US010435673B2 (12 ) United States Patent ( 10 ) Patent No. : US 10 , 435 ,673 B2 Wessjohann et al. (45 ) Date of Patent : Oct. 8 , 2019 ( 54 ) METHOD FOR THE BIOTECHNOLOGICAL ( 56 ) References Cited PRODUCTION OF FLAVONOIDS (71 ) Applicant: Symrise AG , Holzminden (DE ) FOREIGN PATENT DOCUMENTS WO 2005 /084305 AL 9 /2005 (72 ) Inventors: Ludger Wessjohann , Halle (DE ) ; WO 2005 / 115377 Al 12 / 2005 Anne - Kathrin Bauer , Halle (DE ) ; Jakob Ley , Holzminden (DE ) ; Torsten OTHER PUBLICATIONS Geissler , Einbeck (DE ) ; Katrin Lee at al , “ Biotransformation of Flavonoids with O -Methyltransferase Geissler , Einbeck (DE ) from Bacillus cereus, " J . Microbiol. Biotechnol . (2008 ) , 16 ( 7 ) , pp . 1090 - 1096 ( 73 ) Assignee : SYMRISE AG , Holzminden (DE ) Kitamura et al, “ Production of Hydroxlated Flavonoids with Cytochrome P450 BM3 Variant F87V and Their Antioxidative Activities ,” Biosci . Biotechnol . Biochem . 77 (6 ) , pp . 1340 - 1343 (2013 ) . ( * ) Notice: Subject to any disclaimer, the term of this Wessjohann et al, “ Alkylating enzymes, ” Current Opinion in Chemi patent is extended or adjusted under 35 cal Biology 2013 , 17 :229 - 235 . U . S .C . 154 (b ) by 87 days. Kasai et al, “ Enzymatic properties of cytochrome P450 catalyzing 3 ' -hydroxylation of naringenin from the white- rot fungus Phanerochaete chrysosporium ,” Biochemical and Biophysical Research Commu (21 ) Appl. No. : 15 /514 , 120 nications 387 (2009 ), pp . 103 - 108 . Kabumoto et al, “ Directed Evolution of the Actinomycete Cytochrome ( 22 ) PCT Filed : Sep . 27 , 2015 P450 MOXA (CYP105 ) for Enhanced Activity , ” Biosci. Biotechnol . Biochem . 73 ( 9 ) , pp . 1922 - 1927 ( 2009 ) . European Search Report dated May 19 , 2015 in corresponding ( 86 ) PCT No .: PCT/ EP2015 /072189 European Application No . 14187583 . 1 . $ 371 ( c ) ( 1 ) , First Examination Report dated Dec . 22 , 2017 in corresponding European Application No . 14187583 . 1 . ( 2 ) Date : Mar. 24 , 2017 Bhuiya , M ., et al . , “ Engineering Monolignol 4 - 0 -Methyltransferases to Modulate Lignin Biosynthesis ,” Journal of Biological Chemistry , (87 ) PCT Pub . No .: WO2016 / 050656 vol . 285, No . 1 , pp . 277 - 285 ( Jan . 1 , 2010 ) . PCT Pub . Date : Apr . 7 , 2016 Ravichandran , K ., et al ., “ Crystal Structure of Hemoprotein Domain of P450BM - 3 , a Prototype for Microsomal P450 ' s , ” Science, vol . 261 , pp . 731 -736 ( Aug . 6 , 1993 ) . (65 ) Prior Publication Data Wils , C . , et al ., “ A Single Amino Acid Determines Position Speci ficity of an Arabidopsis thaliana CCOAOMT- like O -methyltransferase ." US 2018 / 0135029 A1 May 17 , 2018 FEBS Letters , vol. 587 , pp . 683 -689 (Feb . 14 , 2013 — on - line ) . (30 ) Foreign Application Priority Data Primary Examiner - Jennifer E Graser (74 ) Attorney , Agent, or Firm — Polsinelli PC Oct. 3 , 2014 (EP ) .. .. .. 14187583 (57 ) ABSTRACT A method is proposed for producing flavonoids , comprising (51 ) Int. Ci. the steps: (a ) providing of a transgenic microorganism , C12P 17 /06 ( 2006 .01 ) containing ( i ) a first nucleic acid section ( A ) , comprising or C12N 9 / 10 ( 2006 .01 ) consisting of a gene coding for a CYP450 oxidase , ( ii ) a CI2N 9 /02 ( 2006 . 01 ) second nucleic acid section ( B ), comprising or consisting of C12N 15 /64 ( 2006 .01 ) a gene coding for a plant O -methyltransferase , and ( b ) ( 52 ) U .S . CI. adding of one or more flavanones to the transgenic micro CPC . .. C12N 9 / 1007 (2013 . 01 ) ; C12N 9 /0071 organism , ( c ) the conversion of the substrate flavanones by ( 2013 .01 ) ; C12N 15 /64 ( 2013 . 01 ) ; C12P 17 / 06 the transgenic microorganism to the corresponding fla ( 2013 .01 ) ; C12Y 114 / 14001 ( 2013 .01 ) ; C12Y vonoids, and optionally ( d ) isolating and purifying of the 201/ 01 ( 2013 . 01 ) final products . ( 58 ) Field of Classification Search None 6 Claims, 8 Drawing Sheets See application file for complete search history . Specification includes a Sequence Listing . atent Oct. 8 , 2019 Sheet 1 of 8 US 10 ,435 ,673 B2 2000 CYP450 -Oxiclase pSYM _ BM3 8 ,519 bp Figure 1 Plasmid pSYM _ BM3 with CYP450 oxidase gene atent Oct. 8 , 2019 Sheet 2 of 8 US 10 ,435 ,673 B2 4,900 3.$ 90 2000 Sob MCPFOMT 'zo40 wito M 4 , 142 bp TTP H al z000 ' Figure 2 Plasmid pSYM _ PFOMT gene coding for O -methyltransferase atent Oct. 8 , 2019 Sheet 3 of 8 US 10 ,435 ,673 B2 . 600 800 .. 4,400 4 ,517 1,009 AtCOMTCO wat !OOV 4,000 PSYM _ ADCOMT code 4 , 517 bp noo olto M Octe 00bc 0092 Figure 3 PlasmidFigure pSYMPSY __ AtCOMTAICOMT genegene coding for cation - independent O -methyltransferase atent Oct. 8 , 2019 Sheet 4 of 8 US 10 ,435 ,673 B2 100 Substanceconcentration(%) 0 200 400 800 900 1000 1200 1400 1600 Incubation time (min ) eriodictyot - 0 . naringenin Figure 4 Biocatalytic transformation from naringenin to eriodictyol U . S . Patent Oct. 8 , 2019 Sheet 5 of 8 US 10 ,435 ,673 B2 120 * . ! Substanceconcentration(%) * . * * 0 20wwwwwwwwwwwwwwwwwwwwwwwwwwwwww 40 60bangsa wwwwwwwwwwwwwali80 WWWWWW 100 120 Incubation time ( s ) - Moeodictyo - 0 . eriodictyok Figure 5 Biocatalytic transformation from eriodictyol to homoeriodictyol BM3 [GVQC ) /PFOMT * Naringenin 49 . .. .. www friodictyol Range/106 Homoeriodictyol 1.4 . hennarration through there t hecharged pidid 25 Time ( s ) Figure 6 Fermentative transformation from naringenin to homoeriodictyol U .S . Patentatent Oct. 8 , 2019 Sheet 6 of 8 US 10 ,435 ,673 B2 . ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' 8M3 [GVQC ) /PFOMT + Eriodictyol Naringenin * Eriodictyol Range/106 « Homoeriodictyol 22 III 5 10 15 20 25 Time ( h ) Figure 7 Fermentative transformation from eriodictyol to homoeriodictyol BM3( GVQC ) + Naringenin - Naringenin Eriodictyol Range/106 O Homoeriodictyol EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE . ". ' . ' . ' . ' .' . ". 1 . 1 . 1 . 1 .1 . 1 . 1 . 1 . 1 . 1 .1 . 1 . 1 . 1 . 1 . 1 .1 . 1 . 1 . 1 . 1 . 1 .1 . 1. ' . ' . ' . ' .' . ". ' . ' . ' . " . 5 10 15 20 TIME (MY Figure 8 Fermentative transformation from naringenin to eriodictyol U . S . Patent Oct . 8 , 2019 Sheet 7 of 8 US 10 , 435 ,673 B2 M inin Figure 9 Isolation of CYP450 oxidase BM3 eeeeeeee Figure 10 Isolation of O -methyltransferase POMT Figure 11 Isolation of O -methyltransferase AtCOMT atent Oct. 8 , 2019 Sheet 8 of 8 US 10 ,435 ,673 B2 : : Substance(%) : . : :. : : : : : : : : GV VQ V GVQS GVQ BM3 variant . naringenin (Substrat ) M eriodictyol w apigenin Figure 12 Comparison of product formation of various variants of BM3 US 10 , 435 ,673 B2 METHOD FOR THE BIOTECHNOLOGICAL FIG . 6 shows the fermentative transformation from nar PRODUCTION OF FLAVONOIDS ingenin to homoeriodictyol; FIG . 7 shows the fermentative transformation from erio FIELD OF THE INVENTION dictyol to homoeriodictyol; 5 FIG . 8 shows the fermentative transformation from narin The invention is in the field of biotechnology and relates qenin to eriodictyol; to a process in which flavanone is converted to the corre FIG . 9 shows the isolation of CYP450 oxidase BM3; sponding flavonoids without requiring any intermediate FIG . 10 shows isolation of O -methyltransferase PfOMT; chemical steps , a corresponding microorganism , a vector , FIG . 11 shows isolation of O -methyltransferase AtCOMT; and a host cell . and STATE OF THE ART FIG . 12 compares product formation of various variants of BM3. Flavonoids such as , for example, homoeriodictyol, erio dictyol ( EP 1258200 B1) and hesperetin ( EP 1998636 B1) 15 DESCRIPTION OF THE INVENTION represent important flavouring and aroma substances . These substances are usually produced by an aldol reaction of free A first subject -matter of the invention relates to a process or partially methylated acetophloroglucinol with free or for the production of flavonoids, comprising the following partially methlylated protocatechualdehyde . Although the steps : processes have been established on an industrial scale , they 20 ( a ) Providing a transgenic microorganism , comprising exhibit significant disadvantages : during the course of reac tion it is necessary to provide the phenolic functions with ( i ) A first nucleic acid fragment ( A ), comprising or protective groups which need to be removed at a later point consisting of a gene coding for a CYP450 oxidase , in time. As a result , the syntheses become multi - stage , i. e . ( ii ) A second nucleic acid fragment ( B ) , comprising or technically complex and obviously also costly . 25 consisting of a gene coding for a plant O -methyltrans The subject matter of WO 2005 115377 A1 is a food ferase, and composition . comprising flavonoids and tocotrienol. ( b ) Adding one or more flavanones to the transgenic micro wherein the flavonoids may represent naringenin , hesperi organism , din , nobiletin or tangeretin . ( c ) Converting the flavanones contained in the substrate by However , it is particularly disadvantageous that these are 30 the transgenic microorganism to the corresponding fla chemical methods of production , i. e . for regulatory reasons vonoids , and , optionally the food additives , flavouring substances and aromas so ( d ) Isolating and purifying the final products . produced may not be designated as natural . However , a Surprisingly , it was found that by insertion of two differ designation as natural or nature - identical is crucial for many ent nucleic acid fragments , which contain a gene coding for consumers when making their purchase decision ; it is , 35 a bacterial CYP450 oxidase on the one hand and a gene therefore , obvious that there is a particular demand for coding for a plant