US009359.622B2

(12) United States Patent (10) Patent No.: US 9,359,622 B2 Hilmer et al. (45) Date of Patent: Jun. 7, 2016

(54) METHOD FOR BOTECHNOLOGICAL (56) References Cited PRODUCTION OF DIHYDROCHALCONES U.S. PATENT DOCUMENTS

(71) Applicant: Symrise AG, Holzminden (DE) 2005/02O8643 A1 9, 2005 Schmidt-Dannert et al. (72) Inventors: Jens Michael Hilmer, Holzminden OTHER PUBLICATIONS (DE); Egon Gross, Holzminden (DE); Chaparro-Riggers et al., Comparison of Three Enoate Reductases Gerhard Krammer, Holzminden (DE); and their Potential Use for Biotransformations, Adv. Synth. Catal. Jakob Peter Ley, Holzminden (DE); 2007, 349, 1521-31.* Mechthild Gall, Greifswald (DE); Uwe Ngaki et al., Evolution of the chalcone- fold from fatty-acid Bornscheuer, Griefswald (DE); Maren binding to stereospecific catalysis, Nature, May 2012, 485, 530-33 Thomsen, Greifswald (DE); Christin and Supplemental Information.* Peters, Greifswald (DE); Patrick Baldocket al., A Mechanism of Drug Action Revealed by Structural Jonczyk, Hannover (DE); Sascha Studies of Enoyl Reductase, Science, 1996, 274, 2107-10.* Uniprot, Accession No. V9P0A9, 2014. www.uniprot.org.* Beutel, Hannover (DE); Thomas Uniprot, Accession No. V9P074, 2014. www.uniprot.org.* Scheper, Hannover (DE) European Search Report dated Sep. 30, 2013. Schmidt, et al., “Biocatalytic Formation of a Bioactive (73) Assignee: SYMRISE AG, Holzminden (DE) Dihydrochalcone by Eubacterium Ramulus,” Journal of Biotechnol ogy, Elsevier Science Publishers, Amsterdam, NL, B.D. 150, Nov. 1, (*) Notice: Subject to any disclaimer, the term of this 2010, p. 150, XPO27489759. patent is extended or adjusted under 35 Schneider, et al., “Anaerobic Degradation of Flavonoids by U.S.C. 154(b) by 48 days. Eubacterium Ramulus.” Archives of Microbiology, Springer, DE, Bd. 173, Nr. 1, Jan. 1, 2000, pp. 71-75, XP008115183. (21) Appl. No.: 13/954,957 Herles, et al., “First Bacterial Chalcone Isomerase isolated from Eubacterium Ramulus.” Archives of Microbiology, Bd. 181, Nr. 6. (22) Filed: Jul. 30, 2013 Jun. 2004, pp. 428-434, XP002713878. Hwang, et al., “Production of Plant-Specific Flavonones by Prior Publication Data Escherichia coli Containing an Artificial Gene Cluster.” Applied and (65) Environmental Microbiology, American Society for Microbiology, US 2014/OO45233 A1 Feb. 13, 2014 US, Bd. 69, Nr. 5, May 1, 2003, pp. 2699-2706, XP008117399. Databse UniProt, Feb. 1, 1995, “RecName: Full=Chalcone (30) Foreign Application Priority Data flavonone isomerase 1: Short=Chalcone isomerase 1; EC=5.5.1.6; AltName: Full=Protein Transparent Testa 5.” XP002713879. Jul. 31, 2012 (DE) ...... 10 2012 213 492 * cited by examiner (51) Int. C. CI2P 7/26 (2006.01) Primary Examiner — Robert Mondesi CI2N L/21 (2006.01) Assistant Examiner — Todd M Epstein CI2N 9/02 (2006.01) (74) Attorney, Agent, or Firm — Polsinelli PC CI2N 9/90 (2006.01) CI2N 15/53 (2006.01) (57) ABSTRACT CI2N I5/6 (2006.01) A method for production of a dihydrochalcone, especially of CI2N 15/63 (2006.01) phloretin, using a transgenic microorganism, containing a CI2R I/OI (2006.01) nucleic acid section (a), comprising or consisting of a gene CI2R L/19 (2006.01) coding for a bacterial chalcone isomerase, and/or a nucleic CI2R L/645 (2006.01) acid section (a'), comprising or consisting of a gene coding for CI2R L/865 (2006.01) a plant chalcone isomerase, and a nucleic acid section (b). CI2R L/84 (2006.01) comprising or consisting of a gene coding for a bacterial (52) U.S. C. enoate reductase, corresponding transgenic microorganisms, CPC. CI2P 7/26 (2013.01): CI2N 9/001 (2013.01); containing a nucleic acid section (a), comprising or consisting CI2N 9/90 (2013.01); CI2R I/01 (2013.01); of a gene coding for a bacterial chalcone isomerase, and/or a CI2R 1/19 (2013.01): CI2R I/645 (2013.01); nucleic acid section (a'), comprising or consisting of a gene CI2R 1/84 (2013.01): CI2R 1/865 (2013.01); coding for a plant chalcone isomerase, and/or a nucleic acid CI2Y 103/0.1031 (2013.01); C12Y505/01006 section (b), comprising or consisting of a gene coding for a (2013.01); Y02P 20/52 (2015.11) bacterial enoate reductase, and host cells, containing one or (58) Field of Classification Search more identical or different such vectors. None See application file for complete search history. 39 Claims, 6 Drawing Sheets U.S. Patent Jun. 7, 2016 Sheet 1 of 6 US 9,359,622 B2

Figure U.S. Patent Jun. 7, 2016 Sheet 2 of 6 US 9,359,622 B2

::::::: 38:8; 3. of 3:38.

Figure 2 U.S. Patent Jun. 7, 2016 Sheet 3 of 6 US 9,359,622 B2

3arri (98)

sERED pE-238(+)

340

Figure 3 U.S. Patent Jun. 7, 2016 Sheet 4 of 6 US 9,359,622 B2

388x3

Figure 4 U.S. Patent Jun. 7, 2016 Sheet 5 of 6 US 9,359,622 B2

R. EAE - C - Resource ( R EAE - C - Resote: { extak G F Pic

SOS-Page der Affeinigung SOS-Page der Aufreinigung {gefärbi mit Coomassie Bluie igefartt imit Siber)

Figure 5 U.S. Patent Jun. 7, 2016 Sheet 6 of 6 US 9,359,622 B2

-8- -- HPP-8- Phloretin-8- OD :400 16 40

350 i - 4-a-a-? - -8-Y. 4 35

300 2 30 s E. a > 250 10 25 s

st w s : 5 200 0.8 2O s & S. 8x S 50 06 S2 15 a. S s wr 2. 2 OO O4 C O s s h SO 0.2 5

() O,t) )

Zeith

Figure 6 US 9,359,622 B2 1. 2 METHOD FOR BOTECHNOLOGICAL Further aspects of the present invention and preferred con PRODUCTION OF DIHYDROCHALCONES figurations thereof can be seen from the following descrip tion, the exemplary embodiments and the claims. CROSS-REFERENCE TO RELATED APPLICATIONS BRIEF DESCRIPTION OF THE DRAWINGS This application claims the benefit of DE Patent Applica FIG. 1: Plasmid pET52b EREDstrep for heterologous tion Serial No. 10 2012 213 492.1, filed on 31 Jul. 2012, the expression and characterization of the ERED from E. ramu benefit of the earlier filing date of which is hereby claimed lus DSM 16296; under 35 USC S119(a)-(d) and (f). The application is hereby FIG. 2: Plasmid plT28b CHI for heterologous expression incorporated in its entirety as if fully set forth herein. 10 and characterization of the CHI from E. ramulus DSM 16296; FIG. 3: Plasmid pET22b ERED for heterologous expres SUBMISSION OF SEQUENCE LISTING sion and characterization of the ERED from E. ramulus DSM 16296; The Sequence Listing associated with this application is FIG. 4 is a graph showing lines representing without cell filed in electronic format via EFS-Web and hereby incorpo 15 extract, with cell extract E. coli Rosetta (without plasmid), rated by reference into the specification in its entirety. The and with cell extract E. coli Rosetta (with pBT28 HI); name of the text file containing the Sequence Listing is Sub FIG. 5 provides examples of the results of the individual stitute Sequence Listing 34430 7. The size of the text file is purification steps following expression of the vector 22 KB, and the text file was created on Dec. 3, 2015. pET28b CHI in E. coli; and FIG. 6 is a graph of Naringenin/HPP/Concentration uM BACKGROUND OF THE INVENTION against Phloretin concentration uM over time h. 1. Field of the Invention DETAILED DESCRIPTION OF THE PREFERRED The present invention primarily concerns a method for EMBODIMENTS production of a dihydrochalcone, especially of phloretin, or a 25 To facilitate an understanding of the principles and features method for reduction of using a transgenic micro of the various embodiments of the invention, various illustra organism, containing a nucleic acid section (a), comprising or tive embodiments are explained below. Although exemplary consisting of a gene coding for a bacterial chalcone embodiments of the invention are explained in detail, it is to isomerase, and/or a nucleic acid section (a'), comprising or be understood that other embodiments are contemplated. consisting of a gene coding for a plant chalcone isomerase, 30 Accordingly, it is not intended that the invention is limited in and a nucleic acid section (b), comprising or consisting of a its scope to the details of construction and arrangement of gene coding for a bacterial enoate reductase. components set forth in the following description or illus 2. Description of Related Art trated in the drawings. The invention is capable of other Dihydrochalcones, especially phloretin, are normally pro embodiments and of being practiced or carried out in various duced either by chemical reduction of chalcones or by 35 ways. Also, in describing the exemplary embodiments, spe Friedel–Crafts acylation of phenols with dihydrocinammic cific terminology will be resorted to for the sake of clarity. acids. The disadvantage of this method is that food additives, It must also be noted that, as used in the specification and flavourings or aromatic Substances produced in this way can the appended claims, the singular forms “a,” “an and “the not be described as natural. In addition, dihydrochalcones, include plural references unless the context clearly dictates especially phloretin, can be obtained by extraction of for 40 otherwise. For example, reference to a component is intended example the corresponding (E.g. from Malus spp. also to include composition of a plurality of components. raw materials) with Subsequent generation of the aglycone. References to a composition containing “a” constituent is This process is time-consuming and cost-intensive, however, intended to include other constituents in addition to the one named. and is also dependent on the season. Also, in describing the exemplary embodiments, terminol Important flavourings and aromatic Substances with a dihy 45 ogy will be resorted to for the sake of clarity. It is intended that drochalcone structure are for example phloretin (E.g. accord each term contemplates its broadest meaning as understood ing EP 1998,636-B1), phloridzin, trilobtain (see Tanaka, T.; by those skilled in the art and includes all technical equiva Yamasaki, K. Kohda, H.; Tanaka, O.; Mahato, S. B., Dihy lents which operate in a similar manner to accomplish a drochalcone-glucosides as Sweet principles of Symplocos similar purpose. ssp. Planta Medica 1980, (Suppl.), 81-83), dihydro 50 Ranges may be expressed herein as from “about or chalcone and neohesperidine dihydrochalcone (Crosby, G. “approximately” or “substantially one particular value and/ A., New Sweeteners. Critical Reviews in Food Science and or to “about' or “approximately or “substantially' another Nutrition 1976, (June), 297-323). particular value. When such a range is expressed, other exem Due to the existing and also the future need for advanta plary embodiments include from the one particular value geous dihydrochalcones, especially for phloretin, the primary 55 and/or to the other particular value. problem for the present invention was to provide an efficient Similarly, as used herein, “substantially free” of some and preferably cost-effective that can be used on an industrial thing, or 'substantially pure', and like characterizations, can scale for producing dihydrochalcones, especially phloretin. include both being “at least substantially free” of something, A further problem was to provide suitable or necessary or “at least substantially pure', and being “completely free” means for performing Such a method. 60 of something, or "completely pure'. Further problems for the present invention are apparent By “comprising or “containing” or “including is meant from the following description and especially the attached that at least the named compound, element, particle, or claims. method step is present in the composition or article or These and other objects, features, and advantages of the method, but does not exclude the presence of other com present invention will become more apparent upon reading 65 pounds, materials, particles, method steps, even if the other the following specification in conjunction with the accompa Such compounds, material, particles, method steps have the nying drawing figures. same function as what is named. US 9,359,622 B2 3 4 It is also to be understood that the mention of one or more The primary problem for the present invention is solved by method steps does not preclude the presence of additional an innovative biotechnological method for producing a dihy method steps or intervening method steps between those drochalcone, especially phloretin, using a transgenic micro steps expressly identified. Similarly, it is also to be under organism, comprising the following steps: stood that the mention of one or more components in a com 5 position does not preclude the presence of additional compo (i) Providing a transgenic microorganism, containing (as nents than those expressly identified. the transgene) The materials described as making up the various elements a nucleic acid section (a), comprising or consisting of a of the invention are intended to be illustrative and not restric gene coding for a bacterial chalcone isomerase, tive. Many suitable materials that would perform the same or and/or a nucleic acid section (a'), comprising or consist a similar function as the materials described herein are 10 ing of a gene coding for a plant chalcone isomerase, intended to be embraced within the scope of the invention. and Such other materials not described hereincan include, but are a nucleic acid section (b), comprising or consisting of a not limited to, for example, materials that are developed after gene coding for a bacterial enoate reductase. the time of the development of the invention. (ii) Adding one or more flavanones, especially adding nar The present invention primarily concerns a method for 15 ingin, and/or one or more one or more precursors or one production of a dihydrochalcone, especially of phloretin, or a or more derivatives thereof, especially a precursor or a method for reduction of flavanones using a transgenic micro derivative of naringin, to the transgenic microorganism organism, containing a nucleic acid section (a), comprising or and cultivation of the transgenic microorganism under consisting of a gene coding for a bacterial chalcone conditions which allow the conversion of the flava isomerase, and/or a nucleic acid section (a'), comprising or none(s) and/or precursor(s) or derivative(s) thereof, consisting of a gene coding for a plant chalcone isomerase, especially maringin and/or the precursor or derivative of and a nucleic acid section (b), comprising or consisting of a naringin, into a dihydrochalcone, especially into phlo gene coding for a bacterial enoate reductase. retin. The present invention further concerns a transgenic micro (iii) Optionally: isolating and if necessary purifying the organism, containing a nucleic acid section (a), comprising or 25 dihydrochalcone, especially phloretin. consisting of a gene coding for a bacterial chalcone The or one, more or all the flavanones or precursors or isomerase, and/or a nucleic acid section (a'), comprising or derivatives thereof to be used according to the invention are consisting of a gene coding for a plant chalcone isomerase, preferably selected from the group consisting of: and a nucleic acid section (b), comprising or consisting of a naringenin, maringin, , or other maringenin glyco gene coding for a bacterial enoate reductase. 30 sides, , , , hesperetin-7-O- The present invention also concerns a vector, especially a glucoside, or other hesperetin , . erio plasmid vector, containing a nucleic acid section (a), com citrin, or other eriodictyol glycoside, Sterubin, Sterubin prising or consisting of a gene coding for a bacterial chalcone glycoside, , Sakuranetinglycosides, isosakurane isomerase, and/or a nucleic acid section (a), comprising or tin, glycosides, 4',7-dihydroxy- or consisting of a gene coding for a plant chalcone isomerase, 35 glycosides thereof, 4,7-dihydroxy-3-methoxy-flavanone or and/or a nucleic acid section (b), comprising or consisting of glycosides thereof, 3',7-dihydroxy-4-methoxy-flavanone or a gene coding for a bacterial enoate reductase. glycosides thereof, 3',4',7-trihydroxy-flavanone or glyco In addition, the present invention concerns a host cell, sides thereof, wherein the flavanones with regard to the 2-po containing one or more identical or different vectors accord sition of the flavanone structure can be present as (S)-, as ing to the invention. 40 (R)-enantiomer, as racemate or as any mixture of the two Further aspects of the invention are apparent from the enantiomers. following description, the examples and especially the In the following examples are provided of a number of attached claims. flavanones that are used by preference:

OH O OH O

O O beta-D-Glucopyranosid OH OH Narirutin (2-O-alpha-L-rhamnosyl-beta-D-glucosid) Naringenin Naringin

OH O OH O OH O

OH OH O O OH HO O O O O rC o1 o1 o1 (6-O-alpha-L-rhamnosyl-beta-D-glucosid) (2-O-alpha-L-rhamnosyl-beta-D-glucosid) Hesperetin Hesperidin Neohesperidin US 9,359,622 B2

-continued

OH O OH O OH O

OH OH O O O OH HO O o1 solo OH OH (6-O-alpha-L-rhamnosyl-beta-D-glucosid) Sterubin Eriodictyol

OH O OH O

No O OH colo OH 4,7-Dihydroxy-flavanon Sakuranetin Isosakura.netin

O O O 4,7-Dihydroxy-flavanon

HO OH O OH HO O HO O o1 OH 4,7-Dihydroxy-3'-methoxyflavanon 34.7-Trihydroxy-flavanon 3,7-Dihydroxy-4-methoxyflavanon Naringenin (2-O-alpha-L-rhamnosyl-beta-D-glucoside) beta-D-glucopyranoside Naringin Narirutin Hesperitin (6-O-alpha-L-rhamnosyl-beta-D-glucoside) (2-O-alpha-L-rhamnosyl-beta-D-glucoside) Hesperidin Neohesperidin Eriodictyol (6-O-alpha-L-rhamnosyl-beta-D-glucoside) Sterubin Eriocitrin Sakuranetin IsoSakuranetin 4',7-dihydroxy-flavanone 4,7-dihydroxy-flavanone 3',7-dihydroxy-4'-methoxyflavanone 3'47-trihydroxyflavanone 4',7-dihydroxy-3'-methoxyflavanone

The dihydrochalcone to be produced according to the 45 alcone, Sakuranetin dihydrochalcone glycosides, isos invention is preferably selected from the group consisting of akuranetin dihydrochalcone, isosakuranetin dihydrochal phloretin, maringin dihydrochalcone, phloridzin or other CO glycosides, 2',4',4-trihydroxydihydrochalcone phloretin-glycosides, hesperetin dihydrochalcone, hesperi (davidigenin) or glycosides thereof, 3-methoxy-2',4',4-trihy din dihydrochalcone, neohesperidine dihydrochalcone, or droxydihydrochalcone or glycosides thereof 4-methoxy-2", other hesperetin dihydrochalcone glycosides, eriodictyol 50 3,4'-trihydroxydihydrochalcone or glycosides thereofor 24, dihydrochalcone (3-hydroxyphloretin), or other eriodictyol 4,3-tetrahydroxydihydrochalcone or glycosides thereof. dihydrochalcone glycosides, Sterubin dihydrochalcone, In the following examples are provided of preferred dihy Sterubin dihydrochalcone glycoside, Sakuranetin dihydroch drochalcones:

OH OH O

OH O OH

Phloretin OH beta-D-Glucopyranosid (2-O-alpha-L-rhamnosyl-beta-D-glucosid) Phloridzin Naringindihydrochalkon US 9,359,622 B2

-continued

OH O OH O OH O

OH HO O OH OH OH O OH O OH o1 o1 Hesperetindihydrochalkon o1 (2-O-alpha-L-rhamnosyl-beta-D-glucosid) (6-O-alpha-L-rhamnosyl-beta-D-glucosid) Neohesperidindihydrochalkon Hesperidindihydrochalkon

OH O OH O OH O

OH OH OH HO OH O OH solo OH OH o1 Sterubindihydrochalkon 3-Hydroxyphloretin (6-O-alpha-L-rhamnosyl-beta-D-glucosid) Eriocitrindihydrochalkon

OH O OH O O

solo OH colo OH Sakuranetindihydrochalkon OH Davidigenin coolIsosakuranetiindihydrochalkon

O O

OH OH HO OH HO OH O HO OH o1 OH OH 3-Methoxy-2',4',4-trihydroxydihydrochalkon 4-Methoxy-2,3,4'-trihydroxydihydrochalkon 2'44".3-Tetrahydroxydihydrochalkon

Phloretin (2-O-alpha-L-rhamnosyl-beta-D-glucoside) beta-D-glucopyranoside Naringin dihydrochalcone Phloridzin Hesperitin dihydrochalcone (6-O-alpha-L-rhamnosyl-beta-D-glucoside) (2-O-alpha-L-rhamnosyl-beta-D-glucoside) Hesperidin dihydrochalcone Neohesperidin dihydrochalcone 3-hydroxyphloretin (6-O-alpha-L-rhamnosyl-beta-D-glucoside) Sterubin dihydrochalcone Eriocitrin dihydrochalcone Sakuranetin IsoSakura.netin dihydrochalcone Davidgenin dihydrochalcone Dihydrochalcone, Dihydrochalcone, Dihydrochalcone, 2-4,4',3-tetrahydroxy 3-methoxy-2',4',4-trihydroxy 4-methoxy-2',3'4-trihydroxy

Especially preferred flavanones and the respective dihy- 55 using a bacterial chalcone isomerase (especially preferably drochalcones formed from these are: naringenin and phlore from microorganisms as described further below) and/or a tin, naringin and naringin dihydrochalcone, narirutin and plant chalcone isomerase (especially preferably from plants phloridzin, hesperetin and hesperetin dihydrochalcone, hes as described further below) in combination with a bacterial peridin and hesperidin dihydrochalcone, neohesperidin and enoate reductase (preferably from microorganisms as neohesperidine dihydrochalcone, and eriodictyol and 3-hy 60 described below) in a transgenic microorganism. droxyphloretin. A preferred embodiment concerns a biotechnological A first aspect of the present invention accordingly concerns method for production of (natural) phloretin, starting from a biotechnological method for production of (natural) dihy naringin and/or a precursor or a derivative of naringin, espe drochalcones, especially of (natural) phloretin, starting from cially of maringin, narirutin or maringenin, using one or more one or more corresponding flavanones and/or a precursor or a 65 chalcone (as described herein) in combination derivative thereof, especially ofnaringin and/or a precursor or with an enoate reductase (as described herein) using a trans a derivative of naringin, especially of naringin or maringenin, genic microorganism (as similarly described herein). US 9,359,622 B2 9 10 An especially preferred embodiment concerns a biotech yeasts (E. g. saccharomyces). To date there has been little nological method for the production of (natural) phloretin, research into enzymatic ether splitting, however. starting from naringin and/or a precursor or a derivative of In connection with the present invention, reference is basi naringin, especially of naringin, narirutin or maringenin, cally made to the following publications: Schoefer et al. using a bacterial chalcone isomerase (preferably from the Anaerobic Degradation of Flavonoids by Clostridium Orbis anaerobic organism Eubacterium ramulus, as described fur cindens, Appl. Environ. Microbiol., October 2003, p. 5849 ther below) in combination with plant chalcone isomerase 5854; and Herles et al, First bacterial chalcone isomerase (preferably from Arabidopsis thaliana or Medicago sativa, as isolated from Eubacterium ramulus, Arch Microbiol (2004) described further below) and a bacterial enoate reductase 181: 428-434. Findings in connection with the degradation of (similarly preferably from the anaerobic organism Eubacte 10 ligninhave for example been described by Masaietal. (Masai rium ramulus, as described further below) using a transgenic et al., 1993; Otsuka et al., 2003; see also JP 2002034557). microorganism (as described herein). In WO 200601.0117 from Koffas et al. and WO In the state of the art it was known that the anaerobic 2005084305 from Schmidt-Dannert et al. the application of microorganism Eubacterium ramulus is able to degrade nar heterologous expression for the formation of flavonoids is ingenin, wherein intermediate phloretin is formed. However, 15 described. In these (exclusively) plant genes are described, this is not understood to be a (biotechnological) method for which can be used for heterologous expression of various production of phloretin within the meaning of the present Substances (starting from L-phenylalanine, tyrosine and cin invention, especially not a method (as described above), Suit namic acid). able for the industrial production of phloretin, E.g. for pro Surprisingly, by means of the genome sequence of Eubac duction of phloretin on an industrial scale. For the interme terium ramulus the gene coding (a) for a chalcone isomerase diate phloretin formed is immediately further metabolised and (b) for an enoate reductase (for conversion of naringin or into Eubacterium ramulus(see Schneider et al. Anaerobic a precursor a derivative of naringin to phloretin; see on this degradation of flavonoids by Eubacterium ramulus, Arch point the reaction diagram shown above) could be identified Microbiol (2000) 173: 71-75), as shown in the following and thereupon expressed in transgenic microorganisms. The reaction diagram, (see especially the reaction brought about 25 heterologous expression of this in a transgenic by the phloretin (PhH)): microorganism is able to advantageously avoid or circumvent

OH OH CHI HO OH O 2H) HO OH O -- Reduktase

OH O OH O OH O Naringenin Naringeninchalcon Phloretin pit O HO OH

HO OH 3-(4-Hydroxyphenyl)propansâure Phloroglucinol HPP

Naringinin 50 the secondary reaction that normally takes place in E. ramu Naringinin chalcone lus of phloretin to phloroglucinol and 3-(4-hydroxyphenyl) Reductase propanic acid (HPP) through the phloretin hydrolase (on this Phloretin point see the reaction diagram shown above), in order ulti hEH mately to allow the production of phloretin on an industrial p (4-hydroxyphenyl)propanic acid HPP scale or a significant increase in yield. Ph lucinol 55 A “transgenic microorganism' in connection with the orogluc1no present invention is understood to be a genetically engineered In the context of the investigations in connection with the or modified microorganism, in which specifically through present invention it was possible for the purposes of the biotechnological methods nucleic acid sections (see nucleic method described herein to clarify and characterize crucial acid sections (a) and (b) as described herein) or genes are molecular biological and biochemical principles of the 60 introduced into another organism (so-called transgene). biotransformation, for the purposes of production of phlore A “chalcone isomerase” (CHI) within the meaning of the tin on an industrial scale (without immediate degrading of the present invention is an enzyme that catalyses the phloretin formed). “chalconegsflavanone' reaction. CHI especially catalyses In the state of the art a number of different possibilities for the reaction of to maringenin to/of naringenin chalcone (see use of enzyme systems and methods of microbial biotrans 65 the reaction diagram shown above), for the purposes of the formation are indeed described; for example, it is known to be present invention especially the reaction of naringenin to possible for double bonds to be simply reduced by means of naringenin chalcone. US 9,359,622 B2 11 12 An “enoate reductase' (ERED) within the meaning of the more for SEQID NO:6 or SEQID NO:7, in particular of 50% present invention is an enzyme, that catalyses the dehydration or more, 60% or more or 80% or more, especially preferably of certain compounds especially the reaction of maringenin of 95% or more, chalcone to phloretin (see the reaction diagram shown above). and/or In view of the relationships explained above, the transgenic microorganism used in connection with the method accord the nucleic acid section (b) is comprised or consists of a ing to the invention is in particular not Eubacterium ramulus, nucleotide sequence according to SEQID NO:2 (nucleotide in particular not a microorganism of the Clostridiales order, sequence of the gene coding for the bacterial ERED from E. more preferably not a microorganism of the Clostridia class, ramulus DSM 16296) or SEQ ID NO:5 (codon optimised especially preferably not a microorganism of the phylum 10 nucleotide sequence of the gene coding for bacterial ERED (section) of Firmicutes. Rather, the microorganism is prefer from E. ramulus DSM 16296, especially for expression in E. ably selected from the group consisting of facultative anaero coli BL21, integrated in pET 22b (clones by means of Ndel bic microorganisms, especially facultative aerobic bacteria, and BamH1)) or a nucleotide sequence with a nucleotide preferably proteobacteria, especially enterobacteria, for sequence identity of 40% or more for SEQID NO:2 or SEQ example of the genus Escherichia, preferably E. coli, espe 15 ID NO:5, in particular of 50% or more, 60% or more or 80% cially E. coli BL21, E. coli Rosetta (derivative of E. coli BL or more, especially preferably of 95% or more. 21) and E. coli SE1, and yeasts, for example S. cerevesiae and Preference according to the invention is further for a P. pastoris. According to a preferred aspect for the purposes method (as described above), wherein of the invention described herein basically those microorgan the bacterial chalcone isomerase is comprised or consists isms are preferred which grow under aerobic conditions and ofan amino acid sequence according to SEQID NO:3 (amino (also) under exclusion of oxygen are able to express the acid sequence of the bacterial CHI from E. ramulus DSM introduced gene (transgene; see above). 16296) or an amino acid sequence with an amino acid Preference according to the invention is for a method (as sequence identity of 40% or more for SEQ ID NO:3, in described above), wherein in step (ii) maringin and/or an 25 particular of 50% or more, 60% or more or 80% or more, aglycone thereof is or are added. especially preferably of 95% or more, Coding in particular takes place as follows the gene coding for a bacterial chalcone isomerase for a and/or chalcone isomerase from a microorganism from the phylum the plant chalcone isomerase is comprised or consists of an Firmicutes, in particular the Clostridia class, especially of the 30 amino acid sequence according to SEQID NO:8 (amino acid Clostridiales order, especially preferably for a chalcone sequence of the chalcone isomerase from Medicago sativa) or isomerase from E. ramulus, SEQ ID NO:9 (amino acid sequence of the chalcone fla and/or Vanone isomerase 1 from Arabidopsis thaliana) or an amino the gene coding for a plant chalcone isomerase for a chal acid sequence with an amino acid sequence identity of 40% or cone isomerase from a plant of the order Brassicales, in 35 particular from the family of Brassicaceae, preferably the more for SEQID NO:8 or SEQID NO:9, in particular of 50% tribe Camelineae, especially the genus Arabidopsis, above all or more, 60% or more or 80% or more, especially preferably of the type Arabidopsis thaliana, or the order Fabales, in of 95% or more, particular the family Fabaceae, preferably the sub-family and/or Faboidae, especially the genus Medicago, above all of the 40 the bacterial enoate reductase is comprised or consists of type Medicago sativa, thus especially preferably for a chal an amino acid sequence according to SEQID NO:4 (amino cone isomerase from A. thaliana or M. Sativa (see on this acid sequence of the bacterial ERED from E. ramulus DSM point WO2005084305 A2 (Schmidt-Dannert) and 16296) or an amino acid sequence with an amino acid WO2006010117A2 (Koffas)), sequence identity of 40% or more for SEQ ID NO:4, in and/or 45 the gene coding for a bacterial enoate reductase for an particular of 50% or more, 60% or more or 80% or more, enoate reductase from a microorganism from the phylum especially preferably of 95% or more. Firmicutes, in particular the class Clostridia, especially the In the context of the present invention the “amino acid order Clostridiales, especially preferably for an enoate reduc sequence identity” is preferably determined using the Water tase from E. ramulus. 50 man-Smith algorithm with a gap open penalty of 10, a gap Especially preferable is a method according to the inven extension penalty of 0.5 and the BLOSUM62 matrix (regard tion (as described herein), wherein ing the Waterman-Smith algorithm, see for example Smith, T. the nucleic acid section (a) is comprised or consists of a F. and Waterman, M. S., Identification of common molecular nucleotide sequence according to SEQID NO: 1 (nucleotide subsequences, Journal of Molecular biology (1981), 147: sequence of the gene coding for the bacterial CHI from E. 55 195-197; implemented online via the corresponding tool page ramulus DSM 16296) or a nucleotide sequence with a nucle of the EMBL). otide sequence identity of 40% or more for SEQID NO:1, in particular of 50% or more, 60% or more or 80% or more, For the purposes of the present invention especially pre especially preferably of 95% or more, ferred is the use of a chalcone isomerase, having one, several and/or 60 or all the following characteristics and/or a temperature and the nucleic acid section (a') is comprised or consists of a pH stability according to Tables 1-3: nucleotide sequence according to SEQID NO:6 (nucleotide sequence of the CHI from M. sativa (cultivar Iroquois) (Ms CHI-1) mRNA, complete cds) or SEQID NO:7 (nucleotide K. Imol/l) V, U/mg k, s' k/K II* mol'*s' sequence of the chalcone flavanone isomerase 1 (TT5) 65 36.83 107.3 416.7 1.13 * 107 mRNA from Arabidopsis thaliana, complete cds) or a nucle otide sequence with a nucleotide sequence identity of 40% or US 9,359,622 B2 13 14 TABLE 1. TABLE 3-continued activity measurements for determination of the of results of the activity measurements for temperature optimum for CHI determination of the pH optimum Temperature Spec. activity Umg Standard deviation Spec. activity Standard pH value Umg deviation RT (23° C.) 158.39 26.19 30° C. 373.47 5.77 6.4 177.59 2.44 37o C. 795.04 45.62 6.55 169.79 1.38 40° C. 887.73 37.95 6.8 185.86 2.46 6.93 175.37 2.47 45° C. 1133.38 76.26 10 7.12 174.86 1.15 50° C. 748.66 37.37 7.45 173.01 1.73 7.7 168.79 1.37 8 152.55 5.97 TABLE 2 15 For the purposes of the present invention preference is for for temperature stability: the use of an enoate reductase, having one, more or all the Spec. activity Standard following characteristics: Temp Timeh Umg deviation protein size of 74.455 kDa RT (23° C.) O 88.56 1.97 expressed both in the soluble and in the insoluble protein O.S 45.67 2.61 fraction after up to 20 hours under anoxic conditions at 1 48.67 6.89 various temperatures. 2 44.05 2.04 4 46.39 2.85 In the following further details preferred according to the 6 41.06 2.64 invention, of a method according to the invention for the 24 25.68 .44 25 production of phloretin are described. 25o C. O 88.56 97 Concerning the provision of a transgenic microorganism O.S 36.70 2.89 1 41.10 25 (as described herein) it should be stated that basically any 2 S2.08 2.61 method familiar to a person skilled in the art can be used, in 4 52.48 71 order to introduce the nucleic acid sections (a), (a") and (b) 6 46.46 38 30 24 16.37 O.22 described herein or the transgenes described herein into the 30° C. O 88.56 97 microorganisms, E. g. basically conjugation, transduction or O.S 69.66 9.66 transformation, in particular by heat-shock treatment, elec 1 S8.01 7.76 troporation, conjugation, gene-gun, the lithium-acetate 2 54.62 3.SO 4 45.77 63 method or transduction. Within the context of the present 35 6 47.18 4.45 invention, however, it is generally preferred for the nucleic 24 1996 .14 acid sections (a), (a") and (b) or the transgenes described to be 37o C. O 88.56 97 introduced by means of a vector, especially a plasmid vector, O.S 55.20 0.44 in particular a vector according to the invention as described 1 64.39 3.68 2 56.17 .87 herein (see below). Methods for this are sufficiently known to 4 56.84 O.38 40 a person skilled in the art. 6 56.21 3.88 A transgenic microorganism within the meaning of the 24 93.93 3.89 41° C. O 88.56 97 present invention can contain one or more copies of the intro O.S S1.96 19 duced nucleic acid sections or the transgenes described 1 S422 O.SO herein. 2 49.12 0.57 45 Methods allowing, on the basis of the introduced nucleic 4 46.59 2.73 acid sections or transgenes, an expression of the desired acid 6 49.39 3.36 24 93.30 2.71 sequences or the desired enzyme, are similarly sufficiently 44° C. O 88.56 1.97 known to a person skilled in the art, E. g. using a regulatory O.S 58.12 O.S2 element, especially a promoter (see on this point also the 1 50.15 O.34 50 attached examples). 2 29.07 0.75 4 15.07 O.24 As described above in step (ii) of a method according to the 6 83.35 6.13 invention one or more flavanones and/or one or more precur 50° C. O 88.56 1.97 sor(s) or derivative(s) thereof are added to the transgenic O.S SO.49 S.61 microorganism, wherein the transgenic microorganism is cul 1 3.05 1.55 55 tivated under conditions which allow conversion of the fla 2 2.35 O.63 vanone(s) and/or the precursor(s) or of the derivative or derivative thereof, especially ofnaringin and/or the precursor or the derivative ofnaringin, to a dihydrochalcone, especially TABLE 3 to phloretin. 60 of results of the activity measurements for As described above in step (ii) of a method according to the determination of the pH optimum invention especially preferably naringin and/or a precursor or a derivative of naringin is added to the transgenic microor Spec. activity Standard ganism, wherein the transgenic microorganism is cultivated pH value Umg deviation under conditions which allow conversion of the naringin and/ 6.13 82.O7 5.97 65 or the precursor or the derivative of maringin to phloretin. 6.35 163.43 S.62 According to a preferred execution of a method according to the invention (as described herein) the (transgenic) micro US 9,359,622 B2 15 16 organisms are initially, that is to say prior to step (ii), culti section (a'), comprising or consisting of a gene coding for a vated under aerobic conditions, in particular in order to plant chalcone isomerase (as the transgene), and a nucleic achieve a maximum biomass concentration. In doing so the acid section (b), comprising or consisting of a gene coding for ODoo should preferably beat least in the range 8-15 or above, a bacterial enoate reductase (as a further transgene). in particular in the range 5-190, especially in the range For the terms employed here, that stated above for these 10-180, preferably in the range 15-170.Then the microorgan same terms applies by analogy. Preferred microorganisms isms in step (ii) are preferably cultivated under anaerobic according to the invention are apparent from the correspond conditions, wherein the expression of the desired amino acid ing statements above in connection with a preferred microor sequences or the desired takes place on the basis of ganism in the context of the method according to the inven the introduced nucleic acid sections or the introduced trans 10 genes, for example stimulated by means of induction by IPTG tion. and/or Lactose (when using a corresponding, Suitable pro The microorganism according to the invention for the pur moter or a corresponding, Suitable expression system). poses of the present invention preferably has at least one Basically it is preferred according to the invention if the chalcone isomerase and one enoate reductase activity, but no incubation in step (ii) takes place at least in part or completely 15 phloretin hydrolase activity. The same applies to microorgan under anaerobic conditions. isms (as described above) to be used by preference in the Depending on the microorganism a person skilled in the art context of a method according to the invention. in step (ii) for the purposes of present invention can create Particularly preferably the microorganism is not a Eubac Suitable ambient conditions and especially provide a Suitable terium ramulus, preferably not a microorganism of the order (cultivation). The cultivation preferably takes place in LB or Clostridiales, more preferably nor not a microorganism of the TB medium. Alternatively a (more complex) medium com class Clostridia, especially preferably not a microorganism of prising or consisting of plant raw materials, especially from the phylum Firmicutes, and is particularly preferably selected citrus, grapefruit and orange plants, can be used. The cultiva from the group comprising facultative anaerobic microorgan tion takes place for example at a temperature of more than 20° isms, especially facultative aerobic bacteria, preferably pro C., preferably of more than 25°C., especially of more than 25 teobacteria, especially enterobacteria, for example of the 30°C. (preferably in the range 30-40°C.), which can espe genus Escherichia, preferably E. coli, especially E. coli cially favour the phloretin formation or increase the yield. Rosetta, E. coli BL21 and E. coli SE1, and yeasts, for example Furthermore, an induction temperature (see above) of less S. cerevesiae and P. pastoris. Otherwise that stated above for than 40°C., especially of less than 35°C. (preferably in the microorganisms to be used by preference in the context of a range 20-30°C.), can favour phloretin formation or increase 30 method according to the invention applies by analogy. the yield. Accordingly, a microorganism according to the invention Naringin or the precursors or derivatives thereof in relation is especially preferred wherein to the (cultivation) medium, containing the transgenic micro the gene coding for a bacterial chalcone isomerase codes organisms, will be added in step (ii) in particular in a quantity for a chalcone isomerase from a microorganism from the of 0.1 mM-100 mM (mMol/L), preferably of 0.5-95 mM, 35 phylum Firmicutes, in particular the class Clostridia, espe especially preferably of 1-90 mM, to the transgenic microor cially the order Clostridiales, especially preferably for a chal ganism. Here Suitable (co-)solvents can be used. cone isomerase from E. ramulus, If for induction (E.g. of the lac operon) one or more and/or suitable inductors, E. g. IPTG or lactose, are used (see above), the gene coding for a plant chalcone isomerase codes for a it is preferred that the inductor in relation to the (cultivation-) 40 chalcone isomerase from A. thaliana or M. Sativa (for other medium, containing the transgenic microorganisms, is used preferred sources see above), in step (ii) in a quantity of 0.001-1 mM, preferably of 0.005 and/or 0.9 mM, especially preferably of 0.01-0.8 mM, since in so the gene coding for a bacterial enoate reductase codes for doing particularly good yields can be achieved. an enoate reductase from a microorganism from the phylum Concerning the optional isolation and possible purification 45 Firmicutes, in particular the class Clostridia, especially from of phloretin: Here, for example, extractions can be carried out the order Clostridiales, especially preferably for an enoate with organic solvents (preferably selected from the following reductase from E. ramulus. list: isobutane, 2-propanol, toluene, methyl acetate, cyclo It is further preferred if hexane, 2-butanol, hexane, 1-propanol, light petroleum, 1.1, the nucleic acid section (a) comprises or consists of a 1.2-tetrafluorethane, methanol, propane, 1-butanol, butane, 50 nucleotide sequence according to SEQID NO:1 or a nucle ethyl methyl ketone, ethyl acetate, diethyl ether, ethanol, otide sequence with a nucleotide sequence identity of 40% or dibutyl ether, CO, tert. butyl methyl ether, acetone, dichlo more for SEQID NO:1, in particular of 50% or more, 60% or romethane and N2O), especially preferably those which with more or 80% or more, especially preferably of 95% or more, water develop a visibly discernible phase boundary. Then the and/or removal of the residual water in the solvent and the removal of 55 the nucleic acid section (a') comprises or consists of a the solvent itself are possible, followed in turn by re-dissolu nucleotide sequence according to SEQID NO:6 or SEQ ID tion of the (for example) phloretin in a (possibly other) sol NO:7 or a nucleotide sequence with a nucleotide sequence vent, which is suitable for a possible Subsequent crystallisa identity of 40% or more for SEQID NO:6 or SEQID NO:7, tion and drying of the product. Alternatively or additionally a in particular of 50% or more, 60% or more or 80% or more, purification by adsorption, distillation and/or chromatogra 60 especially preferably of 95% or more, phy can take place. and/or Further details of the method according to the invention are the nucleic acid section (b) comprises or consists of a apparent from the attached examples. nucleotide sequence according to SEQID NO:2 or SEQ ID A further aspect of the present invention concerns a trans NO:5 or a nucleotide sequence with a nucleotide sequence genic microorganism, containing a nucleic acid section (a), 65 identity of 40% or more for SEQID NO:2 or SEQID NO:5, comprising or consisting of a gene coding for a bacterial in particular of 50% or more, 60% or more or 80% or more, chalcone isomerase (as the transgene), and/or a nucleic acid especially preferably of 95% or more. US 9,359,622 B2 17 18 It is also preferable if It is especially preferable if the bacterial chalcone isomerase comprises or consists of the nucleic acid section (a) comprises or consists of a an amino acid sequence according to SEQ ID NO:3 or an nucleotide sequence according to SEQID NO:1 or a nucle amino acid sequence with an amino acid sequence identity of otide sequence with a nucleotide sequence identity of 40% or 40% or more for SEQID NO:3, in particular of 50% or more, 5 more for SEQID NO:1, in particular of 50% or more, 60% or 60% or more or 80% or more, especially preferably of 95% or more or 80% or more, especially preferably of 95% or more, more, and/or and/or the nucleic acid section (a') comprises or consists of nucle the plant chalcone isomerase comprises or consists of an otide sequence according to SEQID NO:6 or SEQID NO:7 amino acid sequence according to SEQID NO:8 or SEQID 10 or a nucleotide sequence with a nucleotide sequence identity NO:9 oranamino acid sequence with an amino acid sequence of 40% or more for SEQ ID NO:6 or SEQ ID NO:7, in identity of 40% or more for SEQID NO:8 or SEQID NO:9, particular of 50% or more, 60% or more or 80% or more, in particular of 50% or more, 60% or more or 80% or more, especially preferably of 95% or more, especially preferably of 95% or more, 15 and/or and/or the nucleic acid section (b) comprises or consists of a the bacterial enoate reductase comprises or consists of an nucleotide sequence according to SEQID NO:2 or SEQ ID amino acid sequence according to SEQID NO:4 or an amino NO:5 or a nucleotide sequence with a nucleotide sequence acid sequence with an amino acid sequence identity of 40% or identity of 40% or more for SEQID NO:2 or SEQID NO:5, more for SEQID NO:4, in particular of 50% or more, 60% or in particular of 50% or more, 60% or more or 80% or more, more or 80% or more, especially preferably of 95% or more. especially preferably of 95% or more. For the determination of the nucleotide sequence and It is further preferable if amino acid sequence identity that stated above applies hereby the bacterial chalcone isomerase comprises or consists of analogy. an amino acid sequence according to SEQ ID NO:3 or an A further aspect of the present invention concerns a vector, 25 amino acid sequence with an amino acid sequence identity of E.g. a transport vesicle (“gene shuttle') for transfer of exter 40% or more for SEQID NO:3, in particular of 50% or more, nal nucleic acid(s) into a receiver cell, especially a plasmid 60% or more or 80% or more, especially preferably of 95% or vector, allowing the cloning of one or more nucleic acid more, sections, containing a nucleic acid section (a), comprising or and/or consisting of a gene coding for a bacterial chalcone 30 the plant chalcone isomerase comprises or consists of an amino acid sequence according to SEQID NO:8 or SEQID isomerase, and/or a nucleic acid section (a'), comprising or NO:9 oran amino acid sequence with an amino acid sequence consisting of a gene coding for a plant chalcone isomerase, identity of 40% or more for SEQID NO:8 or SEQID NO:9, and/or a nucleic acid section (b), comprising or consisting of in particular of 50% or more, 60% or more or 80% or more, a gene coding for a bacterial enoate reductase. Here it is 35 especially preferably of 95% or more, preferred if the vector contains both a nucleic acid section (a) and/or and/or (a"), as well as a nucleic acid section (b). the bacterial enoate reductase comprises or consists of an Apart from nucleic acid section(s) (a), (a") and/or (b) a amino acid sequence according to SEQID NO:4 or an amino vector according to the invention may contain for the pur acid sequence with an amino acid sequence identity of 40% or poses of the present invention further normal components, 40 more for SEQID NO:4, in particular of 50% or more, 60% or especially those which improve or possibly actually allow the more or 80% or more, especially preferably of 95% or more. expression of the transgenes described herein in microorgan Here again, for the determination of the nucleotide isms, especially in those as described above. Basically a sequence and amino acid sequence identity that stated above vector according to the invention preferably also contains one applies by analogy. or more further components or elements selected from the 45 Especially preferred, and for the purposes of the invention group consisting of promoter, sequence of origin, sequence especially suitable, vectors and components or elements for affinity chromatography purification, selection marker, thereof are apparent from the attached examples and figures operator sequence, terminator, ribosomal binding sites, pro (FIG. 1: Plasmid pET52b EREDstrep for heterologous tease cleavage sequence, recombination binding sites, expression and characterization of the ERED from E. ramu sequences of fusion proteins and chaperone sequences. 50 lus DSM 16296; FIG. 2: Plasmid pET28b CHI for heterolo With the vectors according to the invention (as described gous expression and characterization of the CHI from E. above) it is also preferred if ramulus DSM 16296; FIG. 3: Plasmid pET22b ERED for the gene coding for a bacterial chalcone codes for a chal heterologous expression and characterization of the ERED cone isomerase from a microorganism from the phylum Fir from E. ramulus DSM 16296). micutes, in particular the class Clostridia, especially the order 55 The present invention also concerns a host cell, containing Clostridiales, especially preferably for a chalcone isomerase one or more identical or different vectors according to the from E. ramulus, invention as described herein. Preference according to the and/or invention is for a host cell, which contains both one or more the gene coding for a plant chalcone isomerase codes for a vectors with a nucleic acid section (a), comprising or consist chalcone isomerase from A. thaliana or M. Sativa (for other 60 ing of a gene coding for a bacterial chalcone isomerase, preferred sources see above), and/or a nucleic acid section (a'), comprising or consisting of and/or a gene coding for a plant chalcone isomerase, and also one or the gene coding for a bacterial enoate reductase codes for more vectors with a nucleic acid section (b), comprising or an enoate reductase from a microorganism from the phylum consisting of a gene coding for a bacterial enoate reductase. Firmicutes, in particular the class Clostridia, especially the 65 Especially preferred is a host cell, containing one or more order Clostridiales, especially preferably for an enoate reduc vectors with both a nucleic acid section (a) and/or (a"), and a tase from E. ramulus. nucleic acid section (b). US 9,359,622 B2 19 20 With a host cell according to the invention it is in particular sections binding directly to the gene are shown in italics; the a case of a microorganism (as described above) according to Kpnl interface inserted in the forward primer (containing no or to be used according to the invention. The host cells or sequence sections binding directly to the gene) were marked microorganisms according to or to be used according to the in bold): invention described herein are or in particular serve as a (production) strain for biotechnological production of the dihydrochalcones described herein, especially of phloretin forward: (as described above). AGTGTGATGGGTACCTGCAGAATTCGCC In the following the present invention is explained in more rewerse : detail using examples, wherein these do not restrict the Sub 10 GATCAAGCTTAGATAATTTCCATIGCTGCGGTCCA ject-matter of the attached claims. (see above) The vector pCRR2.1-TOPOR) similarly contains a SacI EXAMPLE1 interface, which was used from further cloning. 15 Following the PCR this PCR product was digested with Provision of Transgenic Microorganisms (See Step Sac1 and Kpnl and then the ligation in the plasmid plT52b (i)) similarly digested with Sac1 and Kpn1. The gene construct was expressed in E. coli Rosetta. 1.1 CHI: Sequencing was used to confirm that the gene of the enoate Using the identified gene sequence of the chalcone reductase was successfully ligated in the plasmid plT52b. isomerase from E. ramulus two primers were prepared, which wherein the inserted N-terminal strep tag remained available, were used for reproduction of the genomic DNA by means of allowing a highly specific protein purification. PCR. Here, with the help of the primers, in front of the gene In the context of a further approach a codon-optimised a restriction interface was attached for Kpn1 and BamH1 and sequence with the interfaces Ndel and BamH1 was ligated in behind the gene an interface for Not1 to the target sequence, 25 the vector pBT-22b and expressed in E. coli BL21. which were used for ligation of the gene section in the target 1.3 Antibiotic-free expression: Vector. Furthermore, the gene sections of the CHI can be inte Primers used (sequence sections, binding directly to the grated via the interfaces BamH1 and XHO1 in the plasmid gene are shown in italics): pET22b with the synthetic ERED gene. 30 For this the CHI gene is amplified via a PCR with the forward: forward-primer GTCTAGGATCCAGAAATAATTTTGTT CTAATCGGATCCGGTACCATGGCAGATTTCAAATCGAACCAATG TAACTTTAAGAAGGAGA and the pET-rP-primer CTAGT

rewerse : TATTGCTCAGCGG, wherein the Xbal interface before the TCAGTAGCGGCCGCTTATCTCATGGTGATGTATCCACGATAATT 35 CHI gene is mutated via the forward-primer into a BamHI interface. Then the construct from both genes is cut via the The resultant DNA fragment of the chalcone isomerase interfaces Xbal and Xhol from the plasmid and ligated in the gene was inserted by means of TOPO TA Cloning R (from plasmid pStaby 1.2 prepared with Xbal and Xho1. Invitrogen, Carlsbad, Calif., USA) in the vector pCRR2.1- TOPOR). Following successful transformation of this con Then an antibiotic-free expression can take place by means 40 of the StabyExpress-system. struct the chalcone isomerase gene was cut out from this vector via Nco1 and Not1 and inserted in the target vector pET28b likewise cut with Nco1 and Not1. The plasmid was EXAMPLE 2 introduced into E. coli Rosetta and Successfully expressed there. Biotechnological Production of Phloretin (See Step Following Successful transformation the sequence identity 45 (ii)) was confirmed by means of sequencing. 1.2 ERED: TB medium comprising 24 g yeast extract, 12g Tryptone The enoate reductase was amplified with the following and 4g glycerine is made up to 900 ml and autoclaved at 121° specific primers from the genomic DNA (sequence sections C. for 15 minutes. A separately prepared saline solution (0.72 50 M of KHPO and 0.17 M of KHPO) is autoclaved under binding directly to the gene are shown in italics): the same conditions. Then 100 ml of the saline solution is added to 900 ml of the sterile TB medium. LB medium forward: comprising 5 g yeast extract, 10g Tryptone and 10g NaCl is GATCCTCGAGATGGCAGAAAAAAATCAGTATTTTCCACA made up to 1000 ml with distilled water and autoclaved for 15 55 minutes at 121° C. rewerse : The E. coli Rosetta or E. coli BL21 (see above) trans GATCAAGCTTAGATAATTTCCATIGCTGCGGTCCA formed for example according to Example 1 are first culti Here in front of the gene an interface for SacI was inserted vated in 250 ml Erlenmeyer flasks (with baffles), filled with and behind the gene an interface for HindIII, in order to allow 50 ml LB medium, for approximately 8 hours at 37° C. and Subsequent cloning. 60 180 rpm. From this culture 7 ml are then taken and used to This fragment was also processed further with the TOPO inoculate a mini fermenter containing 700 ml of TB medium TA Cloning R-Kit. The sequence identity of the resultant (as described above). The culture is grown overnight at 25°C. clone (with vector pCRR2.1-TOPOR) and gene for enoate and 150-200 rpm with 40% oxygen saturation to achieve a reductase contained therein) was confirmed by means of maximum biomass concentration. sequencing. 65 Before (over)expression of the introduced nucleic acid sec The gene from this plasmid was amplified with primers, tions or the introduced transgene the air Supply is terminated which inserted a Kipnl interface in front of the gene (sequence and the existing oxygen is driven out by nitrogen. US 9,359,622 B2 21 22 To induce (over)expression initially an IPTG concentration TABLE 6 of 1 mM in the medium is set and then 7 ml of a naringenin solution (100 mM in DMSO) added. Anaerobic fermentation with CHI and ERED: 10 nM naringenin Further cultivation then takes place in the expression phase Phloretin for at least 8-20 hours under anoxic conditions. 5 Time in hours in mM OO O.OO8 1.OO O.189 EXAMPLE 3 2.OO O.282 3.00 O404 10 4.OO O485 Characterisation of the Expressed CHI S.OO O494 6.OO O.694 1O.OO O821 Following expression of for example plT28b CHI in E. 22.OO 0.855 coli (see above) CHI can be established both in the soluble 26.OO O840 and in the insoluble fractions. 15 Following purification with for example anion exchange The enzymes CHI and ERED (from E. ramulus DSM chromatography, hydrophobic interaction chromatography, 16296) were placed separately for expression in a vector size exclusion chromatography and/or Resource Q purifica system suitable for E. coli (see above). Then the activity of tion the CHI can be isolated for characterization (FIG. 5 these—following addition of maringenin in a defined culture provides examples of the results of the individual purification medium—could be determined by means of HPLC. steps following expression of the vector pET28b CHI in E. The values in Table 5 show the formation as a function of coli). time of phloretin (with a reduction in the naringenin concen Our own investigations produced the results shown in tration), pointing to the activity of the enzymes CHI and 25 ERED. Table 4: The values in the table in Table 6 show (further) results of the chalcone isomerase activity of the expressed CHI, TABLE 4 obtained by photometric determination (at 368 nm) in rela Characterisation of the enzyme activity of the CHI following tion to the degradation of naringenin chalcone (to form phlo expression of the vector pET28b CHI in E. coli 30 retin). A photometric determination allowed for further inves KM kcat kcat KM tigation of the CHI activity—the reduction over time in the Imoll Vmax Umg S-1 1 * mol-1 *s-1) concentration of naringenin chalcone in a cell-free protein 36.83 107.3 416.7 1.13 * 107 crude extract to be observed. A control experiment (plasmid 35 without CHI) showed a reduction in the naringin chalcone used as the marker Substance.

EXAMPLE 5 EXAMPLE 4 40 Results of (Comparative) Expression Experiments (Further) Characterization of the Expressed Enzymes with E. ramulus CHI and ERED E. ramulus (DSMZ 16296) was cultivated anaerobically according to Herles et al. (Arch Microbiol (2004) 181: 428 Tables 5-6 show (further) results of the characterization of 45 434) in ST medium. For its preparation 9 g of tryptically the expressed enzymes CHI and ERED (from E. ramulus treated meat peptone, 1 g peptone, 3 g meat extract, 4 g yeast DSM 16296 during anaerobic conversion (see above)). extract, 6 g glucose, 3 g sodium chloride, 2 g disodium hydro gen phosphate, 0.5 ml Tween 80, 0.25 g cystine, 0.25 g cys TABLE 5 teine-HCl, 0.1 g magnesium Sulphate heptahydrate, 5 mg iron 50 Sulphate heptahydrate and 3.4 mg manganese Sulphate dehy Anaerobic cultivation drate were adjusted to pH 7.0, made up to 1 1 with distilled Time in h Naringenin in mM Phloretin in mM water and then autoclaved at 121° C. for 15 minutes. O.25 1.OO O.09 Cultivations were performed both in a conventional steel 1.75 0.77 0.44 reactor with stirrer, and in bag reactors with Wipp system 3.25 O.S9 O.62 55 under anoxic conditions, wherein the temperature was main 4.75 O.45 O.68 tained at 37° C. and the pH at 7.0 with acid and alkaline 6.25 0.44 O.68 10.25 O42 O.74 solution (HC1 or NaOH). 21.00 O.29 O.70 In an example fermentation at the start of cultivation 275 26.OO 0.27 O.71 LM maringenin were added to the medium and the growth and 60 the conversion of the determined (for results see FIG. 6).

SEO ID NO: 1 ATGGCAGATTTCAAATTCGAACCAATGAGAAGTCTTATCTACGTTGACTGCGTAAGC

GAAGACTACAGACCAAAACTTCAGAGATGGATTTATAAAGTACATATTCCGGACAG

US 9,359, 622 B2 33 34 - Contin lued gaaaatgcgg taa.ca.gcaat ggacgtatac agcaatgact ttgcaggtot tggaaagagc 162O accatcgtac toggtggcgg tctggttggc tgttgaggcag cc.gcagatta tattgat cac 168O ggtgtagaga Caacgattgt tgaaatgaaa ggtgcgctga tgc.cggagac alaccggtctg 1740 taccgtacag ctgtacatga titt catcgac aaaaacggcg gcaaatacga agtaaatgca 18OO aaagttgtca aagttggcaa. agattttgttg gtagcggaac aagatgggaa agagattacc 1860 atcaaag.cag attctgttgt Caatgcaatg ggacgc.cgtg cgcatgcgac agalagcactt 1920 gaga cagcta t caaagaa.gc tgg tatt cc.g gtatggalaga tcggtgactg 198O cgtcagat.cg gtgatgcggit aagagaaggc tggaccgcag Caatggaaat tat Ctaa 2O37

<210s, SEQ ID NO 3 &211s LENGTH: 283 212. TYPE: PRT &213s ORGANISM: Eubacterium ramu lus

<4 OOs, SEQUENCE: 3 Met Ala Asp Phe Llys Phe Glu Pro Met Arg Ser Lieu. Ile Tyr Val Asp 1. 1O 15

Cys Wall Ser Glu Asp Tyr Arg Pro Llys Lieu. Glin Arg Trp Ile Tyr Lys 25 3O

Wall His Ile Pro Asp Ser Ile Ser Glin Phe Glu Pro Tyr Val Thr Lys 35 4 O 45

Ala Phe Tyr Pro Ser Phe Pro Ile Pro Pro Glin Gly Asp Arg Phe SO 55 6 O Gly Ala Arg Met Gln Lieu. Thr Glu. His His Trp Lieu Val Ser Asp 65 70 7s 8O

Lell Asp Pro Arg Lieu. Glu Ile Llys Ala Ile Ala Glu Thir Phe Pro Met 85 90 95

Asp Wall Lieu Val Trp Glin Gly Glin Ile Pro Ala Ala Ala His Thir Asp 11 O

Ala Glin Ile Asp Ser Asp Gly Asp Ala Gly Asn Ala Ala Arg Llys Ser 115 12 O 125

Asn Asn Ala Glu Gly Asn Pro Phe Ile Phe Ala Phe Leu Pro Met Trp 13 O 135 14 O

Trp Glu Lys Asp Lieu Lys Gly Lys Gly Arg Thr Ile Glu Asp Gly Ala 145 150 155 160

Asn Arg Phe Asn Met Thr Ile Gly Phe Pro Glu Gly Val Asp Llys 1.65 17O 17s

Ala Glu Gly Glu Lys Trp Lieu. Phe Glu Lys Val Wall Pro Ile Lieu. Glin 18O 19 O

Ala Ala Pro Glu. Cys Thr Arg Val Lieu Ala Ser Ala Wall Lys 195 2O5

Ile Asn Gly Cys Val Met Asp Trp Wall Lieu. Glu Ile Trp Phe Glu Asn 21 O 215 22O

Glin Ser Gly Trp Tyr Lys Val Met Val Asp Asp Met Lys Ala Lieu. Glu 225 23 O 235 24 O

Pro Ser Trp Ala Glin Glin Asp Ala Phe Pro Phe Lieu Lys Pro Tyr 245 250 255

His Asn Val Cys Ser Ala Ala Val Ala Asp Tyr Thir Pro Ser Asn. Asn 26 O 265 27 O

Lell Ala Asn Tyr Arg Gly Tyr Ile Thr Met Arg 28O US 9,359,622 B2 35 36 - Continued

<210s, SEQ ID NO 4 &211s LENGTH: 678 212. TYPE: PRT <213> ORGANISM: Eubacterium ramulus

<4 OOs, SEQUENCE: 4 Met Ala Glu Lys Asn Glin Tyr Phe Pro His Leu Phe Glu Pro Leu Lys 1. 5 1O 15 Val Gly Ser Lys Thir Ile Lys Asn Arg Ile Glu Ala Ala Pro Ala Lieu 2O 25 3O Phe Ala Phe Glu. His Tyr Ile Glu Lieu. Asn Pro Asp Pro Phe Gly Tyr 35 4 O 45 Thir Thr Pro Val Pro Glu Arg Ala Phe Arg Met Lieu. Glu Ala Lys Ala SO 55 6 O Lys Gly Gly Ala Gly Ile Val Cys Lieu. Gly Glu Lieu. Ser Pro Asn His 65 70 7s 8O Glu Tyr Asp Lys Arg Phe Pro Phe Glu Pro Tyr Lieu. Asp Phe Thr Ser 85 90 95 Arg Ser Asp Llys Glin Phe Glu Ile Met Lys Glu Thir Ala Glu Met Ile 1OO 105 11 O Lys Ser Tyr Gly Ala Phe Pro Met Gly Glu Lieu Lleu Ser Cys Gly Glu 115 12 O 125 Ile Llys Thr Asn. Ile Gly Asp Gly Ile Asn Pro Llys Gly Pro Ser Glu 13 O 135 14 O Lys Asp Lieu Pro Asp Gly Ser His Val Glu Ala Phe Thr Lys Glu Glu 145 150 155 160 Ile Leu Ser Cys Tyr Glin Asp Tyr Val Thr Ala Cys Lys Trp Phe Glin 1.65 17O 17s Ala Ala Gly Trp Glu Gly Ile Met Ile His Cys Gly. His Gly Trp Lieu 18O 185 19 O Pro Ala Glin Phe Leu Ser Pro Glin Tyr Asn Lys Arg Thr Asp Glu Tyr 195 2OO 2O5 Gly Gly Ser Phe Glu Asn Arg Ala Arg Phe Thr Val Asp Lieu. Lieu Lys 21 O 215 22O Thr Val Arg Glu Ala Met Gly Pro Asp Phe Val Ile Glu Ile Arg Val 225 23 O 235 24 O Ser Ser Ser Glu. His Lieu Pro Gly Gly Lieu. Glu Lieu. Glu Asp Ala Val 245 250 255 Asn Tyr Cys Llys Lieu. Cys Glu Pro Tyr Ile Asp Met Ile His Val Ser 26 O 265 27 O Cys Gly His Tyr Lieu. Ser Ser Ser Arg Ser Trp Glu Phe Thr Thr Ala 27s 28O 285 Tyr Ala Pro His Gly Pro Asn Ile Glu Pro Ala Ala Val Ile Lys Glin 29 O 295 3 OO Asn Val Ser Ile Pro Val Ala Ala Val Gly Gly Ile Asn. Ser Pro Glu 3. OS 310 315 32O

Glin Ala Glu Glu Ala Ile Ala Ser Gly Lys Ile Asp Met Val Ser Met 3.25 330 335

Gly Arg Glin Phe Phe Ala Asp Pro Ala Phe Pro Asn Lys Ala Lys Glu 34 O 345 35. O Gly His Ala Asp Glu Ile Arg Arg Cys Lieu. Arg Cys Gly Arg Cys Tyr 355 360 365 Pro Gly Pro Ser Gly Glu. His Glu Thr Glu Ile Trp Thr Val Llys Phe 37 O 375 38O US 9,359,622 B2 37 38 - Continued

Pro Pro Luell Asp Ser Cys Thr Ile Asn Pro Tyr Asp Val Trp Pro Ala 385 390 395 4 OO

Ser His His Llys Val Lieu Pro Asp Arg Met Pro Lys Pro Glu Ala Ser 4 OS 41O 415

Arg Wall Lieu Val Val Gly Gly Gly Cys Gly Gly Lell Glin Thir Ala 425 43 O

Ile Thir Ala Ser Asp Arg Gly His Glin Wall Ile Lell Cys Glu Llys Ser 435 44 O 445

Gly Wall Luell Gly Gly Lieu. Ile Asn Phe Thr Asp His Thir Asp His Lys 450 45.5 460

Wall Asp Ile Arg Asn. Phe Lys Asp Lieu. Lieu. Ile Arg Asp Wall Glu Lys 465 470 47s 48O

Arg Pro Ile Glu Val Arg Lieu. Asn Cys Glu Val Thir Pro Glu Lieu. Ile 485 490 495

Arg Glu Ile Ala Pro Glu Ala Wall Wall Lieu Ala Wall Gly Ser Asp Asp SOO 505

Lell Ile Luell Pro Ile Glu Gly Ile Glu Asn Ala Wall Thir Ala Met Asp 515 525

Wall Tyr Ser Asn Asp Phe Ala Gly Lieu. Gly Lys Ser Thir Ile Wall Lieu 53 O 535 54 O

Gly Gly Gly Lieu Val Gly Cys Glu Ala Ala Ala Asp Ile Asp His 5.45 550 555 560

Gly Wall Glu Thir Thir Ile Wall Glu Met Lys Gly Ala Lell Met Pro Glu 565 st O sts

Thr Thr Gly Lieu. Tyr Arg Thr Ala Val His Asp Phe Ile Asp Lys Asn 585 59 O

Gly Gly Lys Tyr Glu Val Asn Ala Llys Val Val Wall Gly 595 605

Phe Wall Wall Ala Glu Glin Asp Gly Lys Glu Ile Thir Ile Ala Asp 610 615 62O

Ser Wall Wall Asn Ala Met Gly Arg Arg Ala His Ala Thir Glu Ala Lieu 625 630 635 64 O

Glu Thir Ala Ile Lys Glu Ala Gly Ile Pro Wall Trp Ile Gly Asp 645 650 655

Wall Arg Ala Arg Glin Ile Gly Asp Ala Val Arg Glu Gly Trp Thr 660 665 67 O

Ala Ala Met Glu Ile Ile 675

SEO ID NO 5 LENGTH: 2O37 TYPE: DNA ORGANISM: Eubacterium ramulus

< 4 OOs SEQUENCE: 5 atggcagaaa agaaccalata citt cocqcac ctgtttgaac cgctgaaagt cggctictaaa 6 O acCattaaaa. atcgcatcga agcagcaccg gcc ctgtttg catt.cgaaca ttatat cqaa 12 O

Ctgaac ccgg accc.gtttgg ttacaccacg CC9gtgc.cgg aacgtgcatt ccg tatgctg 18O gaagccaaag caaaaggcgg tgc.cggcatt gtttgtctgg gtgaactgag cc.cgaat cac 24 O gaatatgata aacgctitt co gttctgaaccg tacctggatt ttaccagcc.g ttctgacaaa 3OO

Cagttcgaaa titatgaaaga aacggcagaa atgat caaaa gctatggcgc ttitt.ccgatg 360 ggtgaactgc tgaaatcaaa accaa.cattg gcgatggitat caatc.cgaaa

US 9,359,622 B2 43 44 - Continued

Ala Glu Ala Met Gln Llys Phe Ala Glu Ala Phe Llys Pro Val Asn. Phe 13 O 135 14 O

Pro Pro Gly Ala Ser Val Phe Tyr Arg Glin Ser Pro Asp Gly Ile Leu 145 150 155 160

Gly Leu Ser Phe Ser Pro Asp Thr Ser Ile Pro Glu Lys Glu Ala Ala 1.65 17O 17s

Lieu. Ile Glu Asn Lys Ala Val Ser Ser Ala Val Lieu. Glu Thir Met Ile 18O 185 19 O Gly Glu. His Ala Val Ser Pro Asp Lieu Lys Arg Cys Lieu Ala Ala Arg 195 2OO 2O5

Lieu Pro Ala Lieu. Lieu. Asn. Glu Gly Ala Phe Lys Ile Gly Asn 21 O 215 22O

<210s, SEQ ID NO 9 &211s LENGTH: 246 212. TYPE: PRT <213> ORGANISM: Arabidopsis thaliana

<4 OOs, SEQUENCE: 9 Met Ser Ser Ser Asn Ala Cys Ala Ser Pro Ser Pro Phe Pro Ala Val 1. 5 1O 15

Thr Lys Lieu. His Val Asp Ser Val Thr Phe Val Pro Ser Val Lys Ser 2O 25 3O

Pro Ala Ser Ser Asn Pro Lieu. Phe Lieu. Gly Gly Ala Gly Val Arg Gly 35 4 O 45

Lieu. Asp Ile Glin Gly Llys Phe Val Ile Phe Thr Val Ile Gly Val Tyr SO 55 6 O

Lieu. Glu Gly Asn Ala Val Pro Ser Lieu. Ser Val Llys Trp Llys Gly Lys 65 70 7s 8O

Thir Thr Glu Glu Lieu. Thr Glu Ser Ile Pro Phe Phe Arg Glu Ile Val 85 90 95

Thr Gly Ala Phe Glu Lys Phe Ile Llys Val Thr Met Lys Lieu. Pro Leu 1OO 105 11 O

Thr Gly Glin Glin Tyr Ser Glu Lys Val Thr Glu Asn Cys Val Ala Ile 115 12 O 125

rop LivsLly Glin Lieu. Glyy Lieu. Tvry Thir ASOp CVSCy Glu Ala Livsy Ala Wall Glu 13 O 135 14 O

Llys Phe Leu Glu Ile Phe Lys Glu Glu Thr Phe Pro Pro Gly Ser Ser 45 150 155 160

le Leu Phe Ala Leu Ser Pro Thr Gly Ser Lieu. Thr Val Ala Phe Ser 1.65 17O 17s

Lys Asp Asp Ser Ile Pro Glu Thr Gly Ile Ala Val Ile Glu Asn Lys 18O 185 19 O

Lieu. Lieu Ala Glu Ala Val Lieu. Glu Ser Ile Ile Gly Lys Asn Gly Val 195 2OO 2O5

Ser Pro Gly. Thir Arg Lieu. Ser Val Ala Glu Arg Lieu. Ser Glin Lieu Met 21 O 215 22O

Met Lys Asn Lys Asp Glu Lys Glu Val Ser Asp His Ser Val Glu Glu 225 23 O 235 24 O

Llys Lieu Ala Lys Glu Asn 245 US 9,359,622 B2 45 46 What is claimed is: the nucleic acid section (a') comprises a nucleotide 1. A transgenic microorganism containing transgenes, said sequence according to SEQID NO:6 or SEQID NO:7, transgenes comprising O a nucleic acid section (a), comprising a gene coding for a the nucleic acid section (b) comprises a nucleotide bacterial chalcone isomerase, or sequence according to SEQID NO:2 or SEQID NO:5. a nucleic acid section (a'), comprising a gene coding for a 14. The vector according to claim 10, wherein plant chalcone isomerase, or both nucleic acid section the bacterial chalcone isomerase comprises an amino acid (a) and nucleic acid section (a'); sequence according to SEQID NO:3, or and the plant chalcone isomerase comprises an amino acid a nucleic acid section (b), comprising a gene coding for an 10 enoate reductase from E. ramulus operative to form a sequence according to SEQID NO:8 or SEQID NO:9, dihydrochalcone as a product. O 2. The microorganism according to claim 1, wherein the the bacterial enoate reductase is comprised comprises an microorganism has chalcone isomerase and enoate reductase amino acid sequence according to SEQID NO:4. activity, but no phloretin hydrolase activity. 15 15. A host cell containing one or more identical or different 3. The microorganism according to claim 1, wherein the vectors according to claim 10. microorganism is not a microorganism of the phylum Firmi 16. A host cell containing one or more identical or different Cutes. vectors according to claim 11, and wherein said host cell is a 4. Microorganism according to claim3, wherein the micro microorganism. organism is not Eubacterium ramulus. 17. A host cell containing one or more vectors with a 5. The microorganism according to claim 1, wherein the nucleic acid section microorganism is selected from the group consisting of fac (a), comprising a gene coding for a bacterial chalcone ultative anaerobic microorganisms. isomerase, or a nucleic acid section (a'), comprising a 6. The microorganism according to claim 5, wherein the gene coding for a plant chalcone isomerase, or both microorganism is selected from the group consisting of 25 nucleic acid section (a) and nucleic acid section (a'); and enterobacteria and yeasts. one or more vectors with a nucleic acid section (b), com 7. The microorganism according to claim 1, wherein prising a gene coding for an enoate reductase from E. the gene coding for a bacterial chalcone isomerase codes ramulus operative to form a dihydrochalcone as a prod for a chalcone isomerase from E. ramulus, or uct. the gene coding for a plant chalcone isomerase codes for a 30 18. Method for production of a dihydrochalcone using a chalcone isomerase from A. thaliana or M. sativa. transgenic microorganism comprising: 8. The microorganism according to claim 1, wherein (i) providing a transgenic microorganism containing trans the nucleic acid section (a) comprises a nucleotide genes, said transgenes comprising sequence according to SEQID NO:1, or a nucleic acid section (a), comprising a gene coding for the nucleic acid section (a') comprises a nucleotide 35 a bacterial chalcone isomerase, or a nucleic acid sec sequence according to SEQID NO:6 or SEQID NO:7, tion (a'), comprising a gene coding for a plant chal O cone isomerase, or both nucleic acid section (a) and the nucleic acid section (b) comprises a nucleotide nucleic acid section (a'); sequence according to SEQID NO:2 or SEQID NO:5. and 9. The microorganism according to claim 1, wherein the 40 a nucleic acid section (b), comprising a gene coding for the bacterial chalcone isomerase comprises an amino acid an enoate reductase from E. ramulus operative to form sequence according to SEQID NO:3, or a dihydrochalcone as a product; the plant chalcone isomerase comprises an amino acid (ii) adding one or more flavanones and optionally one or sequence according to SEQID NO:8 or SEQID NO:9, more precursors or one or more derivatives thereof to the O 45 transgenic microorganism and cultivation of the trans the bacterial enoate reductase comprises an amino acid genic microorganism under conditions which allow the sequence according to SEQID NO:4. conversion of the flavanone(s) and/or the precursor(s) or 10. A vector containing: of the derivative(s) thereof to a dihydrochalcone; and a nucleic acid section (a), comprising a gene coding for a (iii) optionally isolating and purifying the dihydrochal bacterial chalcone isomerase, or a nucleic acid section 50 COC. (a"), comprising a gene coding for a plant chalcone 19. The method of claim 18, wherein: the dihydrochalcone isomerase, or both nucleic acid section (a) and nucleic is phloretin; and the flavanones are naringin. acid section (a'); 20. Method of claim 18, wherein the transgenic microor and ganism is not a microorganism of the phylum Firmicutes. a nucleic acid section (b), comprising a gene coding for an 55 21. Method of claim 18, wherein the transgenic microor enoate reductase from E. ramulus operative to form a ganism is selected from the group consisting of facultative dihydrochalcone as a product. anaerobic microorganisms, facultative aerobic bacteria, pro 11. The vector of claim 10, wherein the vector is a plasmid teobacteria, enterobacteria, E. coli, E. coli Rosetta, E. coli Vector. BL21 and E. coli SE1, yeasts, S. cerevesiae and P. pastoris. 12. The vector according to claim 10, wherein 60 22. Method of claim 18, wherein the gene coding for a the gene coding for a bacterial chalcone isomerase codes bacterial chalcone isomerase codes for a chalcone isomerase for a chalcone isomerase from E. ramulus, or from a microorganism from the phylum Firmicutes. the gene coding for a plant chalcone isomerase codes for a 23. Method of claim 18, wherein the gene coding for a plant chalcone isomerase from A. thaliana or M. sativa. chalcone isomerase codes for a chalcone isomerase from A. 13. The vector according to claim 10, wherein 65 thaliana or M. Sativa. the nucleic acid section (a) comprises a nucleotide 24. Method of claim 18, wherein the nucleic acid section sequence according to SEQID NO:1, or (a) is comprised of a nucleotide sequence according to SEQ US 9,359,622 B2 47 48 IDNO: 1 or a nucleotide sequence with a nucleotide sequence 33. Method according to claim 30, wherein identity of 95% or more for SEQID NO:1. the gene coding for a bacterial chalcone isomerase codes 25. Method of claim 18, wherein the nucleic acid section for a chalcone isomerase from a microorganism from the (a") is comprised of a nucleotide sequence according to SEQ phylum Firmicutes, and/or ID NO:6 or SEQ ID NO:7 or a nucleotide sequence with a the gene coding for a plant chalcone isomerase codes for a nucleotide sequence identity of 95% or more for SEQ ID chalcone isomerase from A. thaliana or M. sativa. NO:6 or SEQID NO:7. 34. Method according to claim 30, wherein 26. Method of claim 18, wherein the nucleic acid section the nucleic acid section (a) is comprised of a nucleotide (b) is comprised of a nucleotide sequence according to SEQ sequence according to SEQ ID NO:1 or a nucleotide ID NO:2 or SEQ ID NO:5 or a nucleotide sequence with a 10 nucleotide sequence identity of 95% or more for SEQ ID sequence with a nucleotide sequence identity of 95% or NO:2 or SEQID NO:5. more for SEQID NO:1, 27. Method of claim 18, wherein the bacterial chalcone and/or isomerase is comprised of an amino acid sequence according the nucleic acid section (a') is comprised or consists of a to SEQ ID NO:3 or an amino acid sequence with an amino 15 nucleotide sequence according to SEQID NO: 6 or SEQ acid sequence identity of 95% or more for SEQID NO:3. ID NO:7 or a nucleotide sequence with a nucleotide 28. Method of claim 18, wherein the plant chalcone sequence identity of 95% or more for SEQID NO:6 or isomerase is comprised of an 20 amino acid sequence accord SEQID NO:7, ing to SEQ ID NO:8 or SEQ ID NO:9 or an amino acid and/or sequence with an amino acid sequence identity of 95% or the nucleic acid section (b) is comprised of a nucleotide more for SEQID NO:8 or SEQID NO:9. sequence according to SEQID NO:2 or SEQID NO:5 or 29. Method of claim 18, wherein the bacterial enoate a nucleotide sequence with a nucleotide sequence iden reductase is comprised of an amino acid sequence according tity of 95% or more for SEQID NO:2 or SEQID NO:5. to SEQ ID NO:4 or an amino acid sequence with an amino 35. Method according to claim 30, wherein acid sequence identity of 95% or more for SEQID NO:4. 25 the bacterial chalcone isomerase is comprised of an amino 30. Method for production of a dihydrochalcone using a acid sequence according to SEQID NO:3 or an amino transgenic microorganism, comprising the following steps: acid sequence with an amino acid sequence identity of (i) providing a transgenic microorganism containing trans 95% or more for SEQID NO:3, genes, said transgenes comprising and/or a nucleic acid section (a), comprising a gene coding for 30 the plant chalcone isomerase is comprised of an amino acid a bacterial chalcone isomerase, or a nucleic acid sec sequence according to SEQID NO: 8 or SEQID NO:9 tion (a'), comprising a gene coding for a plant chal or an amino acid sequence with an amino acid sequence identity of 95% or more for SEQID NO:8 or SEQ ID cone isomerase, or both nucleic acid section (a) and NO:9, nucleic acid section (a'); and and/or a nucleic acid section (b), comprising a gene coding 35 for an enoate reductase from E. ramulus operative the bacterial enoate reductase is comprised of an amino to form a dihydrochalcone as a product acid sequence according to SEQ ID NO:4 or an amino (ii) adding one or more flavanones and optionally one or acid sequence with an amino acid sequence identity of more precursors or one or more derivatives thereof, to 95% or more for SEQID NO:4. the transgenic microorganism and cultivation of the 40 36. The method according to claim 30, wherein the micro transgenic microorganism under conditions which allow organism is not Eubacterium ramulus. the conversion of the flavanone(s) and/or the prec 37. The method according to claim 30, wherein the micro ursor(s) or of the derivative(s) thereof to a dihydrochal organism is selected from the group consisting of facultative cone; and anaerobic microorganisms. (iii) isolating and purifying the dihydrochalcone. 45 38. The method according to claim 30, wherein the micro 31. Method according to claim 30, wherein the transgenic organism is selected from the group consisting of enterobac microorganism is not a microorganism of the phylum Firmi teria and yeasts. Cutes. 39. The method of claim 30, wherein the dihydrochalcone 32. Method according to claim 30, wherein in step (ii) is phloretin; and the flavanones are naringin. naringin and/or an aglycone thereof is or are added. ck ck ck ck ck