(19) TZZ _T (11) EP 2 645 847 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: A01H 5/00 (2018.01) A01H 13/00 (2006.01) 17.01.2018 Bulletin 2018/03 C12N 15/82 (2006.01) A23L 2/60 (2006.01) (21) Application number: 11793960.3 (86) International application number: PCT/US2011/062428 (22) Date of filing: 29.11.2011 (87) International publication number: WO 2012/075030 (07.06.2012 Gazette 2012/23) (54) MICROBIAL PRODUCTION OF NATURAL SWEETENERS, DITERPENOID STEVIOL GLYCOSIDES MIKROBIELLE HERSTELLUNG VON NATÜRLICHEN SÜSSSTOFFEN, DITERPENOIDEN STEVIOSIDEN PRODUCTION MICROBIENNE D’ÉDULCORANTS NATURELS, GLYCOSIDES DE STÉVIOL DITERPÉNOÏDES (84) Designated Contracting States: • AJIKUMAR PARAYIL KUMARAN ET AL: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB "Isoprenoid Pathway Optimization for Taxol GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO Precursor Overproduction in Escherichia coli", PL PT RO RS SE SI SK SM TR SCIENCE (WASHINGTON D C), vol. 330, no. 6000, October 2010 (2010-10) , pages 70-74, (30) Priority: 30.11.2010 US 418357 P XP002618146, ISSN: 0036-8075 30.09.2011 US 201113249388 • BRANDLE ET AL: "Steviol glycoside biosynthesis", PHYTOCHEMISTRY, PERGAMON (43) Date of publication of application: PRESS, GB, vol. 68, no. 14, 1 July 2007 09.10.2013 Bulletin 2013/41 (2007-07-01), pages 1855-1863, XP022145443, ISSN: 0031-9422, DOI: (73) Proprietor: Massachusetts Institute of Technology 10.1016/J.PHYTOCHEM.2007.02.010 Cambridge, MA 02139 (US) • TANIA V HUMPHREY ET AL: "Spatial Organisation of Four Enzymes from Stevia (72) Inventors: rebaudiana that are Involved in Steviol Glycoside • STEPHANOPOULOS, Gregory Synthesis", PLANT MOLECULAR BIOLOGY, Winchester KLUWER ACADEMIC PUBLISHERS, MA 01890 (US) DORDRECHT, NL, vol. 61, no. 1-2, 1 May 2006 • AJIKUMAR, Parayil, K. (2006-05-01), pages 47-62, XP019405427, ISSN: Cambridge 1573-5028, DOI: 10.1007/S11103-005-5966-9 MA 02141 (US) • PARAYIL KUMARAN AJIKUMAR ET AL: "Terpenoids: opportunities for biosynthesis of (74) Representative: Carpmaels & Ransford LLP natural product drugs using engineered One Southampton Row microorganisms", MOLECULAR London WC1B 5HA (GB) PHARMACEUTICS, AMERICAN CHEMICAL SOCIETY, US, vol. 5, no. 2, 1 March 2008 (56) References cited: (2008-03-01), pages167-190, XP002617175, ISSN: US-A1- 2004 072 323 US-A1- 2010 297 722 1543-8384, DOI: 10.1021/MP700151B [retrieved on 2008-03-21] 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 2 645 847 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 2 645 847 B1 • KIRBY JAMES ET AL: "Biosynthesis of plant • KLEIN-MARCUSCHAMER ET AL: "Engineering isoprenoids: perspectives for microbial microbial cell factories for biosynthesis of engineering", ANNUAL REVIEW OF PLANT isoprenoid molecules: beyond lycopene", BIOLOGY,, vol. 60, 1 January 2009 (2009-01-01), TRENDS IN BIOTECHNOLOGY, ELSEVIER pages 335-355, XP009143317, DOI: PUBLICATIONS, CAMBRIDGE, GB, vol. 25, no. 9, 10.1146/ANNUREV.ARPLANT.043008.091955 23 August 2007 (2007-08-23), pages 417-424, • EFFENDI LEONARDA ET AL: "Combining XP022210242, ISSN: 0167-7799, DOI: metabolic and protein engineering of a terpenoid 10.1016/J.TIBTECH.2007.07.006 biosynthetic pathway for overproduction and selectivity control", PROCEEDINGS OF THE Remarks: NATIONAL ACADEMY OF SCIENCES OF USA, Thefile contains technical information submitted after NATIONAL ACADEMY OF SCIENCE, the application was filed and not included in this WASHINGTON, DC; US, vol. 107, no. 31, 3 August specification 2010 (2010-08-03), pages 13654-13659, XP002617176, ISSN: 0027-8424, DOI: 10.1073/PNAS.1006138107 [retrieved on 2010-07-19] 2 EP 2 645 847 B1 Description Field of the Invention 5 [0001] The invention relates to the production of steviol in E. coli through genetic engineering. Background of the Invention [0002] US 2010/0297722 provides a transgenic moss cell that produces or accumulates a terpenoid compound and 10 methods of producing a terpenoid compound through culturing of the transgenic moss. Ajikumar et al. (Science. 2010 Oct 1;330(6000):70-4) discloses isoprenoid pathway optimization for taxol precursor overproduction in E. coli. [0003] Steviol glycosides are natural constituents of the plant Stevia rebaudiana Bertoni, referred to as Stevia. Stevia is native to the Amambay region of Northeastern Paraguay and has been reported to grow in neighboring parts of Brazil and Argentina. Although Stevia continues to be a rare plant in its native habitat, it is now farmed in South America and 15 Asia. Stevia leaves have been used to sweeten beverages and make tea. In addition, the leaves are also used for their medicinal benefits in high blood pressure, obesity, topical dressing of wounds and other skin disorders (1). [0004] The crushed Stevia leaves are about 30 times sweeter than sugar (2). The sweet tasting components of the Stevia plant are called steviol glycosides. Steviol glycosides are obtained from the leaves of Stevia rebaudiana Bertoni. The leaves are processed with hot water and aqueous extraction to concentrate and purify the steviol glycosides. The 20 final product may be spray dried. Steviol glycosides preparations are available as white or slightly yellowish white crystalline odorless soluble powders. Summary of the Invention 25 [0005] The current production of steviol glycoside sweeteners solely relies on cultivation of the plantStevia and extraction of steviol glycosides from the plant, which yields variable mixtures with undesirable taste profiles, and the yield is severely limited by cultivation and extraction procedures. A promising solution to this problem is to engineer fast growing microorganisms such as bacteria and yeast to synthesize steviol glycosides or its precursor molecule steviol that can be chemically converted to steviol glycosides through established inexpensive methods. 30 The present invention relates to a method for the synthesis of steviol comprising recombinantly expressing a copalyl diphosphate synthase (CPS), a kaurene synthase (KS), and a geranylgeranyl diphosphate synthase (GGPPS) as a first expression module and a Stevia rebaudiana kaurene oxidase (KO) and a Stevia rebaudiana kaurenoic acid 13-hydrox- ylase (KAH) as a second expression module in an E. coli cell that expresses an endogenous non-mevalonate pathway (MEP) and overexpresses the dxs, idi, ispD, and ispF genes of said pathway, wherein the KO enzyme and/or the KAH 35 enzyme are truncated to remove all or part of the N-terminal transmembrane region and/or are expressed as fusion proteins with a cytochrome P450 reductase (CPR); and culturing the E. coli in a culture medium to produce steviol. [0006] In some embodiments, the copalyl diphosphate synthase (CPS) enzyme is a Stevia enzyme such as a Stevia rebaudiana Bertoni enzyme. In some embodiments, the kaurene synthase (KS) enzyme is a Stevia enzyme such as a Stevia rebaudiana Bertoni enzyme. In some embodiments, the GGPPS enzyme is aTaxus enzyme such as a Taxus 40 canadenis enzyme or Stevia enzyme such as a Stevia rebaudiana Bertoni enzyme. In some embodiments, the gene encoding the copalyl diphosphate synthase (CPS) enzyme and/or the gene encoding the kaurene synthase (KS) enzyme and/or the gene encoding the GGPPS enzyme and/or the genes encoding the one or more components of the MEP pathway is/are expressed from one or more plasmids. In some embodiments, the gene encoding the copalyl diphosphate synthase (CPS) enzyme and/or the gene encoding the kaurene synthase (KS) enzyme and/or the gene encoding the 45 GGPPS enzyme and/or the genes encoding the one or more components of the MEP pathway is/are incorporated into the genome of the cell. [0007] In some embodiments, one or more overexpressed components of the non-mevalonate (MEP) pathway are selected from dxs, ispC, ispD, ispE, ispF, ispG, ispH, idi, ispA and ispB. In certain embodiments, dxs, idi, ispD and ispF are overexpressed in the cell. For example, dxs, idi, ispD and ispF can be expressed or overexpressed on the operon 50 dxs-idi-iSpDF, or ispC, ispE, ispG and ispH can be expressed or overexpressed on the operon ispC-ispE-ispG-ispH. In some embodiments, the gene encoding the copalyl diphosphate synthase (CPS) enzyme, the gene encoding the kaurene synthase (KS) enzyme and the gene encoding the GGPPS enzyme are expressed together on an operon. In some embodiments, the operon is KS-CPS-GGPPS. [0008] In some embodiments, the gene encoding the kaurene oxidase (KO) synthase enzyme, and/or, the gene 55 encoding the kaurenoic acid 13-hydroxylase (KAH) enzyme is expressed from one or more plasmids. In some embod- iments, the gene encoding the kaurene oxidase (KO) synthase and/or the gene encoding the kaurenoic acid 13-hydrox- ylase (KAH) enzyme is incorporated into the genome of the cell. [0009] Insome embodiments, thecell further expresses one ormore UDP-glycosyltransferases (UGTs) or a catalytically 3 EP 2 645 847 B1 active portion thereof. In some embodiments, the UDP-glycosyltransferase (UGT) enzyme(s) is a Stevia enzyme such as a Stevia rebaudiana Bertoni enzyme. In some embodiments, the gene encoding for one or more of the UDP-glyco- syltransferases (UGTs) or a catalytically active portion are expressed together on an operon. In some embodiments, the gene encoding for the UDP-glycosyltransferases (UGTs) or a catalytically active portion is expressed from one or 5 more plasmids. In some embodiments, the gene encoding for the UDP-glycosyltransferases (UGTs) or a catalytically active portion is incorporated into the genome of the cell. [0010] The expression of the copalyl diphosphate synthase (CPS), kaurene synthase (KS), a geranylgeranyl diphos- phate synthase (GGPPS) enzyme, and the one or more components of the MEP pathway can be balanced to maximize production of kaurene.