Method for biotechnological production of methylized cinnamic acids and cinnamic acid esters, methylized phenethylamines and the coupling products thereof, particularly of cinnamic acid amides

Patent metadata

Field	Value
Publication number	US-2016281118-A1
Application number	US-201615011855-A
Country	US
Kind code	A1
Filing date	Feb 1, 2016
Priority date	Jan 30, 2015
Publication date	Sep 29, 2016
Grant date	—

Abstract

The invention relates to providing both fermentative and biotechnological methods for producing 3,4-methylized cinnamic acids, 3,4-methylized cinnamic acid esters, 3,4-dimethoxyphenethylamine, and 4-methylized cinnamic acid amides using a 4′-O-methyltransferase, optionally in combination with further enzymes, wherein the enzymes are selected by means of metabolic engineering and operation have been adapted by targeted optimization, and compositions obtained by means of the method.
The invention further relates to vector systems, recombinant microorganisms or fungi, and specific nucleic acid segments and polypeptides.

First claim

1 . A method for producing a 3,4-methylized cinnamic acid or a 3,4-methylized cinnamic acid ester using a recombinant microorganism or fungus and comprising the following steps:
(i) providing a recombinant microorganism or fungus comprising
(a1) a nucleic acid segment comprising or made of at least one gene coding for a 4′-O-methyltransferase, and
(a2) optionally a nucleic acid segment comprising or made of at least one gene coding for a 3′-O-Methyltransferase, and
(b) optionally for fermentative production, a nucleic acid segment comprising or made of a gene coding for an S-adenosylmethionine synthase (SAMS);
(ii) cultivating the recombinant microorganism or fungus under conditions allowing the expression of the nucleic acid segment(s) for obtaining the corresponding product(s) of expression; (iii) optionally isolating and optionally purifying the product(s) of expression obtained; (iv) adding one or more hydroxycinnamic acid(s), preferably adding caffeic acid or ferulic acid, optionally esterified on a hemicellulose, and/or adding one or more precursors or one or more derivatives thereof, optionally esterified on a hemicellulose, to the cultivated recombinant microorganism or fungus according to step (ii) for a fermentative conversion or to the product(s) of expression according to step (iii) for an enzymatic conversion; (v) cultivating or incubating the recombinant microorganism or fungus or the product(s) of expression under conditions enabling the conversion of the hydroxycinnamic acid or the precursor(s) or the derivative or derivatives thereof to a 3,4-methylized cinnamic acid or a 3,4-methylized cinnamic acid ester, obtaining the corresponding 3,4-methylized cinnamic acid or 3,4-methylized cinnamic acid ester; and (vi) optionally isolating and optionally purifying the obtained 3,4-methylized cinnamic acid or the 3,4-methylized cinnamic acid ester and further byproducts that are optionally present.

2 . The method for producing 3,4-dimethoxyphenethylamine according to claim 1 using a recombinant microorganism or fungus and comprising the following steps:
(i) providing a recombinant microorganism or fungus comprising
(a1) a nucleic acid segment comprising or made of at least one gene coding for a 4′-O-Methyltransferase, and
(a2) a nucleic acid segment comprising or made of at least one gene coding for a 3′-O-Methyltransferase, and
(b) optionally a nucleic acid segment comprising or made of a gene coding for an S-adenosylmethionine synthase, and
(c) optionally a nucleic acid segment comprising or made of a gene coding for a DOPA-Decarboxylase,

(ii) cultivating the recombinant microorganism or fungus under conditions allowing the expression of the nucleic acid segments for obtaining the corresponding products of expression;
(iii) optionally isolating and optionally purifying the products of expression obtained;
(iv) adding dopamine and/or L-dihydroxyphenylalanine and/or one or more precursors or one or more derivatives thereof to the cultivated recombinant microorganism according to step (ii) for a fermentative conversion or to the products of expression according to step (iii) for an enzymatic conversions, wherein for the case of enzymatic conversion S-adenosylmethionine is also preferably added; and
(v) cultivating or incubating the recombinant microorganism or fungus or the products of expression under conditions enabling the conversion of dopamine or L-dihydroxyphenylalanine and/or the precursor(s) or the derivative or derivatives thereof to 3,4-dimethoxyphenethylamine for obtaining 3,4-dimethoxyphenethylamine;
(vi) optionally isolating and optionally purifying the obtained 3,4-dimethoxyphenethylamine and further byproducts that are optionally present.

3 . A method for producing a 4-methylized cinnamic acid amide using a recombinant microorganism or fungus and comprising the following steps:
(i) providing a recombinant microorganism according to claim 1 and additionally comprising:
(d) a nucleic acid segment comprising or made of at least one gene coding for a 4-coumarat:CoA-ligase, and
(e) a nucleic acid segment comprising or made of at least one gene coding for a tyramine-N-hydroxycinnamoyltransferase, and
(f) optionally a nucleic acid segment comprising or made of a gene coding for a DOPA-decarboxylase,
(ii) cultivating the recombinant microorganism or fungus under conditions allowing the expression of the nucleic acid segments for obtaining the corresponding products of expression; (iii) optionally isolating and optionally purifying the products of expression obtained; (iv) adding dopamine and/or L-dihydroxyphenylalanine and a hydroxycinnamic acid, preferably caffeic acid or ferulic acid, to the cultivated recombinant microorganism or fungus according to step (ii) for a fermentative conversion, or a phenethylamine to the products of expression according to step (iii) for an enzymatic conversion, wherein for the case of enzymatic conversion S-adenosylmethionine is preferably also added; and (v) cultivating or incubating the recombinant microorganism or fungus or the products of expression, optionally adding a lipase under conditions enabling the conversion of dopamine and/or L-dihydroxyphenylalanine and of a hydroxycinnamic acid to a 4-methylized cinnamic acid amide; (vi) optionally isolating and optionally purifying the obtained 4-methylized cinnamic acid amide and further byproducts that are optionally present.

4 . The method according to claim 3 , wherein the 4-methylized cinnamic acid amide is selected from the group of rubemamine [(2E)-3-(3,4-dimethoxyphenyl)-N-[2-(3,4-dimethoxyphenyl)ethyl]prop-2-enamide], feruloyl-3-methoxytyramide [(2E)-3-(4-hydroxy-3-methoxyphenyl)-N-[2-(4-hydroxy-3-methoxyphenyl)ethyl]prop-2-enamide], (2E)-3-(3,4-dimethoxyphenyl)-N-[2-(4-hydroxy-3-methoxy-phenyl)ethyl]prop-2-enamide], and 3,4-dimethoxycinnamoylmethoxytyramide [(2E)-3-(3,4-dimethoxyphenyl)-N-[2-(4-methoxyphenyl)ethyl]prop-2-enamide].

5 . The method according to claim 1 , wherein the 4′-O-methyltransferase, or the nucleic acid segment coding the 4′-O-methyltransferase, is selected from SEQ ID Nos: 5, 6, 7, 8, 9, 15, 17, 18, 25, 26, 27, 28, 29, 35, 37, 38, 85, and 86, and/or the 3′-O-methyltransferase, or the nucleic acid segment coding the 3′-O-methyltransferase, is selected from SEQ ID Nos: 3, 4, 16, 19, 20, 23, 24, 36, 39, and 40, and/or the S-adenosylmethionine synthase, or the nucleic acid segment coding the S-adenosylmethionine synthase is selected from SEQ ID Nos: 10, 30, and 65 through 72, and/or the DOPA-decarboxylase, or the nucleic acid segment coding the DOPA-decarboxylase is selected from SEQ ID Nos: 1, 2, 21, 22, and 59 through 64, and/or the tyramine-N-hydroxycinnamoyltransferase, or the nucleic acid segment coding the tyramine-N-hydroxycinnamoyltransferase, is selected from SEQ ID Nos: 13, 14, 33, 34, and 79 through 82, and/or the 4-coumarat:CoA-ligase, or the nucleic acid segment coding the 4-coumarat:CoA-ligase, is selected from SEQ ID NOs:11, 12, 31, 32, and 73 through 78, and the lipase is a lipase B selected from SEQ ID NOs:83 and 84; wherein the polypeptides and the sequences coding the polypeptides comprise sequences having at least 80% sequence homology with the corresponding SEQ ID Nos, under the condition that a polypeptide sequence thus modified and having a corresponding degree of homology still fulfills the same enzymatic function as the corresponding non-modified polypeptide having the same SEQ ID NO.

6 . A nucleic acid segment suitable for use in performing the method according to claim 1 , comprising or made of at least one coding gene selected from the group of SEQ ID NOs: 8, 16, 17, 20, and 85.

7 . A polypeptide coded by a nucleic acid segment according to claim 6 , selected from the group of SEQ ID Nos: 28, 36, 37,

Assignees

• Symrise Ag

Inventors

• Krammer Gerhard
• Ley Jakob Peter
• Geissler Katrin
• Geissler Torsten
• Gomoll Frauke
• Welters Peter
• Jach Guido
• Wessjohann Ludger

Classifications

• C12Y205/01006

  Methionine adenosyltransferase (2.5.1.6), i.e. adenosylmethionine synthetase · CPC title

• C12P7/22

  aromatic · CPC title

• A23L27/204

  Aromatic compounds · CPC title

• C12P13/02 Primary

  Amides, e.g. chloramphenicol {or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes (peptides C12P21/00 or C07K)} · CPC title

• C12P7/40

  containing a carboxyl group {including Peroxycarboxylic acids} · CPC title