGSELNGELLLNSIQQAGFIFGDMNIYHRHLSPDGSGPALFSLANMVK

PGTFDPEMKDFTTPGVTIFMQVPSYGDELQNFKLMLQSAQHIADEVGGVV

LDDQRRMMTPQKLREYQDIIREVKDANA(SEQ ID NO: 21)

>ycgG [Escherichia coli]

MRNTLIPILVAICLFITGVAILNIQLWYSAKAEYLAGARYAANNINHILE

EASQATQTAVNIAGKECNLEEQYQLGTEAALKPHLRTIIILKQGIVWCTS

LPGNRVLLSRIPVFPDSNLLLAPAIDTVNRLPILLYQNQFADTRILVTIS

DQHIRGALNVPLKGVRYVLRVADDIIGPTGDVMTLNGHYPYTEKVHSTKY

HFTIIFNPPPLFSFYRLIDKGFGILIFILLIACAAAFLLDRYFNKSATPE

EILRRAINNGEIVPFYQPVVNGREGTLRGVEVLARWKQPHGGYISPAAFI

PLAEKSGLIVPLTQSLMNQVARQMNAIASKLPEGFHIGINFSASHIISPT

FVDECLNFRDSFTRRDLNLVLEVTEREPLNVDESLVQRLNILHENGFVIA

LDDFGTGYSGLSYLHDLHIDYIKIDHSFVGRVNADPESTRILDCVLDLAR

KLSISIVAEGVETKEQLDYLNQNYITFQQGYYFYKPVTYIDLVKIILSKP

KVKVVVE (SEQ ID NO: 22)

>djlA [Escherichia coli]

MQYWGKIIGVAVALLMGGGFWGVVLGLLIGHMFDKARSRKMAWFANQRER

QALFFATTFEVMGHLTKSKGRVTEADIHIASQLMDRMNLHGASRTAAQNA

FRVGKSDNYPLREKMRQFRSVCFGRFDLIRMFLEIQIQAAFADGSLHPNE

RAVLYVIAEELGISRAQFDQFLRMMQGGAQFGGGYQQQTGGGNWQQAQRG

PTLEDACNVLGVKPTDDATTIKRAYRKLMSEHHPDKLVAKGLPPEMMEMA

KQKAQEIQQAYELIKQQKGFK(SEQ ID NO: 23)

>sohB [Escherichia coli]

MELLSEYGLFLAKIVTVVLAIAAIAAIIVNVAQRNKRQRGELRVNNLSEQ

YKEMKEELAAALMDSHQQKQWHKAQKKKHKQEAKAAKAKAKLGEVATDSK

PRVWVLDFKGSMDAHEVNSLREEITAVLAAFKPQDQVVLRLESPGGMVHG

YGLAASQLQRLRDKNIPLTVTVDKVAASGGYMMACVADKIVSAPFAIVGS

IGVVAQMPNFNRFLKSKDIDIELHTAGQYKRTLTLLGENTEEGREKFREE

LNETHQLFKDFVKRMRPSLDIEQVATGEHWYGQQAVEKGLVDEINTSDEV

ILSLMEGREVVNVRYMQRKRLIDRFTGSAAESADRLLLRWWQRGQKPLM(

SEQ ID NO: 24)

>1pxK [Escherichia coli]

MIEKIWSGESPLWRLLLPLSWLYGLVSGAIRLCYKLKLKRAWRAPVPVVV

VGNLTAGGNGKTPVVVWLVEQLQQRGIRVGVVSRGYGGKAESYPLLLSAD

TTTAQAGDEPVLIYQRTDAPVAVSPVRSDAVKAILAQHPDVQIIVTDDGL

QHYRLARDVEIVVIDGVRRFGNGWWLPAGPMRERAGRLKSVDAVIVNGGV

PRSGEIPMHLLPGQAVNLRTGTRCDVAQLEHVVAMAGIGHPPRFFATLKM

CGVQPEKCVPLADHQSLNHADVSALVSAGQTLVMTEKDAVKCRAFAEENW

WYLPVDAQLSGDEPAKLLTQLTLLASGN(SEQ ID NO: 25)

>fliO [Escherichia coli]

MNNHATVQSSAPVSAAPLLQVSGALIAIIALILAAAWLVKRLGFAPKRTG

VNGLKISASASLGARERVVVVDVEDARLVLGVTAGQINLLHKLPPSAPTE

EIPQTDFQSVMKNLLKRSGRS (SEQ ID NO:26)

>motA [Escherichia coli]

MLILLGYLVVLGTVFGGYLMTGGSLGALYQPAELVIIAGAGIGSFIVGNN

GKAIKGTLKALPLLFRRSKYTKAMYMDLLALLYRLMAKSRQMGMFSLERD

IENPRESEIFASYPRILADSVMLDFIVDYLRLIISGHMNTFEIEALMDEE

IETHESEAEVPANSLALVGDSLPAFGIVAAVMGVVHALGSADRPAAELGA

LIAHAMVGTFLGILLAYGFISPLATVLRQKSAETSKMMQCVKVTLLSNLN

GYAPPIAVEFGRKTLYSSERPSFIELEEHVRAVKNPQQQTTTEEA (SEQ

ID NO: 27)

>htpx [Escherichia coli]

MMRIALFLLTNLAVMVVFGLVLSLTGIQSSSVQGLMIMALLFGFGGSFVS

LLMSKWMALRSVGGEVIEQPRNERERWLVNTVATQARQAGIAMPQVAIYH

APDINAFATGARRDASLVAVSTGLLQNMSPDEAEAVIAHEISHIANGDMV

TMTLIQGVVNTFVIFISRILAQLAAGFMGGNRDEGEESNGNPLIYFAVAT

VLELVFGILASIITMWFSRHREFHADAGSAKLVGREKMIAALQRLKTSYE

PQEATSMMALCINGKSKSLSELFMTHPPLDKRIEALRTGEYLK (SEQ I

D NO: 28)

>pgaC [Escherichia coli]

MINRIVSFFILCLVLCIPLCVAYFHSGELMMRFVFFWPFFMSIMWIVGGV

YFWVYRERHWPWGENAPAPQLKDNPSISIIIPCFNEEKNVEETIHAALAQ

RYENIEVIAVNDGSTDKTRAILDRMAAQIPHLRVIHLAQNQGKAIALKTG

AAAAKSEYLVCIDGDALLDRDAAAYIVEPMLYNPRVGAVTGNPRIRTRST

LVGKIQVGEYSSIIGLIKRTQRIYGNVFTVSGVIAAFRRSALAEVGYWSD

DMITEDIDISWKLQLNQWTIFYEPRALCWILMPETLKGLWKQRLRWAQGG

AEVFLKNMTRLWRKENFRMWPLFFEYCLTTIWAFTCLVGFIIYAVQLAGV

PLNIELTHIAATHTAGILLCTLCLLQFIVSLMIENRYEHNLTSSLFWIIW

FPVIFWMLSLATTLVSFTRVMLMPKKQRARWVSPDRGILRG (SEQ ID

NO: 29)

>ygdD [Escherichia coli]

MTSRFMLIFAAISGFIFVALGAFGAHVLSKTMGAVEMGWIQTGLEYQAFH

TLAILGLAVAMQRRISIWFYWSSVFLALGTVLFSGSLYCLALSHLRLWAF

VTPVGGVSFLAGWALMLVGAIRLKRKGVSHE(SEQ ID NO: 30)

>hemr [Escherichia coli]

MNVIKTAICTLITLPVGLQAATSHSSSMTKDTITVVATGNQNTVFETPSM

VSVVTNDTPWSKNAVTSAGMLRGVAGLSQTGAGRTNGQTFNLRGYDKSGV

LVLVDGVRQLSDMAKSSGTYLDPALVKRIEVVRGPNSSLYGSGGLGGVVD

FRTADAADFLPPGETNGVSLWGNIASGDHSTGSGLTWFGKTEKTDALLSV

IMRKRGSIYQSDGERAPNKEKPAALFAKGSVSITDSNKAGASLRLYRNST

TEPGNPTLTHGDSGLRDRKTAQNDMQFWYQYAPADNSLINVKSTLYLSDI

TVKTNGHNKTAEWRNNRTSGVNVVNRSHSLIFPGAHQLSYGAEYYRQQQK

PEGTATLYPEGHIDFTSLYFQDEMTMESYPVNIIVGSRYDRYNSFNARAG

ELNAERLSPRAAMSVSPTDWLMMYGSISSAFRAPTMAEMYRDDVHFYRKG

KPNYWVPNLNLKPENNTTREIGAGIQLDSLLTDNDRLQLKGGYFGTDARN

YIATRVDMKRMRSYSYNVSRARIWGWDIQGNYQSDYVDWMLSYNRTESMD

ASSREWLGSGNPDTLISDISIPVGHRGVYAGWRAELSAPATHVKKGDPCQ

DGYAIHSFSLSYKPVSVKGFEASVTLDNAFNKLAMNGKGVPLSGRTVNLY

TRYQW(SEQ ID NO: 31)

>yciS [Escherichia coli]

MKYLLIFLLVLAIFVISVTLGAQNDQQVTFNYLLAQGEYRISTLLAVLFA

AGFAIGWLICGLFWLRVRVSLARAERKIKRLENQLSPATDVAVVPHSSAA

KE (SEQ ID NO: 32)

Cytochrome P450 Reductase Partners

>SrCPR [Stevia rebaudiana]

MQSDSVKVSPFDLVSAAMNGKAMEKLNASESEDPTTLPALKMLVENRELL

TLFTTSFAVLIGCLVFLMWRRSSSKKLVQDPVPQVIVVKKKEKESEVDDG

KKKVSIFYGTQTGTAEGFAKALVEEAKVRYEKTSFKVIDLDDYAADDDEY

EEKLKKESLAFFFLATYGDGEPTDNAANFYKWFTEGDDKGEWLKKLQYGV

FGLGNRQYEHFNKIAIVVDDKLTEMGAKRLVPVGLGDDDQCIEDDFTAWK

ELVWPELDQLLRDEDDTSVTTPYTAAVLEYRVVYHDKPADSYAEDQTHTN

GHVVHDAQHPSRSNVAFKKELHTSQSDRSCTHLEFDISHTGLSYETGDHV

GVYSENLSEVVDEALKLLGLSPDTYFSVHADKEDGTPIGGASLPPPFPPC

TLRDALTRYADVLSSPKKVALLALAAHASDPSEADRLKFLASPAGKDEYA

QWIVANQRSLLEVMQSFPSAKPPLGVFFAAVAPRLQPRYYSISSSPKMSP

NRIHVTCALVYETTPAGRIHRGLCSTWMKNAVPLTESPDCSQASIFVRTS

NFRLPVDPKVPVIMIGPGTGLAPFRGFLQERLALKESGTELGSSIFFFGC

RNRKVDFIYEDELNNFVETGALSELIVAFSREGTAKEYVQHKMSQKASDI

WKLLSEGAYLYVCGDAKGMAKDVHRTLHTIVQEQGSLDSSKAELYVKNLQ

MSGRYLRDVW (SEQ ID NO: 33)

>AtCPR [Arabidopsis thaliana]

MTSALYASDLFKQLKSIMGTDSLSDDVVLVIATTSLALVAGFVVLLWKKT

TADRSGELKPLMIPKSLMAKDEDDDLDLGSGKTRVSIFFGTQTGTAEGFA

KALSEEIKARYEKAAVKVIDLDDYAADDDQYEEKLKKETLAFFCVATYGD

GEPTDNAARFSKWFTEENERDIKLQQLAYGVFALGNRQYEHFNKIGIVLD

EELCKKGAKRLIEVGLGDDDQSIEDDFNAWKESLWSELDKLLKDEDDKSV

ATPYTAVIPEYRVVTHDPRFTTQKSMESNVANGNTTIDIHHPCRVDVAVQ

KELHTHESDRSCIHLEFDISRTGITYETGDHVGVYAENHVEIVEEAGKLL

GHSLDLVFSIHADKEDGSPLESAVPPPFPGPCTLGTGLARYADLLNPPRK

SALVALAAYATEPSEAEKLKHLTSPDGKDEYSQWIVASQRSLLEVMAAFP

SAKPPLGVFFAAIAPRLQPRYYSISSCQDWAPSRVHVTSALVYGPTPTGR

IHKGVCSTWMKNAVPAEKSHECSGAPIFIRASNFKLPSNPSTPIVMVGPG

TGLAPFRGFLQERMALKEDGEELGSSLLFFGCRNRQMDFIYEDELNNFVD

QGVISELIMAFSREGAQKEYVQHKMMEKAAQVWDLIKEEGYLYVCGDAKG

MARDVHRTLHTIVQEQEGVSSSEAEAIVKKLQTEGRYLRDVW (SEQ ID

NO: 34)

>TcCPR [Taxus cuspidata]

MQANSNTVEGASQGKSLLDISRLDHIFALLLNGKGGDLGAMTGSALILTE

NSQNLMILTTALAVLVACVFFFVWRRGGSDTQKPAVRPTPLVKEEDEEEE

DDSAKKKVTIFFGTQTGTAEGFAKALAEEAKARYEKAVFKVVDLDNYAAD

DEQYEEKLKKEKLAFFMLATYGDGEPTDNAARFYKWFLEGKEREPWLSDL

TYGVFGLGNRQYEHFNKVAKAVDEVLIEQGAKRLVPVGLGDDDQCIEDDF

TAWREQVWPELDQLLRDEDDEPTSATPYTAAIPEYRVEIYDSVVSVYEET

HALKQNGQAVYDIHHPCRSNVAVRRELHTPLSDRSCIHLEFDISDTGLIY

ETGDHVGVHTENSIETVEEAAKLLGYQLDTIFSVHGDKEDGTPLGGSSLP

PPFPGPCTLRTALARYADLLNPPRKAAFLALAAHASDPAEAERLKFLSSP

AGKDEYSQWVTASQRSLLEIMAEFPSAKPPLGVFFAAIAPRLQPRYYSIS

SSPRFAPSRIHVTCALVYGPSPTGRIHKGVCSNWMKNSLPSEETHDCSWA

PVFVRQSNFKLPADSTTPIVMVGPGTGFAPFRGFLQERAKLQEAGEKLGP

AVLFFGCRNRQMDYIYEDELKGYVEKGILTNLIVAFSREGATKEYVQHKM

LEKASDTWSLIAQGGYLYVCGDAKGMARDVHRTLHTIVQEQESVDSSKAE

FLVKKLQMDGRYLRDIW (SEQ IDNO: 35)

>AaCPR [Artemisia annua]

MAQSTTSVKLSPFDLMTALLNGKVSFDTSNTSDTNIPLAVFMENRELLMI

LTTSVAVLIGCVVVLVWRRSSSAAKKAAESPVIVVPKKVTEDEVDDGRKK

VTVFFGTQTGTAEGFAKALVEEAKARYEKAVFKVIDLDDYAAEDDEYEEK

LKKESLAFFFLATYGDGEPTDNAARFYKWFTEGEEKGEWLDKLQYAVFGL

GNRQYEHFNKIAKVVDEKLVEQGAKRLVPVGMGDDDQCIEDDFTAWKELV

WPELDQLLRDEDDTSVATPYTAAVAEYRVVFHDKPETYDQDQLTNGHAVH

DAQHPCRSNVAVKKELHSPLSDRSCTHLEFDISNTGLSYETGDHVGVYVE

NLSEVVDEAEKLIGLPPHTYFSVHADNEDGTPLGGASLPPPFPPCTLRKA

LASYADVLSSPKKSALLALAAHATDSTEADRLKFLASPAGKDEYAQWIVA

SHRSLLEVMEAFPSAKPPLGVFFASVAPRLQPRYYSISSSPRFAPNRIHV

TCALVYEQTPSGRVHKGVCSTWMKNAVPMTESQDCSWAPIYVRTSNFRLP

SDPKVPVIMIGPGTGLAPFRGFLQERLAQKEAGTELGTAILFFGCRNRKV

DFIYEDELNNFVETGALSELVTAFSREGATKEYVQHKMTQKASDIWNLLS

EGAYLYVCGDAKGMAKDVHRTLHTIVQEQGSLDSSKAELYVKNLQMAGRY

LRDVW (SEQ ID NO: 36)

>AtCPR1 [Arabidopsis thaliana]

MATSALYASDLFKQLKSIMGTDSLSDDVVLVIATTSLALVAGFVVLLWKK

TTADRSGELKPLMIPKSLMAKDEDDDLDLGSGKTRVSIFFGTQTGTAEGF

AKALSEEIKARYEKAAVKVIDLDDYAADDDQYEEKLKKETLAFFCVATYG

DGEPTDNAARFYKWFTEENERDIKLQQLAYGVFALGNRQYEHFNKIGIVL

DEELCKKGAKRLIEVGLGDDDQSIEDDFNAWKESLWSELDKLLKDEDDKS

VATPYTAVIPEYRVVTHDPRFTTQKSMESNVANGNTTIDIHHPCRVDVAV

QKELHTHESDRSCIHLEFDISRTGITYETGDHVGVYAENHVEIVEEAGKL

LGHSLDLVFSIHADKEDGSPLESAVPPPFPGPCTLGTGLARYADLLNPPR

KSALVALAAYATEPSEAEKLKHLTSPDGKDEYSQWIVASQRSLLEVMAAF

PSAKPPLGVFFAAIAPRLQPRYYSISSSPRLAPSRVHVTSALVYGPTPTG

RIHKGVCSTWMKNAVPAEKSHECSGAPIFIRASNFKLPSNPSTPIVMVGP

GTGLAPFRGFLQERMALKEDGEELGSSLLFFGCRNRQMDFIYEDELNNFV

DQGVISELIMAFSREGAQKEYVQHKMMEKAAQVWDLIKEEGYLYVCGDAK

GMARDVHRTLHTIVQEQEGVSSSEAEAIVKKLQTEGRYLRDVW (SEQ I

D NO: 37)

>AtCPR2 [Arabidopsis thaliana]

MASSSSSSSTSMIDLMAAIIKGEPVIVSDPANASAYESVAAELSSMLIEN

RQFAMIVTTSIAVLIGCIVMLVWRRSGSGNSKRVEPLKPLVIKPREEEID

DGRKKVTIFFGTQTGTAEGFAKALGEEAKARYEKTRFKIVDLDDYAADDD

EYEEKLKKEDVAFFFLATYGDGEPTDNAARFYKWFTEGNDRGEWLKNLKY

GVFGLGNRQYEHFNKVAKVVDDILVEQGAQRLVQVGLGDDDQCIEDDFTA

WREALWPELDTILREEGDTAVATPYTAAVLEYRVSIHDSEDAKFNDINMA

NGNGYTVFDAQHPYKANVAVKRELHTPESDRSCIHLEFDIAGSGLTYETG

DHVGVLCDNLSETVDEALRLLDMSPDTYFSLHAEKEDGTPISSSLPPPFP

PCNLRTALTRYACLLSSPKKSALVALAAHASDPTEAERLKHLASPAGKDE

YSKWVVESQRSLLEVMAEFPSAKPPLGVFFAGVAPRLQPRFYSISSSPKI

AETRIHVTCALVYEKMPTGRIHKGVCSTWMKNAVPYEKSENCSSAPIFVR

QSNFKLPSDSKVPIIMIGPGTGLAPFRGFLQERLALVESGVELGPSVLFF

GCRNRRMDFIYEEELQRFVESGALAELSVAFSREGPTKEYVQHKMMDKAS

DIWNMISQGAYLYVCGDAKGMARDVHRSLHTIAQEQGSMDSTKAEGFVKN

LQTSGRYLRDVW (SEQ ID NO:38)

>ATR2 [Arabidopsis thaliana]

MASSSSSSSTSMIDLMAAIIKGEPVIVSDPANASAYESVAAELSSMLIEN

RQFAMIVTTSIAVLIGCIVMLVWRRSGSGNSKRVEPLKPLVIKPREEEID

DGRKKVTIFFGTQTGTAEGFAKALGEEAKARYEKTRFKIVDLDDYAADDD

EYEEKLKKEDVAFFFLATYGDGEPTDNAARFYKWFTEGNDRGEWLKNLKY

GVFGLGNRQYEHFNKVAKVVDDILVEQGAQRLVQVGLGDDDQCIEDDFTA

WREALWPELDTILREEGDTAVATPYTAAVLEYRVSIHDSEDAKFNDITLA

NGNGYTVFDAQHPYKANVAVKRELHTPESDRSCIHLEFDIAGSGLTMKLG

DHVGVLCDNLSETVDEALRLLDMSPDTYFSLHAEKEDGTPISSSLPPPFP

PCNLRTALTRYACLLSSPKKSALVALAAHASDPTEAERLKHLASPAGKDE

YSKWVVESQRSLLEVMAEFPSAKPPLGVFFAGVAPRLQPRFYSISSSPKI

AETRIHVTCALVYEKMPTGRIHKGVCSTWMKNAVPYEKSEKLFLGRPIFV

RQSNFKLPSDSKVPIIMIGPGTGLAPFRGFLQERLALVESGVELGPSVLF

FGCRNRRMDFIYEEELQRFVESGALAELSVAFSREGPTKEYVQHKMMDKA

SDIWNMISQGAYLYVCGDAKGMARDVHRSLHTIAQEQGSMDSTKAEGFVK

NLQTSGRYLRDVW (SEQ ID NO:39)

>SrCPR1 [Stevia rebaudiana]

MAQSDSVKVSPFDLVSAAMNGKAMEKLNASESEDPTTLPALKMLVENREL

LTLFTTSFAVLIGCLVFLMWRRSSSKKLVQDPVPQVIVVKKKEKESEVDD

GKKKVSIFYGTQTGTAEGFAKALVEEAKVRYEKTSFKVIDLDDYAADDDE

YEEKLKKESLAFFFLATYGDGEPTDNAANFYKWFTEGDDKGELLKKLQYG

VFGLGNRQYEHFNKIAIVVDDKLTEMGAKRLVPVGLGDDDQCIEDDFTAW

KELVWPELDQLLRDEDDTSVTTPYTAAVLEYRVVYHDKPADSYAEDQTHT

NGHVVHDAQHPSRSNVAFKKELHTSQSDRSCTHLEFDISHTGLSYETGDH

VGVYSENLSEVVDEALKLLGLSPDTYFSVHADKEDGTPIGGASLPPPFPP

CTLRDALTRYADVLSSPKKVALLALAAHASDPSEADRLKFLASPAGKDEY

AQWIVANQRSLLEVMQSFPSAKPPLGVFFAAVAPRLQPRYYSISSSPKMS

PNRIHVTCALVYETTPAGRIHRGLCSTWMKNAVPLTESPDCSQASIFVRT

SNFRLPVDPKVPVIMIGPGTGLAPFRGFLQERLALKESGTELGSSIFFFG

CRNRKVDFIYEDELNNFVETGALSELIVAFSREGTAKEYVQHKMSQKASD

IWKLLSEGAYLYVCGDAKGMAKDVHRTLHTIVQEQGSLDSSKAELYVKNL

QMSGRYLRDVW (SEQ ID NO:40)

>SrCPR2 [Stevia rebaudiana]

MAQSESVEASTIDLMTAVLKDTVIDTANASDNGDSKMPPALAMMFEIRDL

LLILTTSVAVLVGCFVVLVWKRSSGKKSGKELEPPKIVVPKRRLEQEVDD

GKKKVTIFFGTQTGTAEGFAKALFEEAKARYEKAAFKVIDLDDYAADLDE

YAEKLKKETYAFFFLATYGDGEPTDNAAKFYKWFTEGDEKGVWLQKLQYG

VFGLGNRQYEHFNKIGIVVDDGLTEQGAKRIVPVGLGDDDQSIEDDFSAW

KELVWPELDLLLRDEDDKAAATPYTAAIPEYRVVFHDKPDAFSDDHTQTN

GHAVHDAQHPCRSNVAVKKELHTPESDRSCTHLEFDISHTGLSYETGDHV

GVYCENLIEVVEEAGKLLGLSTDTYFSLHIDNEDGSPLGGPSLQPPFPPC

TLRKALTNYADLLSSPKKSTLLALAAHASDPTEADRLRFLASREGKDEYA

EWVVANQRSLLEVMEAFPSARPPLGVFFAAVAPRLQPRYYSISSSPKMEP

NRIHVTCALVYEKTPAGRIHKGICSTWMKNAVPLTESQDCSWAPIFVRTS

NFRLPIDPKVPVIMIGPGTGLAPFRGFLQERLALKESGTELGSSILFFGC

RNRKVDYIYENELNNFVENGALSELDVAFSRDGPTKEYVQHKMTQKASEI

WNMLSEGAYLYVCGDAKGMAKDVHRTLHTIVQEQGSLDSSKAELYVKNLQ

MSGRYLRDVW (SEQ ID NO: 41)

>SrCPR3 [Stevia rebaudiana]

MAQSNSVKISPLDLVTALFSGKVLDTSNASESGESAMLPTIAMIMENREL

LMILTTSVAVLIGCVVVLVWRRSSTKKSALEPPVIVVPKRVQEEEVDDGK

KKVTVFFGTQTGTAEGFAKALVEEAKARYEKAVFKVIDLDDYAADDDEYE

EKLKKESLAFFFLATYGDGEPTDNAARFYKWFTEGDAKGEWLNKLQYGVF

GLGNRQYEHFNKIAKVVDDGLVEQGAKRLVPVGLGDDDQCIEDDFTAWKE

LVWPELDQLLRDEDDTTVATPYTAAVAEYRVVFHEKPDALSEDYSYTNGH

AVHDAQHPCRSNVAVKKELHSPESDRSCTHLEFDISNTGLSYETGDHVGV

YCENLSEVVNDAERLVGLPPDTYFSIHTDSEDGSPLGGASLPPPFPPCTL

RKALTCYADVLSSPKKSALLALAAHATDPSEADRLKFLASPAGKDEYSQW

IVASQRSLLEVMEAFPSAKPSLGVFFASVAPRLQPRYYSISSSPKMAPDR

IHVTCALVYEKTPAGRIHKGVCSTWMKNAVPMTESQDCSWAPIYVRTSNF

RLPSDPKVPVIMIGPGTGLAPFRGFLQERLALKEAGTDLGLSILFFGCRN

RKVDFIYENELNNFVETGALSELIVAFSREGPTKEYVQHKMSEKASDIWN

LLSEGAYLYVCGDAKGMAKDVHRTLHTIVQEQGSLDSSKAELYVKNLQMS

GRYLRDVW (SEQ ID NO: 42)

>PgCPR [Pelargonium graveolens]

MAQSSSGSMSPFDFMTAIIKGKMEPSNASLGAAGEVTAMILDNRELVMIL

TTSIAVLIGCVVVFIWRRSSSQTPTAVQPLKPLLAKETESEVDDGKQKVT

IFFGTQTGTAEGFAKALADEAKARYDKVTFKVVDLDDYAADDEEYEEKLK

KETLAFFFLATYGDGEPTDNAARFYKWFLEGKERGEWLQNLKFGVFGLGN

RQYEHFNKIAIVVDEILAEQGGKRLISVGLGDDDQCIEDDFTAWRESLWP

ELDQLLRDEDDTTVSTPYTAAVLEYRVVFHDPADAPTLEKSYSNANGHSV

VDAQHPLRANVAVRRELHTPASDRSCTHLEFDISGTGIAYETGDHVGVYC

ENLAETVEEALELLGLSPDTYFSVHADKEDGTPLSGSSLPPPFPPCTLRT

ALTLHADLLSSPKKSALLALAAHASDPTEADRLRHLASPAGKDEYAQWIV

ASQRSLLEVMAEFPSAKPPLGVFFASVAPRLQPRYYSISSSPRIAPSRIH

VTCALVYEKTPTGRVHKGVCSTWMKNSVPSEKSDECSWAPIFVRQSNFKL

PADAKVPIIMIGPGTGLAPFRGFLQERLALKEAGTELGPSILFFGCRNSK

MDYIYEDELDNFVQNGALSELVLAFSREGPTKEYVQHKMMEKASDIWNLI

SQGAYLYVCGDAKGMARDVHRTLHTIAQEQGSLDSSKAESMVKNLQMSGR

YLRDVW (SEQ ID NO: 43)

LINKER SEQUENCES

GSGGGGS (SEQ ID NO: 44)
GSGEAAAK (SEQ ID NO: 45)
GSGEAAAKEAAAK (SEQ ID NO: 46)
GSGMGSSSN (SEQ ID NO: 47).

P450 Enzymes With Transmembrane Domain Deleted

>t22ZzHO [Zingiber zerumbet]

LLIKRSSRSSVHKQQVLLASLPPSPPRLPLIGNIHQLVGGNPHRILLQLA

RTHGPLICLRLGQVDQVVASSVEAVEEIIKRHDLKFADRPRDLTFSRIFF

YDGNAVVMTPYGGEWKQMRKIYAMELLNSRRVKSFAAIREDVARKLTGEI

AHKAFAQTPVINLSEMVMSMINAIVIRVAFGDKCKQQAYFLHLVKEAMSY

VSSFSVADMYPSLKFLDTLTGLKSKLEGVHGKLDKVFDEIIAQRQAALAA

EQAEEDLIIDVLLKLKDEGNQEFPITYTSVKAIVMEIFLAGTETSSSVID

WVMSELIKNPKAMEKVQKEMREAMQGKTKLEESDIPKFSYLNLVIKETLR

LHPPGPLLFPRECRETCEVMGYRVPAGARLLINAFALSRDEKYWGSDAES

FKPERFEGISVDFKGSNFEFMPFGAGRRICPGMTFGISSVEVALAHLLFH

FDWQLPQGMKIEDLDMMEVSGMSATRRSPLLVLAKLIIPLP (SEQ ID

NO:48)

>t20BsGAO [Barnadesia spinosa]

LLTGSKSTKNSLPEAWRLPIIGHMHHLVGTLPHRGVTDMARKYGSLMHLQ

LGEVSTIVVSSPRWAKEVLTTYDITFANRPETLTGEIVAYHNTDIVLSPY

GEYWRQLRKLCTLELLSAKKVKSFQSLREEECWNLVKEVRSSGSGSPVDL

SESIFKLIATILSRAAFGKGIKDQREFTEIVKEILRLTGGFDVADIFPSK

KILHHLSGKRAKLTNIHNKLDSLINNIVSEHPGSRTSSSQESLLDVLLRL

KDSAELPLTSDNVKAVILDMFGAGTDTSSATIEWAISELIRCPRAMEKVQ

TELRQALNGKERIQEEDIQELSYLKLVIKETLRLHPPLPLVMPRECREPC

VLAGYEIPTKTKLIVNVFAINRDPEYWKDAETFMPERFENSPINIMGSEY

EYLPFGAGRRMCPGAALGLANVELPLAHILYYFNWKLPNGARLDELDMSE

CFGATVQRKSELLLVPTAYKTANNSA (SEQ ID NO:49)

>t16HmPO [Hyoscyamus muticus]

FLLRKWKNSNSQSKKLPPGPWKLPLLGSMLHMVGGLPHHVLRDLAKKYGP

LMHLQLGEVSAVVVTSPDMAKEVLKTHDIAFASRPKLLAPEIVCYNRSDI

AFCPYGDYWRQMRKICVLEVLSAKNVRSFSSIRRDEVLRLVNFVRSSTSE

PVNFTERLFLFTSSMTCRSAFGKVFKEQETFIQLIKEVIGLAGGFDVADI

FPSLKFLHVLTGMEGKIMKAHHKVDAIVEDVINEHKKNLAMGKTNGALGG

EDLIDVLLRLMNDGGLQFPITNDNIKAIIFDMFAAGTETSSSTLVWAMVQ

MMRNPTILAKAQAEVREAFKGKETFDENDVEELKYLKLVIKETLRLHPPV

PLLVPRECREETEINGYTIPVKTKVMVNVWALGRDPKYWDDADNFKPERF

EQCSVDFIGNNFEYLPFGGGRRICPGISFGLANVYLPLAQLLYHFDWKLP

TGMEPKDLDLTELVGVTAARKSDLMLVATPYQPSRE (SEQ ID NO:50

)

>t19LsGAO [Lactuca sativa]

KLATRPKSTKKQLPEASRLPIIGHMHHLIGTMPHRGVMDLARKHGSLMHL

QLGEVSTIVVSSPKWAKEILTTYDITFANRPETLTGEIIAYHNTDIVLAP

YGEYWRQLRKLCTLELLSVKKVKSFQSIREEECWNLVKEVKESGSGKPIN

LSESIFTMIATILSRAAFGKGIKDQREFTEIVKEILRQTGGFDVADIFPS

KKFLHHLSGKRARLTSIHKKLDNLINNIVAEHHVSTSSKANETLLDVLLR

LKDSAEFPLTADNVKAIILDMFGAGTDTSSATVEWAISELIRCPRAMEKV

QAELRQALNGKEKIQEEDIQDLAYLNLVIRETLRLHPPLPLVMPRECREP

VNLAGYEIANKTKLIVNVFAINRDPEYWKDAEAFIPERFENNPNNIMGAD

YEYLPFGAGRRMCPGAALGLANVQLPLANILYHFNWKLPNGASHDQLDMT

ESFGATVQRKTELLLVPSF (SEQ ID NO:51)

>t16NtEAO [Nicotiani tabacum]

FLLRKWKNSNSQSKKLPPGPWKIPILGSMLHMIGGEPHHVLRDLAKKYGP

LMHLQLGEISAVVVTSRDMAKEVLKTHDVVFASRPKIVAMDIICYNQSDI

AFSPYGDHWRQMRKICVMELLNAKNVRSFSSIRRDEVVRLIDSIRSDSSS

GELVNFTQRIIWFASSMTCRSAFGQVLKGQDIFAKKIREVIGLAEGFDVV

DIFPTYKFLHVLSGMKRKLLNAHLKVDAIVEDVINEHKKNLAAGKSNGAL

GGEDLIDVLLRLMNDTSLQFPITNDNIKAVIVDMFAAGTETSSTTTVWAM

AEMMKNPSVFTKAQAEVREAFRDKVSFDENDVEELKYLKLVIKETLRLHP

PSPLLVPRECREDTDINGYTIPAKTKVMVNVWALGRDPKYWDDAESFKPE

RFEQCSVDFFGNNFEFLPFGGGRRICPGMSFGLANLYLPLAQLLYHFDWK

LPTGIMPRDLDLTELSGITIARKGGLYLNATPYQPSRE (SEQ ID NO:

52)

>t26CpVO [Citrus x paradisi]

WVWLRPKKLEKFLRQQGLKGNSYRLLFGDLKENSIELKEAKARPLSLDDD

IAIRVNPFLHKLVNDYGKNSFMWFGPTPRVNIMNPDQIKAIFTKINDFQK

VNSIPLARLLIVGLATLEGEKWAKHRKLINPAFHQEKLKLMLPAFYLSCI

EIITKWEKQMSVEGSSELDVWPYLANLTSDVISRTAFGSSYEEGRRIFQL

QAELAELTMQVFRSVHIPGWRFLPTKRNRRMKEIDKEIRASLMGIIKNRE

KAMRAGEAANNDLLGILMETSFREIEEHGNNKNVGFSMNDVIEECKLFYF

AGQETTSVLLNWTMVLLSKHQDWQERARQEVLQVFGNNKPDYDGLNHLKI

VQMILYEVLRLYPPVTVLSRAVFKETKLGNLTLPAGVQIGLPMILVHQDP

ELWGDDAVEFKPERFAEGISKAAKNQVSYFPFALGPRICVGQNFALVEAK

MATAMILQNYSFELSPSYVHAPTAVPTLHPELGTQLILRKLWCKNN (SE

Q ID NO:53)

>t23AaAO [Artemesia annua]

FVYKFATRSKSTKKSLPEPWRLPIIGHMHHLIGTTPHRGVRDLARKYGSL

MHLQLGEVPTIVVSSPKWAKEILTTYDITFANRPETLTGEIVLYHNTDVV

LAPYGEYWRQLRKICTLELLSVKKVKSFQSLREEECWNLVQEIKASGSGR

PVNLSENVFKLIATILSRAAFGKGIKDQKELTEIVKEILRQTGGFDVADI

FPSKKFLHHLSGKRARLTSLRKKIDNLIDNLVAEHTVNTSSKTNETLLDV

LLRLKDSAEFPLTSDNIKAIILDMFGAGTDTSSSTIEWAISELIKCPKAM

EKVQAELRKALNGKEKIHEEDIQELSYLNMVIKETLRLHPPLPLVLPREC

RQPVNLAGYNIPNKTKLIVNVFAINRDPEYWKDAEAFIPERFENSSATVM

GAEYEYLPFGAGRRMCPGAALGLANVQLPLANILYHFNWKLPNGVSYDQI

DMTESSGATMQRKTELLLVPSF (SEQ ID NO:54)

>t21AtKO [Arabidopsis thaliana]

FFFKKLLSFSRKNMSEVSTLPSVPVVPGFPVIGNLLQLKEKKPHKTFTRW

SEIYGPIYSIKMGSSSLIVLNSTETAKEAMVTRFSSISTRKLSNALTVLT

CDKSMVATSDYDDFHKLVKRCLLNGLLGANAQKRKRHYRDALIENVSSKL

HAHARDHPQEPVNFRAIFEHELFGVALKQAFGKDVESIYVKELGVTLSKD

EIFKVLVHDMMEGAIDVDWRDFFPYLKWIPNKSFEARIQQKHKRRLAVMN

ALIQDRLKQNGSESDDDCYLNFLMSEAKTLTKEQIAILVWETIIETADTT

LVTTEWAIYELAKHPSVQDRLCKEIQNVCGGEKFKEEQLSQVPYLNGVFH

ETLRKYSPAPLVPIRYAHEDTQIGGYHVPAGSEIAINIYGCNMDKKRWER

PEDWWPERFLDDGKYETSDLHKTMAFGAGKRVCAGALQASLMAGIAIGRL

VQEFEWKLRDGEEENVDTYGLTSQKLYPLMAIINPRRS (SEQ ID NO:

55)

>t30SrKO [Stevia rebaudiana]

WYLKSYTSARRSQSNHLPRVPEVPGVPLLGNLLQLKEKKPYMTFTRWAAT

YGPIYSIKTGATSMVVVSSNEIAKEALVTRFQSISTRNLSKALKVLTADK

TMVAMSDYDDYHKTVKRHILTAVLGPNAQKKHRIHRDIMMDNISTQLHEF

VKNNPEQEEVDLRKIFQSELFGLAMRQALGKDVESLYVEDLKITMNRDEI

FQVLVVDPMMGAIDVDWRDFFPYLKWVPNKKFENTIQQMYIRREAVMKSL

IKEHKKRIASGEKLNSYIDYLLSEAQTLTDQQLLMSLWEPIIESSDTTMV

TTEWAMYELAKNPKLQDRLYRDIKSVCGSEKITEEHLSQLPYITAIFHET

LRRHSPVPIIPLRHVHEDTVLGGYHVPAGTELAVNIYGCNMDKNVWENPE

EWNPERFMKENETIDFQKTMAFGGGKRVCAGSLQALLTASIGIGRMVQEF

EWKLKDMTQEEVNTIGLTTQMLRPLRAIIKPRI (SEQ ID NO:56)

>t52PpKO [Physcomitrella patens]

FVARTCLRNKKRLPPAIPGGLPVLGNLLQLTEKKPHRTFTAWSKEHGPIF

TIKVGSVPQAVVNNSEIAKEVLVTKFASISKRQMPMALRVLTRDKTMVAM

SDYGEEHRMLKKLVMTNLLGPTTQNKNRSLRDDALIGMIEGVLAELKASP

TSPKVVNVRDYVQRSLFPFALQQVFGYIPDQVEVLELGTCVSTWDMFDAL

VVAPLSAVINVDWRDFFPALRWIPNRSVEDLVRTVDFKRNSIMKALIRAQ

RMRLANLKEPPRCYADIALTEATHLTEKQLEMSLWEPIIESADTTLVTSE

WAMYEIAKNPDCQDRLYREIVSVAGTERMVTEDDLPNMPYLGAIIKETLR

KYTPVPLIPSRFVEEDITLGGYDIPKGYQILVNLFAIANDPAVWSNPEKW

DPERMLANKKVDMGFRDFSLMPFGAGKRMCAGITQAMFIIPMNVAALVQH

CEWRLSPQEISNINNKIEDVVYLTTHKLSPLSCEATPRISHRLP (SEQ

ID NO:57)

>t15PsVO [Pleurotus sapidus]

MGKLHPLAIIPDYKGSMAASVTIFNKRTNPLDISVNQANDWPWRYAKTCV

LSSDWALHEMIIHLNNTHLVEEAVIVAAQRKLSPSHIVFRLLEPHWVVTL

SLNALARSVLIPEVIVPIAGFSAPHIFQFIRESFTNFDWKSLYVPADLES

RGFPVDQLNSPKFHNYAYARDINDMWTTLKKFVSSVLQDAQYYPDDASVA

GDTQIQAWCDEMRSGMGAGMTNFPESITTVDDLVNMVTMCIHIAAPQHTA

VNYLQQYYQTFVSNKPSALFSPLPTSIAQLQKYTESDLMAALPLNAKRQW

LLMAQIPYLLSMQVQEDENIVTYAANASTDKDPIIASAGRQLAADLKKLA

AVFLVNSAQLDDQNTPYDVLAPEQLANAIVI (SEQ ID NO:58)

>t20CiVO [Cichorium intybus]

LLTRTTSKKNLLPEPWRLPIIGHMHHLIGTMPHRGVMELARKHGSLMHLQ

LGEVSTIVVSSPRWAKEVLTTYDITFANRPETLTGEIVAYHNTDIVLAPY

GEYWRQLRKLCTLELLSNKKVKSFQSLREEECWNLVKDIRSTGQGSPINL

SENIFKMIATILSRAAFGKGIKDQMKFTELVKEILRLTGGFDVADIFPSK

KLLHHLSGKRAKLTNIHNKLDNLINNIIAEHPGNRTSSSQETLLDVLLRL

KESAEFPLTADNVKAVILDMFGAGTDTSSATIEWAISELIRCPRAMEKVQ

TELRQALNGKERIQEEDLQELNYLKLVIKETLRLHPPLPLVMPRECREPC

VLGGYDIPSKTKLIVNVFAINRDPEYWKDAETFMPERFENSPITVMGSEY

EYLPFGAGRRMCPGAALGLANVELPLAHILYFNWKLPNGKTFEDLDMTES

FGATVQRKTELLLVPTDFQTLTAST (SEQ ID NO:59)

>t20HaGAO [Helianthus annuus]

LLTRPTSSKNRLPEPWRLPIIGHMHHLIGTMPHRGVMDLARKYGSLMHLQ

LGEVSAIVVSSPKWAKEILTTYDIPFANRPETLTGEIIAYHNTDIVLAPY

GEYWRQLRKLCTLELLSVKKVKSFQSLREEECWNLVQEIKASGSGTPFNL

SEGIFKVIATVLSRAAFGKGIKDQKQFTEIVKEILRETGGFDVADIFPSK

KFLHHLSGKRGRLTSIHNKLDSLINNLVAEHTVSKSSKVNETLLDVLLRL

KNSEEFPLTADNVKAIILDMFGAGTDTSSATVEWAISELIRCPRAMEKVQ

AELRQALNGKERIKEEEIQDLPYLNLVIRETLRLHPPLPLVMPRECRQAM

NLAGYDVANKTKLIVNVFAINRDPEYWKDAESFNPERFENSNTTIMGADY

EYLPFGAGRRMCPGSALGLANVQLPLANILYYFKWKLPNGASHDQLDMTE

SFGATVQRKTELMLVPSF (SEQ ID NO:60)

Claims

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.