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Methods of Developing Terpene Synthase Variants Verfahren Zur Entwicklung Von Terpensynthasevarianten Procédés De Développement De Variants De Terpène Synthase

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Methods of Developing Terpene Synthase Variants Verfahren Zur Entwicklung Von Terpensynthasevarianten Procédés De Développement De Variants De Terpène Synthase (19) TZZ Z_T (11) EP 2 670 846 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 9/88 (2006.01) C12Q 1/527 (2006.01) 19.08.2015 Bulletin 2015/34 (86) International application number: (21) Application number: 12742056.0 PCT/US2012/023446 (22) Date of filing: 01.02.2012 (87) International publication number: WO 2012/106405 (09.08.2012 Gazette 2012/32) (54) METHODS OF DEVELOPING TERPENE SYNTHASE VARIANTS VERFAHREN ZUR ENTWICKLUNG VON TERPENSYNTHASEVARIANTEN PROCÉDÉS DE DÉVELOPPEMENT DE VARIANTS DE TERPÈNE SYNTHASE (84) Designated Contracting States: (56) References cited: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB US-A1- 2002 035 058 US-A1- 2009 053 797 GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO US-A1- 2010 281 846 PL PT RO RS SE SI SK SM TR • YOSHIKUNI Y ET AL: "Engineering Cotton (30) Priority: 02.02.2011 US 201161438948 P (+)-delta-Cadinene Synthase to an Altered Function: Germacrene D-4-ol Synthase", (43) Date of publication of application: CHEMISTRY AND BIOLOGY, CURRENT 11.12.2013 Bulletin 2013/50 BIOLOGY, LONDON, GB, vol. 13, no. 1, 1 January 2006 (2006-01-01), pages 91-98, XP025131704, (73) Proprietor: Amyris, Inc. ISSN: 1074-5521, DOI: 10.1016/J.CHEMBIOL. Emeryville, CA 94608 (US) 2005.10.016 [retrieved on 2006-01-01] • YASUO YOSHIKUNI ET AL: "Designed divergent (72) Inventors: evolutionof enzyme function",NATURE, vol. 440, • ZHAO, Lishan no. 7087, 22 February 2006 (2006-02-22), pages Albany, CA 94706-1409 (US) 1078-1082, XP055087632, ISSN: 0028-0836, DOI: •XU,Lan 10.1038/nature04607 Orinda, CA 94563 (US) • YOSHIKUNI ET AL: "Pathway engineering by • WESTFALL, Patrick J. designed divergent evolution", CURRENT Westfield, IN 46074-9358 (US) OPINION IN CHEMICAL BIOLOGY, CURRENT • MAIN, Andrew BIOLOGYLTD, LONDON, GB,vol. 11, no. 2, 5 April Alameda, CA 94501 (US) 2007 (2007-04-05), pages 233-239, XP022022270, ISSN: 1367-5931, DOI: 10.1016/J.CBPA. (74) Representative: Brasnett, Adrian Hugh 2007.02.033 Mewburn Ellis LLP City Tower 40 Basinghall Street London EC2V 5DE (GB) Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 670 846 B1 Printed by Jouve, 75001 PARIS (FR) EP 2 670 846 B1 Description 1. FIELD OF THE INVENTION 5 [0001] The present disclosure relates to methods of developing terpene synthase variants through engineered host cells. Particularly, the disclosure provides methods of developing terpene synthase variants with improved in vivo per- formance that are useful in the commercial production of terpene products. Further encompassed in the present disclosure are superior terpene synthase variants, and host cells comprising such terpene synthase variants. 10 2. BACKGROUND [0002] Terpenes are a large class of hydrocarbons that are produced in many organisms. They are derived by linking units of isoprene (C5H8), and are classified by the number of isoprene units present. Hemiterpenes consist of a single isoprene unit. Isoprene itself is considered the only hemiterpene. Monoterpenes are made of two isoprene units, and 15 have the molecular formula C10H16. Examples of monoterpenes are geraniol, limonene, and terpineol. Sesquiterpenes are composed of three isoprene units, and have the molecular formula C15H24. Examples of sesquiterpenes are far- nesenes, farnesol and patchoulol. Diterpenes are made of four isoprene units, and have the molecular formula C 20H32. Examples of diterpenes are cafestol, kahweol, cembrene, and taxadiene. Sesterterpenes are made of five isoprene units, and have the molecular formula C 25H40. An example of a sesterterpenes is geranylfarnesol. Triterpenes consist 20 of six isoprene units, and have the molecular formula C 30H48. Tetraterpenes contain eight isoprene units, and have the molecular formula C40H64. Biologically important tetraterpenes include the acyclic lycopene, the monocyclic gamma- carotene, and the bicyclic alpha- and beta-carotenes. Polyterpenes consist of long chains of many isoprene units. Natural rubber consists of polyisoprene in which the double bonds are cis. [0003] Whenterpenes are chemically modified(e.g., via oxidation orrearrangement of thecarbon skeleton)the resulting 25 compoundsare generally referred to as terpenoids, which arealso known as isoprenoids. Isoprenoids play many important biological roles, for example, as quinones in electron transport chains, as components of membranes, in subcellular targeting and regulation via protein prenylation, as photosynthetic pigments including carotenoids, chlorophyll, as hor- mones and cofactors, and as plant defense compounds with various monoterpenes, sesquiterpenes, and diterpenes. They are industrially useful as antibiotics, hormones, anticancer drugs, insecticides, and chemicals. 30 [0004] Terpenes are biosynthesized through condensations of isopentenyl pyrophosphate (isopentenyl diphosphate or IPP) and its isomer dimethylallyl pyrophosphate (dimethylallyl diphosphate or DMAPP). Two pathways are known to generate IPP and DMAPP, namely the mevalonate-dependent (MEV) pathway of eukaryotes, and the mevalonate- independent or deoxyxylulose-5-phosphate (DXP) pathway of prokaryotes. Plants use both the MEV pathway and the DXP pathway. IPP and DMAPP in turn are condensed to polyprenyl diphosphates (e.g., geranyl disphosphate or GPP, 35 farnesyl diphosphate or FPP, and geranylgeranyl diphosphate or GGPP) through the action of prenyl disphosphate synthases (e.g., GPP synthase, FPP synthase, and GGPP synthase, respectively). [0005] The polyprenyl diphosphate intermediates are converted to more complex isoprenoid structures by terpene synthases. Terpene synthases are organized into large gene families that form multiple products. Examples of terpene synthases include sesquiterpene synthases, which convert FPP into sesquiterpenes. An example of a sesquiterpene 40 synthase is farnesene synthase, which converts FPP to farnesene. The reaction mechanism of terpene synthases has been extensively investigated and is well understood. Overall, three steps are required to convert a diphosphate substrate such as FPP to its isoprenoid product: a) formation of enzyme-substrate complex (ES), b) formation of an enzyme-bound reactive carbocation intermediate, subsequent rearrangements, and the formation of product (EP), and c) release of product from the enzyme-product complex. In vitro kinetic and pre-steady state kinetic studies on terpene synthase 45 catalyzed reactions have shown that the overall rate-limiting step for the reactions is the release of product (Cane et al. (1997) Biochemistry, 36(27):8332-9, and Mathis et al. (1997) Biochemistry 36(27):8340-8). The turnover rates of terpene synthases are low, generally measured at less than 0.5 per second (Cane, D. C. (1990) Chem. Rev. 90:1089-1103). [0006] Terpene synthases are important in the regulation of pathway flux to an isoprenoid because they operate at metabolic branch points and often compete with other metabolic enzymes for a prenyl diphosphate pool. For example, 50 FPP is the precursor to many cellular molecules including squalene, dolichols, and the cofactor heme. In engineered microbes where the production of sesquiterpenes such as farnesene is desired, the terpene synthases hold the key to high yield production of such terpenes. However, because they are slow enzymes, terpene synthases are often the bottlenecks in the metabolic pathways. In addition, they can suffer from other shortcomings such as substrate inhibition that limit the kinetic capacity required for efficient production of terpenes in engineered microbial hosts (Crock et al. 55 (1997) Proc. Natl. Acad. Sci. USA 94:12833-12838). [0007] Hence, there are potentially enormous benefits to improving the catalytic efficiency of terpene synthases so that these enzymes would no longer limit the overall metabolic flux to an isoprenoid. Attempts to engineer terpene synthases for altered product specificity as well as the use of rational approaches such as those based on structural 2 EP 2 670 846 B1 guidance or adaptive evolution have been described previously (Greenhagen et al. (2006) Proc. Natl. Acad. Sci. USA 103:9826-9831; O’Mailleet al. (2008)Nat. Chem. Biol.4:617-623; Yoshikuni et al.(2006) Nature 440:1078-1082;Yoshiku- ni et al. (2006) Chem. Biol. 13:91-98);Yoshikuni et al. (2008) Chem. Biol. 15:607-618). However, these studies have fallen short of improving the kinetic capacity of terpene synthases while also maintaining their product specificity. In 5 addition, the application of conventional protein engineering strategies, such as directed evolution, has been devoid for terpene synthases primarily because of the lack of available and effective high throughput screening methods (Yoshikuni et al. (2008) (supra)). There thus remains a need for reliable and high throughput methods for improving the catalytic efficiency of terpene synthases, and for terpene synthase variants that have such improved catalytic efficiency. 10 3. SUMMARY OF THE INVENTION [0008] The present disclosure relates to methods of developing terpene synthase variants through engineered host cells. Particularly, the disclosure provides methods of developing terpene synthase variants with improved in vivo per- formance. The methods
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