USOO8927241B2

(12) United States Patent (10) Patent No.: US 8,927.241 B2 Ajikumar et al. (45) Date of Patent: *Jan. 6, 2015

(54) MICROBIAL ENGINEERING FOR THE 2010/0297722 A1 11/2010 Anterola et al. PRODUCTION OF CHEMICAL AND 38: i h S. A. s3. Fi Sharks et al. PHARMACEUTICAL PRODUCTS FROM THE Jikumar et al. ISOPRENOID PATHWAY FOREIGN PATENT DOCUMENTS (75) Inventors: Parayil K. Ajikumar, Cambridge, MA WO WO97,385.71 A1 10, 1997 (US); Gregory Stephanopoulos, Int (US); Too Heng Phon, OTHER PUBLICATIONS 73) Assi : M h Insti fTechnol Broun et al., Catalytic plasticity of fatty acid modification enzymes (73) SS1gnees: C s t it's R nology, underlying chemical diversity of plant lipids. Science, 1998, vol. 282: ambridge, ; Nationa 1315-1317. liversity of Singapore, Singapore Chica et al., Semi-rational approaches to engineering enzyme activ (SG) ity: combining the benefits of directed evolution and rational design. (*) Notice: Subject to any disclaimer, the term of this Curr. Opi. --Biotechnol. 200 5, vol. 16. 378RSRRSRA 384. sk atent is extended or adjusted under 35 Devos et al., Practical limits of function prediction. Proteins: Struc p S.C. 154(b) by 354 days ture, Function, and Genetics. 2000, vol. 41: 98-107. M YW- y yS. Kisselev L., Polypeptide release factors in prokaryotes and This patent is Subject to a terminal dis- eukaryotes: same function, different structure. Structure, 2002, vol. claimer. 10:8-9. Seffernicket al., Melamine deaminase and Atrazine chlorohydrolase: (21) Appl. No.: 13/249,388 98 percent identical but functionally different. J. Bacteriol., 2001, vol. 183 (8): 2405-2410.* (22) Filed: Sep. 30, 2011 Sen et al., Developments in directed evolution for improving enzyme functions. Appl. Biochem. Biotechnol., 2007, vol. 143: 212-223.* (65) Prior Publication Data Whisstock et al., Prediction of protein function from protein US 2012/O1 O7893 A1 May 3, 2012 sequence. Q. Rev. Biophysics., 2003, vol. 36 (3): 307-340.* Wishart et al. A single mutation converts a novel phosphotyrosine binding domain into a dual-specificity phosphatase. J. Biol. Chem. Related U.S. Application Data 1995, vol. 270(45): 267.82-26785.* Witkowski et al., Conversion of b-ketoacyl synthase to a Malonyl (63) Continuation-in-part of application No. 12/943,477, Decarboxylase by replacement of the active cysteine with glutamine. filed on Nov. 10, 2010, now Pat. No. 8,512,988. Biochemistry, 1999, vol. 38: 11643-1 1650.* (60) Provisional application No. 61/280,877, filed on Nov. SENN Sign,NIHNCB. Accession No. AANO1134;

10, 2009, provisional application No. 61/388,543, CSN. St.ubmission, NIH NCBI , AAccession On No. U87908 filed on Sep. 30, 2010. Bohlmann et al.; Sep. 24, 2007. (51) Int. Cl. GENBANK Submission; NIH/NCBI, Accession No. AY195608; CI2P 5/00 (2006.01) Dudareva et al.; May 3, 2003. CI2P 5/02 (2006.01) GENBANK Submission; NIH/NCBI, Accession No. AF271259; CI2N 15/74 (2006.01) Hosoi et al.; Feb. 3, 2005. CI2N L/20 (2006.01) Ajikumar et al Isoprenoid pathway optimization for Taxol precursor CI2N 9/02 (2006.01) SES :7). Escherichia coli. Science. Oct. 1, CI2P 15/00 (2006.01) 10,330(6000):70-4. CI2P 17/02 (2006.01) producE. gS al., using Terpenoids: engineered ept microorganisms. for ity Mol Fnarm.of nyl Mar.- (52) st 00 (2006.01) Apr. 2008:5(2):167-90. Epub Mar. 21, 2008. CPC ...... CI2P 15/00 (2013.01); C12P 17/02 (Continued) (2013.01); CI2P 23/00 (2013.01); CI2P5/007 (2013.01) USPC ...... 435/166; 435/167; 435/471; 435/252.3: Primary Examiner — Ganapathirama Raghu 435/252.33:435/189 (74) Attorney, Agent, or Firm — Wolf, Greenfield & Sacks, (58) Field of Classification Search P.C. USPC ...... 435/166, 167, 471,252.3, 252.33, 189 See application file for complete search history. (57) ABSTRACT (56) References Cited The invention relates to the production of one or more terpe U.S. PATENT DOCUMENTS noids through microbial engineering, and relates to the manu 8,512,988 B2 * 8/2013 Ajikumar et al...... 435,166 facture of products comprising terpenoids. 2004f0072323 A1 4/2004 Matsuda et al. 2007/0026484 A1 2/2007 Kinney et al. 2008/0274523 A1 11/2008 Renninger et al. 9 Claims, 24 Drawing Sheets US 8,927.241 B2 Page 2

(56) References Cited Hoffmann et al., Stress induced by recombinant protein production in Escherichia coli. Adv Biochem Eng Biotechnol. 2004:89:73-92. OTHER PUBLICATIONS Holton et al., First total synthesis of taxol. 2. Completion of the Cand D rings. J. Am. Chem. Soc. Feb. 1994; 116 (4): 1599-1600. Alper et al., Construction of lycopene-overproducing E. coli strains Huang et al., Engineering Escherichia coli for the synthesis of by combining systematic and combinatorial gene knockout targets. taxadiene, a key intermediate in the biosynthesis of taxol. Bioorg Nat Biotechnol. May 2005:23(5):612-6. Epub Apr. 10, 2005. Med Chem. Sep. 2001:9(9):2237-42. Behr et al.. Myrcene as a natural base chemical in Sustainable chem Hughes et al., Metabolic engineering of the indole pathway in istry: a critical review. ChemSusChem. 2009:2(12): 1072-95. doi: Catharanthus roseus hairy roots and increased accumulation of 10.1002/cSSc.200900186. tryptamine and serpentine. Metab Eng. Oct. 2004;6(4):268-76. Bohlmann et al., Monoterpene synthases from grand fir (Abies Humphrey et al., Spatial organisation of four enzymes from Stevia grandis). cDNA isolation, characterization, and functional expres rebaudiana that are involved in Steviolglycoside synthesis. Plant Mol sion of myrcene synthase, (-)-(4S)-limonene synthase, and (-)- Biol. May 2006:61(1-2):47-62. (1S,5S)-pinene synthase. J Biol Chem. Aug. 29. Jennewein et al., Coexpression in yeast of Taxus cytochrome P450 1997:272(35):21784-92. reductase with cytochrome P450 oxygenases involved in Taxol Brandle et al., Steviol glycoside biosynthesis. Phytochemistry. Jul. biosynthesis. Biotechnol Bioeng. Mar. 5, 2005;89(5):588-98. 2007:68(14): 1855-63. Epub Mar. 29, 2007. Jennewein et al., Cytochrome p450 taxadiene 5alpha-hydroxylase, a Brosius et al., Spacing of the -10 and -35 regions in the tac promoter. mechanistically unusual monooxygenase catalyzing the first oxygen Effect on its in vivo activity. J Biol Chem. Mar. 25. ation step of taxolbiosynthesis. Chem Biol. Mar. 2004; 11(3):379-87. 1985:260(6):3539-41. Brunner et al., Promoter recognition and promoter strength in the Jennewein et al., Random sequencing of an induced Taxus cell cDNA Escherichia coli system. EMBO J. Oct. 1987:6(10):3139-44. library for identification of clones involved in Taxol biosynthesis. Burke et al., Geranyl diphosphate synthase from Abies grandis: Proc Natl AcadSci USA. Jun. 15, 2004; 101(24):9149-54. Epub Jun. cDNA isolation, functional expression, and characterization. Arch 3, 2004. Biochem Biophys. Sep. 1, 2002:405(1): 130-6. Jennewein et al., Taxol: biosynthesis, molecular genetics, and Chang et al., Engineering Escherichia coli for production of biotechnological applications. Appl Microbiol Biotechnol. Oct. functionalized terpenoids using plant P450s. Nat Chem Biol. May 2001:57(1-2): 13-9. 2007:3(5): 274-7. Epub Apr. 15, 2007. Jin et al., Multi-dimensional gene target search for improving Chang et al., Gene expression profiling of Escherichia coli growth lycopene biosynthesis in Escherichia coli. Metab Eng. Jul. transitions: an expanded stringent response model. Mol Microbiol. 2007:9(4):337-47. Epub Apr. 12, 2007. Jul. 2002:45(2): 289-306. Jones et al., Low-copy plasmids can perform as well as or better than Chang et al., Production of isoprenoid pharmaceuticals by engi high-copy plasmids for metabolic engineering of bacteria. Metab neered microbes. Nat Chem Biol. Dec. 2006:2(12):674-81. Eng. Oct. 2000:2(4):328-38. Chau et al., Taxol biosynthesis: Molecular cloning and characteriza Kaspera et al., Cytochrome P450 oxygenases of Taxol biosynthesis. tion of a cytochrome P450 taxoid 7 beta-hydroxylase. Chem Biol. Phytochem Rev. Jun. 2006; 5(2-3): 433-444. doi:10.1007/s11101 May 2004; 11(5):663-72. OO6-9006-4. Croteau et al., Taxolbiosynthesis and molecular genetics. Phytochem Khosla et al., Metabolic engineering for drug discovery and devel Rev. Feb. 2006:5(1):75-97. opment. Nat Rev Drug Discov. Dec. 2003:2(12): 1019-25. Das et al., an update on microbial carotenoid production: application Kim et al., Metabolic engineering of the nonmevalonate isopentenyl of recent metabolic engineering tools. Appl Microbiol Biotechnol. diphosphate synthesis pathway in Escherichia coli enhances Dec. 2007;77(3):505-12. Epub Oct. 3, 2007. lycopene production. Biotechnol Bioeng. Feb. 20, 2001:72(4):408 Datsenko et al., One-step inactivation of chromosomal genes in 15. Escherichia coli K-12 using PCR products. Proc Natl AcadSci USA. Kimchi-Sarfaty et al. A "silent” polymorphism in the MDR1 gene Jun. 6, 2000:97(12):6640-5. changes substrate specificity. Science. Jan. 26, 2007:315(5811):525 Dejong et al., Genetic engineering of taxol biosynthetic genes in 8. Epub Dec. 21, 2006. Erratum in: Science. Oct. 7, Saccharomyces cerevisiae. Biotechnol Bioeng. Feb. 5, 2001:334(6052);39. Science. Nov. 30, 2007:318(5855): 1382-3. 2006:93(2):212-24. Kingston, The shape of things to come: structural and synthetic Engels et al., Metabolic engineering of taxadiene biosynthesis in studies of taxol and related compounds. Phytochemistry. Jul. yeast as a first step towards Taxol (Paclitaxel) production. Metab Eng. 2007:68(14): 1844-54. Epub Dec. 20, 2006. May-Jul. 2008:10(3–4):201-6. doi: 10.1016/j.ymben. 2008.03.001. Kirby et al., Biosynthesis of plant isoprenoids: perspectives for Epub Mar. 26, 2008. microbial engineering. Annu Rev Plant Biol. 2009:60:335-55. Farmer et al., Improving lycopene production in Escherichia coli by Klein-Marcuschamer et al., Engineering microbial cell factories for engineering metabolic control. Nat Biotechnol. May biosynthesis of isoprenoid molecules: beyond lycopene. Trends 2000:18(5):533-7. Farmer et al., Precursor balancing for metabolic engineering of Biotechnol. Sep. 2007:25(9):417-24. Epub Aug. 2, 2007. lycopene production in Escherichia coli. Biotechnol Prog. Jan.-Feb. Kodumal et al., Total synthesis of long DNA sequences: synthesis of 2001:17(1):57-61. a contiguous 32-kb polyketide synthase gene cluster. Proc Natl Acad Frense, Taxanes: perspectives for biotechnological production. Appl Sci U S A. Nov. 2, 2004: 101(44): 15573-8. Epub Oct. 20, 2004. Microbiol Biotechnol. Jan. 2007:73(6): 1233-40. Epub Nov. 24, Leonard et al., Combining metabolic and protein engineering of a 2006. terpenoid biosynthetic pathway for overproduction and selectivity Hefner et al., Cloning and functional expression of a cDNA encoding control. Proc Natl Acad Sci U S A. Aug. 3, 2010; 107(31): 13654-9. geranylgeranyl diphosphate synthase from Taxus canadensis and Epub Jul. 19, 2010. assessment of the role of this prenyltransferase in cells induced for Leonard et al., Engineering of artificial plant cytochrome P450 taxol production. Arch Biochem Biophys. Dec. 1, 1998:360(1):62 enzymes for synthesis of isoflavones by Escherichia coli. Appl T4. Environ Microbiol. Nov. 2007:73(22):7246-51. Epub Sep. 28, 2007. Heinig et al., Taxol. A complex diterpenoid natural product with an Liao, Molecular biology of the biosynthetic pathways of taxol pre evolutionarily obscure origin. African J Biotechnol. 2009;8:1370-85. cursors and metabolic engineering of anti-tumor terpenoid indole Hoffmann et al., Metabolic adaptation of Escherichia coli during alkaloids. China doctor dissertation. Jan. 31, 2006. 1-157. temperature-induced recombinant protein production: 1. Readjust Martin et al., Engineering a mevalonate pathway in Escherichia coli ment of metabolic enzyme synthesis. Biotechnol Bioeng. Nov. 5, for production of terpenoids. Nat Biotechnol. Jul. 2003:21(7):796 2002:80(3):313-9. 802. Epub Jun. 1, 2003. US 8,927.241 B2 Page 3

(56) References Cited Sorensen et al., Advanced genetic strategies for recombinant protein expression in Escherichia coli. J. Biotechnol. Jan. 26. OTHER PUBLICATIONS 2005; 115(2): 113-28. Trapp et al., Genomic organization of plant terpene synthases and Morrone et al., Increasing diterpene yield with a modular metabolic molecular evolutionary implications. Genetics. Jun. engineering system in E. coli: comparison of MEV and MEP 2001;158(2):811 32. isoprenoid precursor pathway engineering. Appl Microbiol Tyo et al., Expanding the metabolic engineering toolbox: more options to engineer cells. Trends Biotechnol. Mar. 2007:25(3): 132-7. Biotechnol. Feb. 2010:85(6): 1893-906. doi: 10.1007/s00253-009 Epub Jan. 24, 2007. 2219-X. Epub Sep. 24, 2009. Tyo et al., Stabilized gene duplication enables long-term selection Nackley et al., Human catechol-O-methyltransferase haplotypes free heterologous pathway expression. Nat Biotechnol. Aug. modulate protein expression by altering mRNA secondary structure. 2009:27(8):760-5. doi:10.1038/nbt. 1555. Epub Jul. 26, 2009. Science. Dec. 22, 2006:314(5807):1930-3. Waljiet al., Strategies to Bypass the Taxol Problem: Enantioselective Nelson, Cytochrome P450 and the individuality of species. Arch Cascade Catalysis, a New Approach for the Efficient Construction of Biochem Biophys. Sep. 1, 1999:369(1): 1-10. Molecular Complexity. Synlett. 2007:10:1477-1489. Nicolaou, et al. Total synthesis of taxol. Nature. Feb. 17. Walker et al., Taxol biosynthetic genes. Phytochemistry. Sep. 2001:58(1):1-7. 1994:367(6464): 630-4. Wang et al., Programming cells by multiplex genome engineering Nishizaki et al., Metabolic Engineering of Carotenoid Biosynthesis and accelerated evolution. Nature. Aug. 2009:460:894-898. in Escherichia coli by Ordered Gene Assembly in Bacillus subtilis. Wani et al., Plantantitumor agents. VI. The isolation and structure of Appl Environ Microbiol. Feb. 2007; 73(4): 1355-1361. taxol, a novel antileukemic and antitumor agent from Taxus Roberts, Production and engineering of terpenoids in plant cell cul brevifolia. J Am ChemSoc. May 5, 1971:93(9): 2325-7. ture. Nat Chem Biol. Jul. 2007:3(7):387-95. Whitmer et al., Influence of Precursor Availability on Alkaloid Accu Rontein et al., CYP725A4 from yew catalyzes complex structural mulation by Transgenic Cell Line of Catharanthus roseus. Plant rearrangement of taxa-4(5), 11(12)-diene into the cyclic ether 5(12)- Physiol. Feb. 1, 1998; 116(2):853-7. oxa-3(11)-cyclotaxane. J Biol Chem. Mar. 7, 2008:28.3(10):6067-75. Wildung et al. A cDNA clone for taxadiene synthase, the diterpene doi:10.1074/bc.M708950200. Epub Dec. 31, 2007. cyclase that catalyzes the committed step of taxolbiosynthesis. JBiol Rost et al., The PredictProtein server. Nucleic Acids Res. Jul. 1, Chem. Apr. 19, 1996:271(16): 920 1-4. 2004:32(Web Server issue):W321-6. Williams et al., Heterologous expression and characterization of a Sandmann, Combinatorial biosynthesis of carotenoids in a “Pseudomature” form of taxadiene synthase involved in paclitaxel heterologous host: a powerful approach for the biosynthesis of novel (Taxol) biosynthesis and evaluation of a potential intermediate and structures. Chembiochem. Jul. 2, 2002:3(7):629-35. inhibitors of the multistep diterpene cyclization reaction. Arch Sauna et al., Silent polymorphisms speak: how they affect Biochem Biophys. Jul. 1, 2000:379(1):137-46. pharmacogenomics and the treatment of cancer. Cancer Res. Oct. 15, Xu et al., Strain improvement and optimization of the media of 2007:67(20):9609-12. taxol-producing fungus Fusarium maire. Biochem Engineer J. Aug. Schuler et al., Functional genomics of P450s. Annu Rev Plant Biol. 2006:31(1):67-73. 2003:54:629-67. Yang et al., Metabolic engineering of Escherichia coli for the Shalel-Levanon et al. Effect of ArcA and FNR on the expression of biosynthesis of alpha-pinene. Biotechnol Biofuels. Apr. 30, genes related to the oxygen regulation and the glycolysis pathway in 2013;6(1):60. doi:10.1 186/1754-6834-6-60. Escherichia coli under microaerobic growth conditions. Biotechnol Yuan et al., Chromosomal promoter replacement of the isoprenoid Bioeng. Oct. 20, 2005:92(2): 147-59. pathway for enhancing carotenoid production in E. coli. Metab Eng. Shigemori et al., Biological activity and chemistry of taxoids from Jan. 2006:8(1):79-90. Epub Oct. 28, 2005. the Japanese yew, Taxus cuspidata JNat Prod. Feb. 2004:67(2):245 56. * cited by examiner U.S. Patent Jan. 6, 2015 Sheet 1 of 24 US 8,927.241 B2

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-- 3:38::::: -8-8:::::::::: *x-8:::::::::: US 8,927,241 B2 1. 2 MICROBAL ENGINEERING FOR THE ing strains have eluded prior attempts aiming at the transfer of PRODUCTION OF CHEMICAL AND this complex biosynthetic machinery into a microbial PHARMACEUTICAL PRODUCTS FROM THE host.'" Yet, as with other natural products, microbial pro ISOPRENOID PATHWAY duction through metabolically engineered strains, offers attractive economics and great potential for synthesizing a RELATED APPLICATIONS diverse array of new compounds with anti-cancer and other pharmaceutical activity.'” This application is a continuation-in-part of U.S. applica The metabolic pathway for Taxol and its analogs consists tion Ser. No. 12/943,477, filed on Nov. 10, 2010, now U.S. of an upstream isoprenoid pathway that is native to E. coli, Pat. No. 8,512,988, which claims the benefit under 35 U.S.C. 10 and a heterologous downstream terpenoid pathway (FIG. 6). S119(e) of U.S. Provisional Application Ser. No. 61/280,877, The upstream mevalonic acid (MVA) or methylerythritol entitled “Microbial Engineering for the Production of Chemi phosphate (MEP) pathways can produce the two common cal and Pharmaceutical Products from Isoprenoid Pathway.” building blocks, isopentenyl pyrophosphate (IPP) and dim filed on Nov. 10, 2009 and U.S. Provisional Application Ser. ethylallyl pyrophosphate (DMAPP), from which Taxol and No. 61/388,543, entitled “Microbial Engineering for the Pro 15 other isoprenoid compounds are formed." Recent studies duction of Chemical and Pharmaceutical Products from Iso have highlighted the engineering of the above upstream path prenoid Pathway.” filed on Sep. 30, 2010, the entire disclo ways to Support biosynthesis of heterologous isoprenoids sures of which are incorporated by reference herein in their such as lycopene and artemisinic acid. The downstream entireties. taxadiene pathway has been reconstructed in E. coli, but, GOVERNMENT INTEREST to-date, titers have not exceeded 1.3 mg/L. The above rational metabolic engineering approaches This invention was made with government Support under focused on either the upstream (MVA or MEP) or the down Grant No. GMO85323 awarded by the National Institutes of stream terpenoid pathway, implicitly assuming that modifi Health. The government has certain rights in this invention. 25 cations are additive, i.e. a linear behavior. While this approach can yield moderate increases in flux, it generally FIELD OF THE INVENTION ignores non-specific effects, such as toxicity of intermediate metabolites, cellular effects of the vectors used for expres The invention relates to the production of one or more Sion, and hidden unknown pathways that may compete with terpenoids through microbial engineering. 30 the main pathway and divert flux away from the desired target. Combinatorial approaches can avoid Such problems as BACKGROUND OF THE INVENTION they offer the opportunity to adequately sample the parameter space and elucidate these complex non-linear interac Terpenoids are a class of compounds with diverse uses, tions.'''''' However, they require a high throughput including in fragrances, cosmetics, food products, pharma 35 screen, which is often not available for many desirable natural ceuticals, and other products. Thus, methods for their effi products. Yet another class of pathway optimization meth cient and cost effective production are needed. ods has explored the combinatorial space of different sources For example, Taxol and its structural analogs have been of the heterologous genes comprising the pathway of inter recognized as the most potent and commercially Successful est. Still dependent on a high throughput assay, these meth anticancer drugs introduced in the last decade." Taxol was 40 ods generally ignore the need for determining an optimal first isolated from the bark of the Pacific Yew tree, and early level of expression for the individual pathway genes and, as stage production methods required sacrificing two to four Such, have proven less effective in structuring an optimal fully grown trees to supply sufficient dosage for one patient. pathway. Taxol's structural complexity necessitated a complex chemi In the present work, as an example of aspects of the inven cal synthesis route requiring 35-51 steps with highest yield of 45 tion, we focus on the optimal balancing between the 0.4%.'" However, a semi-synthetic route was devised upstream, IPP-forming pathway with the downstream terpe whereby the biosynthetic intermediate baccatin III was first noid pathway of taxadiene synthesis. This is achieved by isolated from plant Sources and was Subsequently converted grouping the nine-enzyme pathway into two modules—a to Taxol. While this approach and subsequent plant cell four-gene, upstream, native (MEP) pathway module and a culture-based production efforts have decreased the need for 50 two-gene, downstream, heterologous pathway to taxadiene harvesting the yew tree, production still depends on plant (FIG. 1). Using this basic configuration, parameters such as based processes with accompanying limitations of produc the effect of plasmid copy number on cell physiology, gene tivity and scalability, and constraints on the number of Taxol order and promoter strength in an expression cassette, and derivatives that can be synthesized in search for more effica chromosomal integration are evaluated with respect to their cious drugs." 55 effect on taxadiene production. This modular and multivari Methods for the production ofterpenoids and derivatives of able combinatorial approach allows us to efficiently sample these compounds, and methods of making products that com the main parameters affecting pathway flux without the need prise Such compounds, are needed. for a high throughput screen. The multivariate search across multiple promoters and copy numbers for each pathway mod SUMMARY OF THE INVENTION 60 ule reveals a highly non-linear taxadiene flux landscape with a global maximum exhibiting a 15,000 fold increase in taxa Recent developments in metabolic engineering and syn diene production over the control, yielding 300 mg/L produc thetic biology offer new possibilities for the overproduction tion of taxadiene in small-scale fermentations. Further, we of complex natural products through more technically ame have engineered the P450 based oxidation chemistry in Taxol nable microbial hosts.'" Although exciting progress has 65 biosynthesis in E. coli, with our engineered strains improving been made in the elucidation of the biosynthetic mechanism the taxadien-5C.-ol production 2400-fold over the state of the of Taxol in Taxus.''' commercially relevant Taxol-produc art. These improvements unlock the potential for the large US 8,927,241 B2 3 4 scale production of thousands of valuable terpenoids by well embodiments, the cell is a yeast cell Such as a Saccharomyces established microbial systems. cell or a Yarrowia cell. In some embodiments, the cell is an Aspects of the invention relate to methods involving algal cell or a plant cell. recombinantly expressing a taxadiene synthase enzyme and a In some embodiments, the taxadiene synthase enzyme is a geranylgeranyl diphosphate synthase (GGPPS) enzyme in a Taxus enzyme Such as a Taxus brevifolia enzyme. In some cell that overexpresses one or more components of the non embodiments, the GGPPS enzyme is a Taxus enzyme such as mevalonate (MEP) pathway. In some embodiments the cell is a Taxus Canadenis enzyme. In some embodiments, the gene a bacterial cell such as an Escherichia coli cell. In some encoding for the taxadiene synthase enzyme and/or the gene embodiments, the bacterial cell is a Gram-positive cell such encoding for the GGPPS enzyme and/or the genes encoding as a Bacillus cell. In some embodiments, the cell is a yeast cell 10 for the one or more components of the MEP pathway is Such as a Saccharomyces cell or a Yarrowia cell. In some expressed from one or more plasmids. In some embodiments, embodiments, the cell is an algal cell or a plant cell. the gene encoding for the taxadiene synthase enzyme and/or In some embodiments, the taxadiene synthase enzyme is a the gene encoding for the GGPPS enzyme and/or the genes Taxus enzyme Such as a Taxus brevifolia enzyme. In some encoding for the one or more components of the MEP is embodiments, the GGPPS enzyme is a Taxus enzyme such as 15 incorporated into the genome of the cell. a Taxus Canadenis enzyme. In some embodiments, the gene In some embodiments, the one or more components of the encoding for the taxadiene synthase enzyme and/or the gene non-mevalonate (MEP) pathway is selected from the group encoding for the GGPPS enzyme and/or the genes encoding consisting of dxS. ispC. isp), ispE. ispF. ispG. ispH, idi, ispA for the one or more components of the MEP pathway is and ispB. In certain embodiments, dxs, idi, isp) and ispF are expressed from one or more plasmids. In some embodiments, overexpressed. For example, dxS, idi, isp) and ispF can be the gene encoding for the taxadiene synthase enzyme and/or overexpressed on the operon dxs-idi-idpDF. In some embodi the gene encoding for the GGPPS enzyme and/or the genes ments, the gene encoding for the taxadiene synthase enzyme encoding for the one or more components of the MEP is and the gene encoding for the GGPPS enzyme are expressed incorporated into the genome of the cell. together on an operon. In some embodiments, the expression In some embodiments, one or more components of the 25 of the taxadiene synthase enzyme, the GGPPS enzyme and non-mevalonate (MEP) pathway are selected from the group the one or more components of the MEP pathway are bal consisting of dxS. ispC. isp), ispE. ispF. ispG. ispH, idi, ispA anced to maximize production of the taxadiene. and ispB. In certain embodiments, dxs, idi, isp) and ispF are In some embodiments, the cell further expresses a taxadi overexpressed. For example, dxS, idi, isp) and ispF can be ene 5C-hydroxylase (T5OOH) or a catalytically active por overexpressed on the operon dxs-idi-idpDF. In some embodi 30 tion thereof. In certain embodiments, the T5C.OH enzyme or ments, the gene encoding for the taxadiene synthase enzyme a catalytically active portion thereof is fused to a cytochrome and the gene encoding for the GGPPS enzyme are expressed P450 reductase enzyme or a catalytically active portion together on an operon. thereof. For example, the T5C.OH enzyme can be In some embodiments, the cell further expresses a taxadi At24T5C.OH-tTCPR. In some embodiments, the cell pro ene 5C-hydroxylase (T5OOH) or a catalytically active por 35 duces taxadiene and/or taxadiene-5C-ol. tion thereof. In certain embodiments, the T5C.OH enzyme or Aspects of the invention relate to methods for selecting a a catalytically active portion thereof is fused to a cytochrome cell that exhibits enhanced production of a terpenoid, includ P450 reductase enzyme or a catalytically active portion ing creating or obtaining a cell that overexpresses one or more thereof. For example, the T5C.OH enzyme can be components of the non-mevalonate (MEP) pathway, produc At24TSOOH-TCPR. 40 ing terpenoid from the cell, comparing the amount of terpe The expression of the taxadiene synthase enzyme, the noid produced from the cell to the amount of terpenoid pro GGPPS enzyme and the one or more components of the MEP duced in a control cell, and selecting a first improved cell that pathway can be balanced to maximize production of the taxa produces a higher amount of terpenoid than a control cell, diene. Methods associated with the invention can further whereina first improved cell that produces a higher amount of encompass culturing a cell to produce taxadiene or taxadiene 45 terpenoid than the control cell is a cell that exhibits enhanced 5C-ol. In some embodiments, at least 10 mg L' of taxadiene production of terpenoid. is produced. In certain embodiments, at least 250 mg L' of In some embodiments, the cell recombinantly expresses a taxadiene is produced. In some embodiments, at least 10 mg terpenoid synthase enzyme and/or a geranylgeranyl diphos L' of taxadiene-5C.-ol is produced. In certain embodiments, phate synthase (GGPPS) enzyme. Methods can further com at least 50 mg L' of taxadiene-5C-ol is produced. In some 50 prise altering the level of expression of one or more of the embodiments, the percentage of taxadiene conversion to components of the non-mevalonate (MEP) pathway, the ter taxadiene-5C.-ol and the byproduct 5(12)-Oxa-3 (11)-cyclo penoid synthase enzyme and/or the geranylgeranyl diphos taxane is at least 50%, at least 75% or at least 95%. phate synthase (GGPPS) enzyme in the first improved cell to Methods associated with the invention can further com produce a second improved cell, and comparing the amount prise recovering the taxadiene or taxadiene-5C.-ol from the 55 of terpenoid produced from the second improved cell to the cell culture. In some embodiments, the taxadiene or taxadi amount of terpenoid produced in the first improved cell, ene-5C.-ol is recovered from the gas phase while in other wherein a second improved cell that produces a higher embodiments, an organic layer is added to the cell culture, amount of terpenoid than the first improved cell is a cell that and the taxadiene or taxadiene-5C.-ol is recovered from the exhibits enhanced production of terpenoid. In some embodi organic layer. 60 ments, the terpenoid synthase enzyme is a taxadiene synthase Aspects of the invention relate to cells that overexpress one enzyme. The cell can further recombinantly express any of or more components of the non-mevalonate (MEP) pathway, the polypeptides associated with the invention. and that recombinantly expresses a taxadiene synthase Aspects of the invention relate to isolated polypeptides enzyme and a geranylgeranyl diphosphate synthase (GGPPS) comprising a taxadiene 5C.-hydroxylase (T5C.OH) enzyme or enzyme. In some embodiments the cell is a bacterial cell Such 65 a catalytically active portion thereof fused to a cytochrome as an Escherichia coli cell. In some embodiments, the bacte P450 reductase enzyme or a catalytically active portion rial cell is a Gram-positive cell Such as a Bacillus cell. In some thereof. In some embodiments, the cytochrome P450 reduc US 8,927,241 B2 5 6 tase enzyme is a Taxus cytochrome P450 reductase (TCPR). least in part by balancing an upstream non-mevalonate iso In certain embodiments, the taxadiene 5C.-hydroxylase and prenoid pathway with a downstream heterologous terpenoid TCPR are joined by a linker such as GSTGS (SEQ ID synthesis pathway. In some embodiments, the product is a food product, food NO:50). In some embodiments, the taxadiene 5C.-hydroxy additive, beverage, chewing gum, candy, or oral care product. lase and/or TCPR are truncated to remove all or part of the In such embodiments, the terpenoid or derivative may be a transmembrane region. In certain embodiments, 8, 24, or 42 flavor enhancer or Sweetener. In some embodiments, the N-terminal amino acids of taxadiene 5C.-hydroxylase are product is a food preservative. truncated. In certain embodiments, 74 amino acids of TCPR In various embodiments, the product is a fragrance prod are truncated. In some embodiments, an additional peptide is uct, a cosmetic, a cleaning product, or a soap. In Such embodi fused to taxadiene 5C.-hydroxylase. In certain embodiments, 10 ments, the terpenoid or derivative may be a fragrance. the additional peptide is from bovine 17C. hydroxylase. In In still other embodiments, the product is a vitamin or certain embodiments, the peptide is MALLLAVF (SEQ ID nutritional Supplement. NO:51). In certain embodiments, the isolated polypeptide is In some embodiments, the product is a solvent, cleaning product, lubricant, or Surfactant. At24T5C.OH-tTCPR. Aspects of the invention also encom 15 In some embodiments, the product is a pharmaceutical, and pass nucleic acid molecules that encode for any of the the terpenoid or derivative is an active pharmaceutical ingre polypeptides associated with the invention and cells that dient. recombinantly express any of the polypeptides associated In some embodiments, the terpenoid or derivative is poly with the invention. merized, and the resulting polymer may be elastomeric. Aspects of the invention relate to methods for increasing In some embodiments, the product is an insecticide, pesti terpenoid production in a cell that produces one or more cide orpest control agent, and the terpenoid or derivative is an terpenoids. The methods include controlling the accumula active ingredient. tion of indole in the cell or in a culture of the cells, thereby In some embodiments, the product is a cosmetic or per increasing terpenoid production in a cell. Any of the cells Sonal care product, and the terpenoid or derivative is not a described herein can be used in the methods, including bac 25 fragrance. terial cells, such as Escherichia coli cells; Gram-positive These and other aspects of the invention, as well as various cells. Such as Bacillus cells; yeast cells, such as Saccharomy embodiments thereof, will become more apparent in refer ces cells or Yarrowia cells; algal cells; plant cell; and any of ence to the drawings and detailed description of the invention. the engineered cells described herein. In some embodiments, the step of controlling the accumu 30 BRIEF DESCRIPTION OF THE DRAWINGS lation of indole in the cell or in a culture of the cells includes balancing the upstream non-mevalonate isoprenoid pathway The accompanying drawings are not intended to be drawn with the downstream product synthesis pathways and/or to scale. In the drawings, each identical or nearly identical modifying or regulating the indole pathway. In other embodi component that is illustrated in various figures is represented ments, the step of controlling the accumulation of indole in 35 by a like numeral. For purposes of clarity, not every compo the cell or in a culture of the cells includes or further includes nent may be labeled in every drawing. In the drawings: removing the accumulated indole from the fermentation FIG. 1. Multivariate-modular isoprenoid pathway engi through chemical methods. Such as by using absorbents or neering reveals strong non-linear response interpenoid accu Scavengers. mulation. To increase the flux through the upstream MEP The one or more terpenoids produced by the cell(s) or in the 40 pathway, we targeted reported bottleneck enzymatic steps culture can be a monoterpenoid, a sesquiterpenoid, a diterpe (dxs, idi, isp) and ispF) for overexpression by an operon noid, a triterpenoid or a tetraterpenoid. In certain embodi (dxs-idi-ispDF). To channel the overflow flux from the uni ments, the terpenoids is taxadiene or any taxol precursor. versal isoprenoid precursors, IPP and DMAPP, towards Taxol Aspects of the invention relate to methods that include biosynthesis, a synthetic operon of downstream genes GGPP measuring the amount or concentration of indole in a cell that 45 synthase (G) and Taxadiene synthase (T)' was constructed. produces one or more terpenoids or in a culture of the cells The upstream isoprenoid and downstream synthetic taxadi that produce one or more terpenoids. The methods can ene pathways were placed under the control of inducible include measuring the amount or concentration of indole two promoters to control their relative gene expression. (a) Sche or more times. In some embodiments, the measured amount matic of the two modules, the native upstream MEP iso or concentration of indole is used to guide a process of pro 50 prenoid pathway (left) and synthetic taxadiene pathway ducing one or more terpenoids. In some embodiments, the (right). In E. coli biosynthetic network, the MEP isoprenoid measured amount or concentration of indole is used to guide pathway is initiated by the condensation of the precursors strain construction. glyceraldehydes-3 phosphate (G3P) and pyruvate (PYR) Thus, in Some aspects the invention provides a method for from glycolysis. The Taxol pathway bifurcation starts from making a product containing a terpenoid or terpenoid deriva 55 the universal isoprenoid precursors IPP and DMAPP to form tive. The method according to this aspect comprises increas first the “linear precursor Geranylgeranyl diphosphate, and ing terpenoid production in a cell that produces one or more then the “cyclic' taxadiene, a committed and key intermedi terpenoids by controlling the accumulation of indole in the ate to Taxol. The cyclic olefin taxadiene undergoes multiple cell or in a culture of the cells. The terpenoid, or a derivative rounds of stereospecific oxidations, acylations, benzoylation of the terpenoid prepared through one or more chemical or 60 with side chain assembly to, ultimately, form Taxol. (b) Sche enzymatic steps, is incorporated into a product to thereby matic of the multivariate-modular isoprenoid pathway engi make the product containing a terpenoid or terpenoid deriva neering approach for probing the non-linear response inter tive. According to this aspect, the cell expresses components penoid accumulation from upstream and downstream of the MEP pathway, and may be a bacterial cell such as E. pathway engineered cells. Expression of upstream and down coli or B. subtilis. In various embodiments, the accumulation stream pathways is modulated by varying the promoter of indole in the cell or in a culture of the cells is controlled at strength (Trc, T5 and T7) or increasing the copy number using US 8,927,241 B2 7 8 different plasmids. Variation of upstream and downstream expressions. Our gene expression analysis directly Supported pathway expression gives different maxima in taxadiene the hypothesis, with increase in plasmid copy number (5, 10 accumulation. and 20) and promoter strength (Trc, T5 and T7) the expression FIG.2. Optimization of taxadiene production by regulating of the upstream and downstream pathways can be modulated. the expression of the up- and down-stream modular path (c) Cell growth of the engineered strains 25-29. The growth ways. (a) Response in taxadiene accumulation to the increase phenotype was affected from activation of isoprenoid in upstream pathway strengths for constant values of the metabolism (strain 26), recombinant protein expression downstream pathway. (b) the dependence on the downstream (strain 25) and plasmid born metabolic burden (control vs pathway for constant increases in the upstream pathway engineered strains) and (d) growth phenotypes of strains 17. strength. Observed multiple local maxima in taxadiene 10 22, 25-32. The black color lines are the taxadiene producing response depends on the increase in the pathway expression engineered strains and the gray color lines are control strains strength upstream or downstream. (c) Taxadiene response without downstream expression carrying an empty plasmid from strains engineered (17-24) with high upstream pathway with promoter and multi cloning sites. The growth was cor overexpressions (20-100) with two different downstream related to the activation of the terpenoid metabolism, plasmid expressions (-30 and ~60) to identify taxadiene response 15 born metabolic burden as well the recombinant protein with balanced expressions. Expression of downstream path expression. way from the low copy plasmid (p5 and p10) under strong FIG. 5. Engineering Taxol p450 oxidation chemistry in E. promoter T7TG operon was used to modulate these expres coli. (a) Schematics of the conversion of taxadiene to taxadi sions. Note that both upstream and downstream pathway ene 5C-ol to Taxol. (b) Transmembrane engineering and con expressed from different plasmids with different promoters struction of one-component chimera protein from taxadiene can impose plasmid born metabolic burden. (d) Modulating 5C-ol hydroxylsase (T5C.OH) and Taxus cytochrome p450 the upstream pathway with increasing promoter strength reductase (TCPR). 1 and 2 represents the full length proteins from chromosome with two different downstream expres of T5OOH and TCPR identified with 42 and 74 amino acid sions (-30 and ~60) to identify the missing search space with TM regions respectively, 3-chimera enzymes generated reduced toxic effects (strains 25-32). (e) Genetic details of the 25 from the three different TM engineered T5C.OH constructs, taxadiene producing Strains. The numbers corresponding to (At8T5COH, At24T5C.OH and Atá2T5COH constructed by different Strains and its corresponding genotype, E-E. coli fusing 8 residue synthetic peptide (A) to 8, 24 and 42 AA K12mG 1655 ArecAAendA, EDE3-E. coli K12mG 1655 truncated T5OOH) through a translational fusion with 74AA Areca AendA with T7 RNA polymerase DE3 construct in the truncated TCPR (tTCPR) using 5 residue GSTGS linker pep chromosome, MEP-dxs-idi-ispDF operon, GT-GPPS-TS 30 tide. (c) Functional activity of At8T5COH-tTCPR, operon, TG-TS-GPPS operon, Ch1-1 copy in chromosome, At24T5OOH-tTCPR and Ata-2T5COH-tTCPR constructs Trc-Trc promoter, T5-T5 promoter, T7-T7 promoter, p5, p.10, transformed into taxadiene producing strain 18. (d) Time p20-5 (SC101), ~10 (p15), and ~20 (p3R322) copy plas course profile of taxadien-5C.-ol accumulation and growth mid. profile of the strain 18-At24T5COH-tTCPR fermented in a 1 FIG.3. Metabolite inversely correlates with taxadiene pro 35 L. bioreactor. duction. (a) Mass spectrum of metabolite that was detected to FIG. 6. Biosynthetic scheme for taxol production in E. coli. correlate inversely with taxadiene production in the strain Schematics of the two modules, native upstream isoprenoid constructs of FIG. 2. The observed characteristic peaks of the pathway (left) and synthetic Taxol pathway (right). In E. coli metabolite are 233,207, 178, 117, 89 and 62. (b) Correlation biosynthetic network, divergence of MEP isoprenoid path between the isoprenoid byproduct of FIG. 3a and taxadiene. 40 way initiates from the precursors glyceraldehyde-3 phos Strains 26-29 and 30-32, all with chromosomally integrated phate (G3P) and Pyruvate (PYR) from glycolysis (I-V). The upstream pathway expression, were chosen for consistent Taxol pathway bifurcation starts from the E. coli isoprenoid comparison. In strains 26-29 and 30-32, upstream expression precursor IPP and DMAPP to “linear precursor Gera increased by changing the promoters from Trc, to T5 and T7 nylgeranyl diphosphate (VIII), “cyclic' taxadiene (IX), “oxi respectively. The two sets of strains differ only in the expres 45 dized’ taxadiene 5C.-ol (X) to multiple rounds of stereospe sion of the downstream pathway with the second set (30-32) cific oxidations, acylations, benzoylations and epoxidation having twice the level of expression of the first. With the first for early precursor Baccatin III (XII) and finally with side set, optimal balancing is achieved with Strain 26, which uses chain assembly to Taxol (XIII). DXP-1-deoxy-D-xylulose-5- the Trc promoter for upstream pathway expression and also phosphate, MEP-2C-methyl-D-erythritol-4-phosphate, shows the lowest metabolite accumulation. With strains 50 CDP-ME-4-diphosphocytidyl-2C-methyl-D-erythritol, 30-32, strain 31 shows the lowest accumulation of metabolite CDP-MEP-4-diphosphocytidyl-2C-methyl-D-erythritol-2- and highest production of taxadiene. The data demonstrate phosphate, ME-cPP-2C-methyl-D-erythritol-2,4-cyclo the inverse correlation observed between the unknown diphosphate, IPP-isopentenyl diphosphate, DMAPP-dim metabolite and taxadiene production. ethylallyl diphosphate. The genes involved biosynthetic FIG. 4. Upstream and downstream pathway transcriptional 55 pathways from G3P and PYR to Taxol. DXS-1-deoxy-D- gene expression levels and changes in cell physiology of Xylulose-5-phosphate synthase, ispC-1-Deoxy-D-xylulose engineered strains. Relative expression of the first genes in 5-phosphate reductoisomerase, IspD-4-diphosphocytidyl the operon of upstream (DXS) and downstream (TS) pathway 2C-methyl-D-erythritol synthase, IspE-4-diphosphocytidyl is quantified by qPCR. Similar expression profiles were 2-C-methyl-D-erythritol kinase, IspF-2C-Methyl-D- observed with the genes in the downstream of the operons. 60 erythritol-2,4-cyclodiphosphate Synthase, IspG-1-hydroxy The corresponding strain numbers are shown in the graph. (a) 2-methyl-2-(E)-butenyl-4-diphosphate synthase, IspH-4- Relative transcript level DXS gene expression quantified hydroxy-3-methyl-2-(E)-butenyl-4-diphosphate reductase from different upstream expressions modulated using pro IDI-isopentenyl-diphosphate isomerase, GGPPS-geranyl moters and plasmids under two different downstream expres geranyldiphosphate synthase, Taxadiene synthase, Taxoid sions. (b) Relative transcript level TS gene expression quan 65 5C-hydroxylase, Taxoid-5C.-O-acetyltransferase, Taxoid tified from two different downstream expression modulated 13C-hydroxylase, Taxoid 103-hydroxylase, Taxoid 2C-hy using p5T7 and p10T7 plasmids under different upstream droxylase, Taxoid 2-O-benzoyltransferase, Taxoid 7B-hy US 8,927,241 B2 10 droxylase, Taxoid 10-O-acetyltransferase, Taxoid 13-hy FIG. 15. Impact of metabolite byproduct Indole accumu droxylase, Taxoid 9C.-hydroxylase, Taxoid 9-keto-oxidase, lation on taxadiene production and growth. (a) Inverse corre Taxoid C4C20-B-epoxidase, Phenylalanine aminomutase, lation between taxadiene and Indole. Strains 26 to 28 and 30 Side chain CoA-ligase, Taxoid O-phenylpropanoyltrans to 32, all with chromosomally integrated upstream pathway ferase, Taxoid 2'-hydroxylase, Taxoid 3'-N-benzoyltrans expression, were chosen for consistent comparison. The two ferase.''' marked genes are yet to be identified or char sets of strains differ only in the expression of the downstream acterized. pathway with the second set (30 to 32) having twice the level FIG. 7. Fold improvements in taxadiene production from of expression of the first. In strains 26 to 28 and 30 to 32, the modular pathway expression search. (a) Taxadiene upstream expression increased by changing the promoters response in fold improvements from all the observed maxi 10 from Trc, to T5 and T7, respectively. With the first set, optimal mas from FIG. 2a, b, and c compared to strain 1. The 2.5 fold balancing is achieved with strain 26, which uses the Trc differences between two highest maximas (strain 17 and 26) promoter for upstream pathway expression and also shows and 23 fold (strain 26 and 10) with lowest one indicates that the lowest indole accumulation. With strains 30 to 32, strain missing an optimal response results in significantly lower 31 shows the lowest accumulation of indole and highest pro 15 duction of taxadiene. The fold improvements are relative to titers. strain 25 and 29, respectively, for the two sets. (b) Effect of FIG. 8. Metabolite (a) Correlation between taxadiene to externally-introduced indole on taxadiene production for the metabolite accumulation. The metabolite accumulation from high-producing strain 26. Different concentrations of indole the engineered Strain is anti-proportionally related to the taxa were introduced into cultures of cells cultured in minimal diene production in an exponential manner. The correlation media with 0.5% yeast extract. Taxadiene production was coefficient for this relation was determined to 0.92 (b) Rep significantly reduced as indole concentration increased from resentative GC-profile from the strains 26-28 to demonstrate 50 mg/L to 100 mg/L (c) Effect of externally-introduced the change in taxadiene and metabolite accumulation. Num indole on cell growth for engineered strains of E. coli. Data bers in the chromatogram 1 and 2 corresponding to metabo are mean+/-SD for three replicates. Strains devoid of the lite and taxadiene peak respectively. (c) GC-MS profile of 25 downstream pathway and with different strengths of the metabolite (1) and taxadiene (2) respectively. The observed upstream pathway (1, 2, 6, 21, 40 and 100) were selected. characteristic peaks of the metabolite are 233,207, 178, 117. Strain 26, the high taxadiene producer, exhibits the strongest 89 and 62. Taxa-4(20), 11,12-diene characteristic ion m/z 272 inhibition. (Pt), 257 (P. CH3), 229 (P" CH): 121, 122, 123 (C-ring FIG. 16. Unknown metabolite identified as indole. (A) and fragment cluster).' The peak marked with a star is the inter 30 (a) Gas chromatogram and mass spectrum of the unknown nal standard caryophylene. metabolite extracted using hexane from cell culture. (B) and FIG.9. GC-MS profiles and taxadiene/taxadien-5C-ol pro (b) correspond to the gas chromatogram and mass spectrum duction from artificial chimera enzyme engineered in Strain of pure indole dissolved in hexane. Further to confirm the 26. (a) GC profile of the hexane:ether (8:2) extract from three chemical identity, the metabolite was extracted from the fer constructs (A-At8T5C.OH-tTCPR, t24T5COH-tTCPR and 35 mentation broth using hexane extraction and purified by silica At42T5C.OH-tTCPR) transferred to strain 26 and fermented column chromatography using hexane:ethylacetate (8:2) as for 5 days. 1, 2 and 3 labels in the peaks corresponding to the eluent. The purity of the compound was confirmed by TLC taxadiene, taxadien-5C.-ol and 5(12)-Oxa-3(11)-cyclotaxane and GC-MS. 'HNMR and 'CNMR spectra confirmed the (OCT) respectively. (b) The production of taxa-4(20), 11,12 chemical identity of the metabolite as indole. (c) 'HNMR dien-5C-ol and OCT quantified from the three strains. (c) and 40 spectrum of indole extracted from cell culture (CDC1, 400 (d) GC-MS profile of taxa-4(20), 11,12-dien-5C-ol and OCT MHz) 8: 6.56 (d. 1H, Ar C H), 7.16 (m, 3H, Ar C H), 7.38 and the peaks corresponding to the fragmentation was com (d. 1H, Ar C-H), 7.66 (d. 1H, Ar C H), 8.05 (b. 1H, Indole pared with the authentic standards and previous reports' NH). (d) CNMR 8: 135.7, 127.8, 124.2, 122, 120.7, 119.8, GC-MS analysis confirmed the mass spectrum identity to 111, 102.6. (e) is the 'HNMR spectrum of pure indole. authentic taxa-4(20), 11,12-dien-5C.-ol with characteristic ion 45 FIG. 17. Fed batch cultivation of engineered strains in 1 m/z 288(Pt), 273 (P" HO), 255 (P" HO CH3). L-bioreactor. Time courses of taxadiene accumulation (a), FIG. 10 presents a schematic depicting the terpenoid bio cell growth (b), acetic acid accumulation (c) and total Sub synthetic pathway and natural products produced by this strate (glycerol) addition (d) for strains 22, 17 and 26 during pathway. 5 days of fed batch bioreactor cultivation in 1 L-bioreactor FIG. 11 presents a schematic depicting modulation of the 50 vessels under controlled pH and oxygen conditions with upstream pathway for amplifying taxadiene production. minimal media and 0.5% yeast extract. After glycerol FIG. 12 presents a schematic depicting modulation of the depletes to ~0.5 to 1 g/L in the fermentor, 3 g/L of glycerol downstream pathway for amplifying taxadiene production. was introduced into the bioreactor during the fermentation. FIG. 13 presents a schematic indicating that the newly Data are mean of two replicate bioreactors. identified pathway diversion is not the characteristic of down 55 stream synthetic pathway. DETAILED DESCRIPTION OF THE INVENTION FIG. 14. Pathway strength correlates to transcriptional gene expression levels. (c) relative expression of idi, isp) and Taxol is a potent anticancer drug first isolated as a natural ispF genes with increasing upstream pathway strength and product from the Taxus brevifolia Pacific yew tree. However, downstream strength at 31 arbitrary units, and (d) relative 60 reliable and cost-efficient production of Taxol or Taxol ana expression of idi, isp) and ispF genes with increasing logs by traditional production routes from plant extracts is upstream pathway strength and downstream strength at 61 limited. Here, we report a multivariate-modular approach to arbitrary units. As expected the gene expression increased as metabolic pathway engineering to amplify by 15000 fold the the upstream pathway strength increased. The corresponding production of taxadiene in an engineered Escherichia coli. strain numbers are indicated in the bar graph. The relative 65 Taxadiene, the first committed Taxol intermediate, is the bio expression was quantified using the expression of the house synthetic product of the non-mevalonate pathway in E. coli keeping rrSA gene. Data are mean+/-SD for four replicates. comprising two modules: the native upstream pathway form US 8,927,241 B2 11 12 ing Isopentenyl Pyrophosphate (IPP) and a heterologous Described herein are methods and compositions for opti downstream terpenoid-forming pathway. Systematic multi mizing production of terpenoids in cells by controlling variate search identified conditions that optimally balance the expression of genes or proteins participating in an upstream two pathway modules to minimize accumulation of inhibi pathway and a downstream pathway. The upstream pathway tory intermediates and flux diversion to side products. We also involves production of isopentyl pyrophosphate (IPP) and engineered the next step, after taxadiene, in Taxol biosynthe dimethylallyl pyrophosphate (DMAPP), which can be sis, a P450-based oxidation step, that yielded >98% substrate achieved by two different metabolic pathways: the mevalonic conversion and present the first example of in Vivo production acid (MVA) pathway and the MEP (2-C-methyl-D-erythritol of any functionalized Taxol intermediates in E. coli. The 4-phosphate) pathway, also called the MEP/DOXP (2-C-me modular pathway engineering approach not only highlights 10 thyl-D-erythritol 4-phosphate? 1-deoxy-D-xylulose 5-phos the complexity of multi-step pathways, but also allowed accu phate) pathway, the non-mevalonate pathway or the meva mulation of high taxadiene and taxadien-5C.-ol titers (-300 lonic acid-independent pathway. mg/L and 60 mg/L, respectively) in Small-scale fermenta The downstream pathway is a synthetic pathway that leads tions, thus exemplifying the potential of microbial production to production of a terpenoids and involves recombinant gene of Taxol and its derivatives. 15 expression of a terpenoid synthase (also referred to as terpene This invention is not limited in its application to the details cyclase) enzyme, and a geranylgeranyl diphosphate synthase of construction and the arrangement of components set forth (GGPPS) enzyme. In some embodiments, a terpenoid syn in the following description or illustrated in the drawings. The thase enzyme is a diterpenoid synthase enzyme. Several non invention is capable of other embodiments and of being prac limiting examples of diterpenoid synthase enzymes include ticed or of being carried out in various ways. Also, the phrase casbene synthase, taxadiene synthase, levopimaradiene Syn ology and terminology used herein is for the purpose of thase, abietadiene synthase, isopimaradiene synthase, ent description and should not be regarded as limiting. The use of copalyl diphosphate synthase, Syn-stemar-13-ene synthase, “including.” “comprising.” or “having.” “containing.” Syn-stemod-13(17)-ene synthase, Syn-pimara-7, 15-diene “involving, and variations thereofherein, is meant to encom synthase, ent-sandaracopimaradiene synthase, ent-cassa-12, pass the items listed thereafter and equivalents thereofas well 25 15-diene synthase, ent-pimara-8014), 15-diene synthase, ent as additional items. kaur-15-ene synthase, ent-kaur-16-ene synthase, aphidi Microbial production of terpenoids such as taxadiene is collan-163-ol synthase, phyllocladan-16C-ol synthase, demonstrated herein. When expressed at satisfactory levels, fusicocca-2,10(14)-diene synthase, and terpentetriene microbial routes reduce dramatically the cost of production of cyclase. Such compounds. Additionally, they utilize cheap, abundant 30 Surprisingly, as demonstrated in the Examples section, and renewable feedstocks (such as Sugars and other carbohy optimization of terpenoid synthesis by manipulation of the drates) and can be the source for the synthesis of numerous to upstream and downstream pathways described herein, was derivatives that may exhibit far superior properties than the not a simple linear or additive process. Rather, through com original compound. A key element in the cost-competitive plex combinatorial analysis, optimization was achieved production of compounds of the isoprenoid pathway using a 35 through balancing components of the upstream and down microbial route is the amplification of this pathway in order to stream pathways. Unexpectedly, as demonstrated in FIGS. 1 allow the overproduction of these molecules. Described and 2, taxadiene accumulation exhibited a strong non-linear herein are methods that enhance or amplify the flux towards dependence on the relative strengths of the upstream MEP terpenoid production in Escherichia coli (E. coli). Specifi and downstream synthetic taxadiene pathways. cally, methods are provided to amplify the metabolic flux to 40 Aspects of the invention relate to controlling the expres the synthesis ofisopentenyl pyrophosphate (IPP) (a key inter sion of genes and proteins in the MEP pathway for optimized mediate for the production of isoprenoid compounds), dim production of a terpenoid such as taxadiene. Optimized pro ethylallyl pyrophosphate (DMAPP), geranyl diphosphate duction of a terpenoid refers to producing a higher amount of (GPP), farnesyl diphosphate (FPP), geranylgeranyl diphos a terpenoid following pursuit of an optimization Strategy than phate (GGPP), and farnesyl geranyl diphosphate (FGPP), 45 would be achieved in the absence of such a strategy. It should paclitaxel (Taxol), ginkolides, geraniol, farnesol, geranylge be appreciated that any gene and/or protein within the MEP raniol, linalool, isoprene, monoterpenoids such as menthol, pathway is encompassed by methods and compositions carotenoids Such as lycopene, polyisoprenoids such as poly described herein. In some embodiments, a gene within the isoprene or natural rubber, diterpenoids such as eleutherobin, MEP pathway is one of the following: dxs, ispC. isp), ispE, and sesquiterpenoids such as artemisinin. 50 ispF. ispG. ispH, idi, ispA or ispB. Expression of one or more Aspects of the invention relate to the production of terpe genes and/or proteins within the MEP pathway can be noids. As used herein, a terpenoid, also referred to as an upregulated and/or downregulated. In certain embodiments, isoprenoid, is an organic chemical derived from a five-carbon upregulation of one or more genes and/or proteins within the isoprene unit. Several non-limiting examples of terpenoids, MEP pathway can be combined with downregulation of one classified based on the number of isoprene units that they 55 or more genes and/or proteins within the MEP pathway. contain, include: hemiterpenoids (1 isoprene unit), monoter It should be appreciated that genes and/or proteins can be penoids (2 isoprene units), sesquiterpenoids (3 isoprene regulated alone or in combination. For example, the expres units), diterpenoids (4 isoprene units), Sesterterpenoids (5 sion of dxS can be upregulated or downregulated alone or in isoprene units), triterpenoids (6 isoprene units), tetraterpe combination with upregulation or downregulation of expres noids (8 isoprene units), and polyterpenoids with a larger 60 sion of one or more ofispC. isp), ispE. ispF. ispG. ispH, idi, number of isoprene units. In some embodiments, the terpe ispA and ispB. The expression ofispC can be upregulated or noid that is produced is taxadiene. In some embodiments, the downregulated alone or in combination with upregulation or terpenoid that is produced is Citronellol, Cubebol, Nootka downregulation of expression of one or more of dxS. isp), tone, Cineol, Limonene, Eleutherobin, Sarcodictyin, ispE. ispF. ispG. ispH, idi, isp A and ispB. The expression of Pseudopterosins, Ginkgolides, Stevioside, Rebaudioside A, 65 isp) can be upregulated or downregulated alone or in com Sclareol, labdenediol, levopimaradiene, Sandracopimaradi bination with upregulation or downregulation of expression ene or isopemaradiene. of one or more of dxS, ispC. ispE, ispF. ispG. ispH, idi, ispA US 8,927,241 B2 13 14 and ispB. The expression ofispE can be upregulated or down operons can be regulated through manipulation of the copy regulated alone or in combination with upregulation or down number of the gene or operon in the cell. For example, in regulation of expression of one or more of dxS, ispC. isp), certain embodiments, a strain containing an additional copy ispF, ispG. ispH, idi, isp A and ispB. The expression of ispF of the dxs-idi-isp)F operon on its chromosome under Trc can be upregulated or downregulated alone or in combination promoter control produces an increased amount of taxadiene with upregulation or downregulation of expression of one or relative to one overexpressing only the synthetic downstream more of dxs, ispC. isp), ispE. ispG. ispH, idi, isp A and ispB. pathway. In some embodiments, expression of genes or oper The expression ofispG can be upregulated or downregulated ons can be regulated through manipulating the order of the alone or in combination with upregulation or downregulation genes within a module. For example, in certain embodiments, of expression of one or more of dxS. ispC. isp), ispE. ispF. 10 changing the order of the genes in a downstream synthetic ispH, idi, isp A and ispB. The expression of ispH can be operon from GT to TG results in a 2-3 fold increase in taxa upregulated or downregulated alone or in combination with diene production. In some embodiments, expression of genes upregulation or downregulation of expression of one or more or operons is regulated through integration of one or more of dxS, ispC. isp), ispE, ispF. ispG, idi, isp A and ispB. The genes or operons into a chromosome. For example, in certain expression of idi can be upregulated or downregulated alone 15 embodiments, integration of the upstream dxs-idi-isp F or in combination with upregulation or downregulation of operon into the chromosome of a cell results in increased expression of one or more of dxs, ispC. isp), ispE. ispF. ispG. taxadiene production. ispH, isp A and ispB. The expression ofispA can be upregu It should be appreciated that the genes associated with the lated or downregulated alone or in combination with upregu invention can be obtained from a variety of sources. In some lation or downregulation of expression of one or more of dxS, embodiments, the genes within the MEP pathway are bacte ispC. isp), ispE. ispF, ispG. ispH, idi and ispB. The expres rial genes such as Escherichia coli genes. In some embodi sion ofispB can be upregulated or downregulated alone or in ments, the gene encoding for to GGPPS is a plant gene. For combination with upregulation or downregulation of expres example, the gene encoding for GGPPS can be from a species sion of one or more of dxS. ispC. isp), ispE. ispF. ispG. ispH, of Taxus Such as Taxus Canadensis (T. Canadensis). In some idi and ispA. In some embodiments, expression of the gene 25 embodiments, the gene encoding for taxadiene synthase is a and/or protein of one or more of dxS. ispC. isp), ispE. ispF. plant gene. For example, the gene encoding for taxadiene ispG. ispH, and idi is upregulated while expression of the synthase can be from a species of Taxus Such as Taxus brevi gene and/or protein of ispA and/or ispB is down-regulated. folia (T. brevifolia). Representative GenBank Accession Expression of genes within the MEP pathway can be regu numbers for T. Canadensis GGPPS and T. brevifolia taxadiene lated in a modular method. As used herein, regulation by a 30 synthase are provided by AF081514 and U48796, the modular method refers to regulation of multiple genes sequences of which are incorporated by reference herein in together. For example, in some embodiments, multiple genes their entireties. within the MEP pathway are recombinantly expressed on a As one of ordinary skill in the art would be aware, homolo contiguous region of DNA. Such as an operon. It should be gous genes for use in methods associated with the invention appreciated that a cell that expresses such a module can also 35 can be obtained from other species and can be identified by express one or more other genes within the MEP pathway homology searches, for example through a protein BLAST either recombinantly or endogenously. search, available at the National Center for Biotechnology A non-limiting example of a module of genes within the Information (NCBI) internet site (www.ncbi.nlm.nih.gov). MEP pathway is a module containing the genes dxs, idi, isp) Genes and/or operons associated with the invention can be and ispF, as presented in the Examples section, and referred to 40 cloned, for example by PCR amplification and/or restriction hereinas dxs-idi-isplF. It should be appreciated that modules digestion, from DNA from any source of DNA which con of genes within the MEP pathway, consistent with aspects of tains the given gene. In some embodiments, a gene and/or the invention, can contain any of the genes within the MEP operon associated with the invention is synthetic. Any means pathway, in any order. of obtaining a gene and/or operon associated with the inven Expression of genes and proteins within the downstream 45 tion is compatible with the instant invention. synthetic terpenoid synthesis pathway can also be regulated In some embodiments, further optimization of terpenoid in order to optimize terpenoid production. The synthetic production is achieved by modifying a gene before it is downstream terpenoid synthesis pathway involves recombi recombinantly expressed in a cell. In some embodiments, the nant expression of a terpenoid synthase enzyme and a GGPPS GGPPS enzyme has one or more of the follow mutations: enzyme. Any terpenoid synthase enzyme, as discussed above, 50 A162V, G140C, L 182M, F218Y, D160G, C184S, K367R, can be expressed with GGPPS depending on the downstream A151 T, M185I, D264Y, E368D, C184R, L331 I, G262V, product to be produced. For example, taxadiene synthase is R365S, A114D, S239C, G295D, 1276V, K343N, P183S, used for the production of taxadiene. Recombinant expres I172T, D267G, 1149V, T234I, E153D and T259A. In some sion of the taxadiene synthase enzyme and the GGPPS embodiments, the GGPPS enzyme has a mutation in residue enzyme can be regulated independently or together. In some 55 S239 and/or residue G295. In certain embodiments, the embodiments the two enzymes are regulated together in a GGPPS enzyme has the mutation S239C and/or G295D. modular fashion. For example the two enzymes can be In some embodiments, modification of a gene before it is expressed in an operon in either order (GGPPS-TS, referred recombinantly expressed in a cell involves codon optimiza to as “GT or TS-GGPPS, referred to as “TG). tion for expression in a bacterial cell. Codon usages for a Manipulation of the expression of genes and/or proteins, 60 variety of organisms can be accessed in the Codon Usage including modules such as the dxs-idi-isp F operon, and the Database (www.kazusa.or.jp/codon/). Codon optimization, TS-GGPPS operon, can beachieved through methods known including identification of optimal codons for a variety of to one of ordinary skill in the art. For example, expression of organisms, and methods for achieving codon optimization, the genes or operons can be regulated through selection of are familiar to one of ordinary skill in the art, and can be promoters, such as inducible promoters, with different 65 achieved using standard methods. strengths. Several non-limiting examples of promoters In some embodiments, modifying a gene before it is include Trc, T5 and T7. Additionally, expression of genes or recombinantly expressed in a cell involves making one or US 8,927,241 B2 15 16 more mutations in the gene before it is recombinantly cell that recombinantly expresses genes associated with the expressed in a cell. For example, a mutation can involve a invention, including prokaryotic and eukaryotic cells. In substitution or deletion of a single nucleotide or multiple some embodiments the cell is a bacterial cell, such as Escheri nucleotides. In some embodiments, a mutation of one or more chia spp., Streptomyces spp., Zymonas spp., Acetobacter nucleotides in a gene will result in a mutation in the protein 5 spp., Citrobacter spp., Synechocystis spp., Rhizobium spp., produced from the gene, such as a Substitution or deletion of Clostridium spp., Corynebacterium spp., Streptococcus spp., one or more amino acids. Xanthomonas spp., Lactobacillus spp., Lactococcus spp., In some embodiments, it may be advantageous to use a cell Bacillus spp., Alcaligenes spp., Pseudomonas spp., Aeromo that has been optimized for production of a terpenoid. For nas spp., Azotobacter spp., Comamonas spp., Mycobacte example, in some embodiments, a cell that overexpresses one 10 rium spp., Rhodococcus spp., Gluconobacter spp., Ralstonia or more components of the non-mevalonate (MEP) pathway spp., Acidithiobacillus spp., Microlunatus spp., Geobacter is used, at least in part, to amplify isopentyl diphosphate (IPP) spp., Geobacillus spp., Arthrobacter spp., Flavobacterium and dimethylallyl diphosphate (DMAPP), substrates of spp., Serratia spp., Saccharopolyspora spp., Thermus spp., GGPPS. In some embodiments, overexpression of one or Stenotrophomonas spp., Chromobacterium spp., Sinorhizo more components of the non-mevalonate (MEP) pathway is 15 bium spp., Saccharopolyspora spp., Agrobacterium spp. and achieved by increasing the copy number of one or more Pantoea spp. The bacterial cell can be a Gram-negative cell components of the non-mevalonate (MEP) pathway. For Such as an Escherichia coli (E. coli) cell, or a Gram-positive example, copy numbers of components at rate-limiting steps cell such as a species of Bacillus. In other embodiments, the in the MEP pathway such as (dxs, isp), ispF, idi) can be cell is a fungal cell Such as a yeast cell, e.g., Saccharomyces amplified, such as by additional episomal expression. spp., Schizosaccharomyces spp., Pichia spp., Pafia spp., In Some embodiments “rational design” is involved in con Kluyveromyces spp., Candida spp., Talaromyces spp., Bret structing specific mutations in proteins such as enzymes. As tanomyces spp., Pachysolen spp., Debaryomyces spp., Yar used herein, "rational design” refers to incorporating knowl rowia spp., and industrial polyploid yeast strains. Preferably edge of the enzyme, or related enzymes, such as its three the yeast strain is a S. cerevisiae strain or a Yarrowia spp. dimensional structure, its active site(s), its Substrate(s) and/or 25 strain. Other examples of fungi include Aspergillus spp., to the interaction between the enzyme and substrate, into the Pennicilium spp., Fusarium spp., Rhizopus spp., Acremonium design of the specific mutation. Based on a rational design spp., Neurospora spp., Sordaria spp., Magnaporthe spp., approach, mutations can be created in an enzyme which can Allomyces spp., Ustilago spp., Botrytis spp., and Tricho then be screened for increased production of a terpenoid derma spp. In other embodiments, the cell is an algal cell, or relative to control levels. In some embodiments, mutations 30 a plant cell. It should be appreciated that some cells compat can be rationally designed based on homology modeling. As ible with the invention may express an endogenous copy of used herein, "homology modeling” refers to the process of one or more of the genes associated with the invention as well constructing an atomic resolution model of one protein from as a recombinant copy. In some embodiments, if a cell has an its amino acid sequence and a three-dimensional structure of endogenous copy of one or more of the genes associated with a related homologous protein. 35 the invention then the methods will not necessarily require In Some embodiments, random mutations can be made in a adding a recombinant copy of the gene(s) that are endog gene. Such as a gene encoding for an enzyme, and these enously expressed. In some embodiments the cell may endog mutations can be screened for increased production of a ter enously express one or more enzymes from the pathways penoid relative to control levels. For example, screening for described herein and may recombinantly express one or more mutations in components of the MEP pathway, or compo 40 other enzymes from the pathways described herein for effi nents of other pathways, that lead to enhanced production of cient production of a terpenoid. a terpenoid may be conducted through a random mutagenesis Further aspects of the invention relate to screening for screen, or through screening of known mutations. In some bacterial cells or strains that exhibit optimized terpenoid pro embodiments, shotgun cloning of genomic fragments could duction. As described above, methods associated with the be used to identify genomic regions that lead to an increase in 45 invention involve generating cells that overexpress one or production of a terpenoid, through screening cells or organ more genes in the MEP pathway. Terpenoid production from isms that have these fragments for increased production of a culturing of Such cells can be measured and compared to a terpenoid. In some cases one or more mutations may be control cell wherein a cell that exhibits a higher amount of a combined in the same cell or organism. terpenoid production relative to a control cell is selected as a In some embodiments, production of a terpenoid in a cell 50 first improved cell. The cell can be further modified by recom can be increased through manipulation of enzymes that act in binant expression of a terpenoid synthase enzyme and a the same pathway as the enzymes associated with the inven GGPPS enzyme. The level of expression of one or more of the tion. For example, in Some embodiments it may be advanta components of the non-mevalonate (MEP) pathway, the ter geous to increase expression of an enzyme or other factor that penoid synthase enzyme and/or the GGPPS enzyme in the acts upstream of a target enzyme such as an enzyme associ 55 cell can then be manipulated and terpenoid production can be ated with the invention. This could be achieved by over measured again, leading to selection of a second improved expressing the upstream factor using any standard method. cell that produces greater amounts of a terpenoid than the first Optimization of protein expression can also be achieved improved cell. In some embodiments, the terpenoid synthase through selection of appropriate promoters and ribosome enzyme is a taxadiene synthase enzyme. binding sites. In some embodiments, this may include the 60 Further aspects of the invention relate to the identification selection of high-copy number plasmids, or low or medium and characterization (via GC-MS) of a previously unknown copy number plasmids. The step of transcription termination metabolite in bacterial E. coli cells (FIGS. 3 and 6). The level can also be targeted for regulation of gene expression, of accumulation of the newly identified metabolite, indole, through the introduction or elimination of structures such as can be controlled by genetically manipulating the microbial stem-loops. 65 pathway by the overexpression, down regulation or mutation Aspects of the invention relate to expression of recombi of the isoprenoid pathway genes. The metabolite indole anti nant genes in cells. The invention encompasses any type of correlates as a direct variable to the taxadiene production in US 8,927,241 B2 17 18 engineered strains (FIGS. 3, 6 and 15). Further controlling the p450's.' In addition, the structural and functional diver accumulation of indole for improving the flux towards terpe sity with the possible evolutionary analysis implicit that the noid biosynthesis in bacterial systems (specifically in cells, taxadiene-5C-ol gene can be the parental sequence from such as E. coli cells) or other cells, can be achieved by bal which the other hydroxylase genes in the Taxol biosynthetic ancing the upstream non-mevalonate isoprenoid pathway pathway evolved, reflecting the order of hydroxylations." with the downstream product synthesis pathways or by modi Further aspects of the invention relate to chimeric P450 fications to or regulation of the indole pathway. In so doing, enzymes. Functional expression of plant cytochrome P450 the skilled person can reduce or control the accumulation of has been considered challenging due to the inherent limita indole and thereby reduce the inhibitory effect of indole on tions of bacterial platforms, such as the absence of electron the production of taxadiene, and other terpenoids derived 10 transfer machinery, cytochrome P450 reductases, and trans from the described pathways. Such as: monoterpenoids, ses lational incompatibility of the membrane signal modules of quiterpenoids (including amorphadiene), diterpenoids (in P450 enzymes due to the lack of an endoplasmic reticulum. cluding levopimaradiene), triterpenes, and tetraterpenes. In some embodiments, the taxadiene-5C.-hydroxylase Other methods for reducing or controlling the accumulation associated with methods of the invention is optimized of indole include removing the accumulated indole from the 15 through N-terminal transmembrane engineering and/or the fermentation through chemical methods such as by using generation of chimeric enzymes through translational fusion absorbents, Scavengers, etc. with a CPR redox partner. In some embodiments, the CPR In other embodiments, methods are provided that include redox partner is a Taxus cytochrome P450 reductase (TCPR: measuring the amount or concentration of indole in a cell that FIG. 5b). In certain embodiments, cytochrome P450 taxadi produces one or more terpenoids or in a culture of the cells ene-5C.-hydroxylase (T5C.OH) is obtained from Taxus cuspi that produce one or more terpenoids. The amount or concen date (GenBank Accession number AY289209, the sequence tration of indole can be measured once, or two or more times, of which is incorporated by reference herein). In some as Suitable, using methods known in the art and as described embodiments, NADPH:cytochrome P450 reductase (TCPR) herein. Such methods can be used to guide processes of is obtained from Taxus cuspidate (GenBank Accession num producing one or more terpenoids, e.g., in process improve 25 ber AY571340, the sequence of which is incorporated by ment. Such methods can be used to guide Strain construction, reference herein). e.g., for strain improvement. The taxadiene 5C-hydroxylase and TCPR can be joined by The identification of the means to achieve this balancing a linker such as GSTGS (SEQID NO:50). In some embodi yielded a 15000 fold improvement in the overproduction of ments, taxadiene 5C-hydroxylase and/or TCPR are truncated terpenoids Such as taxadiene, compared to wildtype bacterial 30 to remove all or part of the transmembrane region of one or cells, expressed with a heterologous taxadiene biosynthetic both proteins. For example, taxadiene 5C-hydroxylase in pathway. The production was further increased through some embodiments is truncated to remove 8, 24, or 42 N-ter modified fermentation methods that yielded concentrations minal amino acids. In some embodiments, the N-terminal 74 of approximately 2 g/L, which is 1500 fold higher compared amino acids of TCPR are truncated. An additional peptide can to any prior reported taxadiene production. As demonstrated 35 also be fused to taxadiene 5C.-hydroxylase. For example, one herein, by genetically engineering the non-mevalonate iso or more amino acids from bovine 17C. hydroxylase can be prenoid pathway in E. coli the accumulation of this metabo added to taxadiene 5C.-hydroxylase. In certain embodiments, lite can now be controlled which regulates the flux towards the peptide MALLLAVF (SEQID NO:51) is added to taxa the isoprenoid biosynthesis in bacterial E. coli cells. Also diene 5C.-hydroxylase. A non-limiting example of polypep demonstrated herein is further channeling of the taxadiene 40 tide comprising taxadiene 5C.-hydroxylase fused to TCPR is production into the next key precursor to Taxol, taxadien-5C.- At24TSOOH-tTCPR. ol, achieved through engineering the oxidation chemistry for In some embodiments, the chimeric enzyme is able to carry Taxol biosynthesis. Example 5 presents the first successful out the first oxidation step with more than 10% taxadiene extension of the synthetic pathway from taxadiene to taxa conversion to taxadiene-5C-ol and the byproduct 5(12)-Oxa dien-5C-ol. Similar to the majority of other terpenoids, the 45 3(11)-cyclotaxane. For example, the percent taxadiene con Taxol biosynthesis follows the unified fashion of “two phase' version to taxadiene-5C.-ol and the byproduct 5(12)-Oxa-3 biosynthetic process, (i) the “cyclase phase' of linear cou (11)-cyclotaxane can be at least 20%, at least 30%, at least pling of the prenyl precursors (IPP and DMAPP) to GGPP 40%, at least 50%, at least 60%, at least 70%, at least 80%, at followed by the molecular cyclization and rearrangement for least 90%, at least 95%, at least 98%, approximately 99% or the committed precursor taxadiene (FIG. 6, VIII-IX).7 50 approximately 100%. After the committed precursor, (ii) the “oxidation phase', the In certain embodiments, the chimeric enzyme is cyclic olefin taxadiene core structure is then functionalized At245C.OH-tTCPR, which was found to be capable of carry by seven cytochrome P450 oxygenases together with its ing out the first oxidation step with more than 98% taxadiene redox partners, decorated with two acetate groups and a ben conversion to taxadiene-5C-ol and the byproduct 5(12)-Oxa Zoate group by acyl and aroyl CoA-dependent transferases, 55 3(11)-cyclotaxane (OCT FIG. 9a). Engineering of the step of keto group by keto-oxidase, and epoxide group by epoxidase taxadiene-5C-ol production is critical in the production of lead to the late intermediate baccatin III, to which the C13 Taxol and was found to be limiting in previous efforts to side chain is attached for Taxol (FIG. 6, X-XIII)." Although construct this pathway in yeast. The engineered construct a rough sequential order of the early oxidation phase reac developed herein demonstrated greater than 98% conversion tions are predicted, the precise timing/order of some of the 60 of taxadiene in vivo with a 2400 fold improvement over hydroxylations, acylations and benzoylation reactions are previous heterologous expression in yeast. Thus, in addition uncertain. However it is clear that the early bifurcation starts to synthesizing significantly greater amounts of key Taxol from the cytochrome p450 mediated hydroxylation of taxa intermediates, this study also provides the basis for the syn diene core at C5 position followed the downstream hydroxy thesis of subsequent metabolites in the pathway by similar lations using a homologous family of cytochrome p450 65 P450 chemistry. enzymes with high deduced similarity to each other (>70%) As used herein, the terms “protein’ and “polypeptide' are but with limited resemblance (<30%) to other plant used interchangeably and thus the term polypeptide may be US 8,927,241 B2 19 20 used to refer to a full-length polypeptide and may also be used As used herein, a coding sequence and regulatory to refer to a fragment of a full-length polypeptide. As used sequences are said to be “operably joined when they are herein with respect to polypeptides, proteins, or fragments covalently linked in Such a way as to place the expression or thereof, "isolated' means separated from its native environ transcription of the coding sequence under the influence or ment and present in Sufficient quantity to permit its identifi control of the regulatory sequences. If it is desired that the cation or use. Isolated, when referring to a protein or polypep coding sequences be translated into a functional protein, two tide, means, for example: (i) selectively produced by DNA sequences are said to be operably joined if induction of expression cloning or (ii) purified as by chromatography or a promoter in the 5' regulatory sequences results in the tran electrophoresis. Isolated proteins or polypeptides may be, but Scription of the coding sequence and if the nature of the need not be, substantially pure. The term “substantially pure' 10 linkage between the two DNA sequences does not (1) result in means that the proteins or polypeptides are essentially free of the introduction of a frame-shift mutation, (2) interfere with other substances with which they may be found in production, the ability of the promoter region to direct the transcription of nature, or in vivo systems to an extent practical and appropri the coding sequences, or (3) interfere with the ability of the ate for their intended use. Substantially pure polypeptides corresponding RNA transcript to be translated into a protein. may be obtained naturally or produced using methods 15 Thus, a promoter region would be operably joined to a coding described herein and may be purified with techniques well sequence if the promoter region were capable of effecting known in the art. Because an isolated protein may be admixed transcription of that DNA sequence such that the resulting with other components in a preparation, the protein may transcript can be translated into the desired protein or comprise only a small percentage by weight of the prepara polypeptide. tion. The protein is nonetheless isolated in that it has been When the nucleic acid molecule that encodes any of the separated from the Substances with which it may be associ enzymes of the claimed invention is expressed in a cell, a ated in living systems, i.e. isolated from other proteins. variety of transcription control sequences (e.g., promoter/ The invention also encompasses nucleic acids that encode enhancer sequences) can be used to direct its expression. The for any of the polypeptides described herein, libraries that promoter can be a native promoter, i.e., the promoter of the contain any of the nucleic acids and/or polypeptides 25 gene in its endogenous context, which provides normal regu described herein, and compositions that contain any of the lation of expression of the gene. In some embodiments the nucleic acids and/or polypeptides described herein. promoter can be constitutive, i.e., the promoter is unregulated In Some embodiments, one or more of the genes associated allowing for continual transcription of its associated gene. A with the invention is expressed in a recombinant expression variety of conditional promoters also can be used, such as vector. As used herein, a “vector” may be any of a number of 30 promoters controlled by the presence or absence of a mol nucleic acids into which a desired sequence or sequences may ecule. be inserted by restriction and ligation for transport between The precise nature of the regulatory sequences needed for different genetic environments or for expression in a host cell. gene expression may vary between species or cell types, but Vectors are typically composed of DNA, although RNA vec shall in general include, as necessary, 5' non-transcribed and tors are also available. Vectors include, but are not limited to: 35 5' non-translated sequences involved with the initiation of plasmids, fosmids, phagemids, virus genomes and artificial transcription and translation respectively, such as a TATA chromosomes. box, capping sequence, CAAT sequence, and the like. In A cloning vector is one which is able to replicate autono particular, such 5' non-transcribed regulatory sequences will mously or integrated in the genome in a host cell, and which include a promoter region which includes a promoter is further characterized by one or more endonuclease restric 40 sequence for transcriptional control of the operably joined tion sites at which the vector may be cut in a determinable gene. Regulatory sequences may also include enhancer fashion and into which a desired DNA sequence may be sequences or upstream activator sequences as desired. The ligated such that the new recombinant vector retains its ability vectors of the invention may optionally include 5' leader or to replicate in the host cell. In the case of plasmids, replication signal sequences. The choice and design of an appropriate of the desired sequence may occur many times as the plasmid 45 vector is within the ability and discretion of one of ordinary increases in copy number within the host cell Such as a host skill in the art. bacterium or just a single time per host before the host repro Expression vectors containing all the necessary elements duces by mitosis. In the case of phage, replication may occur for expression are commercially available and knownto those actively during a lytic phase or passively during a lysogenic skilled in the art. See, e.g., Sambrook et al., Molecular Clon phase. 50 ing: A Laboratory Manual, Second Edition, Cold Spring An expression vector is one into which a desired DNA Harbor Laboratory Press, 1989. Cells are genetically engi sequence may be inserted by restriction and ligation Such that neered by the introduction into the cells of heterologous DNA it is operably joined to regulatory sequences and may be (RNA). That heterologous DNA (RNA) is placed under oper expressed as an RNA transcript. Vectors may further contain able control of transcriptional elements to permit the expres one or more marker sequences Suitable for use in the identi 55 sion of the heterologous DNA in the host cell. Heterologous fication of cells which have or have not been transformed or expression of genes associated with the invention, for pro transfected with the vector. Markers include, for example, duction of a terpenoid, Such as taxadiene, is demonstrated in genes encoding proteins which increase or decrease either the Examples section using E. coli. The novel method for resistance or sensitivity to antibiotics or other compounds, producing terpenoids can also be expressed in other bacterial genes which encode enzymes whose activities are detectable 60 cells, fungi (including yeast cells), plant cells, etc. by Standard assays known in the art to (e.g., B-galactosidase, A nucleic acid molecule that encodes an enzyme associ luciferase or alkaline phosphatase), and genes which visibly ated with the invention can be introduced into a cell or cells affect the phenotype of transformed or transfected cells, using methods and techniques that are standard in the art. For hosts, colonies or plaques (e.g., green fluorescent protein). example, nucleic acid molecules can be introduced by stan Preferred vectors are those capable of autonomous replica 65 dard protocols such as transformation including chemical tion and expression of the structural gene products present in transformation and electroporation, transduction, particle the DNA segments to which they are operably joined. bombardment, etc. Expressing the nucleic acid molecule US 8,927,241 B2 21 22 encoding the enzymes of the claimed invention also may be tion in an aerated reaction vessel Such as a stirred tank reactor accomplished by integrating the nucleic acid molecule into can be used to produce large quantities ofterpenoids, such as the genome. taxadiene, that can be recovered from the cell culture. In some In some embodiments one or more genes associated with embodiments, the terpenoid is recovered from the gas phase the invention is expressed recombinantly in a bacterial cell. of the cell culture, for example by adding an organic layer Bacterial cells according to the invention can be cultured in Such as dodecane to the cell culture and recovering the terpe media of any type (rich or minimal) and any composition. As noid from the organic layer. would be understood by one of ordinary skill in the art, Terpenoids, such as taxadiene, produced through methods routine optimization would allow for use of a variety of types described herein have widespread applications including of media. The selected medium can be supplemented with 10 pharmaceuticals such as paclitaxel (Taxol), artemisinin, various additional components. Some non-limiting examples ginkolides, eleutherobin and pseudopterosins, and many of Supplemental components include glucose, antibiotics, other potential pharmaceutical compounds. Further applica IPTG for gene induction, ATCC Trace Mineral Supplement, tions include compounds used in flavors and cosmetics Such and glycolate. Similarly, other aspects of the medium, and as geraniol, farnesol, geranly geraniol, linalool, limonene, growth conditions of the cells of the invention may be opti 15 pinene, cineol and isoprene. Further applications include mized through routine experimentation. For example, pH and compounds for use as biofuels such as alcohols of 5, 10, and temperature are non-limiting examples of factors which can 15 carbon atom length. It is noted that the above compounds be optimized. In some embodiments, factors such as choice of are presently produced as extracts of various plants. Plant media, media Supplements, and temperature can influence extract-based methods are tedious, yield very Small amounts production levels of terpenoids, such as taxadiene. In some and are limited as to the actual molecules that can be so embodiments the concentration and amount of a Supplemen obtained, namely, they do not allow the easy production of tal component may be optimized. In some embodiments, how derivatives that may possess far Superior properties than the often the media is Supplemented with one or more Supple original compounds. mental components, and the amount of time that the media is In one aspect, the invention provides a method for making cultured before harvesting a terpenoid, Such as taxadiene, is 25 a product containing a terpenoid or terpenoid derivative. The optimized. method according to this aspect comprises increasing terpe According to aspects of the invention, high titers of a noid production in a cell that produces one or more terpenoids terpenoid (such as but not limited to taxadiene), are produced by controlling the accumulation of indole in the cell or in a through the recombinant expression of genes as described culture of the cells. The terpenoid, or a derivative of the herein, in a cell expressing components of the MEP pathway, 30 terpenoid prepared through one or more chemical or enzy and one or more downstream genes for the production of a matic steps, is incorporated into a product to thereby make the terpenoid (or related compounds) from the products of the product containing a terpenoid or terpenoid derivative. MEP pathway. As used herein “high titer refers to a titer in According to this aspect, the cell expressed components of the milligrams per liter (mg L') scale. The titer produced for the MEP pathway, and may be a bacterial cell such as E. coli a given product will be influenced by multiple factors includ 35 or B. subtilis. In various embodiments, the accumulation of ing choice of media. In some embodiments, the total titer of a indole in the cell or in a culture of the cells is controlled at terpenoid or derivative is at least 1 mg L'. In some embodi least in part by balancing an upstream non-mevalonate iso ments, the total terpenoid or derivative titer is at least 10 mg prenoid pathway with a downstream heterologous terpenoid L'. In some embodiments, the total terpenoid or derivative synthesis pathway. For example, the upstream non-meva titer is at least 250 mg L'. For example, the total terpenoid or 40 lonate isoprenoid pathway may be balanced with respect to derivative titer can beat least 1,2,3,4,5,6,7,8,9, 10, 11, 12, the downstream heterologous terpenoid synthesis pathway by 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, one or more of: 30, 31, 32,33, 34,35, 36, 37,38, 39, 40, 41,42, 43,44, 45,46, (1) increasing gene copy number for one or more upstream 47, 48,49, 50, 51, 52,53,54, 55,56, 57,58, 59, 60, 61, 62,63, or downstream pathway enzymes, 64, 65, 66, 67, 68, 69, 70, 75, 80, 85,90, 95, 100, 125, 150, 45 (2) increasing or decreasing the expression level of the 175, 200, 225, 250, 275,300, 325, 350, 375,400, 425,450, upstream and downstream pathway, as individual genes 475,500, 525,550, 575, 600, 625, 650, 675, 700, 725,750, or as an operon, using promoters with different 775, 800, 825, 850, 875, 900 or more than 900 mg L-1 strengths, including any intermediate values. In some embodiments, the (3) increasing or decreasing the expression level of the total terpenoid or derivative titer can be at least 1.0, 1.1, 1.2, 50 upstream and downstream pathways, as individual 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, genes or as an operon, using modifications to ribosomal 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, binding sites, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, or more than 5.0 (4) replacing native genes in the MEP pathway with heter g L' including any intermediate values. ologous genes coding for homologous enzymes, In some embodiments, the total taxadiene 5C-ol titer is at 55 (5) codon-optimization of one or more heterologous least 1 mg L'. In some embodiments, the total taxadiene enzymes in the upstream or downstream pathway, 5C-ol titer is at least 10 mg L'. In some embodiments, the (6) mutation of amino acids in one or more genes of the total taxadiene 5C-oltiter is at least 50 mg L'. For example, downstream and/or upstream pathway, or the total taxadiene 5C-oltiter can be at least 1, 2, 3, 4, 5, 6, 7, (7) modifying the order of upstream and downstream path 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 60 way genes in a heterologous operon. 25, 26, 27, 28, 29, 30, 31, 32,33, 34,35, 36, 37,38, 39, 40, 41, In some embodiments, the cell comprises at least one addi 42, 43,44, 45,46, 47, 48,49, 50, 51, 52,53,54, 55,56, 57,58, tional copy of at least one of dxS, idi, isp), and ispF. For 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or more than 70 example, the cell may comprise a heterologous dxs-idi-isp F mg L' including any intermediate values. operon. The liquid cultures used to grow cells associated with the 65 According to embodiments of this aspect, indole accumu invention can be housed in any of the culture vessels known lates in the culture at less than 100 mg/L, or in certain embodi and used in the art. In some embodiments large Scale produc ments, at less than 50 mg/L, at less than 10 mg/L, or at less US 8,927,241 B2 23 24 than 1 mg/L, or less than 0.5 mg/L, or less than 0.1 mg/L. As In Some embodiments, the one or more terpenoids includes an alternative, or in addition to balancing the upstream and Citral, which may be synthesized through a pathway com downstream pathways, the accumulation of indole in the cell prising one or more of Geranyl pyrophosphate synthase (e.g., or in a culture of the cells may comprise modifying or regu AANO1134.1, ACA21458.2), geraniol synthase (e.g., lating the cells indole pathway. In still other embodiments, HM807399, GU136162, AY362553), and geraniol dehydro the step of controlling the accumulation of indole in the cellor genase (e.g., AY879284). in a culture of the cells may comprise or may further comprise In still other embodiments, the one or more terpenoids removing the accumulated indole from the cell culture includes Cubebol, which is synthesized through a pathway through chemical methods, which may include using one or comprising one or more of farnesyl diphosphate synthase more absorbents or scavengers. In some embodiments, the 10 level of indole in the culture or in the cells in monitored, or (e.g., AAK63847.1), and cubebol synthase (e.g., example, by continuously or intermittently measuring the CQ813505.1). amount of indole in the culture. The one or more terpenoids may include Limonene, and The invention according to this aspect can involve the which may be synthesized through a pathway comprising one manufacture of a product containing one or more terpenoids, 15 or more of Geranyl pyrophosphate synthase (e.g., Such as one or more terpenoids selected from a hemiterpe AANO1134.1, ACA21458.2), and limonene synthase (e.g., noid, a monoterpenoid, a sesquiterpenoid, a diterpenoid, a EF426463, JN38.8566, HQ636425). triterpenoid or a tetraterpenoid. The one or more terpenoids may include Menthone or In some embodiments, the product is a food product, food Menthol, which may be synthesized through a pathway com additive, beverage, chewing gum, candy, or oral care product. prising one or more of Geranyl pyrophosphate synthase (e.g., In such embodiments, the terpenoid or derivative may be a AANO1134.1, ACA21458.2), limonene synthase (e.g., flavor enhancer or sweetener. For example, the terpenoid or EF426463, JN1388566, HQ636425), (-)-limonene-3-hy derivative may include one or more of alpha-sinensal; beta droxylase (e.g., EF426464, AY622319). (-)-isopiperitenol Thuj one; Camphor; Carveol; Carvone; Cineole: Citral; Cit dehydrogenase (e.g., EF426465), (-)-isopiperitenone reduc ronellal; Cubebol; Limonene; Menthol; Menthone; myrcene: 25 tase (e.g., EF426466), (+)-cis-isopulegone isomerase, (-)- Nootkatone; Piperitone: Sabinene hydrate; Steviol; Steviol menthone reductase (e.g., EF426467), and for Menthol (-)- glycosides; Thymol; Valencene; or a derivative of one or more menthol reductase (e.g., EF426468). of Such compounds. In other embodiments, the terpenoid or In some embodiments, the one or more terpenoids com derivative is one or more of alpha, beta and y-humulene; prise myrcene, which may be synthesized through a pathway isopinocamphone; (-)-alpha-phellandrene; (+)-1-terpinene 30 comprising one or more of Geranyl pyrophosphate synthase 4-ol; (+)-borneol; (+)-verbenone; 1.3.8-menthatriene: (e.g., AANO1134.1, ACA21458.2) and myrcene synthase 3-carene: 3-Oxo-alpha-Ionone: 4-Oxo-beta-ionone; alpha (e.g., U87908, AY195608, AF271259). sinensal: alpha-terpinolene; alpha-thujene: Ascaridole; Cam The one or more terpenoids may include Nootkatone, phene; Carvacrol; Cembrene; E)-4-decenal; Farnesol; Fen which may be synthesized through a pathway comprising one chone; gamma-Terpinene; Geraniol; hotrienol; Isoborneol; 35 or more of farnesyl diphosphate synthase (e.g., Limonene; myrcene; nerolidol; Ocimene; p-cymene; perilla AAK63847.1), and Valancene synthase (e.g., CO813508, ldehyde; Pulegone; Sabinene; Sabinene hydrate; tagetone: AF441124. 1). Verbenone; or a derivative of one or more of such compounds. The one or more terpenoids may include Sabinene hydrate, Downstream enzymes for the production of Such terpe which may be synthesized through a pathway comprising one noids and derivatives are known. 40 or more of Geranyl pyrophosphate synthase (e.g., For example, the terpenoid may be alpha-sinensal, and AANO1134.1, ACA21458.2), and Sabinene synthase (e.g., which may be synthesized through a pathway comprising one O81193.1). or more of farnesyl diphosphate synthase (e.g., AAK63847.1) The one or more terpenoids may include Steviol or steviol and Valencene synthase (e.g., AF441124 1). glycoside, and which may be synthesized through a pathway In other embodiments, the terpenoid is beta-Thuj one, and 45 comprising one or more of geranylgeranylpyrophosphate which may be synthesized through a pathway comprising one synthase (e.g., AF081514), ent-copalyldiphosphate synthase or more of Geranyl pyrophosphate synthase (e.g., (e.g., AF034545.1), ent-kaurene synthase (e.g., AANO1134.1. ACA21458.2) and (+)-sabinene synthase (e.g., AF0973 11.1), ent-kaurene oxidase (e.g., DQ200952.1), and AF051901.1). kaurenoic acid 13-hydroxylase (e.g., EU722415.1). For ste In other embodiments, the terpenoid is Camphor, which 50 viol glycoside, the pathway may further include UDP-glyco may be synthesized through a pathway comprising one or syltransferases (UGTs) (e.g., AF515727.1, AY345983.1, more of Geranyl pyrophosphate synthase (e.g., AANO1134.1, AY345982.1, AY345979.1, AAN40684.1, ACE87855.1). ACA21458.2), (-)-borneol dehydrogenase (e.g., The one or more terpenoids may include Thymol, which GU253890.1), and bornyl pyrophosphate synthase (e.g., may be synthesized through a pathway comprising one or AF051900). 55 more of Geranyl pyrophosphate synthase (e.g., AANO1134.1, In certain embodiments, the one or more terpenoids ACA21458.2), limonene synthase (e.g., EF426463, include Carveol or Carvone, which may be synthesized JN38.8566, HQ636425), (-)-limonene-3-hydroxylase (e.g., through a pathway comprising one or more of Geranyl pyro EF426464, AY622319). (-)-isopiperitenol dehydrogenase phosphate synthase (e.g., AANO1134.1, ACA21458.2), (e.g., EF426465), and (-)-isopiperitenone reductase (e.g., 4S-limonene synthase (e.g., AAC37366.1), limonene-6-hy 60 EF426466). droxylase (e.g., AAQ18706.1, AAD44150.1), and carveol The one or more terpenoids may include Valencene, which dehydrogenase (e.g., AAU20370.1, ABR15424.1). may be synthesized through a pathway comprising one or In some embodiments, the one or more terpenoids com more of farnesyl diphosphate synthase (e.g., AAK63847.1), prise Cineole, which may be synthesized through a pathway and Valancene synthase (e.g., CO813508, AF441124. 1). comprising one or more of Geranyl pyrophosphate synthase 65 In Some embodiments, the one or more terpenoids includes (e.g., AANO1134.1, ACA21458.2) and 1,8-cineole synthase one or more of alpha, beta and Y-humulene, which may be (e.g., AF051899). synthesized through a pathway comprising one or more of US 8,927,241 B2 25 26 farnesyl diphosphate synthase (e.g., AAK63847.1), and Valencene; Nootkatone; patchouli alcohol; Farnesol; beta humulene synthase (e.g., U92267.1). Ylangene; B-Santalol; B-Santalene; a-Santalene; C.Santalol; In some embodiments, the one or more terpenoids includes B-Vetivone; a-Vetivone,: khusimol; Sclarene, Sclareol; beta (+)-borneol, which may be synthesized through a pathway Damascone; beta-Damascenone; or a derivative thereof. In comprising one or more of Geranyl pyrophosphate synthase these or other embodiments, the one or more terpenoid or (e.g., AANO1134.1, ACA21458.2), and bornyl pyrophos derivative compounds includes one or more of Camphene; phate synthase (e.g., AF051900). Pulegone; Fenchone: Fenchol; Sabinene hydrate; Menthone: The one or more terpenoids may comprise 3-carene, which Piperitone; Carveol; gamma-Terpinene; beta-Thujone; dihy may be synthesized through a pathway comprising one or dro-myrcene; alpha-thujene; alpha-terpineol; ocimene; more of Geranyl pyrophosphate synthase (e.g., AANO1134.1, 10 nerol; nerolidol, E)-4-decenal; 3-carene; (-)-alpha-phelland ACA21458.2), and 3-carene synthase (e.g., HQ336800). rene; hotrienol; alpha-terpinolene; (+)-1-terpinene-4-ol; In some embodiments, the one or more terpenoids include perillaldehyde; verbenone; isopinocamphone; tagetone; 3-OXo-alpha-Ionone or 4-oxo-beta-ionone, which may be trans-myrtanal; alpha-sinensal: 1.3.8-menthatriene; (-)-cis synthesized through a pathway comprising carotenoid cleav rose oxide; (+)-borneol; (+)-verbenone; Germacrene A: Ger age dioxygenase (e.g., ABY60886.1, BAJ054.01.1). 15 macrene B; Germacrene; Germacrene E.; (+)-beta-cadinene; In some embodiments, the one or more terpenoids include epi-cedrol; alpha, beta and y-humulene; alpha-bisabolene; alpha-terpinolene, which may be synthesized through a path beta-aryophyllene; Longifolene; alpha-sinensal; alpha-bis way comprising one or more of Geranyl pyrophosphate Syn abolol, (-)f-Copaene; (-)-C-Copaene, 4(Z).7(Z)-ecadienal; thase (e.g., AANO1134.1, ACA21458.2), and alpha-terpineol cedrol; cedrene; muuroladiene; isopatchoul-3-ene; iso synthase (e.g., AF543529). patchoula-3,5-diene; cedrol; guaiol; (-)-6.9-guaiadiene; bul In some embodiments, the one or more terpenoids include nesol; guaiol; ledene; ledol; lindestrene; alpha-bergamotene; alpha-thujene, which may be synthesized through a pathway maaliol, isovalencenol; muurolol T. beta-Ionone; alpha-Ion comprising one or more of Geranyl pyrophosphate synthase one; Oxo-Edulan I; Oxo-Edulan II: Theaspirone; Dihydroac (e.g., AANO1134.1, ACA21458.2), and alpha-thujene syn tinodiolide: 4-Oxoisophorone; Safranal; beta-Cyclocitral; thase (e.g., AEJ91555.1). 25 (-)-cis-gamma-irone, (-)-cis-alpha-irone; or a derivative In some embodiments, the one or more terpenoids include thereof. Such terpenoids and derivatives may be synthesized Farnesol, which may be synthesized through a pathway com according to a pathway described above. In some embodi prising one or more of farnesyl diphosphate synthase (e.g., ments, the one or more terpenoids include Linalool, which AAK63847.1), and Farnesol synthase (e.g., AF529266.1, may be synthesized through a pathway comprising one or DQ872159.1). 30 more of Geranyl pyrophosphate synthase (e.g., AANO1134.1, In some embodiments, the one or more terpenoids include ACA21458.2), and linalool synthase (e.g., FJ644544, Fenchone, which may be synthesized through a pathway GQ338154, FJ644548). comprising one or more of Geranyl pyrophosphate synthase In some embodiments, the one or more terpenoids include (e.g., AANO1134.1, ACA21458.2), and (-)-endo-fenchol alpha-Pinene, which may be synthesized through a pathway cyclase (e.g., AY693648). 35 comprising one or more of Geranyl pyrophosphate synthase In some embodiments, the one or more terpenoids include (e.g., AANO1134.1, ACA21458.2), and pinene synthase (e.g., gamma-Terpinene, which may be synthesized through a path HQ636424, AF543527, U87909). way comprising one or more of Geranyl pyrophosphate Syn In Some embodiments, the one or more terpenoids includes thase (e.g., AANO1134.1, ACA21458.2), and terpinene syn Germacrene D, which may be synthesized through a pathway thase (e.g., AB1 10639). 40 comprising one or more of farnesyl diphosphate synthase In some embodiments, the one or more terpenoids include (e.g., AAK63847.1), and germacrene synthase (e.g., Geraniol, which may be synthesized through a pathway com AAS66357.1, AAX40665.1, HQ652871). prising one or more of Geranyl pyrophosphate synthase (e.g., In some embodiments, the one or more terpenoids include AANO1134.1, ACA21458.2), and geraniol synthase (e.g. farnesene, which may be synthesized through a pathway HM807399, GU136162, AY362553). 45 comprising one or more of farnesyl diphosphate synthase In still other embodiments, the one or more terpenoids (e.g., AAK63847.1), and farnesene synthase (e.g., include Ocimene, which may be synthesized through a path AAT70237.1, AAS68019.1, AY182241). way comprising one or more of Geranyl pyrophosphate Syn In some embodiments, the one or more terpenoids com thase (e.g., AANO1134.1, ACA21458.2), and beta-ocimene prises patchouli alcohol, which may be synthesized through a synthase (e.g., EU194553.1). 50 pathway comprising one or more of farnesyl diphosphate In certain embodiments, the one or more terpenoids synthase (e.g., AAK63847.1), and Patchoulol synthase (e.g., include Pulegone, which may be synthesized through a path AY508730.1). way comprising one or more of Geranyl pyrophosphate Syn In some embodiments, the one or more terpenoids com thase (e.g., AANO1134.1. ACA21458.2), and pinene synthase prises B-Santalol, which may be synthesized through a path (e.g., HQ636424, AF543527, U87909). 55 way comprising one or more of farnesyl diphosphate Syn In certain embodiments, the one or more terpenoids thase (e.g., AAK63847.1), beta Santalene synthase (e.g., includes Sabinene, which may be synthesized through a path HQ259029.1, HQ343278.1, HQ343277.1). way comprising one or more of Geranyl pyrophosphate Syn In some embodiments, the one or more terpenoids include thase (e.g., AANO1134.1, ACA21458.2), and Sabinene syn B-Santalene, which may be synthesized through a pathway thase (e.g., HQ336804, AF051901, DQ785794). 60 comprising one or more of farnesyl diphosphate synthase In various embodiments, the product is a fragrance prod (e.g., AAK63847.1), and beta Santalene synthase (e.g., uct, a cosmetic, a cleaning product, or a Soap. In Such embodi HQ259029.1, HQ343278.1, HQ343277.1). ments, the terpenoid or derivative may be a fragrance. For In some embodiments, the one or more terpenoids include example, the one or more terpenoid or derivative may include C.-Santalene or C.-Santalol, which may be synthesized one or more of Linalool; alpha-Pinene; Carvone; Citronellal; 65 through a pathway comprising one or more of farnesyl Citronellol; Citral; Sabinene; Limonene; Verbenone; diphosphate synthase (e.g., AAK63847.1), and Santalene Geraniol; Cineole: myrcene; Germacrene D; farnesene; synthase (e.g., HQ343282.1). US 8,927,241 B2 27 28 In other embodiments, the one or more terpenoids include terpenoids include (-)-C.-Copaene, which may be synthe Sclareol, which may be synthesized through a pathway com sized through a pathway comprising one or more of farnesyl prising one or more of geranylgeranylpyrophosphate Syn diphosphate synthase (e.g., AAK63847.1), and Copaene Syn thase (e.g., AF081514), and Sclareol synthase (e.g., thase (e.g., AJO01539.1). HB976923.1). The one or more terpenoids may include cedrolor cedrene, In certain embodiments, the one or more terpenoids which may be synthesized through a pathway comprising one include beta-Damascone or beta-Damacenone, which may be or more of farnesyl diphosphate synthase (e.g., synthesized through a pathway comprising one or more of AAK63847.1), and cedrol synthase (e.g., AJOO1539.1). carotenoid cleavage dioxygenase (e.g., ABY60886.1, The one or more terpenoids may include muuroladiene, BAJ054.01.1). 10 which may be synthesized through a pathway comprising one In some embodiments, the one or more terpenoids include Fenchol, which may be synthesized through a pathway com or more of farnesyl diphosphate synthase (e.g., prising one or more of Geranyl pyrophosphate synthase (e.g., AAK63847.1), and muuroladiene synthase (e.g., AANO1134.1, ACA21458.2), and (-)-endo-fenchol cyclase AJ786641.1). (e.g., AY693648). 15 The one or more terpenoids may include alpha-bergamo In some embodiments, the one or more terpenoids include tene, which may be synthesized through a pathway compris alpha-terpineol, which may be synthesized through a path ing one or more of farnesyl diphosphate synthase (e.g., way comprising one or more of Geranyl pyrophosphate Syn AAK63847.1), and alpha-bergamotene synthase (e.g., thase (e.g., AANO1134.1, ACA21458.2), alpha-terpineol HQ25902.9.1). synthase (e.g., AF543529). The one or more terpenoids may include alpha and/or In some embodiments, the one or more terpenoids include beta-Ionone, which may be synthesized through a pathway Germacrene A, which may be synthesized through a pathway comprising one or more of carotenoid cleavage dioxygenase comprising one or more of farnesyl diphosphate synthase (e.g., ABY60886.1, BAJ054.01.1). (e.g., AAK63847.1), and Germacrene synthase (e.g., In some embodiments of this aspect of the invention, the AEM05858.1, ABE03980.1, AAM11626.1). In other 25 product is a vitamin or nutritional Supplement, and the terpe embodiments, the one or more terpenoids include Germa noid or derivative may be Zexanthin; B-carotene; lycopene; crene B, which may be synthesized through a pathway com Astaxanthin; CoQ10; Vitamin K. Vitamin E: Labdane; Gib prising one or more of farnesyl diphosphate synthase (e.g., berellins: C-carotene; or a derivative thereof. Enzymes and AAK63847.1), and Germacrene synthase (e.g., encoding genes for synthesizing Such terpenoids or deriva ACF9446.9.1). Alternatively or in addition, the one or more 30 tives thereof from products of the MEP pathway are known, terpenoids include Germacrene C, which may be synthesized and some are described above. through a pathway comprising one or more of farnesyl For example, the one or more terpenoids may include Zex diphosphate synthase (e.g., AAK63847.1), and Germacrene anthin, which may be synthesized through a pathway com synthase (e.g., AAC39432.1). prising one or more of geranylgeranylpyrophosphate Syn In still other embodiments, the one or more terpenoids 35 include (+)-beta-cadinene, which may be synthesized thase (e.g., AF081514), phytoene synthase (e.g., through a pathway comprising one or more of farnesyl AAA64982.1), phytoene desaturase (e.g., AAA21263.1, diphosphate synthase (e.g., AAK63847.1), cadinene Syn ACI04664.1), lycopene beta cyclase (e.g., AAA64980.1), thases (e.g., U883.18.1). and beta-carotene hydroxylase (e.g., AAA64983.1). In some embodiments, the one or more terpenoids include 40 In other embodiments, the one or more terpenoids include epi-cedrol, which may be synthesized through a pathway C- and/or B-carotene, which may be synthesized through a comprising one or more of farnesyl diphosphate synthase pathway comprising one or more of geranylgeranylpyrophos (e.g., AAK63847.1), and epicedrol synthase (e.g., phate synthase (e.g., AF081514), phytoene synthase (e.g., AF157059.1). AAA64982.1, phytoene desaturase (e.g., AAA21263.1, In other embodiments, the one or more terpenoids include 45 ACI04664.1), lycopene beta cyclase (e.g., AAA64980.1), alpha-bisabolene, which may be synthesized through a path and lycopene beta cyclase (e.g., NM 001084662.1, way comprising one or more of farnesyl diphosphate Syn AAA64980.1). thase (e.g., AAK63847.1), and bisabolene synthase (e.g., In some embodiments, the one or more terpenoids include HQ343280.1, HQ343279.1). lycopene, which may be synthesized through a pathway com In some embodiments, the one or more terpenoids include 50 prising one or more of geranylgeranylpyrophosphate Syn beta-caryophylene, which may be synthesized through a thase (e.g., AF081514), phytoene synthase (e.g., pathway comprising one or more of farnesyl diphosphate AAA64982.1), phytoene desaturase (e.g., AAA21263.1, synthase (e.g., AAK63847.1), and Caryophyllene synthase ACI04664.1), and lycopene beta cyclase (e.g., (e.g., AF472361). AAA64980.1). The one or more terpenoids may include Longifolene, 55 In some embodiments, the one or more terpenoids include which may be synthesized through a pathway comprising one Astaxanthin, which may be synthesized through a pathway or more of farnesyl diphosphate synthase (e.g., comprising one or more of geranylgeranylpyrophosphate AAK63847.1), and longifolene synthase (e.g., AY473.625.1). synthase (e.g., AF081514), phytoene synthase (e.g., The one or more terpenoids may include alpha-bisabolol. AAA64982.1), phytoene desaturase (e.g., AAA21263.1, which may be synthesized through a pathway comprising one 60 ACI04664.1), lycopene beta cyclase (e.g., AAA64980.1), or more of farnesyl diphosphate synthase (e.g., beta-caroteine hydroxylase (e.g., AAA64983.1), and beta AAK63847.1), and bisabolol synthase (e.g., ADO87004.1). caroteine ketolase (e.g., ABLO9497.1). In some embodiments, the one or more terpenoids include In still other embodiments, the one or more terpenoids (-)-B-Copaene, which may be synthesized through a pathway include Vitamin E, which may be synthesized through a path comprising one or more of farnesyl diphosphate synthase 65 way comprising one or more of geranylgeranylpyrophos (e.g., AAK63847.1), and Copaene synthase (e.g., phate synthase (e.g., AF081514), geranylgeranylpyrophos EU158098). Alternatively, or in addition, the one or more phate reductase (e.g., BAO00022), homogentisate US 8,927,241 B2 29 30 phytyltransferase (e.g., BAO00022), tocopherol-cyclase comprising one or more of geranylgeranylpyrophosphate (e.g., BAO00022), and copalyl pyrophosphate synthase (e.g., synthase (e.g., AF081514), and levopimaradiene synthase AAB87091). (e.g., AAS89668.1). In some embodiments of this aspect of the invention, the In Some embodiments, the one or more terpenoids or product is a solvent or cleaning product. For example, the derivatives may include Abietadiene or Abietic acid, which terpenoid or derivative may be alpha-Pinene; beta-Pinene; may be synthesized through a pathway comprising one or Limonene; myrcene; Linalool; Geraniol; Cineole; or a deriva more of geranylgeranylpyrophosphate synthase (e.g., tive thereof. Some enzymes and encoding genes for synthe AF081514), and abietadiene synthase (e.g., AAK83563.1). sizing such terpenoids orderivatives thereoffrom products of The one or more terpenoids or derivatives may include the MEP pathway are known, and some are described above. 10 beta-amyrin, which may be synthesized through a pathway In some embodiments, the one or more terpenoids include beta-Pinene, which may be synthesized through a pathway comprising one or more of geranylgeranylpyrophosphate comprising one or more of Geranyl pyrophosphate synthase synthase (e.g., AF081514), and beta-Amyrin Synthase (e.g., (e.g., AANO1134.1, ACA21458.2), and pinene synthase (e.g., ACO24697.1). HQ636424, AF543527, U87909). 15 The one or more terpenoids or derivatives may include In some embodiments of this aspect of the invention, the beta-selinene, which may be synthesized through a pathway product is a pharmaceutical, and the terpenoid or derivative is comprising one or more of farnesyl diphosphate synthase to an active pharmaceutical ingredient. For example, the terpe (e.g., AAK63847.1), and selinene synthase (e.g., O64404.1). noid or derivative may be Artemisinin; Taxol; Taxadiene: In still other embodiments, the one or more terpenoids or levopimaradiene; Gingkolides; Abietadiene; Abietic acid; derivatives include 5-epi-aristolochene, which may be syn beta-amyrin; Retinol; or a derivative thereof. In still other thesized through a pathway comprising one or more of far embodiments, the terpenoid or derivative is Thymoquinone; nesyl diphosphate synthase (e.g., AAK63847.1), and 5-epi Ascaridole; beta-selinene; 5-epi-aristolochene; Vetispiradi aristolochene synthase (e.g., AF542544.1). ene; epi-cedrol; alpha, beta and y-humulene: a-cubebene: In Some embodiments, the one or more terpenoids or beta-elemene; Gossypol; Zingiberene: Periplanone B: Cap 25 derivatives include vetispiradiene, which may be synthesized sidiol; Capnellene; illudin; Isocomene; cyperene; Pseudot through a pathway comprising one or more of farnesyl erosins; Crotophorbolone; Englerin: Psiguadial; Stemodi diphosphate synthase (e.g., AAK63847.1), and vetispiradi none; Maritimol; Cyclopamine: Veratramine: Aply violene; ene synthases (e.g., BD227667). macfarlandin E. Betulinic acid, oleanolic acid; Ursoloic acid; In Some embodiments, the one or more terpenoids or Pimaradiene; neo-abietadiene; Squalene; Dolichol; Lupeol; 30 derivatives include epi-cedrol, which may be synthesized Euphol; Kaurene; Gibberellins; Cassaic acid; Erythroxydiol; through a pathway comprising one or more of farnesyl Trisporic acid: Podocarpic acid; Retene: Dehydroleucodine: diphosphate synthase (e.g., AAK63847.1), and epicedrol Phorbol; Cafestol; kahweol; Tetrahydrocannabinol; andros synthase (e.g., AF15705.9.1). tenol; or a derivative thereof. Enzymes and encoding genes The one or more terpenoids or derivatives may include for synthesizing such terpenoids or derivatives thereof from 35 beta-elemene, which may be synthesized through a pathway products of the MEP pathway are known, and some are comprising one or more of farnesyl diphosphate synthase described above. (e.g., AAK63847.1), and elemene synthase (e.g., For example, in some embodiments, the one or more ter DQ872158.1). penoids include Artemisinin, which may be synthesized In still other embodiments, the terpenoid or derivative is through a pathway comprising one or more of farnesyl 40 Gossypol, which may be synthesized through a pathway com diphosphate synthase (e.g., AAK63847.1). Amorphadiene prising one or more of farnesyl diphosphate synthase (e.g., Synthase (e.g., AAF984.44.1), amorpha-4,11-diene AAK63847.1), and (+) delta cadinene synthase (e.g., monooxygenase (e.g., DQ315671), and artemisinic aldehyde U883.18.1). delta-11 (13) reductase (e.g., ACH61780.1). In some embodiments, the terpenoid or derivative is Zin In other embodiments, the one or more terpenoids include 45 giberene, which may be synthesized through a pathway com Taxadiene, which may be synthesized through a pathway prising one or more of farnesyl diphosphate synthase (e.g., comprising one or more of geranylgeranylpyrophosphate AAK63847.1), and Zingiberene synthase (e.g., Q5SBP4.1). synthase (e.g., AF081514), and taxadiene synthase (e.g., In still other embodiments, the one or more terpenoids or U48796.1, AF081514). In some embodiments, the terpenoid derivatives include Capsidiol, which may be synthesized or derivative is Taxol, which may be synthesized through a 50 through a pathway comprising one or more of farnesyl pathway comprising one or more of geranylgeranylpyrophos diphosphate synthase (e.g., AAK63847.1), and epi-aris phate synthase (e.g., AF081514), taxadiene synthase (e.g., tolochene synthase (e.g., Q40577.3). U48796.1, AF081514), Taxus cuspidata taxadiene 5-alpha The one or more terpenoids or derivatives may include hydroxylase (e.g., AY289209.2), Taxus cuspidata 5-alpha Pimaradiene, which may be synthesized through a pathway taxadienol-10-beta-hydroxylase (e.g., AF318211.1), Taxus 55 comprising one or more of geranylgeranylpyrophosphate cuspidata taxoid 10-beta hydroxylase (e.g., AY563635.1), synthase (e.g., AF081514), ent-pimara-8014), 15-diene syn Taxus cuspidata taxane 13-alpha-hydroxylase (e.g., thase (e.g., Q6Z5J6.1), and Syn-pimara-7.15-diene synthase AY056019.1). Taxus cuspidata taxane 14b-hydroxylase (e.g., (e.g., AAU05906.1). AY188177.1), Taxus cuspidata taxoid 7-beta-hydroxylase The one or more terpenoids or derivatives may include (e.g., AY307951.1). 60 neo-abietadiene, which may be synthesized through a path In still other embodiments, the one or more terpenoids or way comprising one or more of geranylgeranylpyrophos derivatives include levopimaradiene, which may be synthe phate synthase (e.g., AF081514), and levopimaradiene Syn sized through a pathway comprising one or more of gera thase (e.g., AAS89668.1). The one or more terpenoids or nylgeranylpyrophosphate synthase (e.g., AF081514), and derivatives may include Squalene, which may be synthesized levopimaradiene synthase (e.g., AAS89668.1). 65 through a pathway comprising one or more of farnesyl In some embodiments, the one or more terpenoids include diphosphate synthase (e.g., AAK63847.1), and squalene Syn Gingkolides, which may be synthesized through a pathway thase (e.g., AEE86403.1). US 8,927,241 B2 31 32 The one or more terpenoids or derivatives may include In some embodiments, the product is a cosmetic or per Lupeol, which may be synthesized through a pathway com Sonal care product, and the terpenoid or derivative is not a prising one or more of geranylgeranylpyrophosphate Syn fragrance. For example, one or more terpenoid or derivative is thase (e.g., AF081514), and lupeol synthase (e.g., Camphor, Linalool; Carvone; myrcene; farnesene; patchouli AAD05032.1). alcohol; alpha-bisabolene; alpha-bisabolol; beta-Ylangene: The one or more terpenoids or derivatives may include B-Santalol; B-Santalene: a-Santalene; C-Santalol; or a deriva Kaurene, which may be synthesized through a pathway com tive thereof. In some embodiments, the terpenoid or deriva prising one or more of geranylgeranylpyrophosphate Syn tive is Camphene; Carvacrol; alpha-terpineol; (Z)-beta thase (e.g., AF081514), ent-copalyl diphosphate synthase ocimene; nerol; (E)-4-decenal; perillaldehyde: (-)-cis-rose (e.g., AF034545.1), and ent-kaurene synthase (e.g., 10 oxide: Copaene, 4(Z).7(Z)-decadienal; isopatchoulenone; AF0973.11.1). (-)-6.9-guaiadiene; Retinol; betulin; (-)-cis-gamma-irone; In some embodiments of this aspect of the invention, the product is a food preservative. In Such embodiments, the (-)-cis-alpha-irone; Phytoene: Phytofluene; or a derivative terpenoid or derivative may be Carvacrol; Carvone; or a thereof. In some embodiments, the one or more terpenoids derivative thereof. For example, the Carvone may be synthe 15 may include alpha-bisabolene, which may be synthesized sized through a pathway comprising one or more of Geranyl through a pathway comprising one or more of farnesyl pyrophosphate synthase (e.g., AANO1134.1, ACA21458.2), diphosphate synthase (e.g., AAK63847.1), and bisabolene 4S-limonene synthase (e.g., AAC37366.1), limonene-6-hy synthase (HQ343280.1, HQ343279.1). droxylase (e.g., AAQ18706.1, AAD44150.1), and carveol In Some embodiments, the one or more terpenoids or dehydrogenase (e.g., AAU20370.1, ABR15424.1). derivatives include (Z)-beta-ocimene, which may be synthe In some embodiments of this aspect, the product is a lubri sized through a pathway comprising one or more of Geranyl cant or surfactant. The terpenoid or derivative may be, for pyrophosphate synthase (e.g., AANO1134.1, ACA21458.2), example, one or more of alpha-Pinene; beta-Pinene; and beta-ocimene synthase (e.g., NM 117775). Limonene; myrcene; farmesene, Squalene; or a derivative In Some embodiments, the one or more terpenoids or thereof. Enzymes and encoding genes for synthesizing Such 25 derivatives include Copaene, which may be synthesized terpenoids or derivatives thereof from products of the MEP through a pathway comprising one or more of farnesyl pathway are known, and some are described above. diphosphate synthase (e.g., AAK63847.1), and Copaene Syn The one or more terpenoids may include farmesene, which thase (e.g., EU158098). may be synthesized through a pathway comprising one or In other embodiments, the terpenoid or derivative is (-)- more of farnesyl diphosphate synthase (e.g., AAK63847.1), 30 cis-gamma-irone or (-)-cis-alpha-irone, which may be Syn and farnesene synthase (e.g., AAT70237.1, AAS68019.1, thesized through a pathway comprising one or more of caro AY182241). The one or more terpenoids may include Squalene, which tenoid cleavage dioxygenase (e.g., ABY60886.1, may be synthesized through a pathway comprising one or BAJ054.01.1). more of farnesyl diphosphate synthase (e.g., AAK63847.1), 35 In still other embodiments, the one or more terpenoids and squalene synthase (e.g., AEE86403.1). include Phytoene or Phytofluene, which may be synthesized In some embodiments, the terpenoid or derivative is poly through a pathway comprising one or more of geranylgera merized, and the resulting polymer may be elastomeric. In nylpyrophosphate synthase (e.g., AF081514), phytoene Syn some such embodiments, the terpenoid or derivative may be thase (e.g., AAA64982.1, and phytoene desaturase (e.g., alpha-Pinene; beta-Pinene; Limonene; myrcene; farnesene; 40 AAA21263.1, ACI04664.1). Squalene; isoprene; or a to derivative thereof. Enzymes and encoding genes for synthesizing Such terpenoids or deriva EXAMPLES tives thereof from products of the MEP pathway are known, and some are described above. In some embodiments, the one Methods or more terpenoids include isoprene, which may be synthe 45 sized through a pathway comprising one or more of Isoprene Strains, Plasmids, oligonucleotides and Genes synthase (e.g., HQ684.728.1). E. coli K12 MG 1655 strain was used as the hoststrain of all In some embodiments of this aspect of the invention, the the taxadiene strain construction. E. coli K12MG 1655 product is an insecticide, pesticide or pest control agent, and A(recAendA) and E. coli K12MG16554(recAendA)ED3 the terpenoid or derivative is an active ingredient. For 50 strains were provided by Professor Kristala Prather's lab at example, the one or more terpenoid or derivative may include MIT (Cambridge, Mass.). Detail of all the plasmids con one or more Carvone; Citronellol; Citral; Cineole; Germa structed for the study is shown in Table 2. All oligonucle crene C; (+)-beta-cadinene; or a derivative thereof. In other otides used in this study are contained in Table 3. embodiments, the one or more terpenoid or derivative is Thy The sequences of geranylgeranyl pyrophosphate synthase mol; Limonene; Geraniol; Isoborneol; beta-Thuj one: 55 (GGPPS), Taxadiene synthase (TS), Cytochrome P450 myrcene; (+)-verbenone; dimethyl-nonatriene; Germacrene Taxadiene 5C-hydroxylase (T5C.OH) and Taxus NADPH: A: Germacrene B; Germacrene D; patchouli alcohol; Guaia cytochrome P450 reductase (TCPR) were obtained from Zulene; muuroladiene; cedrol; alpha-cadinol; d-occidol; AZa Taxus Canadensis, Taxus brevifolia, Taxus cuspidate (Gen dirachtin A; Kaurene; or a derivative thereof. Enzymes and bank accession codes: AF081514, U48796, AY289209 and encoding genes for synthesizing Such terpenoids or deriva 60 AY571340). Genes were custom-synthesized using the plas tives thereof from products of the MEP pathway are known, mids and protocols reported by Kodumal et al. (Supplemen and some are described above. tary details Appendix 1) to incorporate E. coli translation For example, the one or more terpenoids may include Ger codon and removal of restriction sites for cloning purposes. macrene D, which may be synthesized through a pathway Nucleotides corresponding to the 98 and 60 N-terminal comprising one or more of farnesyl diphosphate synthase 65 amino acids of GGPPS and TS (plastid transit peptide) were (e.g., AAK63847.1), and Germacrene synthase (e.g., removed and the translation insertion sequence Met was AAS66357.1, AAX40665.1, HQ652871). inserted.'' US 8,927,241 B2 33 34 Construction of MEP Pathway (dxs-idi-idpDF operon). Kim marker was removed through the action of the FLP dXS-idi-isp F operon was initially constructed by cloning recombinase after Successful gene integration. each of the genes from the genome of Ecoli K12 MG 1655 Construction of Taxadiene 5C.-ol Pathway using the primers dxS(s), dxS(a), idi(s), idi(a), isp)F(s) and The transmembrane region (TM) of the taxadiene 5C-ol isp)FI(a) under pET21C+plasmid with T7 promoter hydroxylase (T5C.OH) and Taxus Cytochrome P450 reduc (p20T7MEP). Using the primers dxsidiisp)FNcoI (s) and tase (TCPR) was identified using PredictProtein software dxsidiisp)FKpnI(a) dxs-idi-ispl)F operon was sub-cloned (www.predictprotein.org). Fortransmembrane engineering into and pTrcHis2B (Invitrogen) plasmid after digested with selective truncation at 8, 24 and 42 amino acid residues on the NcoI and KpnI for pTrcMEP plasmid (p20TreMEP). N-terminal transmembrane region of taxadiene 5C.-ol p20TrcMEP plasmid digested with Mlul and PmeI and 10 hydroxylase (T5C.OH) and 74 amino acid region in the TCPR cloned into Mlul and Pme digested paCYC184-melA(P2A) was performed. The removal of the 8, 24 and 42 residue plasmid to construct p10TrcMEP plasmid.pTrcMEP plasmid N-terminal amino acids of taxadiene 5C-ol hydroxylase digested with Bsta, 17I and Scal and cloned into PyulI (T5C.OH), incorporation of one amino acid substituted digested pCL1920 plasmid to construct pSTrcMEP plasmid. 15 bovine 17a hydroxylase N-terminal 8 residue peptide For constructing p20T5MEP plasmid initially the dxs-idi MALLLAVF (SEQ ID NO:51) to the N-terminal truncated isp)F operon was cloned into pGE plasmid with T5 promoter T5COH sequences' and GSTGS peptide linker was carried (pOE-MEP) using the primers dxsidiisp)FNcoI (s) and dxsi Out using the primer CYP17At8AANdel(s), dispoFXhoI(a). A fraction of the operon DNA with T5 pro CYP17At24AANdel(s), CYP17At42AANdeI(s) and moter was amplified using the primers T5AgeI(s) and T5NheI CYPLinkBamHI(a). Using these primers each modified (a) from pCREMEP plasmid. The DNA fragment was digested DNA was amplified, NdeI/BamHI digested and cloned into with AgeI/NheI and cloned into the p20T7MEP plasmid NdeI/BamHI digested paCYC DUET1 plasmid to construct digested with SGraI/Nhe enzymes. p10At8T5OOH, p10At24T5COH and p10At42T5C.OH plas Construction of Taxadiene Pathway (GT and TG Operons). mids. 74 amino acid truncated TCPR (tTCPR) sequence was The downstream taxadiene pathways (GT and TG operon) 25 amplified using primers CPRBamHI(s) and CPRSalI(a). The were constructed by cloning PCR fragments of GGPS and TS amplified tTCPR sequence and the plasmids, into the Noel-EcoRI and EcoRI-SalI sites of pTrcHIS2B plas p10At8T5OOH, p10At24T5C.OH and p10At42T5COH, was mid to create p20TrcGT and p20TrcTG using the primers digested with BamHI/SalI and cloned to construct the plas GGPPSNcoI(s), GGPPSEcoRI(a), TSEcoRI(s), TSsalI(a), mids p10At8T5C.OH-tTCPR p10At24T5COH-tTCPR and TSNcoI(s)TSEcoRI(a) GGPPSEcoRI(s) and GGPPSSalI(a). 30 p10At42T5C.OH-tTCPR. For constructing p20T5GT, initially the operon was amplified Culture Growth for Screening the Taxadiene and Taxadiene with primers GGPPSNcol(s) and TSXhoI(a) and cloned into 5C-ol Analysis a pCE plasmid under T5 promoter digested with NcoI/XhoI. Single transformants of pre-engineered E. coli strains har Further the sequence was digested with Xbal and Xhol and boring the appropriate plasmid with upstream (MEP), down cloned into the pTrc plasmid backbone amplified using the 35 stream taxadiene pathway and taxadiene 5C.-ol were culti primers pTrcSal(s) and pTrcXba(a). p10T7TG was con vated for 18 h at 30° C. in Luria-Bertani (LB) medium structed by subcloning the NcoI/SalI digested TG operon (Supplemented with appropriate antibiotics, 100 mg/mL car from p20TrcTG into NcoI/SalI digested paCYC-DUET1 benecilin, 34 mg/mL chloramphenicol, 25 mg/L. kanamycin plasmid. p5T7TG was constructed by cloning the BspEI/ or 50 mg/L spectinomycin). For Small scale cultures to Screen Xbal digested fragment to the Xbal/BspEI digested DNA 40 the engineered strains, these preinnoculum were used to seed amplified from pCL1920 plasmid using pCLBspEI(s) and fresh 2-mL rich media (5 g/L yeast extract, 10 g/L Trypton, 15 pCLXbal(a) primers. g/L, glucose, 10 g/L NaCl, 100 mM HEPS, 3 mL/L Antifoam Construction of Chromosomal Integration MEP Pathway B. pH 7.6, 100 ug/mL Carbenicillin and 34 ug/mL chloram Plasmids phenicol), at a starting Asoo of 0.1. The culture was main For constructing the plasmids with FRP-Km-FRP cassette 45 tained with appropriate antibiotics and 100 mM IPTG for for amplifying the sequence for integration, p20T7MEP and gene induction at 22°C. for 5 days. p20T5MEP was digested with XhoI/Scal. FRP-Km-FRP cas Bioreactor Experiments for the Taxadiene 5C.-ol Producing sette was amplified from the Km cassette with FRP sequence Strain. from pkD13 plasmid using the primers KmERPXhoI(s) and The 3-L Bioflo bioreactor (New Brunswick) was KmFRPScal(a). The amplified DNA was digested with XhoI/ 50 assembled as to manufacturer's instructions. One liter of rich Scal and cloned into the XhoI/Scal digested p20T7MEP and media with 1% glycerol (v/v) was inoculated with 50 mL of 8 p20T5MEP plasmid (p20T7MEPKmFRP and h culture (Asoo of -2.2) of the strain 26-At24T5C.OH-tTCPR p20T5MEPKmFRP). Similarly the p20TrcMEP plasmid was grown in LB medium containing the antibiotics (100 mg/mL digested with SacI/Scal and the amplified DNA using the carbenicillin, 34 mg/mL chloramphenicol) at the same con primers KmERPSacI(s) and KmFRPScal(a) was digested, 55 centrations. 1 L-bioreactors with biphasic liquid-liquid fer cloned into the p20TrcMEP plasmid (p20TreMEPKm-FRP). mentation using 20% V/v dodecane. Oxygen was Supplied as Chromosomal Integration of the MEP Pathway Cassette (La filtered air at 0.5 V/v/m and agitation was adjusted to maintain cId-MEP-FRP-Km-FRP) Cassette dissolved oxygen levels above 50%. pH of the culture was The MEP pathways constructed under the promoters T7. controlled at 7.0 using 10% NaOH. The temperature of the T5 and Trc were localized to the ara operon region in the 60 culture in the fermentor was controlled at 30°C. until the cells chromosome with the Kan marker. The PCR fragments were were grown into an optical density of approximately 0.8, as amplified from p20T7MEPKmFRP, p20T5MEPKmFRP and measured at a wavelength of 600 nm (OD600). The tempera p20TrcMEPKm-FRP using the primers IntT7T5(s), IntTrc(s) ture of the fermentor was reduced to 22°C. and the cells were and Int(a) and then electroporated into E. coli MG 1655 recA induced with 0.1 mMIPTG. Dodecane was added aseptically end- and E. coli MG 1655 recA-end-EDE3 cells for chromo 65 to 20% (v/v) of the media volume. During the course of the Somal integration through the Red recombination tech fermentation the concentration of glycerol and acetate accu nique. The site specific localization was confirmed and the mulation was monitored with constant time intervals. During US 8,927,241 B2 35 36 the fermentation as the glycerol concentration depleted below strains. To prevent degradation, RNA was stabilized before 0.5g/L, glycerol (3 g/L) was introduced into the bioreactor. cell lysis using RNAprotect bacterial reagent (Qiagen). Sub The fermentation was further optimized using a fed batch sequently, total RNA was isolated using RNeasy mini kit cultivation with a defined feed medium containing 0.5% yeast (Qiagen) combined with nuclease based removal of genomic extract and 20% (v/v) dodecane (13.3 g/L KHPO, 4 g/L DNA contaminants. cDNA was amplified using iScript (NH4)HPO, 1.7 g/L citric acid, 0.0084 g/L EDTA, 0.0025 cDNA synthesis kit (Biorad). qPCR was carried out on a g/L CoC1, 0.015 g/L MnC1, 0.0015 g/L CuC1, 0.003 g/L Bio-Rad iOycler using the iQ SYBR Green Supermix (Bio HBO, 0.0025 g/L Na MoO, 0.008 g/L Zn(CHCOO), rad). The level of expression of rrsA gene, which is not 0.06g/L Fe(III) citrate, 0.0045 g/L thiamine, 1.3 g/L MgSO, Subject to variable expression, was used for normalization of 10 g/L glycerol, 5 g/L yeast extract, pH 7.0). The same 10 qPCR values. Table 3 has primers used for qPCR. For each medium composition was used for the fermentation of strains primer pair, a standard curve was constructed with mRNA of 17 and 26 with appropriate antibiotics (strain 17: 100 g/mL E. coli as the template. carbenicillin and 50 lug/mL spectinomycin; strain 26: 50 g/mL spectinomycin). Example 1 For the taxadien-5C.-ol producing strain, one liter of com 15 plex medium with 1% glycerol (v/v) was inoculated with 50 Taxadiene Accumulation Exhibits Strong mL of an 8 h culture (OD of -2.2) of strain 26-At24T5C.OH Non-Linear Dependence on the Relative Strengths of tTCPR grown in LB medium containing 50 ug/mL spectino the Upstream MEP and Downstream Synthetic mycin and 34 g/mL chloramphenicol). Oxygen was Sup Taxadiene Pathways plied as filtered air at 0.5 (VVm) and agitation was adjusted to maintain dissolved oxygen levels above 30%. The pH of the FIG. 1b depicts the various ways by which promoters and culture was controlled at 7.0 using 10% NaOH. The tempera gene copy numbers were combined to modulate the relative ture of the culture in the fermentor was controlled at 30° C. flux (or strength) through the upstream and downstream path until the cells were grown into an optical density of approxi ways of taxadiene synthesis. A total of 16 strains were con mately 0.8, as measured at a wavelength of 600 nm (OD600). 25 structed in order to de-bottleneck the MEP pathway, as well as The temperature of the fermentor was reduced to 22°C. and optimally balance it with the downstream taxadiene pathway. the pathway was induced with 0.1 mM IPTG. Dodecane was FIG.2a, b Summarize the results of taxadiene accumulation in added aseptically to 20% (v/v) of the media volume. During each of these strains, with FIG. 2a accentuating the depen the course of the fermentation, the concentration of glycerol dence of taxadiene accumulation on the upstream pathway and acetate accumulation was monitored with constant time 30 for constant values of the downstream pathway, and FIG.2b intervals. During the fermentation as the glycerol concentra the dependence on the downstream pathway for constant tion depleted 0.5-1 g/L, 3 g/L of glycerol was introduced into upstream pathway strength (see also Table 1 for the calcula the bioreactor. tion of the upstream and downstream pathway expression GC-MS Analysis of Taxadiene and Taxadiene-5C.-ol from the reported promoter strengths and plasmid copy num For analysis of taxadiene accumulation from Small scale 35 bers). Clearly, there are maxima exhibited with respect to culture, 1.5 mL of the culture was vortexed with 1 mL hexane both upstream and downstream pathway expression. For con for 30 min. The mixture was centrifuged to separate the stant downstream pathway expression (FIG. 2a), as the organic layer. For bioreactor 1 u, of the dodecane layer was upstream pathway expression increases from very low levels, diluted to 200 uL using hexane. 1 uI of the hexane layer was taxadiene production is increased initially due to increased analyzed by GC-MS (Varian saturn 3800 GC attached to a 40 supply of precursors to the overall pathway. However, after an Varian 2000 MS). The sample was injected into a HP5ms intermediate value, further upstream pathway increases can column (30 mx250 uMx0.25 uM thickness) (Agilent Tech not be accommodated by the capacity of the downstream nologies USA). Helium (ultra purity) at a flow rate 1.0 ml/min pathway. This pathway imbalance leads to the accumulation was used as a carrier gas. The oven temperature was first kept ofan intermediate (see below) that may be either inhibitory to constant at 50° C. for 1 min, and then increased to 220° C. at 45 cells or simply indicate flux diversion to a competing path the increment of 10°C/min, and finally held at this tempera way, ultimately resulting in taxadiene accumulation reduc ture for 10 min. The injector and transfer line temperatures tion. were set at 200° C. and 250° C., respectively. For constant upstream pathway expression (FIG. 2b), a Standard compounds from biological or synthetic sources maximum is similarly observed with respect to the level of for taxadiene and taxadiene 5C-ol was not commercially 50 downstream pathway expression. This is attributed to an ini available. Thus we performed fermentations of taxadiene pro tial limitation of taxadiene production by low expression ducing E. coli in a 2 L bioreactor to extract pure material. levels of the downstream pathway, which is thus rate limiting Taxadiene was extracted by Solvent extraction using hexane, with respect to taxadiene production. At high levels of down followed by multiple rounds of silica column chromatogra stream pathway expression we are likely seeing the negative phy to obtain the pure material for constructing a standard 55 effect of high copy number on cell physiology, hence, a maxi curve for GC-MS analysis. We have compared the GC and mum exists with respect to downstream pathway expression. MS profile of the pure taxadiene with the reported literature to These results demonstrate that dramatic changes in taxadiene confirm the authenticity of the compound". In order to check accumulation can be obtained from changes within a narrow the purity we have performed 'HNMR of taxadiene. Since the window of expression levels for the upstream and down accumulation of taxadiene-5C.-ol was very low level we used 60 stream pathways. For example, a strain containing an addi taxadiene as a measure to quantify the production of this tional copy of the upstream pathway on its chromosome molecule and authentic mass spectral fragmentation charac under Trc promoter control (Strain 8, FIG. 2a) produced 2000 teristics from previous reports'. fold more taxadiene than one overexpressing only the Syn qPCR Measurements for Transcriptional Analysis of Engi thetic downstream pathway (Strain 1, FIG. 2a). Furthermore, neered Strains 65 changing the order of the genes in the downstream synthetic Transcriptional gene expression levels of each gene were operon from GT (GPPS-TS) to TG (TS-GPPS) resulted in detected by qPCR on mRNA isolated from the appropriate 2-3-fold increase (strains 1-4 compared to 5, 8, 11 and 14). US 8,927,241 B2 37 38 The observed results show that the key to taxadiene overpro identity of the metabolite was unknown, we hypothesized that duction is ample downstream pathway capacity and careful it is an isoprenoid side-product, resulting from pathway diver balancing between the upstream precursor pathway with the sion and has been anti-correlated as a direct variable to the downstream synthetic taxadiene pathway. Altogether, the taxadiene production (FIG.3 and FIG.8) from the engineered engineered strains established that the MEP pathway flux can 5 strains. A critical attribute of our optimal strains is the fine be substantial, if a wide range of expression levels for the balancing that alleviates the accumulation of this metabolite, endogenous upstream and synthetic downstream pathway are resulting in higher taxadiene production. This balancing can searched simultaneously. be modulated at different levels from chromosome, or differ ent copy number plasmids, using different promoters, with Example 2 10 significantly different taxadiene accumulation. Subsequently the corresponding peak in the gas chroma Chromosomal Integration and Fine Tuning of the tography-mass spectrometry (GC-MS) chromatogram was Upstream and Downstream Pathways Further identified as indole by GC-MS, "Hand 'C nuclear magnetic Enhances Taxadiene Production resonance (NMR) spectroscopy studies (FIG. 16). We found 15 that taxadiene synthesis by strain 26 is severely inhibited by To provide ample downstream pathway strength while exogenous indole at indole levels higher than ~100 mg/L minimizing the plasmid-borne metabolic burden, two new (FIG. 15b). Further increasing the indole concentration also sets of 4 strains each were engineered (strains 25-28 and inhibited cell growth, with the level of inhibition being very 29-32) in which the downstream pathway was placed under strain dependent (FIG. 15c). Although the biochemical the control of a strong promoter (T7) while keeping a rela mechanism of indole interaction with the isoprenoid pathway tively low number of 5 and 10 copies, respectively. It can be is presently unclear, the results in FIG. 15 suggest a possible seen (FIG. 2C) that, while the taxadiene maximum is main synergistic effect between indole and terpenoid compounds tained at high downstream strength (Strains 21-24), a mono of the isoprenoid pathway in inhibiting cell growth. Without tonic response is obtained at the low downstream pathway knowing the specific mechanism, it appears strain 26 has strength (strains 17-20, FIG.2c). This observation prompted 25 mitigated the indole's effect, which we carried forward for the construction of two additional sets of 4 strains each that further study. maintained the same level of downstream pathway strength as before but expressed very low levels of the upstream pathway Example 4 (strains 25-28 and 29-32, FIG. 2d). Additionally, the operon of the upstream pathway of the latter Strain set was chromo 30 Cultivation of Engineered Strains Somally integrated. It can be seen that not only is the taxadi ene maximum recovered, albeit at very low upstream path In order to explore the taxadiene producing potential under way levels, but a much greater taxadiene maximum is attained controlled conditions for the engineered strains, fed batch (300 mg/L). We believe this significant increase can be attrib cultivations of the three highest taxadiene accumulating uted to a decrease in cell's metabolic burden. This was 35 strains (-60 mg/L from strain 22; ~125 mg/L from strain 17; achieved by 1) eliminating plasmid dependence through inte ~300 mg/L from strain 26) were carried out in 1 L-bioreactors gration of the pathway into the chromosome and 2) attaining (FIG. 17). The fed batch cultivation studies were carried out a fine balance between the upstream and downstream path as liquid-liquid two-phase fermentation using a 20% (v/v) way expression. dodecane overlay. The organic solvent was introduced to The 32 recombinant constructs allowed us to adequately 40 prevent air stripping of secreted taxadiene from the fermen probe the modular pathway expression space and amplify tation medium, as indicated by preliminary findings. In ~15000 fold improvement in taxadiene production. This is by defined media with controlled glycerol feeding, taxadiene far the highest production of terpenoids from E. coli MEP productivity increased to 174+5 mg/L (SD), 210+7 mg/L isoprenoid pathway reported (FIG. 3a). Additionally, the (SD), and 1020-80 mg/L (SD), respectively for strains 22, 17 observed fold improvements interpenoid production are sig 45 and 26 (FIG. 17a). Additionally, taxadiene production sig nificantly higher than those of reported combinatorial meta nificantly affected the growth phenotype, acetate accumula bolic engineering approaches that searched an extensive tion and glycerol consumption (FIG. 17b-17d). genetic space comprising up to a billion combinatorial vari FIG. 17C shows that acetate accumulates in all strains ini ants of the isoprenoid pathway." This suggests that pathway tially, however after ~60 hrs acetate decreases in strains 17 optimization depends far more on fine balancing of the 50 and 26 while it continues to increase in strain 22. This phe expression of pathway modules than multi-source combina nomenon highlights the differences in central carbon metabo torial gene optimization. The multiple maxima exhibited in lism between high MEP flux strains (26 and 17) and low MEP the phenotypic landscape of FIG. 1 underscores the impor flux strain (22). Additionally, this observation is another illus tance of probing the expression space at Sufficient resolution tration of the good physiology that characterizes a well-bal to identify the region of optimum overall pathway perfor 55 anced, -functioning strain. Acetic acid, as product of overflow mance. FIG. 7 depicts the fold improvements in taxadiene metabolism, is initially produced by all strains due to the high production from the modular pathway expression search. initial glycerol concentrations used in these fermentations and corresponding high glycerol pathway flux. This flux is Example 3 sufficient for supplying also the MEP pathway, as well as the 60 other metabolic pathways in the cell. Metabolite Inversely Correlates with Taxadiene At ~48 hrs, the initial glycerol is depleted, and the cultiva Production and Identification of Metabolite tion switches to a fed-batch mode, during which low but constant glycerol levels are maintained. This results in a low Metabolomic analysis of the previous engineered Strains overall glycerol flux, which, for strains with high MEP flux identified an, as yet, unknown, metabolite byproduct that 65 (strains 26 and 17), is mostly diverted to the MEP pathway correlated Strongly with pathway expression levels and taxa while minimizing overflow metabolism. As a result acetic diene production (FIG.3 and FIG. 8). Although the chemical acid production is reduced or even totally eliminated. Regard US 8,927,241 B2 39 40 ing the decline in acetic acid concentration, it is possible that due to significant competition for resources (raw material and acetic acid assimilation may have happened to Some extent, energy) that are withdrawn from the host metabolism for although this was not further investigated from a flux analysis overexpression of both the four upstream and two down standpoint. Some evaporation and dilution due to glycerol stream genes. Compared to the control strain 25c, a 4 fold feed are further contributing to the observed acetic acid con growth inhibition was observed with strain 25 indicated that centration decline. In contrast, for strains with low MEP flux high overexpression of synthetic taxadiene pathway induced (strain 22), flux diversion to the MEP pathway is not very toxicity altering the growth phenotype compared to the over significant, so that glycerol flux still Supplies all the necessary expression of native pathway (FIG. 4c). However, as carbon and energy requirements. Overflow metabolism con upstream expression increased, downstream expression was tinues to occur leading to acetate secretion. 10 reduced, inadvertently in our case, to desirable levels to bal Clearly the high productivity and more robust growth of ance the upstream and downstream pathways, minimizing strain 26 allowed very high taxadiene accumulation. Further growth inhibition (strain 26). improvements should be possible through optimizing condi At the extreme of protein overexpression, T7 promoter tions in the bioreactor, balancing nutrients in the growth driven MEP pathway resulted in severe growth inhibition, due medium, and optimizing carbon delivery. 15 to the synthesis of four proteins at high level (strains 28 and 32). Expression of the TS genes by T7 does not appear to have Example 5 as drastic effect by itself. The high rates of protein synthesis from the T7 induced expression (FIG. 4ab) could lead to the Upstream and Downstream Pathway Expression down regulation of the protein synthesis machinery including Levels and Cell Growth Reveal Underlying components of housekeeping genes from early growth phase Complexity impairs the cell growth and lower the increase in bio mass.' We hypothesized that our observed complex For a more detailed understanding of the engineered bal growth phenotypes are cumulative effects of (1) toxicity ance in pathway expression, we quantified the transcriptional induced by activation of isoprenoid/taxadiene metabolism, gene expression levels of dxS (upstream pathway) and TS 25 and (2) and the effects of high recombinant protein expres (downstream pathway) for the highest taxadiene producing Sion. Altogether our multivariate-modular pathway engineer strains and neighboring strains from FIG.2c and d(strains 17. ing approach generated unexpected diversity in terpenoid 22 and 25-32) (FIG. 4a, b). As we hypothesized, expression of metabolism and its correlation to the pathway expression and the upstream pathway increased monotonically with pro cell physiology. Rational design of microbes for secondary moter strength and copy number for the MEP vector from: 30 metabolite production will require an understanding of path native promoter, Trc, T5, T7, and 10 copy and 20 copy plas way expression that goes beyond a linear/independent under mids, as seen in the DXS expression (FIG. 4a). Thus we found standing of promoter strengths and copy numbers. However, that dxs expression level correlates well with the upstream simple, multivariate approaches, as employed here, can intro pathway strength. Similar correlations were found for the duce the necessary diversity to both (1) find high producers, other genes of the upstream pathway, idi, isp) and ispF (FIG. 35 and (2) provide a landscape for the systematic investigation of 14a, b). In the downstream gene expression, a fold improve higher order effects that are dominant, yet underappreciated, ment was quantified after transferring the pathway from 5 to in metabolic pathway engineering. 10 copy plasmid (25-28 series and 29-32 series) (FIG. 4b). While promoter and copy number effects influenced the Example 6 gene expressions, secondary effects on the expression of the 40 other pathway were also prominent. FIG. 4a shows that for Engineering Taxol P450-based Oxidation Chemistry the same dxS expression cassettes, by increasing the copy in E. coli number of the TS plasmid from 5 to 10, dxs expression was increased. Interestingly, the 5 copy TS plasmid (strains 25-28 A central feature in the biosynthesis of Taxol is oxygen series) contained substantially higher taxadiene yields (FIG. 45 ation at multiple positions of the taxane core structure, reac 2d) and less growth (FIG. 4c,d) than the 10 copy TS plasmid. tions that are considered to be mediated by cytochrome P450 Control plasmids that did not contain the taxadiene heterolo dependent monooxygenases.' After completion of the gous pathway, grew two fold higher densities, implying committed cyclization step of the pathway, the parent olefin, growth inhibition in the strains 25-28 series is directly related taxa-4(5), 11(12)-diene, is next hydroxylated at the C5 posi to the taxadiene metabolic pathway and the accumulation of 50 tion by a cytochrome P450 enzyme, representing the first of taxadiene and its direct intermediates (FIG. 4c). However the eight oxygenation steps (of the taxane core) on route to Taxol strain 29-32 series only showed modest increases in growth (FIG. 6). Thus, a key step towards engineering Taxol-pro yield when comparing the empty control plasmids to the ducing microbes is the development of P450-based oxidation taxadiene expressing strains (FIG. 4d). This interplay chemistry in vivo. The first oxygenation step is catalyzed by between growth, taxadiene production, and expression level 55 a cytochrome P450, taxadiene 5C.-hydroxylase, an unusual can also be seen with the plasmid-based upstream expression monooxygenase catalyzing the hydroxylation reaction along vectors (strain 17 and 22). Growth inhibition was much larger with double bond migration in the diterpene precursor taxa in the 10 copy, high taxadiene producing strain (strain 17) diene (FIG.5a). We report the first successful extension of the compared to the 20 copy, lower taxadiene producing strain synthetic pathway from taxadiene to taxadien-5C-ol and (strain 22) (FIG. 4d). Therefore product toxicity and carbon 60 present the first examples of in vivo production of any func diversion to the heterologous pathway are likely to impede tionalized Taxol intermediates in E. coli. growth, rather than plasmid-maintenance. In general, functional expression of plant cytochrome P450 Also unexpected was the profound effect of the upstream is challenging" due to the inherent limitations of bacterial expression vector on downstream expression. FIG. 4b would platforms, such as the absence of electron transfer machinery, have two straight lines, if there was no cross talk between the 65 cytochrome P450 reductases, and translational incompatibil pathways. However, ~3 fold changes in TS expression are ity of the membrane signal modules of P450 enzymes due to observed for different MEP expression vectors. This is likely the lack of an endoplasmic reticulum. Recently, through US 8,927,241 B2 41 42 transmembrane (TM) engineering and the generation of chi approach seems to be more effective than combinatorial mera enzymes of P450 and CPR reductases, some plant searches of large genetic spaces and also does not depend on P450s have been expressed in E. coli for the biosynthesis of a high throughput screen. functional molecules.’ “Still, every plant cytochrome p450 The MEP-pathway is energetically balanced and thus over is unique in its transmembrane signal sequence and electron 5 all more efficient in converting either glucose or glycerol to transfer characteristics from its reductase counterpart." Our isoprenoids. Yet, during the past 10 years, many attempts at initial studies were focused on optimizing the expression of engineering the MEP-pathway in E. coli to increase the Sup codon-optimized synthetic taxadiene 5C-hydroxylase by ply of the key precursors IPP and DMAPP for carotenoid', N-terminal transmembrane engineering and generating chi sesquiterpenoid’ and diterpenoid' overproduction met with 10 limited success. This inefficiency was attributed to unknown mera enzymes through translational fusion with the CPR regulatory effects associated specifically with the expression redox partner from the Taxus species, Taxus cytochrome of the MEP-pathway in E. Coli. Here we provide evidence P450 reductase (TCPR) (FIG.5b).''' One of the chimera that such limitations are correlated with the accumulation of enzymes generated. At24T5C.OH-tTCPR, was highly effi the metabolite indole, owing to the non-optimal expression of cient in carrying out the first oxidation step with more than 15 the pathway, which inhibits the isoprenoid pathway activity. 98% taxadiene conversion to taxadien-5C.-ol and the byprod Taxadiene overproduction (under conditions of indole forma uct 5(12)-Oxa-3(11)-cyclotaxane (OCT) (FIG. 9a). tion suppression), establishes the MEP-pathway as a very Compared to the other chimeric P450s, At24T5C.OH efficient route for biosynthesis of pharmaceutical and chemi tTCPR yielded two-fold higher (21 mg/L) production of taxa cal products of the isoprenoid family. One simply needs to dien-5C-ol. As well, the weaker activity of At8T5C.OH carefully balance the modular pathways as Suggested by our tTCPR and At24T5OOH-tTCPR resulted in accumulation of multivariate-modular pathway engineering approach. a recently characterized byproduct, a complex structural rear For successful microbial production of Taxol, demonstra rangement of taxadiene into the cyclic ether 5(12)-Oxa-3 tion of the chemical decoration of the taxadiene core by P450 (11)-cyclotaxane (OCT) (FIG.9). The byproduct accumu based oxidation chemistry is essential." Cytochrome P450 lated at approximately equal amounts as the desired product 25 monooxygenases constitute about one half of the 19 distinct taxadien-5C.-ol. The OCT formation was mediated by an enzymatic steps in the Taxol biosynthetic pathway. Charac unprecedented Taxus cytochrome P450 reaction sequence teristically, these genes show unusual high sequence similar involving oxidation and Subsequent cyclizations.' Thus, it ity with each other (>70%) but low similarity (<30%) with seems likely that by protein engineering of the taxadiene other plant P450s.' Due to the apparent similarity among 5C-hydroxylases, termination of the reaction before cycliza 30 Taxol monooxygenases, expressing the proper activity for tion will prevent the accumulation of such undesirable carrying out the specific P450 oxidation chemistry was a byproduct and channeling the flux to taxadien-5C.-ol could be particular challenge. Through TM engineering and construc achieved. tion of an artificial chimera enzyme with redox partner The productivity of strain 26-At24T5COH-tTCPR was (TCPR), the Taxol cytochrome P450, taxadiene 5C.-hydroxy significantly reduced relatively to that of taxadiene produc 35 lase, was functionally expressed in E. coli and shown to tion by the parent strain 26 (-300 mg/L) with a concomitant efficiently convert taxadiene to the corresponding alcohol increase in the accumulation of the previously described product in vivo. Previous in vitro studies have described the uncharacterized metabolite. No taxadiene accumulation was mechanism of converting taxadiene to taxadien-5C.-ol by observed. Apparently, the introduction of an additional native taxadiene 5C.-hydroxylase enzyme, but have not dis medium copy plasmid (10 copy, p10T7) bearing the 40 cussed the same conversion in vivo. This oxygenation and At24T5C.OH-tTCPR construct disturbed the carefully engi rearrangement reaction involves hydrogen abstraction from neered balance in the upstream and downstream pathway of C20 position of the taxadiene to form an allylic radical inter strain 26. Small scale fermentations were carried out in biore mediate, followed by regio- and stereo-specific oxygen inser actors to quantify the alcohol production by Strain tion at C5 position to yield the alcohol derivative (FIG. 5a). 26-At24T5C.OH-tTCPR. The time course profile of taxadien 45 The observed modestabundance of the enzyme in Taxus cells, 5C-ol accumulation (FIG. 5d) indicates alcohol production of and the low k values suggested that the 5C.-hydroxylation up to 58+3 mg/L with an equal amount of the OCT byproduct step of Taxol biosynthesis is slow relative to the downstream produced. The observed alcohol production was 2400 fold oxygenations and acylations in the Taxol pathway.' Thus, higher than previous production in S. cerevisiae.'” Further engineering this step is key to Taxol Synthesis, especially in increases of taxadien-5C-ol production are likely possible 50 the context of functional engineering of Taxol P450s in through pathway optimization and protein engineering. prokaryotic host Such as E. coli. In addition, this step was The multivariate-modular approach of pathway optimiza limiting in previous efforts of constructing the pathway in tion has yielded very high producing strains of a critical Taxol yeast." The engineered construct in this study demonstrated precursor. Furthermore, the recombinant constructs have >98% conversion of taxadiene in vivo with product accumu been equally effective in redirecting flux towards the synthe 55 lation to ~60 mg/L, a 2400 fold improvement over previous sis of other complex pharmaceutical compounds, such as heterologous expression in yeast. This study has therefore mono-, sesqui- and di-terpene (geraniol, linalool, amorpha Succeeded not only in synthesizing significantly greater diene and levopimaradiene) products engineered from the amounts of key Taxol intermediates but also provides the same pathway (unpublished results). Thus, our pathway engi basis for the synthesis of subsequent metabolites in the path neering opens new avenues to bio-synthesize natural prod 60 way by similar P450 chemistry. ucts, especially in the context of microbially-derived terpe Prior studies on structure-activity relationship on Taxol noids for use as chemicals and fuels from renewable have shown that alterations made either by removal or addi resources. By focusing on the universal terpenoid precursors tion of some of its functional groups did not change materially IPP and DMAPP, it was possible to, first, define the critical the activity of the Taxol.'" Such studies, however, were pathway modules and then modulate expression Such as to 65 limited due to the restricted ability to introduce changes by optimally balance the pathway modules for seamless precur chemical synthesis. Availability of a microbial path for Taxol Sor conversion and minimal intermediate accumulation. This synthesis will drastically expand the space of chemical modi US 8,927,241 B2 43 44 fications that can be examined, thus increasing the probability TABLE 2 of identifying more potent drug candidates. This offers excit ing new opportunities for drug development, especially when Detail of all the plasmids constructed for the study considering that such drug candidates will also be associated with an efficient production route. 5 Origin of Antibiotic In the past few decades, Taxol has spawned more interest No Plasmid replication marker within the scientific communities and general public than any 1 b2OT7MEP BR322 Amp other natural product drug candidate. A major Supply crisis 2 b2OTrcMEP BR322 Amp is predicted from the projected increase in the use of Taxolor 3 p2OTSMEP BR322 Amp Taxol analogs for cancer chemotherapy, requiring new pro- 10 4 b2OT7MEPKFRP BR322 Km duction routes. Such as engineering of Taxol biosynthetic S b2OTSMEPKnFRP BR322 Km machinery in microbes. While a few endophytic fungi of 6 b2OTrcMEPKn-FRP BR322 Km Taxus species have been isolated capable of producing Taxol 7 1 OTrcMEP 15A Cm naturally, these microbial systems have yet to demonstrate ME . e suitability for sustainable production of the drug. The 15 10 b2OTrcTG2U C BR322 Ampp results reported here represent a disruptive step towards a 11 b2OTSGT BR322 Amp microbially-derived Taxol or Taxol precursor, by removing 12 1 OT7TG 15A Cm the bottlenecks in the committed precursor pathway. Further- 13 p5T7TG SC101 Spect more, the assembly of a synthetic pathway offers new possi- 14 10At8TSCOH-tTCPR 15A Cm bilities to tailor Taxol analogs by selectively engineering the 20 1S 1 OAlt24TSCOH-tTCPR 15A Cm pathway, thereby altering the taxane structure. These devel- 16 10A42TSCOH-tTCPR 15A Cm opments raise optimism for a microbial route for the cost effective production of Taxol or suitable Taxol precursors. TABLE 1.

Upstrean MEP Downstream GT or TG Construct Expres- Expres (in addition sion Sion Taxadiene Strain to native Strength Copies Strength Ing/L i Pathway engineering copy) Copies Promoter (a.u.) Construct GT/TG Promoter (a.u)" Mean SD 1 E2OTrcGT NA O O 1* BR322 2O 1.00 2O O.O2 O.O1 2 ECh1TrcMEPp2OTrcGT Chri 1 2 BR322 2O 1.00 2O 16.OO 1.59 3 Ep5TrcMEPp2OTrcGT pSC101 5 6 BR322 2O 1.00 2O 2.SS 0.21 4 Ep10TrcMEPp2OTrcGT p15A 10 11 BR322 2O 1.00 2O 1.93 0.323 S E2OTrcTG NA O O 1 BR322 2O 1.20 24 O.19 O.O1 6 Eb2OTSGT NA O O 1 BR322 2O 1.96 39 4.36 O.S33 7 Ep2OTSGTTrcT NA O O 1 BR322 2O 2.96 59 1.74 0.26S 8 ECh1TrcMEPp2OTrcTG Chr. 1 2 BR322 2O 1.20 24 45.44 2.28 9 ECh1TrcMEPp2OTSGT Chr. 1 2 BR322 2O 1.96 39 16.52 0.84 10 ECh1TrcMEPp20T5GT-TrcT Chr. 1 2 BR322 2O 2.96 59th 2.52 O.30 11 Ep5TrcMEPp2OTrcTG pSC101 5 6 BR322 2O 1.20 24 7.41 0.63 12 Ep5TrcMEPp20T5GT pSC101 5 6 BR322 2O 1.96 39 21.23 S.86 13 Ep5TrcMEPp2OTSTG-TrcT pSC101 5 6 BR322 2O 2.96 59 140 (0.10 14 Ep10TrcMEPp2OTrcTG p15A 10 1 BR322 2O 1.20 24 2.36 0.29 15 Ep10TrcMEPp20T5GT p15A 10 1 BR322 2O 1.96 39 8.91 2.94 16 Ep10TrcMEPp20T5GT-TrcT p15A 10 1 BR322 2O 2.96 59 3.40 0.39 17 EDE3p10TrcMEPp5T7TG p15A 10 1 SC101 5 S.96 3 12SOO 8.37 18 EDE3p2OTrcMEPp5T7TG pBR322 2O 2 SC101 5 S.96 3 S8.00 3.07 19 EDE3p2OTSMEPp5T7TG pBR322 2O .96 40 SC101 5 S.96 3 44.OO 2.88. 20 EDE3p20T7MEPp5T7TG pBR322 2O 4.97 1OO SC101 5 S.96 3 32.OO 6.63 21 EDE3p5TrcMEPp10T7TG pSC101 5 6 15A 10 S.96 6 7.OO 140 22 EDE3p2OTrcMEPp10T7TG pBR322 2O 2 15A 10 S.96 6 59.00 5.57 23 EDE3p2OTSMEPp10T7TG pBR322 2O .96 40 15A 10 S.96 6 S8.00 S.68 24 EDE3p20T7MEPp10T7TG pBR322 2O 4.97 1OO 15A 10 S.96 6 2O.OO O.73 25 EDE3p5T7TG NA O O SC101 5 S.96 3 19.00 8.23 26 EDE3Ch1TrcMEPp5T7TG Chr. 1 2 SC101 5 S.96 3 297.00 10.21 27 EDE3Ch1T5MEPp5T7TG Chr 1 .96 3 SC101 5 S.96 3 163.OO 10.84 28 EDE3Ch1T7MEPp5T7TG Chr 1 4.97 6 SC101 5 S.96 3 26.00 0.32 29 EDE3p10T7TG NA O O 15A 10 S.96 6 8.OO O.39 30 EDE3Ch1TrcMEPp10T7TG Chr 1 2 15A 10 S.96 6 3O.OO 1.59 31 EDE3Ch1TSMEPp10T7TG Chr 1 .96 3 15A 10 S.96 6 40.OO O.56 32 EDE3Ch1T7MEPp10T7TG Chr 1 4.97 6 15A 10 S.96 6 17.00 0.41 Table 1. Ranking of upstream and downstream pathway expression in arbitrary units (a.u.), The M EP pathway and GGPP synthaseftaxadiene synthase pathway expression levels were estimated using published values of promoter strengths and copy number, Promoter strengths were calculated as trc = 1, T5 = 1.96, T7 = 4.97, based on Brosius etal, and Brunner et, al.33, 34 Gene copy number was assigned by published copy numbers for origin of replication for the different plasmids used, and one copy was used for integrations, Total expression was calculated as the product of promoter strength and gene copy number, Native expression pf the M EP pathway was arbitrarily assigned a value of one, and changing the operon order of GGPP synthase and taxadiene was assumed to affect taxadiene synthase expression by 20%. These estimates of total expression guided engineering efforts. E—Ecoli K12 MG1655 with two deletions ArecAAendA;EDE3–K12 MG1655ArecAAendA with a T7RNA polymerase (DE3) integrated;M EP—dxs-idi-isp F operon; GT GPPS-TS operon; TG-TS-GPPS operon; Ch1–1 copy in chromosome; Trc-tre promoter; T5 T5 promoter; T7 T7 promoter; p5 -5 copy plasmid (pSC101);, p10–~10 copy plasmid (p15A); and p20–-20 copy plasmid (pBR322). *A value of 1 was given to account for the native copies of the MEP pathway, MEP construct is localized in the chromosome. "p20T5GT-TrcT - An additional copy of gene Tunder separate promoter control (Trc) together operon GT (under T5 promoter) on the same plasmid. For the calculation strength, we have added the value as equivalent two separate operons (TrcT + T5GT = (20 x 1.96 +20 x 1 = 59)) since our studies shows that expression of T was limited compared to G.

US 8,927,241 B2 51 52 5. Holton, R. A. et al. First total synthesis of taxol. 2. Comple 25. Kim, S.W. & Keasling, J. D. Metabolic engineering of the tion of the C and D rings. Journal of the American Chemi nonmevalonate isopentenyl diphosphate synthesis path cal Society 116, 1599-1600 (1994). way in Escherichia coli enhances lycopene production. 6. Walji, A. M. & MacMillan, D. W. C. Strategies to Bypass Biotechnol Bioeng 72, 408-15 (2001). the Taxol Problem. Enantioselective Cascade Catalysis, a 5 26. Farmer, W. R. & Liao, J. C. Improving lycopene produc New Approach for the Efficient Construction of Molecular tion in Escherichia coli by engineering metabolic control. Complexity. Synlett 18, 1477-1489 (2007). Nature Biotechnology 18, 533-537 (2000). 7. Holton RAB. R., Boatman PD. Semisynthesis of taxoland 27. Farmer, W. R. & Liao, J. C. Precursor balancing for taxotere (ed. M. S.) (CRC Press, Boca Raton, 1995). metabolic engineering of lycopene production in Escheri 8. Frense, D. Taxanes: perspectives for biotechnological pro 10 chia coli. Biotechnology progress 17 (2001). duction. Applied microbiology and biotechnology 73, 28. Yuan, L. Z. Rouviere, P. E., Larossa, R. A. & Suh, W. 1233-1240 (2007). Chromosomal promoter replacement of the isoprenoid 9. Roberts, S.C. Production and engineering of terpenoids in pathway for enhancing carotenoid production in E. coli. plant cell culture. Nature Chemical Biology 3, 387-395 15 Metab Eng 8, 79-90 (2006). (2007). 29. Jin, Y. S. & Stephanopoulos, G. Multi-dimensional gene 10. Goodman, J. & Walsh, V. The story of taxol: nature and target search for improving lycopene biosynthesis in politics in the pursuit of an anti-cancer drug (Cambridge Escherichia coli. Metabolic Engineering 9, 337-347 University Press, Cambridge; New York, 2001). (2007). 11. Tyo, K.E., Alper, H. S. & Stephanopoulos, G. N. Expand 30. Wang, H. H. et al. Programming cells by multiplex ing the metabolic engineering toolbox: more options to genome engineering and accelerated evolution. Nature engineer cells. Trends Biotechnol 25, 132-7 (2007). (2009). 12. Ajikumar, P. K. et al. Terpenoids: opportunities for bio 31. Klein-Marcuschamer, D., Ajikumar, P. K. & Stephanopo synthesis of natural product drugs using engineered micro ulos, G. Engineering microbial cell factories for biosyn organisms. Mol Pharm 5, 167-90 (2008). 25 thesis of isoprenoid molecules: beyond lycopene. Trends 13. Jennewein, S. & Croteau, R. Taxol: biosynthesis, molecu in Biotechnology 25, 417-424 (2007). lar genetics, and biotechnological applications. Appl 32. Sandmann, G. Combinatorial biosynthesis of carotenoids Microbiol Biotechnol 57, 13-9 (2001). in a heterologous host: a powerful approach for the biosyn 14. Jennewein, S., Wildung, M. R. Chau, M., Walker, K. & thesis of novel structures. ChemBioChem3 (2002). Croteau, R. Random sequencing of an induced Taxus cell 30 cDNA library for identification of clones involved in Taxol 33. Brosius, J., Erfle, M. & Storella, J. Spacing of the -10 and biosynthesis. Proc Natl Acad Sci USA 101, 9149-54 -35 regions in the tac promoter. Effect on its in vivo activity. (2004). J Biol Chem 260, 3539-41 (1985). 15. Croteau, R. Ketchum, R. E. B., Long, R. M., Kaspera, R. 34. Brunner, M. & Bujard, H. Promoter recognition and pro & Wildung, M. R. Taxol biosynthesis and molecular genet 35 moter strength in the Escherichia coli system. Embo J 6, ics. Phytochemistry Reviews 5, 75-97 (2006). 3139-44 (1987). 16. Walker, K. & Croteau, R. Taxol biosynthetic genes. Phy 35. Nishizaki, T., Tsuge, K., Itaya, M., Doi, N. & Yanagawa, tochemistry 58, 1-7 (2001). H. Metabolic engineering of carotenoid biosynthesis in 17. Dejong, J. M. et al. Genetic engineering of taxol biosyn Escherichia coli by ordered gene assembly in Bacillus thetic genes in Saccharomyces cerevisiae. Biotechnol 40 subtilis. Appl Environ Microbiol 73, 1355-61 (2007). Bioeng 93,212-24 (2006). 36. Sorensen, H. P. & Mortensen, K. K. Advanced genetic 18. Engels, B., Dahm, P. & Jennewein, S. Metabolic engi strategies for recombinant protein expression in Escheri neering of taxadiene biosynthesis in yeast as a first step chia coli. J. Biotechnol 115, 113-28 (2005). towards Taxol (Paclitaxel) production. Metabolic engi 37. Jones, K. L., Kim, S. W. & Keasling, J. D. Low-copy neering 10, 201-206 (2008). 45 plasmids can perform as well as or better than high-copy 19. Chang, M. C. Y. & Keasling, J. D. Production of iso plasmids for metabolic engineering of bacteria. Metab Eng prenoid pharmaceuticals by engineered microbes. Nature 2,328-38 (2000). chemical biology 2, 674-681 (2006). 38. Hoffmann, F. & Rinas, U. Stress induced by recombinant 20. Khosla, C. & Keasling, J. D. Metabolic engineering for protein production in Escherichia coli. Advances in Bio drug discovery and development. Nat Rev Drug Discov 2. 50 chemical Engineering Biotechnology 89, 73-92 (2005). 1019-25 (2003). 39. Hoffmann, F. Weber, J. & Rinas, U. Metabolic adaptation 21. Alper, H., Miyaoku, K. & Stephanopoulos, G. Construc of Escherichia coli during temperature-induced recombi tion of lycopene-overproducing E. coli strains by combin nant protein production: 1. Readjustment of metabolic ing systematic and combinatorial gene knockout targets. enzyme synthesis. Biotechnology and bioengineering 80 Nat Biotechnol 23, 612-6 (2005). 55 (2002). 22. Chang, M. C. Eachus, R. A., Trieu, W., Ro, D. K. & 40. Chang, D. E. Smalley, D. J. & Conway, T. Gene expres Keasling, J. D. Engineering Escherichia coli for produc sion profiling of Escherichia coli growth transitions: an tion of functionalized terpenoids using plant P450s. Nat expanded stringent response model. Molecular microbiol Chem Biol 3, 274-7 (2007). ogy 45, 289-306 (2002). 23. Martin, V.J., Pitera, D.J., Withers, S.T., Newman, J. D. & 60 41. Kaspera, R. & Croteau, R. Cytochrome P450 oxygenases Keasling, J. D. Engineering a mevalonate pathway in of Taxol biosynthesis. Phytochemistry Reviews 5,433-444 Escherichia coli for production ofterpenoids. Nat Biotech (2006). nol 21, 796-802 (2003). 42. Jennewein, S., Long, R. M., Williams, R. M. & Croteau, 24. Huang, Q., Roessner, C. A., Croteau, R. & Scott, A. I. R. Cytochrome p450 taxadiene 5alpha-hydroxylase, a Engineering Escherichia coli for the synthesis of taxadi 65 mechanistically unusual monooxygenase catalyzing the ene, a key intermediate in the biosynthesis of taxol. Bioorg first oxygenation step of taxol biosynthesis. Chem Biol 11, Med Chem 9, 2237-42 (2001). 379-87 (2004). US 8,927,241 B2 53 54 43. Schuler, M.A. & Werck-Reichhart, D. FUNCTIONALG 54. Datsenko, K. A. & Wanner, B. L. 6640-6645 (National ENOMICS OF P450 S. Annual Review of Plant Biology Acad Sciences, 2000). 54, 629-667 (2003). 55. Rost, B., Yachdav, G. & Liu, J. The predict protein server. 44. Leonard, E. & Koffas, M. A. G. Engineering of artificial Nucleic acids research 32. W321 (2004). plant cytochrome P450 enzymes for synthesis of isofla 5 56. Shalel-Levanon, S., San, K.Y. & Bennett, G. N. Effect of ArcA and FNR on the expression of genes related to the vones by Escherichia coli. Applied and Environmental oxygen regulation and the glycolysis pathway in Escheri Microbiology 73, 7246 (2007). chia coli under microaerobic growth conditions. Biotech 45. Nelson, D. R. Cytochrome P450 and the individuality of nology and bioengineering 92 (2005). species. Archives of Biochemistry and Biophysics 369, 57. Heinig, U. & Jennewein, S. Taxol: A complex diterpenoid 1-10 (1999). 10 natural product with an evolutionarily obscure origin. Afri 46. Jennewein, S. et al. Coexpression in yeast of Taxus cyto can Journal of Biotechnology 8, 1370-1385 (2009). chrome P450 reductase with cytochrome P450 oxygenases 58. Walji, A. M. & MacMillan, D. W. C. Strategies to Bypass involved in Taxol biosynthesis. Biotechnol Bioeng 89, the Taxol Problem. Enantioselective Cascade Catalysis, a New Approach for the Efficient Construction of Molecular 588-98 (2005). 15 Complexity. Synlett 18, 1477-1489 (2007). 47. Rontein, D. et al. CYP725A4 from yew catalyzes com 59. Chau, M., Jennewein, S., Walker, K. & Croteau, R. Taxol plex structural rearrangement of taxa-4(5), 11(12)-diene biosynthesis: Molecular cloning and characterization of a into the cyclic ether 5(12)-oxa-3 (11)-cyclotaxane. J Biol cytochrome P450 taxoid 7 beta-hydroxylase. Chem Biol Chem 283,6067-75 (2008). 11, 663-72 (2004). 48. Shigemori, H. & Kobayashi, J. Biological activity and 60. Williams, D.C. et al. Heterologous expression and char chemistry of taxoids from the Japanese yew, Taxus cuspi acterization of a “Pseudomature' form of taxadiene syn data. J Nat Prod 67,245-56 (2004). thase involved in paclitaxel (Taxol) biosynthesis and evalu 49. Xu, F., Tao, W., Cheng, L. & Guo, L. Strain improvement ation of a potential intermediate and inhibitors of the and optimization of the media of taxol-producing fungus multistep diterpene cyclization reaction. Arch Biochem Fusarium maire. Biochemical Engineering Journal 31, 25 Biophys379, 137-46 (2000). 67-73 (2006). 61. Morrone D. et al. Increasing diterpene yield with a modu 50. Hefner, J., Ketchum, R. E. & Croteau, R. Cloning and lar metabolic engineering system in E. coli: comparison of functional expression of a cDNA encoding geranylgeranyl MEV and MEP isoprenoid precursorpathway engineering. diphosphate synthase from Taxus canadensis and assess Appl Microbiol Biotechnol. 85(6):1893-906 (2010) ment of the role of this prenyltransferase in cells induced 30 Having thus described several aspects of at least one for taxol production. Arch Biochem Biophys 360, 62-74 embodiment of this invention, it is to be appreciated various (1998). alterations, modifications, and improvements will readily 51. Wildung, M. R. & Croteau, R. A cDNA clone for taxadi occur to those skilled in the art. Such alterations, modifica ene synthase, the diterpene cyclase that catalyzes the com tions, and improvements are intended to be part of this dis mitted step of taxol biosynthesis. J Biol Chem 271, 9201-4 35 closure, and are intended to be within the spirit and scope of (1996). the invention. Accordingly, the foregoing description and 52. Kodumal, S. J. et al. Total synthesis of long DNA drawings are by way of example only. Those skilled in the art sequences: synthesis of a contiguous 32-kb polyketide syn will recognize, or be able to ascertain using no more than thase gene cluster. Proceedings of the National Academy of routine experimentation, many equivalents to the specific Sciences 101, 15573-15578 (2004). 40 embodiments of the invention described herein. Such equiva 53. Tyo, K. E. J., Ajikumar, P. K. & Stephanopoulos, G. lents are intended to be encompassed by the following claims. Stabilized gene duplication enables long-term selection All references disclosed herein are incorporated by refer free heterologous pathway expression. Nature Biotechnol ence in their entirety for the specific purpose mentioned ogy (2009). herein.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 52

SEO ID NO 1 LENGTH: 33 TYPE: DNA ORGANISM: Artificial Sequence FEATURE; OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs SEQUENCE: 1

cgg catatga gttittgatat tdccaaatac cc.g 33

SEO ID NO 2 LENGTH: 33 TYPE: DNA ORGANISM: Artificial Sequence FEATURE; OTHER INFORMATION: Synthetic Oligonucleotide US 8,927,241 B2 55 56 - Continued

<4 OOs, SEQUENCE: 2 cggctagott atgc.ca.gc.ca ggccttgatt ttg 33

<210s, SEQ ID NO 3 &211s LENGTH: 52 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 3 cgcggctago gaaggagata tacatatgca aacggaacac git catttitat td 52

<210s, SEQ ID NO 4 &211s LENGTH: 32 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 4 cggaatt.cgc ticacalacc cc ggcaaatgtc. g.g 32

<210s, SEQ ID NO 5 &211s LENGTH: 49 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 5 gcgaatticga aggagatata catatggcaa C cact cattt ggatgtttg 49

<210s, SEQ ID NO 6 &211s LENGTH: 37 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 6 gcgcticgagt cattttgttg cct taatgag tagcgc.c 37

<210s, SEQ ID NO 7 &211s LENGTH: 33 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OO > SEQUENCE: 7 taaaccatgg gttittgat at togccaaatac cc.g 33

<210s, SEQ ID NO 8 &211s LENGTH: 36 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 8 cgggg tacct cattttgttg cct taatgag tagcgc 36 US 8,927,241 B2 57 58 - Continued

<210s, SEQ ID NO 9 &211s LENGTH: 36 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 9 cggctcgagt cattttgttg cct taatgag tagcgc 36

<210s, SEQ ID NO 10 &211s LENGTH: 37 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 10 cgta accqigt gcct ctogcta accatgttca tdocttc 37

<210s, SEQ ID NO 11 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 11 citcc titcgct agctitatgcc agcc 24

<210 SEQ ID NO 12 &211s LENGTH: 39 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 12 cgtagaattic act cacaact gacgaaacgc aatgtaatc 39

<210s, SEQ ID NO 13 &211s LENGTH: 47 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 13 cgtagaattic agaaggagat atacatatgg ctagotctac ggg tacg 47

<210s, SEQ ID NO 14 &211s LENGTH: 37 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 14 gatggit cac ttagacctgg attggat.cga tigtaaac 37

<210s, SEQ ID NO 15 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide US 8,927,241 B2 59 60 - Continued

<4 OOs, SEQUENCE: 15 cgtaccatgg ctagotctac ggg tacg 27

<210s, SEQ ID NO 16 &211s LENGTH: 37 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 16 cgtagaattic ttagacctgg attggat.cga tigtaaac 37

<210s, SEQ ID NO 17 &211s LENGTH: 61 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 17 cgtagaattic agaaggagat atacatatgt ttgatttcaa taatatatgaaaagta agg 6 O c 61

<210s, SEQ ID NO 18 &211s LENGTH: 37 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 18 gatggit cac to acaactga caaacgcaa totaatc 37

<210s, SEQ ID NO 19 &211s LENGTH: 37 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 19 gatgct cag ttagacctgg attggat.cga tigtaaac 37

<210s, SEQ ID NO 2 O &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 2O gcc.gtcgacc atcatcatca totatic 25

<210s, SEQ ID NO 21 &211s LENGTH: 35 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 21 gcagtictaga gcc agaaccg titatgatgtc. g.gc.gc 35 US 8,927,241 B2 61 62 - Continued

<210s, SEQ ID NO 22 &211s LENGTH: 35 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 22 cgtgtc.cgga gcatctaacg Cttgagttaa gocgc 35

<210s, SEQ ID NO 23 &211s LENGTH: 32 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 23 gcagtictaga ggaaac Ctgt Citgc.ca.gct gc 32

<210s, SEQ ID NO 24 &211s LENGTH: 91 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 24 gacgct cag gag caataac tag catalacc ccttgggggc tictaaacggg tdttgagggg 6 O titttittgctt gtgtaggctg gagctgct tc g 91

<210s, SEQ ID NO 25 &211s LENGTH: 34 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 25 gacgagtact gaacgt.cgga attgatcc.gt cac 34

<210s, SEQ ID NO 26 &211s LENGTH: 91 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 26 gacggagctic gag caataac tag catalacc ccttgggggc tictaaacggg tdttgagggg 6 O ttttittgctt gtgtaggctg gagctgct tc g 91

<210s, SEQ ID NO 27 &211s LENGTH: 63 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 27 atgacgattt ttgataatta tdaagtgtgg tttgt cattg cattaattgc gttgcgctica 6 O

Ctg 63 US 8,927,241 B2 63 64 - Continued

SEQ ID NO 28 LENGTH: 64 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Oligonucleotide

SEQUENCE: 28 atgacgattt ttgataatta tdaagtgtgg tttgt cattg gcatcc.gctt acagacaagc 6 O

64

SEQ ID NO 29 LENGTH: 64 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Oligonucleotide

SEQUENCE: 29 ttagcgacga aaccogtaat acactitcgtt C cagcgcagc cacgt.cgga attgatc.cgt. 6 O cgac 64

SEQ ID NO 3 O LENGTH: 59 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Oligonucleotide

SEQUENCE: 3 O cgtacatatg gct ctdtt at tag cagttitt tdtggcgaaa tittaacgaag taacccago 59

SEQ ID NO 31 LENGTH: 56 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Oligonucleotide

SEQUENCE: 31 cgtacatatg gct ctdtt at tag cagttitt ttittagcatc gctittgagtg caattg 56

SEQ ID NO 32 LENGTH 58 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Oligonucleotide

SEQUENCE: 32 cgtacatatg gct ctdtt at tag cagttitt tttitcgct cq aaacgt.cat a gtagcctg 58

SEQ ID NO 33 LENGTH: 49 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Oligonucleotide

SEQUENCE: 33 cgcgggat.cc ggtgctgc cc ggacgaggga acagtttgat taaaac cc 49 US 8,927,241 B2 65 66 - Continued

<210s, SEQ ID NO 34 &211s LENGTH: 34 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 34 cgcgggat.cc cqc.cgtggtg gaagtgatac acag 34

<210s, SEQ ID NO 35 &211s LENGTH: 4 O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 35 cgcggit cqac ttaccaaata t ccc.gtaagt agcgt.ccatc 4 O

<210s, SEQ ID NO 36 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 36 attcaaaagc titc.cggtc.ct

<210 SEQ ID NO 37 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OO > SEQUENCE: 37 atctggcgac attcgttitt c

<210s, SEQ ID NO 38 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 38 gacgaactgt cacccgattit

<210s, SEQ ID NO 39 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 39 gctt.cgcggg tagtag acag

<210s, SEQ ID NO 4 O &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide US 8,927,241 B2 67 - Continued

<4 OOs, SEQUENCE: 4 O aggcct tcgg gttgtaaagt

<210s, SEQ ID NO 41 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 41 attic cq atta acgcttgcac

<210s, SEQ ID NO 42 &211s LENGTH: 296 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 42 Met Phe Asp Phe Asn. Glu Tyr Met Lys Ser Lys Ala Val Ala Val Asp 1. 5 1O 15

Ala Ala Lieu. Asp Lys Ala Ile Pro Lieu. Glu Tyr Pro Glu Lys Ile His 2O 25 3O

Glu Ser Met Arg Tyr Ser Lieu. Lieu Ala Gly Gly Lys Arg Val Arg Pro 35 4 O 45

Ala Lieu. Cys Ile Ala Ala Cys Glu Lieu Val Gly Gly Ser Glin Asp Lieu SO 55 6 O

Ala Met Pro Thr Ala Cys Ala Met Glu Met Ile His Thr Met Ser Leu 65 70 7s 8O Ile His Asp Asp Lieu Pro Cys Met Asp Asn Asp Asp Phe Arg Arg Gly 85 90 95

Llys Pro Thr Asn His Llys Val Phe Gly Glu Asp Thr Ala Val Lieu Ala 1OO 105 11 O

Gly Asp Ala Lieu Lleu Ser Phe Ala Phe Glu. His Ile Ala Wall Ala Thr 115 12 O 125

Ser Lys Thr Val Pro Ser Asp Arg Thr Lieu. Arg Val Ile Ser Glu Lieu. 13 O 135 14 O

Gly Lys Thir Ile Gly Ser Glin Gly Lieu Val Gly Gly Glin Val Val Asp 145 150 155 160

Ile Thir Ser Glu Gly Asp Ala Asn Val Asp Lieu Lys Thr Lieu. Glu Trp 1.65 17O 17s

Ile His Ile His Llys Thr Ala Val Lieu. Lieu. Glu. Cys Ser Val Val Ser 18O 185 19 O

Gly Gly Ile Lieu. Gly Gly Ala Thr Glu Asp Glu Ile Ala Arg Ile Arg 195 2OO 2O5

Arg Tyr Ala Arg Cys Val Gly Lieu. Lieu. Phe Glin Val Val Asp Asp Ile 21 O 215 22O

Lieu. Asp Val Thir Lys Ser Ser Glu Glu Lieu. Gly Llys Thr Ala Gly Lys 225 23 O 235 24 O

Asp Lieu. Lieu. Thir Asp Lys Ala Thr Tyr Pro Llys Lieu Met Gly Lieu. Glu 245 250 255

Lys Ala Lys Glu Phe Ala Ala Glu Lieu Ala Thr Arg Ala Lys Glu Glu 26 O 265 27 O US 8,927,241 B2 69 - Continued

Lieu. Ser Ser Phe Asp Glin Ile Lys Ala Ala Pro Lieu. Lieu. Gly Lieu Ala 285 Asp Tyr Ile Ala Phe Arg Glin Asn 29 O 295

SEQ ID NO 43 LENGTH: 888 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Polynucleotide

<4 OOs, SEQUENCE: 43 atgtttgatt toaatgaata tatgaaaagt aaggctgttg cgg tagacgc ggctctggat 6 O aaag.cgattic cgctggaata t ccc.gagaag att cacgaat cgatgcgcta ctic cctdtta 12 O gCaggaggga aacgcgttcg tccggcatta tgcatcgcgg cctgtgaact cgt.cggcggit 18O t cacaggact tagcaatgcc aactgcttgc gcaatggaaa tgatt cacac aatgagcctg 24 O att catgatg atttgccttg Catggacaac gatgactitt c ggcgcggtaa acctactaat 3OO catalaggttt ttggcgaaga tactgcagtg Ctggcgggcg atgcgctgct gtcgtttgcc 360 titcgaacata tcgc.cgt.cgc gacct cqaaa acct cocqt cggaccgtac gct tcgc.gtg attt cogagc tgggaaagac catcggct ct Caaggacticg tgggtggtca gg tagttgat atcacgtctg agggtgacgc gaacgtggac ctgaaaaccc tggagtggat CCatatt CaC 54 O aaaacggc.cg tgctgctgga atgtagcgtg gtgtcagggg ggat Cttggg ggg.cgc.cacg gaggatgaaa tcgc.gc.gt at tcgt.cgittat gCCCCtgtg ttggaCtgtt attt Caggtg 660 gtggatgaca t cctggatgt Cacaaaat CC agcgaagagc ttggcaagac cgcgggcaaa 72 O gaccttctga cggata aggc tacataccc.g aaattgatgg gcttggagaa agccaaggag titcgcagctg aacttgccac gcggg.cgaag gaagaactict cittctitt cqa toaaatcaaa. 84 O

tgctgggc ct cgc.cgattac attgcgtttic gtcagaac 888

SEQ ID NO 44 LENGTH: 8O2 TYPE : PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 44

Met Ser Ser Ser Thr Gly. Thir Ser Llys Val Val Ser Glu Thir Ser Ser 1. 5 1O 15

Thir Ile Wall Asp Asp Ile Pro Arg Lieu. Ser Ala Asn Tyr His Gly Asp 25 3O

Lell Trp His His Asn. Wall Ile Glin Thir Lieu. Glu Thir Pro Phe Arg Glu 35 4 O 45

Ser Ser Thir Tyr Glin Glu Arg Ala Asp Glu Lieu. Wall Wall Ile Llys SO 55 6 O

Asp Met Phe Asn Ala Lieu. Gly Asp Gly Asp Ile Ser Pro Ser Ala Tyr 65 70 7s 8O

Asp Thir Ala Trp Val Ala Arg Lieu Ala Thir Ile Ser Ser Asp Gly Ser 85 90 95

Glu Pro Arg Phe Pro Glin Ala Lieu. Asn Trp Wall Phe Asn ASn Glin 105 11 O

Lell Glin Asp Gly Ser Trp Gly Ile Glu Ser His Phe Ser Luell 115 12 O 125 US 8,927,241 B2 71 72 - Continued

Arg Luell Luell Asn Thir Thir Asn Ser Wall Ile Ala Lell Ser Wall Trp Lys 13 O 135 14 O

Thir Gly His Ser Glin Wall Glin Glin Gly Ala Glu Phe Ile Ala Glu Asn 145 150 155 160

Lell Arg Luell Luell Asn Glu Glu Asp Glu Luell Ser Pro Asp Phe Glin Ile 1.65 17O 17s

Ile Phe Pro Ala Lell Lell Glin Ala Ala Lell Gly Ile Asn Luell 18O 185 19 O

Pro Asp Luell Pro Phe Ile Lys Luell Ser Thir Thir Arg Glu Ala 195

Arg Luell Thir Asp Wall Ser Ala Ala Ala Asp ASn Ile Pro Ala Asn Met 21 O 215 22O

Lell Asn Ala Luell Glu Gly Lell Glu Glu Wall Ile Asp Trp Asn Ile 225 23 O 235 24 O

Met Arg Phe Glin Ser Asp Gly Ser Phe Luell Ser Ser Pro Ala Ser 245 250 255

Thir Ala Wall Lell Met Asn Thir Gly Asp Glu Phe Thir Phe 26 O 265 27 O

Lell Asn Asn Luell Lell Asp Phe Gly Gly Wall Pro Met Tyr 27s 28O 285

Ser Ile Asp Luell Lell Glu Arg Luell Ser Luell Wall Asp Asn Ile Glu His 29 O 295 3 OO

Lell Gly Ile Gly Arg His Phe Glin Glu Ile Gly Ala Luell Asp 3. OS 310 315

Wall Arg His Trp Ser Glu Arg Gly Ile Gly Trp Gly Arg Asp 3.25 330 335

Ser Luell Wall Pro Asp Lell Asn Thir Thir Ala Luell Gly Lell Arg Thir Luell 34 O 345 35. O

Arg Met His Gly Tyr Asn Wall Ser Ser Asp Wall Lell Asn Asn Phe Lys 355 360 365

Asp Glu Asn Gly Arg Phe Phe Ser Ser Ala Gly Glin Thir His Wall Glu 37 O 375

Lell Arg Ser Wall Wall Asn Lell Phe Arg Ala Ser Asp Lell Ala Phe Pro 385 390 395 4 OO

Asp Glu Arg Ala Met Asp Asp Ala Arg Lys Phe Ala Glu Pro Tyr Luell 4 OS 415

Arg Glu Ala Luell Ala Thir Ile Ser Thir ASn Thir Luell Phe Lys 425 43 O

Glu Ile Glu Wall Wall Glu Tyr Pro Trp His Met Ser Ile Pro Arg 435 44 O 445

Lell Glu Ala Arg Ser Ile Asp Ser Asp Asp Asn Wall Trp 450 45.5 460

Glin Arg Thir Lell Tyr Arg Met Pro Ser Luell Ser Asn Ser Cys 465 470

Lell Glu Luell Ala Lys Lell Asp Phe Asn Ile Wall Glin Ser Luell His Glin 485 490 495

Glu Glu Luell Lys Lell Lell Thir Arg Trp Trp Glu Ser Gly Met Ala SOO 505

Asp Ile Asn Phe Thir Arg His Arg Wall Ala Glu Wall Tyr Phe Ser Ser 515 525

Ala Thir Phe Glu Pro Glu Tyr Ser Ala Thir Arg Ile Ala Phe Thir Lys 53 O 535 54 O US 8,927,241 B2 73 74 - Continued

Ile Gly Lieu. Glin Wall Lieu. Phe Asp Asp Met Ala Asp Ile Phe Ala 5.45 550 555 560

Thir Luell Asp Glu Lieu Lys Ser Phe Thr Glu Gly Wall Lys Arg Trp Asp 565 st O sts

Thir Ser Luell Lieu. His Glu. Ile Pro Glu Cys Met Glin Thir Cys Phe Lys 585 59 O

Wall Trp Phe Llys Lieu Met Glu Glu Wall Asn. Asn Asp Wall Wall Llys Val 595 605

Glin Gly Arg Asp Met Lieu Ala His Ile Arg Llys Pro Trp Glu Leu Tyr 610 615

Phe Asn Tyr Val Glin Glu Arg Glu Trp Lieu. Glu Ala Gly 625 630 635 64 O

Pro Thir Phe Glu Glu Tyr Lieu Lys Thr Tyr Ala Ile Ser Wall Gly Lieu. 645 650 655

Gly Pro Thir Lieu. Glin Pro Ile Lieu. Luell Met Gly Glu Luell Val Lys 660 665 67 O

Asp Asp Wall Val Glu Lys Wal His Tyr Pro Ser Asn Met Phe Glu Lieu. 675 685

Wall Ser Luell Ser Trp Arg Lieu. Thr Asn Asp Thr Lys Thir Glin Ala 69 O. 695 7 OO

Glu Ala Arg Gly Glin Glin Ala Ser Gly Ile Ala Met Lys 7 Os 71O 71s 72O

Asp Asn Pro Gly Ala Thr Glu Glu Asp Ala Ile His Ile Cys Arg 72 73 O 73

Wall Wall Asp Arg Ala Lieu Lys Glu. Ala Ser Phe Glu Phe Llys Pro 740 74. 7 O

Ser Asn Asp Ile Pro Met Gly Cys Lys Ser Phe Ile Phe Asn Lieu. Arg 760 765

Lell Cys Wall Glin Ile Phe Tyr Lys Phe Ile Asp Gly Gly Ile Ala 770 775 78O

Asn Glu Glu Ile Lys Asp Tyr Ile Arg Llys Val Ile Asp Pro Ile 79 O 79. 8OO

Glin Wall

SEO ID NO 45 LENGTH: 24 O6 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Polynucleotide

<4 OOs, SEQUENCE: 45 atgtctagot ctacggg tac gtctaaagtic gtgagtgaaa cct catcgac gat.cgtggac 6 O gatatt coac gcttgtcggc gaact atcat ggagatctgt ggcatcatala cgt catt cag 12 O acattggaaa cc.ccgttt cq cgaaagtagc acctaccagg aacgggcaga tgaattagt c 18O gtgaaaatca aagatatgtt taatgcatta ggagatggag a catctogcc cagcgcatat 24 O gatacggcgt. gttggccacg attagct cog atggcagtga aaa.gc.cgcgt. 3OO titc.ccgcagg cgctgaactg ggtgtttaat aat caattgc aggatggcag Ctggggcatt 360 gaat ct cact ttagcct citg tgaccggitta citcaiacacga caaactic cqt aattgcgttg t cagtttgga aaacgggc.ca tagcc aggtt caa.cagggcg cggaattitat cgctgaaaat 48O

tgaacgagga ggacgaactg t cacccgatt ttcagattat ttitt.ccggct 54 O ttactic caga aagccaaagc cittaggcatc aacct gc cat atgatctgcc gttcatcaag

US 8,927,241 B2 77 78 - Continued

Ala Gly Ile Luell Lell Lell Lell Lel Luell Phe Arg Ser Lys Arg His Ser 35 4 O 45

Ser Luell Luell Pro Pro Gly Luell Gly Ile Pro Phe Ile Gly Glu SO 55 6 O

Ser Phe Ile Phe Lell Arg Ala Lel Arg Ser ASn Ser Lell Glu Glin Phe 65 70

Phe Asp Glu Arg Wall Phe Gly Luell Wall Phe Thir Ser Luell 85 90 95

Ile Gly His Pro Thir Wall Wall Lel Cys Gly Pro Ala Gly Asn Arg Luell 105 11 O

Ile Luell Ser Asn Glu Glu Lel Wall Glin Met Ser Trp Pro Ala Glin 115 12 O 125

Phe Met Luell Met Gly Glu Asn Ser Wall Ala Thir Arg Arg Gly Glu 13 O 135 14 O

Asp His Ile Wall Met Arg Ser Ala Luell Ala Gly Phe Phe Gly Pro Gly 145 150 155 160

Ala Luell Glin Ser Tyr Ile Gly Met Asn Thir Glu Ile Glin Ser His 1.65 17O 17s

Ile Asn Glu Lys Trp Gly Asp Glu Wall Asn Wall Luell Pro Luell 18O 185 19 O

Wall Arg Glu Luell Wall Phe Asn Ile Ser Ala Ile Lell Phe Phe Asn Ile 195

Asp Glin Glu Glin Asp Arg Luell His Lell Lell Glu Thir Ile 21 O 215 22O

Lieu Wall Gly Ser Phe Ala Lieu Pro Ile Asp Lieu. Pro Gly Phe Gly Phe 225 23 O 235 24 O

His Arg Ala Luell Glin Gly Arg Ala Luell ASn Ile Met Luell Ser 245 250 255

Lell Ile Lys Arg Glu Asp Luell Glin Ser Gly Ser Ala Thir Ala 26 O 265 27 O

Thir Glin Asp Luell Lell Ser Wall Luell Luell Thir Phe Arg Asp Asp Gly 27s 285

Thir Pro Luell Thir Asn Asp Glu Ile Luell Asp ASn Phe Ser Ser Luell Luell 29 O 295 3 OO

His Ala Ser Asp Thir Thir Thir Ser Pro Met Ala Lell Ile Phe Lys 3. OS 310 315

Lell Luell Ser Ser Asn Pro Glu Glin Lys Wall Wall Glin Glu Glin 3.25 330 335

Lell Glu Ile Luell Ser Asn Glu Glu Gly Glu Glu Ile Thir Trp 34 O 345 35. O

Asp Luell Lys Ala Met Thir Trp Glin Wall Ala Glin Glu Thir Luell 355 360 365

Arg Met Phe Pro Pro Wall Phe Gly Thir Phe Arg Lys Ala Ile Thir Asp 37 O 375

Ile Glin Tyr Asp Gly Tyr Thir Ile Pro Gly Trp Luell Luell Trp 385 390 395 4 OO

Thir Thir Ser Thir His Pro Asp Luell Tyr Phe Asn Glu Pro Glu 4 OS 41O 415

Phe Met Pro Ser Arg Phe Asp Glin Glu Gly His Wall Ala Pro 425 43 O

Thir Phe Luell Pro Phe Gly Gly Gly Glin Arg Ser Cys Wall Gly Trp 435 44 O 445

US 8,927,241 B2 81 82 - Continued

<4 OOs, SEQUENCE: 48

Met Glin Ala Asn Ser Asn Thir Wall Glu Gly Ala Ser Glin Gly Lys Ser 1. 15

Lell Luell Asp Ile Ser Arg Lell Asp His Ile Phe Ala Lell Luell Luell Asn 2O 25

Gly Gly Gly Asp Lell Gly Ala Met Thir Gly Ser Ala Luell Ile Luell 35 4 O 45

Thir Glu Asn Ser Glin Asn Lell Met Ile Luell Thir Thir Ala Luell Ala Wall SO 55 6 O

Lell Wall Ala Wall Phe Phe Phe Wall Trp Arg Arg Gly Gly Ser Asp 65 70

Thir Glin Pro Ala Wall Arg Pro Thir Pro Luell Wall Glu Glu Asp 85 90 95

Glu Glu Glu Glu Asp Asp Ser Ala Lys Lys Wall Thir Ile Phe Phe 105 11 O

Gly Thir Glin Thir Gly Thir Ala Glu Gly Phe Ala Ala Luell Ala Glu 115 12 O 125

Glu Ala Ala Arg Glu Ala Wall Phe Lys Wall Wall Asp Luell 13 O 135 14 O

Asp Asn Tyr Ala Ala Asp Asp Glu Glin Tyr Glu Glu Luell Lys 145 150 155 160

Glu Luell Ala Phe Phe Met Luell Ala Thir Gly Asp Gly Glu Pro 1.65 17O

Thir Asp Asn Ala Ala Arg Phe Lys Trp Phe Lell Glu Gly Lys Glu 18O 185 19 O

Arg Glu Pro Trp Lell Ser Asp Luell Thir Tyr Gly Wall Phe Gly Luell Gly 195 2OO

Asn Arg Glin Tyr Glu His Phe Asn Wall Ala Lys Ala Wall Asp Glu 21 O 215 22O

Wall Luell Ile Glu Glin Gly Ala Arg Luell Wall Pro Wall Gly Luell Gly 225 23 O 235 24 O

Asp Asp Asp Glin Cys Ile Glu Asp Asp Phe Thir Ala Trp Arg Glu Glin 245 250 255

Wall Trp Pro Glu Lell Asp Glin Luell Luell Arg Asp Glu Asp Asp Glu Pro 26 O 265 27 O

Thir Ser Ala Thir Pro Thir Ala Ala Ile Pro Glu Tyr Arg Wall Glu 285

Ile Tyr Asp Ser Wall Wall Ser Wall Glu Glu Thir His Ala Luell 29 O 295 3 OO

Glin Asn Gly Glin Ala Wall Asp Ile His His Pro Arg Ser Asn 3. OS 310 315 32O

Wall Ala Wall Arg Arg Glu Lell His Thir Pro Luell Ser Asp Arg Ser 3.25 330 335

Ile His Luell Glu Phe Asp Ile Ser Asp Thir Gly Lell Ile Tyr Glu Thir 34 O 345 35. O

Gly Asp His Wall Gly Wall His Thir Glu Asn Ser Ile Glu Thir Wall Glu 355 360 365

Glu Ala Ala Lell Lell Gly Tyr Glin Luell Asp Thir Ile Phe Ser Wall 37 O 375

His Gly Asp Glu Asp Gly Thir Pro Luell Gly Gly Ser Ser Luell Pro 385 390 395 4 OO

Pro Pro Phe Pro Gly Pro Thir Luell Arg Thir Ala Lell Ala Arg Tyr 4 OS 41O 415 US 8,927,241 B2 83 84 - Continued

Ala Asp Luell Luell Asn Pro Pro Arg Lys Ala Ala Phe Lell Ala Lieu Ala 42O 425 43 O

Ala His Ala Ser Asp Pro Ala Glu Ala Glu Arg Lell Lys Phe Luell Ser 435 44 O 445

Ser Pro Ala Gly Lys Asp Glu Tyr Ser Glin Trp Wall Thir Ala Ser Glin 450 45.5 460

Arg Ser Luell Lieu. Glu Ile Met Ala Glu Phe Pro Ser Ala Pro Pro 465 470 48O

Lell Gly Wall Phe Phe Ala Ala Ile Ala Pro Arg Lell Glin Pro Arg Tyr 485 490 495

Ser Ile Ser Ser Ser Pro Arg Phe Ala Pro Ser Arg Ile His Wall SOO 505

Thir Ala Lieu Wall Tyr Gly Pro Ser Pro Thir Gly Arg Ile His Lys 515 52O 525

Gly Wall Ser Asn Trp Met Lys Asn Ser Luell Pro Ser Glu Glu Thir 53 O 535 54 O

His Asp Ser Trp Ala Pro Wall Phe Wall Arg Glin Ser Asn Phe Lys 5.45 550 555 560

Lell Pro Ala Asp Ser Thir Thr Pro Ile Wall Met Wall Gly Pro Gly Thr 565 st O sts

Gly Phe Ala Pro Phe Arg Gly Phe Luell Glin Glu Arg Ala Lys Lieu. Glin 58O 585 59 O

Glu Ala Gly Glu Lys Lieu. Gly Pro Ala Wall Luell Phe Phe Gly 595 6OO 605

Asn Arg Gln Met Asp Glu Asp Glu Lieu Gly Tyr Val 610 615

Glu Gly Ile Lieu. Thir Asn Lieu. Ile Wall Ala Phe Ser Arg Glu Gly 625 630 635 64 O

Ala Thir Glu Tyr Wall Glin His Met Luell Glu Ala Ser Asp 645 650 655

Thir Trp Ser Lieu. Ile Ala Glin Gly Gly Luell Wall Cys Gly Asp 660 665 67 O

Ala Gly Met Ala Arg Asp Wall His Thir Lell His Thir Ile Wall 675 68O 685

Glin Glu Glin Glu Ser Val Asp Ser Ser Ala Glu Phe Luell Val Lys 69 O. 695 7 OO

Lys Luell Glin Met Asp Gly Arg Tyr Luell Arg Asp Ile Trp 7 Os 71O 71s

SEQ ID NO 49 LENGTH: 21.51 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Polynucleotide

SEQUENCE: 49 atgcaggcga attictaatac ggttgaaggc gcgagccaag gcaagttctict tctggacatt 6 O agtc.gc.ct cq accatat citt cqc cctogctg ttgaacggga aaggcggaga CCttggtgcg 12 O atgaccgggit cggcct taat tctgacggaa aatagcc aga acttgatgat tctgaccact 18O

ttctggtc.gc titgcgtttitt tttitt cqttt tggaagtgat 24 O acacagaa.gc cc.gc.cgitacg toccacac ct cittgttaaag aagaggacga agaagaagaa 3OO gatgatagcg c caagaaaaa got cacaata ttttittggca CCC agaccgg Caccgc.cgaa 360

US 8,927,241 B2 87 88 - Continued

22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 51

Met Ala Lieu. Lieu. Lieu Ala Wall Phe 1. 5

SEO ID NO 52 LENGTH: 41 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 52 cgtaccatgg ttgatttcaa taatatatgaaaagtalagg C 41

What is claimed is: 4. The method of claim 1, whereinaccumulation of indole 1. A method for making a product containing Myrcene or a in the culture is maintained to below 50 mg/L. derivative thereof, the method comprising: 5. The method of claim 1, whereinaccumulation of indole providing an Escherichia coli (E. coli) that produces iso in the culture is maintained to below 10 mg/L. pentyl pyrophosphate (IPP) and dimethylallyl pyro 6. The method of claim 1, wherein the Myrcene is produced phosphate (DMAPP) through an upstream methyleryth 25 at 10 mg/L or more. 7. The method of claim 1, wherein the Myrcene is produced ritol pathway (MEP) and converts the IPP and DMAPP at 100 mg/L or more. to Myrcene through a recombinantly expressed down 8. The method of claim 1, wherein the E. coli has additional stream synthesis pathway comprising Geranyl Diphos copies of one or more of the dxs, idi, isp) and ispF genes of phate Synthase and Myrcene Synthase; and 30 the MEP pathway. culturing the E. coli to produce the Myrcene, wherein the 9. A method for making a myrcene, the method compris accumulation of indole in the culture is controlled to 1ng: below 100 mg/L to thereby increase Myrcene produc providing an Escherichia coli (E. coli) that produces iso tion; and optionally preparing a derivative of the pentyl pyrophosphate (IPP) and dimethylallyl pyro Myrcene; and 35 phosphate (DMAPP) through an upstream methyleryth incorporating the Myrcene or derivative into a fragrance ritol pathway (MEP) and converts the IPP and DMAPP product, a cosmetic product, oral care product, a pest to myrcene through a recombinantly expressed down control product, a cleaning product, a lubricant or Sur stream synthesis pathway comprising Geranyl Diphos factant, or a Soap product. phate Synthase and Myrcene Synthase; and 2. The method of claim 1, wherein accumulation of indole 40 culturing the E. coli to produce the myrcene, wherein the in the culture is controlled by balancing the upstream MEP accumulation of indole in the culture is controlled to pathway with the downstream synthesis pathway. below 100 mg/L to thereby increase terpenoid produc 3. The method of claim 1, further comprising, measuring tion; and the amount or concentration of indole continuously or inter recovering the myrcene. mittently. k k k k k