FUSION PROTEINS USEFUL FOR MODIFYING TERPENES
20210292797 · 2021-09-23
Inventors
Cpc classification
C12Y114/13106
CHEMISTRY; METALLURGY
C12P5/007
CHEMISTRY; METALLURGY
C12P17/08
CHEMISTRY; METALLURGY
C12N9/0073
CHEMISTRY; METALLURGY
C12P15/00
CHEMISTRY; METALLURGY
C12P17/06
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention provides for a fusion protein comprising: (a) a terpene synthase (TS), or a homolog thereof, (b) a peptide linker, and (c) a P450 enzyme, or a homolog thereof.
Claims
1. A fusion protein comprising: (a) a terpene synthase (TS), or a homolog thereof, (b) a peptide linker, and (c) a P450 enzyme, or a homolog thereof.
2. The fusion protein of claim 1, wherein the TS is 1,8-cineole synthase, sesquiterpene epi-isozizaene synthase, (R)-limonene synthase, (S)-limonene synthase, valencene synthase, (−)-alpha-terpineol synthase, (+)-alpha-pinene synthase, (−)-alpha-pinene synthase, (+)-beta-pinene synthase, (−)-beta-pinene synthase, vetispiradiene synthase, amorphadiene synthase, curcumene synthase, bisabolene synthase, farnesene synthase, cadinene synthase, or aristolochene synthase.
3. The fusion protein of claim 1, wherein the TS comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16.
4. The fusion protein of claim 3, wherein the TS comprises the amino acid sequence Asp-Asp-Xaa-Xaa-Asp (DDXXD) (SEQ ID NO:17).
5. The fusion protein of claim 3, wherein the TS comprises the amino acid sequence Asp-Asp-Xaa-Xaa-Glu (DDXXE) (SEQ ID NO:18).
6. The fusion protein of claim 1, wherein the P450 enzyme is 1,8-cineole 2-endo-monooxygenase or epi-isozizaene 5-monooxygenase.
7. The fusion protein of claim 1, wherein the P450 enzyme comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, or 99% identity with SEQ ID NO:19 or SEQ ID NO:20.
8. The fusion protein of claim 7, wherein the P450 enzyme comprises the amino acid sequence FXXGXRXCXG (SEQ ID NO:21), or CXG, motif and/or EXXR motif.
9. The fusion protein of claim 1, wherein the peptide linker comprises of 0-1000 amino acid residues.
10. The fusion protein of claim 9, wherein the peptide linker comprises of 1-5 repeats of the amino acid sequence GSG.
11. A genetically modified host cell capable of producing a modified terpenes, said genetically modified host cell comprising the fusion protein of claim 1.
12. A nucleic acid comprising a nucleotide sequence encoding the fusion protein of claim 1 operatively linked to a promoter.
13. A vector comprising the nucleic acid of claim 12.
14. A genetically modified host cell capable of producing a modified terpenes, said genetically modified host cell comprising the nucleic acid of claim 14 or a vector of claim 13.
15. The genetically modified host cell of claim 14, wherein the genetically modified host cell is a bacterium.
16. The genetically modified host cell of claim 15, wherein the bacterium is of the genus Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsielia, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, or Paracoccus.
17. The genetically modified host cell of claim 14, wherein the genetically modified host cell is a eukaryotic cell.
18. The genetically modified host cell of claim 17, wherein the eukaryotic cell is a yeast.
19. A method for producing a modified terpene comprising: (a) providing the genetically modified host cell of claim 1, or a culture thereof, (b) culturing or growing the genetically modified host cell to produce the modified terpene, (c) optionally extracting or separating the modified terpene from the culture, and (d) optionally introducing a fuel additive to the extracted or separated the modified terpene.
20. A fuel composition comprising: (a) a modified terpene produced by the fusion protein of claim 1; and (b) a fuel additive.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] The foregoing aspects and others will be readily appreciated by the skilled artisan from the following description of illustrative embodiments when read in conjunction with the accompanying drawings.
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DETAILED DESCRIPTION OF THE INVENTION
[0049] Before the invention is described in detail, it is to be understood that, unless otherwise indicated, this invention is not limited to particular sequences, expression vectors, enzymes, host microorganisms, or processes, as such may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting.
[0050] As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an “expression vector” includes a single expression vector as well as a plurality of expression vectors, either the same (e.g., the same operon) or different; reference to “cell” includes a single cell as well as a plurality of cells; and the like.
[0051] In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:
[0052] The terms “optional” or “optionally” as used herein mean that the subsequently described feature or structure may or may not be present, or that the subsequently described event or circumstance may or may not occur, and that the description includes instances where a particular feature or structure is present and instances where the feature or structure is absent, or instances where the event or circumstance occurs and instances where it does not.
[0053] The term “about” as used herein means a value that includes 10% less and 10% more than the value referred to.
[0054] The terms “host cell” and “host microorganism” are used interchangeably herein to refer to a living biological cell, such as a microbe, that can be transformed via insertion of an expression vector. Thus, a host organism or cell as described herein may be a prokaryotic organism (e.g., an organism of the kingdom Eubacteria) or a eukaryotic cell. As will be appreciated by one of ordinary skill in the art, a prokaryotic cell lacks a membrane-bound nucleus, while a eukaryotic cell has a membrane-bound nucleus.
[0055] The term “heterologous DNA” as used herein refers to a polymer of nucleic acids wherein at least one of the following is true: (a) the sequence of nucleic acids is foreign to (i.e., not naturally found in) a given host microorganism; (b) the sequence may be naturally found in a given host microorganism, but in an unnatural (e.g., greater than expected) amount; or (c) the sequence of nucleic acids comprises two or more subsequences that are not found in the same relationship to each other in nature. The term “heterologous” as used herein refers to a structure or molecule wherein at least one of the following is true: (a) the structure or molecule is foreign to (i.e., not naturally found in) a given host microorganism; or (b) the structure or molecule may be naturally found in a given host microorganism, but in an unnatural (e.g., greater than expected) amount. For example, regarding instance (c), a heterologous nucleic acid sequence that is recombinantly produced will have two or more sequences from unrelated genes arranged to make a new functional nucleic acid. Specifically, the present invention describes the introduction of an expression vector into a host microorganism, wherein the expression vector contains a nucleic acid sequence coding for an enzyme that is not normally found in a host microorganism. With reference to the host microorganism's genome, then, the nucleic acid sequence that codes for the enzyme is heterologous.
[0056] The terms “expression vector” or “vector” refer to a compound and/or composition that transduces, transforms, or infects a host microorganism, thereby causing the cell to express nucleic acids and/or proteins other than those native to the cell, or in a manner not native to the cell. An “expression vector” contains a sequence of nucleic acids (ordinarily RNA or DNA) to be expressed by the host microorganism. Optionally, the expression vector also comprises materials to aid in achieving entry of the nucleic acid into the host microorganism, such as a virus, liposome, protein coating, or the like. The expression vectors contemplated for use in the present invention include those into which a nucleic acid sequence can be inserted, along with any preferred or required operational elements. Further, the expression vector must be one that can be transferred into a host microorganism and replicated therein. Preferred expression vectors are plasmids, particularly those with restriction sites that have been well documented and that contain the operational elements preferred or required for transcription of the nucleic acid sequence. Such plasmids, as well as other expression vectors, are well known to those of ordinary skill in the art.
[0057] The term “transduce” as used herein refers to the transfer of a sequence of nucleic acids into a host microorganism or cell. Only when the sequence of nucleic acids becomes stably replicated by the cell does the host microorganism or cell become “transformed.” As will be appreciated by those of ordinary skill in the art, “transformation” may take place either by incorporation of the sequence of nucleic acids into the cellular genome, i.e., chromosomal integration, or by extrachromosomal integration. In contrast, an expression vector, e.g., a virus, is “infective” when it transduces a host microorganism, replicates, and (without the benefit of any complementary virus or vector) spreads progeny expression vectors, e.g., viruses, of the same type as the original transducing expression vector to other microorganisms, wherein the progeny expression vectors possess the same ability to reproduce.
[0058] As used herein, the terms “nucleic acid sequence,” “sequence of nucleic acids,” and variations thereof shall be generic to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), to polyribonucleotides (containing D-ribose), to any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, and to other polymers containing nonnucleotidic backbones, provided that the polymers contain nucleobases in a configuration that allows for base pairing and base stacking, as found in DNA and RNA. Thus, these terms include known types of nucleic acid sequence modifications, for example, substitution of one or more of the naturally occurring nucleotides with an analog; internucleotide modifications, such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g., aminoalklyphosphoramidates, aminoalkylphosphotriesters); those containing pendant moieties, such as, for example, proteins (including nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.); those with intercalators (e.g., acridine, psoralen, etc.); and those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.). As used herein, the symbols for nucleotides and polynucleotides are those recommended by the IUPAC-IUB Commission of Biochemical Nomenclature (Biochem. 9:4022, 1970).
[0059] The term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
[0060] Terpenes are a large class of organic compounds, primarily produced by plants and constitute the main components of essential oils. Functionalization of the terpene carbon backbone using various downstream processing enzymes, such as cytochrome P450 enzymes, could derive many useful compounds that can be converted to higher value products. Production of terpenes and their functionalized products through microbial metabolic engineering are considered a favorable green strategy compared with isolation from the natural plants or chemical synthesis. However, as terpenes are usually hydrophobic and volatile compounds, the severe evaporation limits their availabilities surrounding downstream processing enzymes (such as cytochrome P450) toward adding functional groups. Particularly, when a solvent overlay is used to trap terpenes and prevent evaporation during the microbial production, it facilitates the isolation of terpenes from the cell and worsens the downstream processing reaction.
[0061] One possible solution is to engineer a fusion protein between terpene synthase and downstream processing enzymes that could improve the spatial favorability for downstream processing enzymes and the terpene substrate, which in turn would improve the substrate availability for downstream conversion. In some embodiments, an enzyme fusion strategy is developed by linking a terpene synthase (or a terpene cyclase) and downstream processing enzyme, specifically cytochrome P450 enzymes. In some embodiments, a series of fusion proteins is engineered between 1,8-cineole synthase and P450cin (CYP176A1) with different lengths of peptide linker to investigate the hydroxylation of 1,8-cineole to produce hydroxycineole. The production of hydroxycineole from both in vitro and in vivo conditions is compared between the fusion and non-fusion proteins. Results show the engineered enzyme fusion is more efficient than non-fused individual enzymes, suggesting a feasible strategy for efficient production of functionalized terpenes as well as possible application during the production of terpene-based bioproducts. Compared with the non-fused enzymes, results show that hydroxycineole production under the in vitro conditions is increased up to 5.2 folds from the enzyme fusion and the production rate is up to 10.5 folds faster. The engineered enzyme fusion is also integrated into a 1,8-cineole overproducing E. coli strain using the mevalonate pathway to investigate the hydroxylation of 1,8-cineole at the in vivo conditions. Results show that the enzyme fusion produced up to 2.8-fold more hydroxycineole than the non-fused enzymes. Thus, the engineered fusion showed higher efficiency during the hydroxylation of 1,8-cineole from both in vitro and in vivo results. This suggests that engineering an enzyme fusion between terpene synthase (or terpene cyclase) and downstream processing enzyme (such as P450) presents a feasible strategy for efficient production of functionalized terpenes, as well as possible applications (i.e. epoxidation, cyclopropanation, and, nitration, and the like) during the production of terpene-based bioproducts (such as, limonene-1,2-epoxide, perillyl alcohol, artemisinic alcohol, and the like).
[0062] In some embodiments, the modifying enzyme is a transaminase, N-oxygenase, or methyl-transferase. In some embodiments, the P450 or other modifying enzyme, or homolog thereof, is any enzyme listed in Table 1.
TABLE-US-00001 TABLE 1 Suitable modifying enzymes and corresponding terpene substrate and products thereof. Potential Terpene reaction substrates Products Enzyme Oxidation Amorphadiene Artemsinic acid 1.14.14.114 Valencene Nootkatone 1.14.14.1 Limonene Perillyl alcohol 1.14.15.1 Limonene Carveol 1.14.14.51 Limonene Isopiperitenol 1.14.14.99 Limonene Limonene-1,2- 1.14.13.107 epoxide Terpineol Sobrerol (Mucolytic 1.14.14.1 agent) Pinene Verbenol (insect 1.14.15.1 pheromone) Amination Ketones Terpene amines transaminase Nitration Terpene amine Nitro terpenoids N-oxygenase Cyclopropanation Methyl transferase
Enzymes, and Nucleic Acids Encoding Thereof
[0063] A homologous enzyme is an enzyme that has a polypeptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to any one of the enzymes described in this specification or in an incorporated reference. The homologous enzyme retains amino acids residues that are recognized as conserved for the enzyme. The homologous enzyme may have non-conserved amino acid residues replaced or found to be of a different amino acid, or amino acid(s) inserted or deleted, but which does not affect or has insignificant effect on the enzymatic activity of the homologous enzyme. The homologous enzyme has an enzymatic activity that is identical or essentially identical to the enzymatic activity any one of the enzymes described in this specification or in an incorporated reference. The homologous enzyme may be found in nature or be an engineered mutant thereof.
[0064] The nucleic acid constructs of the present invention comprise nucleic acid sequences encoding one or more of the subject enzymes. The nucleic acid of the subject enzymes are operably linked to promoters and optionally control sequences such that the subject enzymes are expressed in a host cell cultured under suitable conditions. The promoters and control sequences are specific for each host cell species. In some embodiments, expression vectors comprise the nucleic acid constructs. Methods for designing and making nucleic acid constructs and expression vectors are well known to those skilled in the art.
[0065] Sequences of nucleic acids encoding the subject enzymes are prepared by any suitable method known to those of ordinary skill in the art, including, for example, direct chemical synthesis or cloning. For direct chemical synthesis, formation of a polymer of nucleic acids typically involves sequential addition of 3′-blocked and 5′-blocked nucleotide monomers to the terminal 5′-hydroxyl group of a growing nucleotide chain, wherein each addition is effected by nucleophilic attack of the terminal 5′-hydroxyl group of the growing chain on the 3′-position of the added monomer, which is typically a phosphorus derivative, such as a phosphotriester, phosphoramidite, or the like. Such methodology is known to those of ordinary skill in the art and is described in the pertinent texts and literature (e.g., in Matteuci et al. (1980) Tet. Lett. 521:719; U.S. Pat. Nos. 4,500,707; 5,436,327; and 5,700,637). In addition, the desired sequences may be isolated from natural sources by splitting DNA using appropriate restriction enzymes, separating the fragments using gel electrophoresis, and thereafter, recovering the desired nucleic acid sequence from the gel via techniques known to those of ordinary skill in the art, such as utilization of polymerase chain reactions (PCR; e.g., U.S. Pat. No. 4,683,195).
[0066] Each nucleic acid sequence encoding the desired subject enzyme can be incorporated into an expression vector. Incorporation of the individual nucleic acid sequences may be accomplished through known methods that include, for example, the use of restriction enzymes (such as BamHI, EcoRI, HhaI, Xhol, XmaI, and so forth) to cleave specific sites in the expression vector, e.g., plasmid. The restriction enzyme produces single stranded ends that may be annealed to a nucleic acid sequence having, or synthesized to have, a terminus with a sequence complementary to the ends of the cleaved expression vector. Annealing is performed using an appropriate enzyme, e.g., DNA ligase. As will be appreciated by those of ordinary skill in the art, both the expression vector and the desired nucleic acid sequence are often cleaved with the same restriction enzyme, thereby assuring that the ends of the expression vector and the ends of the nucleic acid sequence are complementary to each other. In addition, DNA linkers may be used to facilitate linking of nucleic acids sequences into an expression vector.
[0067] A series of individual nucleic acid sequences can also be combined by utilizing methods that are known to those having ordinary skill in the art (e.g., U.S. Pat. No. 4,683,195).
[0068] For example, each of the desired nucleic acid sequences can be initially generated in a separate PCR. Thereafter, specific primers are designed such that the ends of the PCR products contain complementary sequences. When the PCR products are mixed, denatured, and reannealed, the strands having the matching sequences at their 3′ ends overlap and can act as primers for each other Extension of this overlap by DNA polymerase produces a molecule in which the original sequences are “spliced” together. In this way, a series of individual nucleic acid sequences may be “spliced” together and subsequently transduced into a host microorganism simultaneously. Thus, expression of each of the plurality of nucleic acid sequences is effected.
[0069] Individual nucleic acid sequences, or “spliced” nucleic acid sequences, are then incorporated into an expression vector. The invention is not limited with respect to the process by which the nucleic acid sequence is incorporated into the expression vector. Those of ordinary skill in the art are familiar with the necessary steps for incorporating a nucleic acid sequence into an expression vector. A typical expression vector contains the desired nucleic acid sequence preceded by one or more regulatory regions, along with a ribosome binding site, e.g., a nucleotide sequence that is 3-9 nucleotides in length and located 3-11 nucleotides upstream of the initiation codon in E. coli. See Shine et al. (1975) Nature 254:34 and Steitz, in Biological Regulation and Development: Gene Expression (ed. R. F. Goldberger), vol. 1, p. 349, 1979, Plenum Publishing, N.Y.
[0070] Regulatory regions include, for example, those regions that contain a promoter and an operator. A promoter is operably linked to the desired nucleic acid sequence, thereby initiating transcription of the nucleic acid sequence via an RNA polymerase enzyme. An operator is a sequence of nucleic acids adjacent to the promoter, which contains a protein-binding domain where a repressor protein can bind. In the absence of a repressor protein, transcription initiates through the promoter. When present, the repressor protein specific to the protein-binding domain of the operator binds to the operator, thereby inhibiting transcription. In this way, control of transcription is accomplished, based upon the particular regulatory regions used and the presence or absence of the corresponding repressor protein. An example includes lactose promoters (LacI repressor protein changes conformation when contacted with lactose, thereby preventing the Lad repressor protein from binding to the operator). Another example is the tac promoter. (See deBoer et al. (1983) Proc. Natl. Acad. Sci. USA, 80:21-25.) As will be appreciated by those of ordinary skill in the art, these and other expression vectors may be used in the present invention, and the invention is not limited in this respect.
[0071] Although any suitable expression vector may be used to incorporate the desired sequences, readily available expression vectors include, without limitation: plasmids, such as pSC101, pBR322, pBBR1MCS-3, pUR, pEX, pMR100, pCR4, pBAD24, pUC19; bacteriophages, such as M13 phage and λ phage. Of course, such expression vectors may only be suitable for particular host cells. One of ordinary skill in the art, however, can readily determine through routine experimentation whether any particular expression vector is suited for any given host cell. For example, the expression vector can be introduced into the host cell, which is then monitored for viability and expression of the sequences contained in the vector. In addition, reference may be made to the relevant texts and literature, which describe expression vectors and their suitability to any particular host cell.
[0072] The expression vectors of the invention must be introduced or transferred into the host cell. Such methods for transferring the expression vectors into host cells are well known to those of ordinary skill in the art. For example, one method for transforming E. coli with an expression vector involves a calcium chloride treatment wherein the expression vector is introduced via a calcium precipitate. Other salts, e.g., calcium phosphate, may also be used following a similar procedure. In addition, electroporation (i.e., the application of current to increase the permeability of cells to nucleic acid sequences) may be used to transfect the host microorganism. Also, microinjection of the nucleic acid sequencers) provides the ability to transfect host microorganisms. Other means, such as lipid complexes, liposomes, and dendrimers, may also be employed. Those of ordinary skill in the art can transfect a host cell with a desired sequence using these or other methods.
[0073] For identifying a transfected host cell, a variety of methods are available. For example, a culture of potentially transfected host cells may be separated, using a suitable dilution, into individual cells and thereafter individually grown and tested for expression of the desired nucleic acid sequence. In addition, when plasmids are used, an often-used practice involves the selection of cells based upon antimicrobial resistance that has been conferred by genes intentionally contained within the expression vector, such as the amp, gpt, neo, and hyg genes.
[0074] The host cell is transformed with at least one expression vector. When only a single expression vector is used (without the addition of an intermediate), the vector will contain all of the nucleic acid sequences necessary.
[0075] Once the host cell has been transformed with the expression vector, the host cell is allowed to grow. For microbial hosts, this process entails culturing the cells in a suitable medium. It is important that the culture medium contain an excess carbon source, such as a sugar (e.g., glucose) when an intermediate is not introduced. In this way, cellular production of the modified terpene ensured. When added, any intermediate is present in an excess amount in the culture medium.
[0076] Any means for extracting or separating the modified terpene from the host cell may be used. For example, the host cell may be harvested and subjected to hypotonic conditions, thereby lysing the cells. The lysate may then be centrifuged and the supernatant subjected to high performance liquid chromatography (HPLC) or gas chromatography (GC).
[0077] In some embodiments, the 1,8-cineole synthase (CS), or a homologous enzyme thereof, has an amino acid sequence having at least 70% identity to the amino acid sequence of Streptomyces clavuligerus 1,8-cineole synthase which is as follows:
TABLE-US-00002 (SEQ ID NO: 1) 10 20 30 40 MPAGHEEFDI PFPSRVNPFH ARAEDRHVAW MRAMGLITGD 50 60 70 80 AAEATYRRWS PAKVGARWFY LAQGEDLDLG CDIFGWFFAY 90 100 110 120 DDHFDGPTGT DPRQTAAFVN RTVAMLDPRA DPTGEHPLNI 130 140 150 160 AFHDLWQRES APMSPLWQRR AVDHWTQYLT AHITEATNRT 170 180 190 200 RHTSPTIADY LELRHRTGFM PPLLDLIERV WRAEIPAPVY 210 220 230 240 TTPEVQTLLH TTNQNINIVN DVLSLEKEEA HGDPHNLVLV 250 260 270 280 IQHERQSTRQ QALATARRMI DEWTDTFIRT EPRLPALCGR 290 300 310 320 LGIPLADRTS LYTAVEGMRA AIRGNYDWCA ETNRYAVHRP 330 TGTGRATTPW
[0078] In some embodiments, the 1,8-cineole synthase comprises the amino acid sequence Asp-Asp-Xaa-Xaa-Asp (DDXXD) (SEQ ID NO:17), or Asp-Asp-Xaa-Xaa-Glu (DDXXE) (SEQ ID NO:18), which is important for the catalytic activity of 1,8-cineole synthase, presumably through binding to Mg.sup.2+.
[0079] In some embodiments, the epi-isozizaene synthase (EIZS), or a homologous enzyme thereof, has an amino acid sequence having at least 70% identity to the amino acid sequence of Streptomyces coelicolor epi-isozizaene synthase which is as follows:
TABLE-US-00003 (SEQ ID NO: 2) 10 20 30 40 MHAFPHGTTA TPTAIAVPPS LRLPVIEAAF PRQLHPYWPK 50 60 70 80 LQETTRTWLL EKRLMPADKV EEYADGLCYT DLMAGYYLGA 90 100 110 120 PDEVLQAIAD YSAWFFVWDD RHDRDIVHGR AGAWRRLRGL 130 140 150 160 LHTALDSPGD HLHHEDTLVA GFADSVRRLY AFLPATWNAR 170 180 190 200 FARHFHTVIE AYDREFHNRT RGIVPGVEEY LELRRLTFAH 210 220 230 240 WIWTDLLEPS SGCELPDAVR KHPAYRRAAL LSQEFAAWYN 250 260 270 280 DLCSLPKEIA GDEVHNLGIS LITHHSLTLE EAIGEVRRRV 290 300 310 320 EECITEFLAV ERDALRFADE LADGTVRGKE LSGAVRANVG 330 340 350 360 NMRNWFSSVY WFHHESGRYM VDSWDDRSTP PYVNNEAAGE K
[0080] In some embodiments, the epi-isozizaene synthase comprises the amino acid sequence Asp-Asp-Xaa-Xaa-Asp (DDXXD) (SEQ ID NO:17), or Asp-Asp-Xaa-Xaa-Glu (DDXXE) (SEQ ID NO:18), which is important for the catalytic activity of epi-isozizaene synthase, presumably through binding to Mg.sup.2+.
[0081] In some embodiments, the (R)-limonene synthase, or a homologous enzyme thereof, has an amino acid sequence having at least 70% identity to the amino acid sequence of Citrus limon (R)-limonene synthase which is as follows:
TABLE-US-00004 (SEQ ID NO: 3) 10 20 30 40 MSSCINPSTL VTSVNAFKCL PLATNKAAIR IMAKYKPVQC 50 60 70 80 LISAKYDNLT VDRRSANYQP SIWDHDFLQS LNSNYTDEAY 90 100 110 120 KRRAEELRGK VKIAIKDVIE PLDQLELIDN LQRLGLAHRF 130 140 150 160 ETEIRNILNN IYNNNKDYNW RKENLYATSL EFRLLRQHGY 170 180 190 200 PVSQEVFNGF KDDQGGFICD DFKGILSLHE ASYYSLEGES 210 220 230 240 IMEEAWQFTS KHLKEVMISK NMEEDVFVAE QAKRALELPL 250 260 270 280 HWKVPMLEAR WFIHIYERRE DKNHLLLELA KMEFNTLQAI 290 300 310 320 YQEELKEISG WWKDTGLGEK LSFARNRLVA SFLWSMGIAF 330 340 350 360 EPQFAYCRRV LTISIALITV IDDIYDVYGT LDELEIFTDA 370 380 390 400 VERWDINYAL KHLPGYMKMC FLALYNFVNE FAYYVLKQQD 410 420 430 440 FDLLLSIKNA WLGLIQAYLV EAKWYHSKYT PKLEEYLENG 450 460 470 480 LVSITGPLII TISYLSGTNP IIKKELEFLE SNPDIVHWSS 490 500 510 520 KIFRLQDDLG TSSDEIQRGD VPKSIQCYMH ETGASEEVAR 530 540 550 560 QHIKDMMRQM WKKVNAYTAD KDSPLTGTTT EFLLNLVRMS 570 580 590 600 HFMYLHGDGH GVQNQETIDV GFTLLFQPIP LEDKHMAFTA SPGTKG
[0082] In some embodiments, the (R)-limonene synthase comprises the amino acid sequence Asp-Asp-Xaa-Xaa-Asp (DDXXD) (SEQ ID NO:17), or Asp-Asp-Xaa-Xaa-Glu (DDXXE) (SEQ ID NO:18), which is important for the catalytic activity of (R)-limonene synthase, presumably through binding to Mg.sup.2+.
[0083] In some embodiments, the (S)-limonene synthase, or a homologous enzyme thereof, has an amino acid sequence having at least 70% identity to the amino acid sequence of Cannabis sativa (S)-limonene synthase which is as follows:
TABLE-US-00005 (SEQ ID NO: 4) 10 20 30 40 MQCIAFHQFA SSSSLPIWSS IDNRFTPKTS ITSISKPKPK 50 60 70 80 LKSKSNLKSR SRSSTCYSIQ CTVVDNPSST ITNNSDRRSA 90 100 110 120 NYGPPIWSFD FVQSLPIQYK GESYTSRLNK LEKDVKRMLI 130 140 150 160 GVENSLAQLE LIDTIQRLGI SYRFENEIIS ILKEKFTNNN 170 180 190 200 DNPNPNYDLY ATALQFRLLR QYGFEVPQEI FNNFKNHKTG 210 220 230 240 EFKANISNDI MGALGLYEAS FHGKKGESIL EEARIFTTKC 250 260 270 280 LKKYKLMSSS NNNNMTLISL LVNHALEMPL QWRITRSEAK 290 300 310 320 WFIEEIYERK QDMNPTLLEF AKLDFNMLQS TYQEELKVLS 330 340 350 360 RWWKDSKLGE KLPFVRDRLV ECFLWQVGVR FEPQFSYFRI 370 380 390 400 MDTKLYVLLT IIDDMHDIYG TLEELQLFTN ALQRWDLKEL 410 420 430 440 DKLPDYMKTA FYFTYNFTNE LAFDVLQEHG FVHIEYFKKL 450 460 470 480 MVELCKHHLQ EAKWFYSGYK PTLQEYVENG WLSVGGQVIL 490 500 510 520 MHAYFAFTNP VTKEALECLK DGHPNIVRHA SIILRLADDL 530 540 550 560 GTLSDELKRG DVPKSIQCYM HDTGASEDEA REHIKYLISE 570 580 590 600 SWKEMNNEDG NINSFFSNEF VQVCQNLGRA SQFIYQYGDG 610 620 HASQNNLSKE RVLGLIITPI PM
[0084] In some embodiments, the (S)-limonene synthase comprises the amino acid sequence Asp-Asp-Xaa-Xaa-Asp (DDXXD) (SEQ ID NO:17), or Asp-Asp-Xaa-Xaa-Glu (DDXXE) (SEQ ID NO:18), which is important for the catalytic activity of (S)-limonene synthase, presumably through binding to Mg.sup.2+.
[0085] In some embodiments, the valencene synthase, or a homologous enzyme thereof, has an amino acid sequence having at least 70% identity to the amino acid sequence of Vitis vinifera valencene synthase which is as follows:
TABLE-US-00006 (SEQ ID NO: 5) 10 20 30 40 MSTQVSASSL AQIPQPKNRP VANFHPNIWG DQFITYTPED 50 60 70 80 KVTRACKEEQ IEDLKKEVKR KLTAAAVANP SQLLNFIDAV 90 100 110 120 QRLGVAYHFE QEIEEALQHI CNSFHDCNDM DGDLYNIALG 130 140 150 160 FRLLRQQGYT ISCDIFNKFT DERGRFKEAL ISDVRGMLGL 170 180 190 200 YEAAHLRVHG EDILAKALAF TTTHLKAMVE SLGYHLAEQV 210 220 230 240 AHALNRPIRK GLERLEARWY ISVYQDEAFH DKTLLELAKL 250 260 270 280 DFNLVQSLHK EELSNLARWW KELDFATKLP FARDRLVEGY 290 300 310 320 FWMHGVYFEP QYLRGRRILT KVIAMTSILD DIHDAYGTPE 330 340 350 360 ELKLFIEAIE RWDINSINQL PEYMKLCYVA LLDVYKEIEE 370 380 390 400 EMEKEGNQYR VHYAKEVMKN QVRAYFAEAK WLHEEHVPAF 410 420 430 440 EEYMRVALAS SGYCLLATTS FVGMGEIATK EAFDWVTSDP 450 460 470 480 KIMSSSNFITRLMDDIKSHK FEQKRGHVTS AVECYMKQYG 490 500 510 520 VSEEQVYSEF QKQIENAWLD INQECLKPTA VSMPLLARLL 530 540 550 NFTRTMDVIY KEQDSYTHVG KVMRDNIASV FINAVI
[0086] In some embodiments, the valencene synthase comprises the amino acid sequence Asp-Asp-Asp-Xaa-Xaa-Asp (DDXXD) (SEQ ID NO:17), or Asp-Asp-Xaa-Xaa-Glu (DDXXE) (SEQ ID NO:18), which is important for the catalytic activity of valencene synthase, presumably through binding to Mg.sup.2+.
[0087] In some embodiments, the (−)-alpha-terpineol synthase, or a homologous enzyme thereof, has an amino acid sequence having at least 70% identity to the amino acid sequence of Vitis vinifera (−)-alpha-terpineol synthase which is as follows:
TABLE-US-00007 (SEQ ID NO: 6) 10 20 30 40 MALSMLSSIP NLITHTRLPI IIKSSSCKAS PRGIKVKIGN 50 60 70 80 SNCEEIIVRR TANYHPTIWD YDYVQSLRSD YVGETYTRRL 90 100 110 120 DKLKRDVKPM LGKVKKPLDQ LELIDVLQRL GIYYHFKDEI 130 140 150 160 KRILNGIYNQ YNRHEEWQKD DLYATALEFR LLRQHGYDVP 170 180 190 200 QDVFSRFKDD TGSFKACLCE DMKGMLCLYE ASYLCVQGES 210 220 230 240 TMEQARDFAH RHLGKGLEQN IDQNLAIEVK HALELPLHWR 250 260 270 280 MPRLEARWFI DVYEKRQDMN PILLEFAKLD FNMVQATHQE 290 300 310 320 DLRHMSSWWS STRLGEKLNF ARDRLMENFL WTVGVIFEPQ 330 340 350 360 YGYCRRMSTK VNTLITIIDD VYDVYGTMDE LELFTDVVDR 370 380 390 400 WDINAMDPLP EYMKLCFLAL YNSTNEMAYD ALKEHGLHIV 410 420 430 440 SYLRKAWSDL CKSYLLEAKW YYSRYTPSLQ EYISNSWISI 450 460 470 480 SGPVILVHAY FLVANPITKE ALQSLERYHN IIRWSSMILR 490 500 510 520 LSDDLGTSLD ELKRGDVPKS IQCYMYETGA SEEDARKHTS 530 540 550 560 YLIGETWKKL NEDGAVESPF PETFIGIAMN LARMAQCMYQ 570 580 590 HGDGHGIEYG ETEDRVLSLL VEPIPSLSSE
[0088] In some embodiments, the (−)-alpha-terpineol synthase comprises the amino acid Asp-Asp-Xaa-Xaa-Asp (DDXXD) (SEQ ID NO:17), or Asp-Asp-Xaa-Xaa-Glu (DDXXE) (SEQ ID NO:18), which is important for the catalytic activity of (−)-alpha-terpineol synthase, presumably through binding to Mg.sup.2+.
[0089] In some embodiments, the (+)-alpha-pinene synthase, or a homologous enzyme thereof, has an amino acid sequence having at least 70% identity to the amino acid sequence of Pinus taeda (+)-alpha-pinene synthase which is as follows:
TABLE-US-00008 (SEQ ID NO: 7) 10 20 30 40 MALVSAVPLN SKLCLRRTLF GFSHELKAIH STVPNLGMCR 50 60 70 80 GGKSIAPSMS MSSTTSVSNE DGVPRRIAGH HSNLWDDDSI 90 100 110 120 ASLSTSYEAP SYRKRADKLI GEVKNIFDLM SVEDGVFTSP 130 140 150 160 LSDLHHRLWM VDSVERLGID RHFKDEINSA LDHVYSYWTE 170 180 190 200 KGIGRGRESG VTDLNSTALG LRTLRLHGYT VSSHVLDHFK 210 220 230 240 NEKGQFTCSA IQTEGEIRDV LNLFRASLIA FPGEKIMEAA 250 260 270 280 EIFSTMYLKD ALQKIPPSGL SQEIEYLLEF GWHTNLPRME 290 300 310 320 TRMYIDVFGE DTTFETPYLI REKLLELAKL EFNIFHSLVK 330 340 350 360 RELQSLSRWW KDYGFPEITF SRHRHVEYYT LAACIANDPK 370 380 390 400 HSAFRLGFGK ISHMITILDD IYDTFGTMEE LKLLTAAFKR 410 420 430 440 WDPSSIECLP DYMKGVYMAV YDNINEMARE AQKIQGWDTV 450 460 470 480 SYARKSWEAF IGAYIQEAKW ISSGYLPTFD EYLENGKVSF 490 500 510 520 GSRITTLEPM LTLGFPLPPR ILQEIDFPSK FNDLICAILR 530 540 550 560 LKGDTQCYKA DRARGEEASA VSCYMKDHPG ITEEDAVNQV 570 580 590 600 NAMVDNLTKE LNWELLRPDS GVPISYKKVA FDICRVFHYG 610 620 YKYRDGFSVA SIEIKNLVTR TVVETVPL
[0090] In some embodiments, the (+)-alpha-pinene synthase comprises the amino acid sequence Asp-Asp-Xaa-Xaa-Asp (DDXXD) (SEQ ID NO:17), or Asp-Asp-Xaa-Xaa-Glu (DDXXE) (SEQ ID NO:18), which is important for the catalytic activity of (+)-alpha-pinene synthase, presumably through binding to Mg.sup.2+.
[0091] In some embodiments, the (−)-alpha-pinene synthase, or a homologous enzyme thereof, has an amino acid sequence having at least 70% identity to the amino acid sequence of Pinus taeda (−)-alpha-pinene synthase which is as follows:
TABLE-US-00009 (SEQ ID NO: 8) 10 20 30 40 MSPVSVISLP SDLCLPTSFI DRSGRELIPL HITIPNVAMR 50 60 70 80 RQGKLMTRAS MSMNLRTAVS DDAVIRRRGD FHSNLWDDDL 90 100 110 120 IQSLSSPYGE PSYRERAERL IGEVKNSFNS MSNEDGESIT 130 140 150 160 PLDDLIQRLW MVDSVERLGI DRHFKKEIKS ALDHVYRYWS 170 180 190 200 EKGIGCGRES VVTDLNSTAL GLRTLRLHGY DVSADVLNHF 210 220 230 240 KNQSGQFACT LKQTEDQIRT VLNLYRASLI AFPGEKVMDE 250 260 270 280 AESFSAKYLK EALQKIPVSS FSREIGDVLE YGWHTYLPRL 290 300 310 320 EARNYIDVFG QDTENSKSYM KTEKLLELAK LEFNIFHALQ 330 340 350 360 KRELEYLVRW WKGSGSPQMT FCRHRHVEYY TLASCIAFEP 370 380 390 400 QHSGFRLGFA KACHIITVLD DMYDTFGTLD ELELFTSAIK 410 420 430 440 RWDPSATECL PEYMKGVYMI VYNTVNEMSQ EADKAQGRDT 450 460 470 480 LNYCRQAWEE YIDAYMQEAK WIASGEVPTF EEYYENGKVS 490 500 510 520 SGHRVSALQP ILTTDIPFPE HVLKEVDIPS QLNDLASAIL 530 540 550 560 RLRGDTRCYQ ADRARGEEAS CISCYMKDNP GTTEEDALNH 570 580 590 600 LNAMISDVIK GLNWELLKPN SSVPISAKKH AFDISRAFHC 610 620 GYKYRDGYSV ANIETKSLVK RTVIDPVTL
[0092] In some embodiments, the (−)-alpha-pinene synthase comprises the amino acid sequence Asp-Asp-Xaa-Xaa-Asp (DDXXD) (SEQ ID NO:17), or Asp-Asp-Xaa-Xaa-Glu (DDXXE) (SEQ ID NO:18), which is important for the catalytic activity of (−)-alpha-pinene synthase, presumably through binding to Mg.sup.2+.
[0093] In some embodiments, the (−)-beta-pinene synthase, or a homologous enzyme thereof, has an amino acid sequence having at least 70% identity to the amino acid sequence of Artemisia annua (−)-beta-pinene synthase which is as follows:
TABLE-US-00010 (SEQ ID NO: 9) 10 20 30 40 MASMCTFSSP FLLCNSSISR TNIVACNKQT STLQAQVKNV 50 60 70 80 ATIETTNRRS ANYAPSLWSY DFVQSLSSKY KGDNYMARSR 90 100 110 120 ALKGVVRTMI LEANGIENPL SLLNLVDDLQ RLGISYHFLD 130 140 150 160 EISNVLEKIY LNFYKSPEKW TNMDLNLRSL GFRLLRQHGY 170 180 190 200 HIPQEIFKDF IDVNGNFKGD IISMLNLYEA SYHSVEEESI 210 220 230 240 LDDAREFTTK YLKETLENIE DQNIALFISH ALVFPLHWMV 250 260 270 280 PRVETSWFIE VYPKKVGMNP TVLEFAKLDF NILQAVHQED 290 300 310 320 MKKASRWWKE TCWEKFGFAR DRLVENFMWT VAENYLPHFQ 330 340 350 360 TGRGVLTKVN AMITTIDDVY DVYGTLPELE LFTNIVNSWD 370 380 390 400 INAIDELPDY LKICFLACYN ATNELSYNTL TNKGFFVHPY 410 420 430 440 LKKAWQDLCN SYIIEAKWFN DGYTPTFNEF IENAYMSIGI 450 460 470 480 APIIRHAYLL TLTSVTEEAL QHIERAESMI RNACLIVRLT 490 500 510 520 NDMGTSSDEL ERGDIPKSIQ CYMHESGATE MEARAYIKQF 530 540 550 560 IVETWKKLNK ERQEIGSEFP QEFVDCVINL PRMGHFMYTD 570 580 GDKHGKPDMF KPYVFSLFVN PI
[0094] In some embodiments, the (−)-beta-pinene synthase comprises the amino acid sequence Asp-Asp-Xaa-Xaa-Asp (DDXXD) (SEQ ID NO:17), or Asp-Asp-Xaa-Xaa-Glu (DDXXE) (SEQ ID NO:18), which is important for the catalytic activity of (−)-beta-pinene synthase, presumably through binding to Mg.sup.2+.
[0095] In some embodiments, the vetispiradiene synthase, or a homologous enzyme thereof, has an amino acid sequence having at least 70% identity to the amino acid sequence of Solanum tuberosum vetispiradiene synthase which is as follows:
TABLE-US-00011 (SEQ ID NO: 10) 10 20 30 40 MTPAAVVMSN YGEEEIVRPI ADFSPSLWGD RFHSFSLDNQ 50 60 70 80 IAGKYAQEIE TLKEQSRIIL SASSRRTLAE KLDLIDIVER 90 100 110 120 LGIAYHFEKQ IDDMLDQFYK ADPNFEAHEY NDLQTLSVQF 130 140 150 160 RLLRQHGYNI SPKLFIRFQD AKGKFKESLC NDIKGLLNLY 170 180 190 200 EASHVRTHGE DILEEALAFS TAHLESAAPH LKSPLSKQVT 210 220 230 240 HALEQSLHKS IPRVETRYFI SIYEEEEQKN DVLLQFAKLD 250 260 270 280 FNLLQMLHKQ ELSEVSRWWK DLDFVTTLPY ARDRAVECYF 290 300 310 320 WTMGVYAEPQ YSQARVMLAK TIAMISIVDD TFDAYGIVKE 330 340 350 360 LEIYTDAIQR WDISQIDRLP DYMKISYKAL LDLYNDYEME 370 380 390 400 LSKDGRSDVV HYAKERMKEI VRNYFVEAKW FIEGYMPPVS 410 420 430 440 EYLSNALATS TYYLLTTTSY LGMKSANKQD FEWLAKNPKI 450 460 470 480 LEANVTLCRV IDDIATYEVE KGRGQIATGI ECYMRDYGVS 490 500 510 520 TEKAMEKFQE MAETAWKDVN EGILRPTPVS TEILTRILNL 530 540 550 ARIIDVTYKH NQDGYTHPEK VLKPHIIALL VDSIEI
[0096] In some embodiments, the vetispiradiene synthase comprises the amino acid sequence Asp-Asp-Xaa-Xaa-Asp (DDXXD) (SEQ ID NO:17), or Asp-Asp-Xaa-Xaa-Glu (DDXXE) (SEQ ID NO:18), which is important for the catalytic activity of vetispiradiene synthase, presumably through binding to Mg.sup.2+.
[0097] In some embodiments, the amorphadiene synthase, or a homologous enzyme thereof, has an amino acid sequence having at least 70% identity to the amino acid sequence of Artemisia annua amorphadiene synthase which is as follows:
TABLE-US-00012 (SEQ ID NO: 11) 10 20 30 40 MSLTEEKPIR PIANFPPSIW GDQFLIYEKQ VEQGVEQIVN 50 60 70 80 DLKKEVRQLL KEALDIPMKH ANLLKLIDEI QRLGIPYHFE 90 100 110 120 REIDHALQCI YETYGDNWNG DRSSLWFRLM RKQGYYVTCD 130 140 150 160 VFNNYKDKNG AFKQSLANDV EGLLELYEAT SMRVPGEIIL 170 180 190 200 EDALGFTRSR LSIMTKDAFS TNPALFTEIQ RALKQPLWKR 210 220 230 240 LPRIEAAQYI PFYQQQDSHN KTLLKLAKLE FNLLQSLHKE 250 260 270 280 ELSHVCKWWK AFDIKKNAPC LRDRIVECYF WGLGSGYEPQ 290 300 310 320 YSRARVFFTK AVAVITLIDD TYDAYGTYEE LKIFTEAVER 330 340 350 360 WSITCLDTLP EYMKPIYKLF MDTYTEMEEF LAKEGRTDLF 370 380 390 400 NCGKEFVKEF VRNLMVEAKW ANEGHIPTTE EHDPVVIITG 410 420 430 440 GANLLTTTCY LGMSDIFTKE SVEWAVSAPP LFRYSGILGR 450 460 470 480 RLNDLMTHKA EQERKHSSSS LESYMKEYNV NEEYAQTLIY 490 500 510 520 KEVEDVWKDI NREYLTTKNI PRPLLMAVIY LCQFLEVQYA 530 540 GKDNFTRMGD EYKHLIKSLL VYPMSI
[0098] In some embodiments, the amorphadiene synthase comprises the amino acid sequence Asp-Asp-Xaa-Xaa-Asp (DDXXD) (SEQ ID NO:17), or Asp-Asp-Xaa-Xaa-Glu (DDXXE) (SEQ ID NO:18), which is important for the catalytic activity of amorphadiene synthase, presumably through binding to Mg.sup.2+.
[0099] In some embodiments, the curcumene synthase, or a homologous enzyme thereof, has an amino acid sequence having at least 70% identity to the amino acid sequence of Pogostemon cablin curcumene synthase which is as follows:
TABLE-US-00013 (SEQ ID NO: 12) 10 20 30 40 MAAFTANAVD MRPPVITIHP RSKDIFSQFS LDDKLQKQYA 50 60 70 80 QGIEALKEEA RSMLMAAKSA KVMILIDTLE RLGLGYHFEK 90 100 110 120 EIEEKLEAIY KKEDGDDYDL FTTALRFRLL RQHQRRVPCS 130 140 150 160 VFDKFMNKEG KFEEEPLISD VEGLLSLYDA AYLQIHGEHI 170 180 190 200 LQEALIFTTH HLTRIEPQLD DHSPLKLKLN RALEFPFYRE 210 220 230 240 IPIIYAHFYI SVYERDDSRD EVLLKMAKLS YNFLQNLYKK 250 260 270 280 ELSQLSRWWN KLELIPNLPY IRDSVAGAYL WAVALYFEPQ 290 300 310 320 YSDVRMAIAK LIQIAAAVDD TYDNYATIRE AQLLTEALER 330 340 350 360 LNVHEIDTLP DYMKIVYRFV MSWSEDFERD ATIKEQMLAT 370 380 390 400 PYFKAEMKKL GRAYNQELKW VMERQLPSFE EYMKNSEITS 410 420 430 440 GVYIMFTVIS PYLNSATQKN IDWLLSQPRL ASSTAIVMRC 450 460 470 480 CNDLGSNQRE SKGGEVMTSL DCYMKQHGAS KQETISKFKL 490 500 510 520 IIEDEWKNLN EEWAATTCLP KVMVEIFRNY ARIAGFCYKN 530 540 NGDAYTSPKI VQQCFDALFV NPLRI
[0100] In some embodiments, the curcumene synthase comprises the amino acid sequence Asp-Asp-Xaa-Xaa-Asp (DDXXD) (SEQ ID NO:17), or Asp-Asp-Xaa-Xaa-Glu (DDXXE) (SEQ ID NO:18), which is important for the catalytic activity of curcumene synthase, presumably through binding to Mg.sup.2+.
[0101] In some embodiments, the bisabolene synthase, or a homologous enzyme thereof, has an amino acid sequence having at least 70% identity to the amino acid sequence of Pogostemon cablin bisabolene synthase which is as follows:
TABLE-US-00014 (SEQ ID NO: 13) 10 20 30 40 MDAFATSPTT ALFETVNCNA HVAPMAGEDS SENRPASNYK 50 60 70 80 PSTWDYEFLQ SLATTNNTVG EKHTRMADKL KEEVKSMMKG 90 100 110 120 TMEPVAKLEL INIVQRLGLK YRFESEIKEE LFSLYKDGTD 130 140 150 160 AWWVGNLHAT ALRFRLLREN GIFVPQDVFE TFKDKSGEFK 170 180 190 200 SQLCKDVRGL LSLYEASYLG WEGEELLDEA KKFSTTNLNN 210 220 230 240 VKESISSNTL GRLVKHALNL PLHWSAARYE ARWFIDEYER 250 260 270 280 EENVIPNLLK YAKLDFNVVQ SIHQKELGNL ARWWVETGLD 290 300 310 320 KLGFVRNTLM QNFMWGCAMA FEPQYGKVRD AAVKLGSLIT 330 340 350 360 MVDDVYDVYG TLEELEIFTD IVDRWDINGI DKLPRNISMI 370 380 390 400 VLTMFNTANQ ISYDLLRDRG FNSIPHIAEA WATLCKTYLK 410 420 430 440 EAKWYHSGYK PTLEEYLENG LVSISFVLSL VTAYLQTERL 450 460 470 480 ENLTYESAAY VNSVPPLVRY SGLLNRLYND LGTSSAEIAR 490 500 510 520 GDTLKSIQCY MTQTGATEEV AREHIKGLVH EAWKGMNRCL 530 540 550 560 FEQTPLAEPF VGFNVNTVRG SQFFYQHGDG YAVTESWTKD 570 LSLSVLIHPI PLNEED
[0102] In some embodiments, the bisabolene synthase comprises the amino acid sequence Asp-Asp-Xaa-Xaa-Asp (DDXXD) (SEQ ID NO:17), or Asp-Asp-Xaa-Xaa-Glu (DDXXE) (SEQ ID NO:18), which is important for the catalytic activity of bisabolene synthase, presumably through binding to Mg.sup.2+.
[0103] In some embodiments, the farnesene synthase, or a homologous enzyme thereof, has an amino acid sequence having at least 70% identity to the amino acid sequence of Zea mays farnesene synthase which is as follows:
TABLE-US-00015 (SEQ ID NO: 14) 10 20 30 40 MDATAFHPSL WGDFFVKYKP PTAPKRGHMT ERAELLKEEV 50 60 70 80 RKTLKAAANQ ITNALDLIIT LQRLGLDHHY ENEISELLRF 90 100 110 120 VYSSSDYDDK DLYVVSLRFY LLRKHGHCVS SDVFTSFKDE 130 140 150 160 EGNFVVDDTK CLLSLYNAAY VRTHGEKVLD EAITFTRRQL 170 180 190 200 EASLLDPLEP ALADEVHLTL QTPLFRRLRI LEAINYIPIY 210 220 230 240 GKEAGRNEAI LELAKLNFNL AQLIYCEELK EVTLWWKQLN 250 260 270 280 VETNLSFIRD RIVECHFWMT GACCEPQYSL SRVIATKMTA 290 300 310 320 LITVLDDMMD TYSTTEEAML LAEAIYRWEE NAAELLPRYM 330 340 350 360 KDFYLYLLKT IDSCGDELGP NRSFRTFYLK EMLKVLVRGS 370 380 390 400 SQEIKWRNEN YVPKTISEHL EHSGPTVGAF QVACSSFVGM 410 420 430 440 GDSITKESFE WLLTYPELAK SLMNISRLLN DTASTKREQN 450 460 470 480 AGQHVSTVQC YMLKHGTTMD EACEKIKELT EDSWKDMMEL 490 500 510 520 YLTPTEHPKL IAQTIVDFAR TADYMYKETD GFTFSHTIKD 530 MIAKLFVDPI SLF
[0104] In some embodiments, the farnesene synthase comprises the amino acid sequence Asp-Asp-Xaa-Xaa-Asp (DDXXD) (SEQ ID NO:17), or Asp-Asp-Xaa-Xaa-Glu (DDXXE) (SEQ ID NO:18), which is important for the catalytic activity of farnesene synthase, presumably through binding to Mg.sup.2+.
[0105] In some embodiments, the cadinene synthase, or a homologous enzyme thereof, has an amino acid sequence having at least 70% identity to the amino acid sequence of Ocimum basilicum cadinene synthase which is as follows:
TABLE-US-00016 (SEQ ID NO: 15) 10 20 30 40 MDVSILRDVR PPVTSYAPNI WADTFSNISL DEEVQKKYAE 50 60 70 80 TIEALKQVVR GMLMAAATPI KQMIFIDTLE RLGLAYHFET 90 100 110 120 EIEHKLQKIY DDNVCGDDCD LFTTALRFRL LRQHRHHVSC 130 140 150 160 DVFDKFLYEE GKFKGDAEGL LSLYEASHVR FHNEKILEEA 170 180 190 200 ERFTRQELSC WIKLQSPLKD KVKRALERPL HREVPILYAR 210 220 230 240 HFISIYEKDE SMDEHLLKLA KFNFNFLQNL YKKELYDLSR 250 260 270 280 WWNKFDLKTK LPYIRDRLAE AYLWGVGYHF EPQYSYVRKG 290 300 310 320 VVLSIKIIGI LDDTYDNYAT VNEAQLFTEI LDRWSMDEID 330 340 350 360 RLPDYMKIVL HFVMSAYEEY ERDAKIVYGK KFASPYFKET 370 380 390 400 IQQLARGYNQ ELKWVMEKQM PPFKDYLKNS EITSCIYIMF 410 420 430 440 ASIIPGLKSF TQEAIDWIKN EPNFAVKAGL IGRYWDDIGS 450 460 470 480 HKRESKGGEM LTVMDCYMKQ YSVSIQETIS EFAKAVEDSW 490 500 510 520 KEVNEGWVYT ISMSKEITVQ FLNYSRMCDA SYNRNNGDGY 530 540 TDPSFAKSNI TALFVDPIII
[0106] In some embodiments, the cadinene synthase comprises the amino acid sequence Asp-Asp-Xaa-Xaa-Asp (DDXXD) (SEQ ID NO:17), or Asp-Asp-Xaa-Xaa-Glu (DDXXE) (SEQ ID NO:18), which is important for the catalytic activity of cadinene synthase, presumably through binding to Mg.sup.2+.
[0107] In some embodiments, the aristolochene synthase, or a homologous enzyme thereof, has an amino acid sequence having at least 70% identity to the amino acid sequence of Penicillium roqueforti aristolochene synthase which is as follows:
TABLE-US-00017 (SEQ ID NO: 16) 10 20 30 40 MATSTETISS LAQPFVHLEN PINSPLVKET IRPRNDTTIT 50 60 70 80 PPPTQWSYLC HPRVKEVQDE VDGYFLENWK FPSFKAVRTF 90 100 110 120 LDAKFSEVTC LYFPLALDDR IHFACRLLTV LFLIDDVLEH 130 140 150 160 MSFADGEAYN NRLIPISRGD VLPDRTKPEE FILYDLWESM 170 180 190 200 RAHDAELANE VLEPTFVFMR AQTDRARLSI HELGHYLEYR 210 220 230 240 EKDVGKALLS ALMRFSMGLR LSADELQDMK ALEANCAKQL 250 260 270 280 SVVNDIYSYD KEEEASRTGH KEGAFLCSAV KVLAEESKLG 290 300 310 320 IPATKRVLWS MTREWETVHD EIVAEKIASP DGCSEAAKAY 330 340 MKGLEYQMSG NEQWSKTTRR YN
[0108] In some embodiments, the aristolochene synthase comprises the amino acid sequence Asp-Asp-Xaa-Xaa-Asp (DDXXD) (SEQ ID NO:17), or Asp-Asp-Xaa-Xaa-Glu (DDXXE) (SEQ ID NO:18), which is important for the catalytic activity of aristolochene synthase, presumably through binding to Mg.sup.2+.
[0109] In some embodiments, any of the synthase described herein comprises the amino acid sequence Asp-Asp-Xaa-Xaa-Asp (DDXXD) (SEQ ID NO:17), or Asp-Asp-Xaa-Xaa-Glu (DDXXE) (SEQ ID NO:18), which is important for the catalytic activity of the synthase, presumably through binding to Mg.sup.2+.
[0110] In some embodiments, the 1,8-cineole 2-endo-monooxygenase, or a homologous enzyme thereof, has an amino acid sequence having at least 70% identity to the amino acid sequence of Citrobacter braakii 1,8-cineole 2-endo-monooxygenase which is as follows:
TABLE-US-00018 (SEQ ID NO: 19) 10 20 30 40 MTATVASTSL FTTADHYHTP LGPDGTPHAF FEALRDEAET 50 60 70 80 TPIGWSEAYG GHWVVAGYKE IQAVIQNTKA FSNKGVTFPR 90 100 110 120 YETGEFELMM AGQDDPVHKK YRQLVAKPFS PEATDLFTEQ 130 140 150 160 LRQSTNDLID ARIELGEGDA ATWLANEIPA RLTAILLGLP 170 180 190 200 PEDGDTYRRW VWAITHVENP EEGAEIFAEL VAHARTLIAE 210 220 230 240 RRTNPGNDIM SRVIMSKIDG ESLSEDDLIG FFTILLLGGI 250 260 270 280 DNTARFLSSV FWRLAWDIEL RRRLIAHPEL IPNAVDELLR 290 300 310 320 FYGPAMVGRL VTQEVTVGDI TMKPGQTAML WFPIASRDRS 330 340 350 360 AFDSPDNIVI ERTPNRHLSL GHGIHRCLGA HLIRVEARVA 370 380 390 400 ITEFLKRIPE FSLDPNKECE WLMGQVAGML HVPIIFPKGK RLSE
[0111] In some embodiments, the 1,8-cineole 2-endo-monooxygenase, or homolog thereof, comprises FXXGXRXCXG (SEQ ID NO:21), or CXG, motif and/or EXXR motif.
[0112] In some embodiments, the epi-isozizaene 5-monooxygenase, or a homologous enzyme thereof, has an amino acid sequence having at least 70% identity to the amino acid sequence of Streptomyces coelicolor epi-isozizaene 5-monooxygenase which is as follows:
TABLE-US-00019 (SEQ ID NO: 20) 10 20 30 40 MTVESVNPET RAPAAPGAPE LREPPVAGGG VPLLGHGWRL 50 60 70 80 ARDPLAFMSQ LRDHGDVVRI KLGPKTVYAV TNPELTGALA 90 100 110 120 LNPDYHIAGP LWESLEGLLG KEGVATANGP LHRRQRRTIQ 130 140 150 160 PAFRLDAIPA YGPIMEEEAH ALTERWQPGK TVDATSESFR 170 180 190 200 VAVRVAARCL LRGQYMDERA ERLCVALATV FRGMYRRMVV 210 220 230 240 PLGPLYRLPL PANRRFNDAL ADLHLLVDEI IAERRASGQK 250 260 270 280 PDDLLTALLE AKDDNGDPIG EQEIHDQVVA ILTPGSETIA 290 300 310 320 STIMWLLQAL ADHPEHADRI RDEVEAVTGG RPVAFEDVRK 330 340 350 360 LRHTGNVIVE AMRLRPAVWV LTRRAVAESE LGGYRIPAGA 370 380 390 400 DIIYSPYAIQ RDPKSYDDNL EFDPDRWLPE RAANVPKYAM 410 420 430 440 KPFSAGKRKC PSDHFSMAQL TLITAALATK YRFEQVAGSN 450 460 DAVRVGITLR PHDLLVRPVA R
[0113] In some embodiments, the epi-isozizaene 5-monooxygenase, or homolog thereof, comprises FXXGXRXCXG (SEQ ID NO:21), or CXG, motif and/or EXXR motif.
Host Cells
[0114] The host cells of the present invention are genetically modified in that heterologous nucleic acid have been introduced into the host cells, and as such the genetically modified host cells do not occur in nature. The suitable host cell is one capable of expressing a nucleic acid construct encoding one or more enzymes described herein. The gene(s) encoding the enzyme(s) may be heterologous to the host cell or the gene may be native to the host cell but is operatively linked to a heterologous promoter and one or more control regions which result in a higher expression of the gene in the host cell.
[0115] The enzyme can be native or heterologous to the host cell. Where the enzyme is native to the host cell, the host cell is genetically modified to modulate expression of the enzyme. This modification can involve the modification of the chromosomal gene encoding the enzyme in the host cell or a nucleic acid construct encoding the gene of the enzyme is introduced into the host cell. One of the effects of the modification is the expression of the enzyme is modulated in the host cell, such as the increased expression of the enzyme in the host cell as compared to the expression of the enzyme in an unmodified host cell.
[0116] Any prokaryotic or eukaryotic host cell may be used in the present method so long as it remains viable after being transformed with a sequence of nucleic acids. Generally, although not necessarily, the host cell is a yeast or a bacterium. In some embodiments, the host cell is a Gram negative bacterium. In some embodiments, the host cell is of the phylum Proteobactera. In some embodiments, the host cell is of the class Gammaproteobacteria. In some embodiments, the host cell is of the order Enterobacteriales. In some embodiments, the host cell is of the family Enterobacteriaceae. Examples of bacterial host cells include, without limitation, those species assigned to the Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsielia, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, and Paracoccus taxonomical classes. In some embodiments, the host cell is not adversely affected by the transduction of the necessary nucleic acid sequences, the subsequent expression of the proteins (i.e., enzymes), or the resulting intermediates required for carrying out the steps associated with the mevalonate pathway. For example, it is preferred that minimal “cross-talk” (i.e., interference) occur between the host cell's own metabolic processes and those processes involved with the mevalonate pathway. Suitable eukaryotic cells include, but are not limited to, fungal, insect or mammalian cells. Suitable fungal cells are yeast cells, such as yeast cells of the Saccharomyces genus.
[0117] The genetically modified host cell can be any microbe capable of production of the modified terpenoid in accordance with the methods of the invention.
[0118] In some embodiments, the host cell is a yeast. Yeast host cells suitable for the invention include, but are not limited to, Yarrowia, Candida, Bebaromyces, Saccharomyces, Schizosaccharomyces and Pichia cells. In one embodiment, Saccharomyces cerevisae is the host cell. In one embodiment, the yeast host cell is a species of Candida, including but not limited to C. tropicalis, C. maltosa, C. apicola, C. paratropicalis, C. albicans, C. cloacae, C. guillermondii, C. intermedia, C. lipolytica, C. panapsilosis and C. zeylenoides. In one embodiment, Candida tropicalis is the host cell. In some embodiments, the yeast host cell is a non-oleaginous yeast. In some embodiments, the non-oleaginous yeast is a Saccharomyces species. In some embodiments, the Saccharomyces species is Saccharomyces cerevisiae. In some embodiments, the yeast host cell is an oleaginous yeast. In some embodiments, the oleaginous yeast is a Rhodosporidium species. In some embodiments, the Rhodosporidium species is Rhodosporidium toruloides.
[0119] In some embodiments the host cell is a bacteria. Bacterial host cells suitable for the invention include, but are not limited to, Escherichia, Corynebacterium, Pseudomonas, Streptomyces, and Bacillus. In some embodiments, the Escherichia cell is an E. coli, E. albertii, E. fergusonii, E. hermanii, E. marmotae, or E. vulneris. In some embodiments, the Corynebacterium cell is Corynebacterium glutamicum, Corynebacterium kroppenstedtii, Corynebacterium alimapuense, Corynebacterium amycolatum, Corynebacterium diphtherias, Corynebacterium efficiens, Corynebacterium jeikeium, Corynebacterium macginleyi, Corynebacterium matruchotii, Corynebacterium minutissimum, Corynebacterium renale, Corynebacterium striatum, Corynebacterium ulcerans, Corynebacterium urealyticum, or Corynebacterium uropygiale. In some embodiments, the Pseudomonas cell is a P. putida, P. aeruginosa, P. chlororaphis, P. fluorescens, P. pertucinogena, P. stutzeri, P. syringae, P. cremoricolorata, P. entomophila, P. fulva, P. monteilii, P. mosselii, P. oryzihabitans, P. parafluva, or P. plecoglossicida. In some embodiments, the Streptomyces cell is a S. coelicolor, S. lividans, S. venezuelae, S. ambofaciens, S. avermitilis, S. albus, or S. scabies. In some embodiments, the Bacillus cell is a B. subtilis, B. megaterium, B. licheniformis, B. anthracis, B. amyloliquefaciens, or B. pumilus.
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[0151] It is to be understood that, while the invention has been described in conjunction with the preferred specific embodiments thereof, the foregoing description is intended to illustrate and not limit the scope of the invention. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.
[0152] All patents, patent applications, and publications mentioned herein are hereby incorporated by reference in their entireties.
[0153] The invention having been described, the following examples are offered to illustrate the subject invention by way of illustration, not by way of limitation.
Example 1
Production of Oxidized Terpenoids Using P450 Enzymes and their Engineered Enzymes Fusions
[0154] Terpenes are a large class of organic compounds, primarily produced by plants and constitute the main components of essential oils. The functionalization of terpene hydrocarbons using cytochrome P450 enzymes can derive many useful compounds that can be converted to diverse valuable products. In this study, to expand the portfolio of bioproducts from biofuel pathways, cytochrome P450 enzymes are investigated for oxidizing monoterpene (e.g. limonene, 1,8-cineole), a few oxidized terpenes are produced, including carveol, isopiperitenol, (1R)-6-hydroxycineole. Based on those oxidized terpenes, possible biosynthetic pathways are explored toward making biological-derived functionalized terpenes, such as carvolactone, a monomer of thermoplastic polyester. On the other hand, terpenes are hydrophobic and volatile compounds that limit the accessibility of P450 enzymes. To optimize the production of oxidized terpenes, an enzyme fusion strategy is developed with linking terpene synthase and P450 enzyme. In an example of producing (1R)-6-hydroxycineole, the engineered fusion shows higher efficiency from both in vitro and in vivo production results. Thus, engineering a fusion between terpene synthase and P450 presents a feasible strategy for producing oxidized terpenes, suggesting broad application during the production of terpene-based bioproducts.
[0155] Production of oxidized terpenes and derived bioproducts using P450 enzymes. The functionalization of terpene hydrocarbons using cytochrome P450 enzymes can derive many useful compounds that can be converted to diverse valuable products. See
[0156] Biosynthesis of carvolactone as a monomer. Biosynthetic pathway is explored based on oxidized terpenes and carvolactone is selected as a functionalized terpene target since it is a new monomer for making thermoplastic polymer. To build the biosynthetic pathway of carvolactone, optimal enzymes (dehydrogenase/reductase) are screened and selected. See
[0157] Engineering fusion between terpene synthase and P450 toward efficient production of oxidized terpene. Enzyme fusions are engineered to improve substrate availability as terpenes are hydrophobic and easily lost by phase separation. See
[0158] In vivo assessment of fusion enzymes toward hydroxycineole production. Fusions are engineered into 1,8-cineole overproducing strain to assess the in vivo production of hydroxycineole. Fusions increase up to 3 folds in hydroxycineole production than non-fused enzymes, a 9 amino-acid linker (G3) shows the highest production of hydroxycineole (56 mg/L). See
[0159] Conclusions. P450 enzymes are used to oxidize terpenes and several oxidized terpenes are produced. Carvolactone is selected and produced as a new monomer using P450 enzymes and limonene synthesis pathway. An enzyme fusion strategy is developed by linking terpene synthase and P450 enzyme toward optimizing oxidized terpene production. Fusion enzymes show higher efficiency during hydroxycineole production.
Example 2
Efficient Production of Oxidized Terpenoids Via Engineering Fusion Proteins of Terpene Synthase and P450
[0160] The functionalization of terpene hydrocarbons using cytochrome P450 enzymes is a versatile route to produce useful derivatives that can be further converted to value-added products. As terpenes are generally hydrophobic and volatile, however, their availability as a substrate for the P450 enzymes is limited especially when a solvent overlay is used to trap terpenes and prevent evaporation during the microbial terpene biosynthesis. In this study, an enzyme fusion strategy is developed by directly linking terpene synthase and P450 enzyme together to improve the accessibility of terpene molecules as a substrate for P450 enzymes. Using the hydroxylation of a monoterpene 1,8-cineole as a model system, a series of fusion proteins between 1,8-cineole synthase and P450.sub.cin (CYP176A1) are engineered with different lengths of peptide linkers to investigate the hydroxylation of 1,8-cineole. Results show that the fusion enzyme increases 5.4 and 3.1 folds in hydroxycineole production than non-fused individual enzymes at the in vitro and in vivo conditions, respectively. The enzyme fusion strategy is also applied to the oxidation of a sesquiterpene epi-isozizaene, in which a 90-fold increase is observed by the fusion in albaflavenol production. Developing fusion enzymes for terpene synthase and P450 presents an efficient strategy toward oxidation of hydrophobic terpene compounds and this strategy will be widely applicable for the functionalization of hydrophobic biosynthetic intermediates.
[0161] Given that the considerable loss of terpene molecules is a critical limitation for the subsequent P450 reaction during the microbial production.sup.5, engineering a fusion protein by linking terpene synthase and P450 to form a chimeric protein could improve the proximity of P450 and the terpene substrate, which in turn would improve the substrate availability for P450.
[0162] In this study, hydroxylation of monoterpene 1,8 cineole is selected as a model system for this approach of terpene synthase-P450 fusion (
Results and Discussion
[0163] While engineering of fusion proteins shows an easy approach for desirable enzymatic characteristics, it is still challenging to achieve the optimal activities, for example, it is difficult to precisely control the distance of enzymes in a fusion protein, the folding of a larger multidomain protein may be inefficient, etc.
Engineering Fusion Enzymes of 1,8-Cineole Synthase and P450.SUB.cin
[0164] 1,8-Cineole, or eucalyptol, is a monoterpene (C10) that naturally found in essential oils.sup.17. 1,8-Cineole is also a potential precursor for high energy density molecules used as jet fuels.sup.18,19, and therefore E. coli is engineered to overproduce 1,8-cineole using the mevalonate (MVA) pathway.sup.20. Hydroxylation of 1,8-cineole introduces a functional group, which decorates this compound with possibilities to further derived valuable products, such as p-cymene.sup.21. P450.sub.cin (CYP176A1) from Citrobacter braakii is found showing a specific activity for 1,8-cineole hydroxylation to (1R)-6β-hydroxycineole (or hydroxycineole).sup.22,23.
[0165] To investigate the hydroxylation of 1,8-cineole as a model system of the fusion of terpene synthase and P450, fusion proteins are prepared between 1,8-cineole synthase (CS) and P450.sub.cin using a widely studied flexible peptide linker (Gly-Ser-Gly).sub.n.sup.24. By adjusting the repeat number (n) of the Gly-Ser-Gly (GSG) linker, five CS-P450.sub.cin fusion proteins have been engineered with different linker lengths (n=1-5) and named as G1 to G5 according to their GSG linker repeats (
In Vitro Production of Hydroxycineole with CS-P450.sub.cin Fusions from GPP
[0166] To investigate the hydroxylation of 1,8-cineole by various engineered CS-P450.sub.cin fusions, equal moles of purified proteins are used for in vitro production of hydroxycineole from GPP (
[0167] According to the in vitro production results, the engineered CS-P450.sub.cin fusions show higher efficiencies than non-fused CS and P450.sub.cin during hydroxycineole production. The improved hydroxylation from fusions can be attributed to the proximity of P450.sub.cin to its hydrophobic substrate 1,8-cineole, which shows the feasibility of linking a P450 enzyme to a biosynthetic pathway enzyme, particularly when terpenes are used as substrates for P450 reactions. Additionally, the increased 1,8-cineole production in the first 3 hours might result from the local hydrophobic environment created by the fusion enzyme and thus facilitated the 1,8-cineole accumulation. It can also suggest that terpene synthase activity is increased in the fusion enzymes as previously reported in a fusion of E. coli beta-galactosidase (LacZ) and the dimeric galactose dehydrogenase (GalDH) from Pseudomonas fluorescens which shows improved enzyme activities when they are linked each other.sup.25.
In Vivo Assessment of CS-P450.sub.cin Fusions for Hydroxycineole Production from Glucose
[0168] The CS-P450.sub.cin fusions are further engineered into the 1,8-cineole overproducing E. coli strain.sup.20 to assess hydroxycineole production under the in vivo conditions. Informed from the previous report.sup.20, a 2-plasmid system is used for hydroxycineole production by inserting P450.sub.cin (CinA) and the reductase (CinC) at the downstream of CS on the plasmid JBEI-15065 (Table 2). For the non-fused expression of CS and P450.sub.cin, an RBS sequence (5′-TTTAAGAAGGAGATATACC-3′) is used for individual expression of both CS and P450.sub.cin (Table 2). For the CS-P450.sub.cin fusions, the same RBS is used for the entire fused protein sequence. As solvent overlay is usually used to enrich terpene molecules and prevent evaporation of the product during fermentation, an overly is used to evaluate the performance of fusion enzymes at the in vivo conditions. While dodecane is used as the overlay for 1,8-cineole production previously.sup.20, it has a similar molecular weight to hydroxycineole (MW=170), as well as a very close retention time in GC analysis. Therefore, nonane, instead of dodecane, is used as the overlay to obtain a better signal of hydroxycineole on GC.
TABLE-US-00020 TABLE 2 Strains and plasmids used in this study. Strains Description Reference CS E. coli BL21 (DE3) with pSKB3-CS This study CinA E. coli BL21 (DES) with pSKB3-CinA This study CinC E. coli BL21 (DE3) with pSKB3-CinC This study G1 E. coli BL21 (DES) with This study pSKB3-CS-G1-CinA G2 E. coli BL21 (DE3) with This study pSKB3-CS-G2-CinA G3 E. coli BL21 (DE3) with This study pSKB3-CS-G3-CinA G4 E. coli BL21 (DE3) with This study pSKB3-CS-G4-CinA G5 E. coli BL21 (DE3) with This study pSKB3-CS-G5-CinA Fpr E. coli BL21 (DE3) with pSKB3-Fpr This study 2pCin_Non- E. coli DH1 with JBEI-3122 + This study fusion pTrc99a-trGPPS-CS-RBS-CinAC 2pCin_G1 E. coli DH1 with JBEI-3122 + This study pTrc99a-trGPPS-CS-G1-CinAC 2pCin_G2 E. coli DH1 with JBEI-3122 + This study pTrc99a-trGPPS-CS-G2-CinAC 2pCin_G3 E. coli DH1 with JBEI-3122 + This study pTrc99a-trGPPS-CS-G3-CinAC 2pCin_G4 E. coli DH1 with JBEI-3122 + This study pTrc99a-trGPPS-CS-G4-CinAC 2pCin_G5 E. coli DH1 with JBEI-3122 + This study pTrc99a-trGPPS-CS-G5-CinAC 2pCin_G3_reverse E. coli DH1 with JBEI-3122 + This study pTrc99a-trGPPS-CinA-G3-CS-CinC 2pEiz E. coli DH1 with JBEI-2704 + This study JBEI-15862 2pEizSC_Non- E. coli DH1 with JBEI-2704 + This study fusion pTrc99a-EizS-RBS-CYP170Al-CinC 2pEizSC_EG1 E. coli DH1 with JBEI-2704 + This study pTrc99a-EizS-G1-CYP170A1-CinC 2pEizSC_EG2 E. coli DH1 with JBEI-2704 + This study pTre99a-EizS-G2-CYPI70A1-CinC 2pEizSC_EG3 E. coli DH1 with JBEI-2704 + This study pTrc99a-EizS-G3-CYP170A1-CinC 2pEizSC_EG4 E. coli DH1 with JBEI-2704 + This study pTrc99a-EizS-G4-CYP170A1-CinC 2pEizSC_EG5 E. coli DH1 with JBEI-2704 + This study pTrc99a-EizS-G5-CYP170A1-CinC pSKB3 Modified pET-28a .sup.30 JBEI-3122 pBbA5c-MTSA-T1-MBI .sup.5 JBEI-15065 pTrc99a-GPPS-CS.sub.Str .sup.20 JBEI-2704 pBbA5c-MevT-T1-MBIS .sup.31 JBEI-15862 pTrc99a-coEizS .sup.29 pSKB3-CS This study pSKB3-CinA This study pSKB3-CinC This study pSKB3-CS-G1-CinA This study pSKB3-CS-G2-CinA This study pSKB3-CS-G3-CinA This study pSKB3-CS-G4-CinA This study pSKB3-CS-G5-CinA This study pSKB3-Fpr This study pTrc99a-trGPPS-CS-RBS-CinAC This study pTrc99a-trGPPS-CS-G1-CinAC This study pTrc99a-trGPPS-CS-G2-CinAC This study pTrc99a-trGPPS-CS-G3-CinAC This study pTrc99a-trGPPS-CS-G4-CinAC This study pTrc99a-trGPPS-CS-G5-CinAC This study pTrc99a-trGPPS-CinA-G3-CS-CinC This study pTrc99a-EizS-RBS-CYP170A1-CinC This study pTrc99a-EizS-G1-CYP170A1-CinC This study pTre99a-EizS-G2-CYP170A1-CinC This study pTrc99a-EizS-G3-CYP170A1-CinC This study pTre99a-EizS-G4-CYP170A1-CinC This study pTrc99a-EizS-G5-CYP170A1-CinC This study
[0169] As shown in
[0170] In addition to the linker length, the orientation of enzymes in a fusion protein is another important factor for the function of a fusion enzyme.sup.12. Given that G3 shows high production of hydroxycineole at both in vitro and in vivo conditions, GSG.sub.3 (3-repeat of Gly-Ser-Gly) is used as the linker length to construct a fusion enzyme with reversely ordered CS and P450.sub.cin (i.e. P450.sub.cin-GSG.sub.3-CS) to test hydroxycineole production. Compared with the regular CS-GSG.sub.3-P450.sub.cin fusion (G3), this reversed fusion produces 51% and 76% less 1,8-cineole and hydroxycineole, respectively when overlay is used (Table 3). These trends are also significant when the overlay is not used. In this case, the reversed fusion produces 80% and 63% less 1,8-cineole and hydroxycineole, respectively, than the fusion with the normal order (Table 3), indicating an inefficient fusion enzyme when CS and P450.sub.cin are linked in the reverse order.
TABLE-US-00021 TABLE 3 Comparison between CS-P450.sub.cin and P450.sub.cin-CS (fusion with reverse order of enzymes). 1,8-Cineole Hydroxycineole With Without With Without overlay overlay overlay overlay CS-P450.sub.cin (mg/L) 86 ± 1 3 ± 1 32 ± 1 56 ± 1 P450.sub.cin-CS (mg/L) 42 ± 10 1 ± 0 8 ± 0 21 ± 1
Production of Oxidized Epi-Isozizaene Using Enzyme Fusions.
[0171] The enzyme fusion strategy is applied to the biosynthetic pathway for oxidized epi-isozizaene (
[0172] Compared with the non-fusion control, epi-isozizaene production level is a little lower in the strains with engineered fusion enzymes. On the other hand, the total oxidized products (albaflavenol and albaflavonone) are notably increased in the fusions with shorter linkers (GSG.sub.1-3) for both conditions with and without overlay (
Conclusions
[0173] The functionalization of terpene molecules using cytochrome P450 enzymes presents opportunities for producing diverse bioproducts via the isoprenoid pathway. In this study, an enzyme fusion strategy is developed by directly linking terpene synthase and P450 enzyme together to facilitate the accessibility of terpene molecules to P450 enzymes.
[0174] The hydroxylation of a monoterpene 1,8-cineole as a model system is selected and engineered a series of fusion proteins between 1,8-cineole synthase and P450.sub.cin (CYP176A1) with different lengths of peptide linker to investigate the hydroxylation of 1,8-cineole. Results show that fusion enzymes increase up to 5.4 and 3.1 folds in hydroxycineole production than non-fused individual enzymes at the in vitro and in vivo conditions, respectively. The enzyme fusion strategy is also applied to the oxidation of a sesquiterpene epi-isozizaene, in which a ˜90-fold increase is observed by the fusion in albaflavenol production. Results suggest engineering fusion enzymes between terpene synthase and P450 shows a feasible strategy toward efficient production of oxidized terpenes, especially when a solvent overlay is used to trap terpene molecules and prevent their evaporation during the production.
Methods
Strains and Plasmid Construction
[0175] All strains and plasmids used in this study are listed in Table 2. E. coli DH1 strain is used for terpene and oxidized terpene production, and E. coli DH5α is used for genetic cloning. Genes of CinA (P450.sub.cin, CYP176A1; GenBank ID: AF456128) and CinC (GenBank ID: AF456128) from Citrobacter braakii, and CYP170A1 (sco5223; GenBank ID: NC_003888) from Streptomyces coelicolor A3(2) are codon-optimized and synthesized by Integrated DNA Technologies, Inc. (San Diego, Calif.). Fpr (GenBank ID: CP032667) is cloned from E. coli genomic DNA. CS from Streptomyces clavuligerus is cloned from plasmid JBEI-15065 (Table 2).
Protein Expression and Purification
[0176] A plasmid pSKB3 encoding interested proteins with N-terminal His-tag is transformed into E. coli BL21 (DE3). BL21 (DE3) strains bearing pSKB3 plasmids are cultured in Lysogeny Broth (LB) medium containing 50 μg/mL kanamycin at 37° C. until the optical density of the culture at 600 nm (OD.sub.600) reaches to 0.5-0.8. The culture is then supplemented with 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for induction and transferred to 18° C. for culturing overnight. Cells are collected by centrifugation and resuspended in 25 mM Tris-HCl (pH 8.0) buffer containing 300 mM NaCl and 10 mM imidazole (pH 8.0). Cells are lysed by sonication and proteins were purified using QIAGEN Ni-NTA Agarose. All purified proteins are desalted in 25 mM Tris-HCl (pH 8.0) buffer containing 100 mM NaCl, and 10% glycerol, and stored at −80° C.
In Vitro Production of Hydroxycineole
[0177] Equal mole of purified proteins, 5 μM CS and 5 μM CinA, or 5 μM fusion protein (G1, G2, G3, G4, G5) with 40 μM CinC and 10 μM Fpr are used for the in vitro reaction in 50 mM Tris-HCl buffer (pH 7.4) containing 5 mM MgCl.sub.2.sup.17,23. NADPH (2 mM) and 1 mM geranyl pyrophosphate (GPP, Sigma-Aldrich 19533) are added to start the reaction. The reaction is conducted in a 1.7-mL microcentrifuge tube at 25° C. for 5 hours.
In Vivo Production of Hydroxycineole
[0178] E. coli DH1 bearing two plasmids is used for hydroxycineole production. Seed cultures of all production strains are prepared by growing single colonies in LB medium containing 30 μg/mL chloramphenicol and 100 μg/mL carbenicillin at 37° C. with 200-rpm shaking for overnight. The seed cultures are diluted in 5 mL EZ-Rich defined medium (Teknova, USA) containing 10 g/L glucose (1%, w/v), 30 μg/mL chloramphenicol, 100 μg/mL carbenicillin, and 0.5 mM IPTG in 50-mL test tubes. 0.5 mL nonane (10%, v/v) is added when required as a solvent overlay. The E. coli cell cultures are incubated in rotary shakers (200 rpm) at 30° C. for 48 h.
In Vivo Production of Oxidized Epi-Isozizaene
[0179] E. coli DH1 bearing two plasmids are used for oxidized epi-isozizaene production. Seed cultures of all production strains are prepared by growing single colonies in LB medium containing 30 μg/mL chloramphenicol and 100 μg/mL carbenicillin at 37° C. with 200-rpm shaking for overnight. The seed cultures are diluted in 5 mL EZ-Rich defined medium (Teknova, USA) containing 10 g/L glucose (1%, w/v), 30 μg/mL chloramphenicol, 100 μg/mL carbenicillin, and 65 mg/L δ-aminolevulinic acid, 0.5 mM IPTG in 50-mL culture tubes. 0.5 mL nonane (10%, v/v) is added when required as a solvent overlay. The E. coli cell cultures are incubated in rotary shakers (200 rpm) at 30° C. for 72 hours.
GC-MS Analysis
[0180] For 1,8-cineole and hydroxycineole, samples are extracted by an equal volume of ethyl acetate containing β-pinene (5 mg/L) as an internal standard. For epi-isozizaene and oxidized products, samples are extracted by an equal volume of ethyl acetate containing guaiazulene (5 mg/L) as an internal standard. The mixture of ethyl acetate and cell culture is vigorously shaken for 15 min and subsequently centrifuged at 21,130 g for 3 min to separate ethyl acetate from the aqueous phase. The ethyl acetate layer is collected and 1 μL was analyzed by Agilent GC-MS equipped with HP-5 column (Agilent, USA). The GC oven is programmed from 40° C. (held for 3 min) to 295° C. at 15° C./min. The solvent delay is set at 3.4 min. Samples are normalized using internal standard (β-pinene or guaiazulene). 1,8-cineole is quantified using an authentic standard. Hydroxycineole is estimated using total ion chromatogram (TIC) areas and 1,8-cineole standards. Epi-isozizaene and oxidized products (albaflavenol, albaflavenone) are estimated using total ion chromatogram (TIC) areas and caryophyllene standards.
[0181] When nonane overlay is used during the production, the solvent delay is set at 6.8 min. Both the nonane overlay and the aqueous phase of the culture re sampled for the GC-MS measurement, respectively. The production titers are the sum of both measured values.
[0182] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.