WO2022122921A1

WO2022122921A1 - Process for the biotechnological production of 2-phenylethanols from plant sources

Info

Publication number: WO2022122921A1
Application number: PCT/EP2021/084999
Authority: WO
Inventors: Michel OELSCHLÄGEL; Anna Stuhr; André Pollender; Dagmar Ganz; Michael SCHLÖMANN
Original assignee: Technische Universität Bergakademie Freiberg
Priority date: 2020-12-09
Filing date: 2021-12-09
Publication date: 2022-06-16
Also published as: DE102020132795A1

Abstract

The invention relates to a method for the biocatalytic synthesis of 2-phenylethanols, in particular 3-hydroxytyrosol, using a whole-cell catalyst, in particular a recombinant microorganism, and to the use thereof for the biocatalytic synthesis of a 2-phenylethanol by means of a biocatalytic conversion of plant starting materials, such as cinnamic acid, in particular caffeic acid.

Description

Process for the biotechnological production of 2-phenylethanols from plant sources

The invention relates to a process for the biocatalytic synthesis of 2-phenylethanols, in particular 3-hydroxytyrosol, a whole-cell catalyst, in particular a recombinant microorganism, and its use for the biocatalytic synthesis of a 2-phenylethanol by biocatalytic conversion from plant starting materials such as cinnamic acids, in particular caffeic acid.

3-Hydroxytyrosol is considered one of the strongest known antioxidants and inhibits cell aging and degeneration processes triggered by free radicals. Thanks to its antioxidant effect, it prevents diseases such as cancer, arteriosclerosis or Alzheimer’s (Hu et al. 2014). Furthermore, positive effects on widespread diseases such as diabetes, on the cardiovascular system, the nervous system and the gastrointestinal tract are proven. Scientific studies have also demonstrated a strong antimicrobial effect against various types of pathogenic bacteria (Tafesh et al. 2011). Antiviral properties, e.g. against HIV, have also been proven (Bedoya et al. 2016).

3-Hydroxytyrosol occurs naturally in some plants, especially olives and parts of the olive tree, but at low levels. In addition to extractive production, there are therefore biotechnological and chemical processes for the production of 3-hydroxytyrosol.

During the extraction process from olive oil, 3-hydroxytyrosol is obtained in the aqueous liquid that is produced when the olives are pressed and is known as vegetation water. A disadvantage here is the complex product preparation for isolating the pure 3-hydroxytyrosol from the complex mixture of substances with structurally similar compounds. Fernandez-Bolanos et al. describe the use of large amounts of organic solvents to isolate 3-hydroxytyrosol from vegetation water (Fernandez-Bolanos et al. 2002).

Alternatively, instead of complex purification, the vegetation water is used directly in further processing. However, consistent product quality is difficult here, since vegetation water is a complex solution. In addition to many other components, it contains 3-hydroxytyrosol in varying concentrations, depending on the batch. Consequently, those made from them differ Products with regard to their ingredients, even if the same content of 3-hydroxytyrosol is always set (Bellumori et al. 2019). This problem can only be avoided by using purified 3-hydroxytyrosol. Another disadvantage of extractive production using olives is the dependence on harvest cycles.

Many biotechnological processes rely on the conversion of the starting material tyrosol. US 2010/0047887 A1 and US 2010/0068775 A1 disclose a method and a microorganism for converting tyrosol to 3-hydroxytyrosol by means of hydroxylases, preferably in the presence of vitamin C (ascorbic acid) and glutathione. US 2010/0047887 A1 describes product amounts of up to 8 mM in 16 h. With a cell concentration with an ODeoo in the range from 21 to 60, 3-hydroxytyrosol concentrations in the range from 1.6 to 1.8 g/l are achieved in a few hours, which corresponds to yields in the range from 58 to 65%. US 2010/0068775 A1 describes conversions with up to 7.8 mM 3-hydroxytyrosol in 18 h or yields of up to 91%. The disadvantage here is that expensive tyrosol is used as the starting material. US 2010/0047887 A1 and US 2010/0068775 A1 also describe the use of expensive co-substrates such as vitamin C or glutathione.

CN 107201331 A and CN 107586794 A describe the use of cheaper glucose, which is first converted into 4-hydroxyphenylpyruvate by the shikimate metabolism in the bacterial cell, converted into tyrosol via 4-hydroxyphenylacetaldehyde and finally hydroxylated into 3-hydroxytyrosol. The disadvantage of the processes starting from glucose is low amounts of 3-hydroxytyrosol, in particular CN 107586794 A discloses a product amount of 0.65 g/l after 48 hours. CN 107201331 A describes conversions of up to 0.4 g/l. WO 2012/135389 A2 discloses the production of 0.08 mM 3-hydroxytyrosol when using glucose (see WO 2012/135389 A2 Fig. 7).

Other methods use the compounds dopamine or L-3,4-dihydroxyphenylalanine, which either serve directly as a substrate or are obtained within the cell from tyrosine or from glucose, which is previously converted into tyrosine in the cells. CN 109295113 A, WO 2012/135389 A2 and WO 2017/223569 A1 describe the use of a decarboxylase or an amino acid transferase and a dehydrogenase; a monoamine oxidase and an alcohol dehydrogenase for the synthesis of 3-hydroxytyrosol from L-3,4-dihydroxyphenylalanine. WO 2012/135389 A2 discloses amounts of product in the range from 0.47 to 0.74 mM with direct use of the intermediates L-3,4-dihydroxyphenylalanine or dopamine as substrate. However, the disadvantage here is that expensive starting materials are used. Shanker et al. disclose the conversion of ferulic acid to acetovanillon (apocynin) using Rhizopus oryzae cells, forming acetovanillon as the major metabolite, dihydroferulic acid, coniferyl alcohol, and dihydroconiferyl alcohol as by-products, and vanillin, vanillyl alcohol, vanillic acid, and phenylethyl alcohol as trace products (<1-3%) ( Shanker et al 2007).

Mabinya et al. disclose the bioconversion of ferulic acid and 4-vinylguaiacol by a white-rot fungus isolated from decaying wood, with 4-vinylguaiacol being further converted to acetovanillon (Mabinya et al. 2010). Furthermore, Mabinya et al. the formation of vanillin and vanillic acid, as well as 2-phenylethanol, 4-ethyl-2-methoxyphenol, 1-(2,3-dihydroxy-4-methoxy-6-methylphenyl)-ethanone, 4,5-dimethoxy-2-methylphenol and vanillic acid ethyl ester as by-products.

Zhou et al. describe an enantioselective one-pot synthesis of D-phenylglycines from racemic mandelic acids, styrenes or bio-based L-phenylalanine via cascade biocatalysis using a recombinant Escherichia coli LZ110, which co-expresses four enzymes (Zhou et al. 2017). Furthermore, Zhou et al. the use of E. coli LZ116 expressing seven enzymes to convert styrene to enantiopure D-phenylglycine, and the use of E. coli LZ143 expressing nine enzymes to convert L-phenylalanine to enantiopure D-phenylglycine.

Chemical processes offer a possible alternative. The most important chemical process developed by Wacker Chemie AG (US Pat. No. 8,822,738 B1) allows the purely chemical production of 3-hydroxytyrosol from 3,4-dimethoxyphenylethanol using aluminum-based catalysts, in particular triisobutylaluminum or diisobutylaluminum hydride. The processing is carried out using an aqueous solution of a hydroxycarboxylic acid and extraction using organic solvents.

EP 2 114 846 B1 or CN 101641316 A describes the preparation of 3-hydroxytyrosol from 4-chloroacetylcatechol using metal formate and formic acid in an aqueous solution and catalytic hydrogenation using a noble metal catalyst, preferably palladium (Pd) or ruthenium (Ru), in particular Pd/C or Ru/C, in an organic solvent, especially a liquid alkyl ester. A five-stage process starting from 3,4-dihydroxybenzene (CN 103664536 A) is also known. CN 103664536 A describes the protection of the two phenol groups by the synthesis of 1,2-methylenedioxybenzene using dichloromethane, a Friedel-Crafts reaction to produce 3,4-methylenedioxyacetophenone, a Wolff-Kishner-Huangminglong reduction to produce 3,4 -methylenedioxyphenylacetic acid, a reduction to produce 3,4-methylenedioxyphenylethanol and a deprotection using boron tribromide or Pd/C.

A disadvantage of chemical processes is that they require expensive starting materials such as 3,4-dimethoxyphenylethanol in addition to expensive catalysts such as palladium. Furthermore, the use of petrochemical feedstocks is not sustainable.

Therefore, the object of the present invention is to provide a method for synthesizing compounds of formula (V), which overcomes the disadvantages of the prior art, in particular to provide a cheaper, biotechnological method for synthesizing compounds of formula (V), with increased product quantities are obtained.

It is also an object of the invention to provide a whole-cell catalyst for the biocatalytic synthesis of a compound of the formula (V).

According to the invention, the object is achieved by the method according to the invention for the biocatalytic synthesis of a compound according to formula (V)

by the biocatalytic conversion of a substance comprising at least one compound

Formula (I)

wherein R ¹ is -H or -OH, wherein R ² is -H, -OH or -OCH3, comprising the following steps: a) providing at least one whole cell catalyst comprising i. a gene coding for an enzyme phenolic acid decarboxylase and ii. at least one gene coding for an enzyme comprising an oxygenase subunit and/or a reductase subunit, preferably a styrene monooxygenase or indole monooxygenase, and iii. a gene coding for an enzyme styrene oxide isomerase, and iv. a gene coding for an enzyme alcohol dehydrogenase, and at least one promoter for the controllable expression of genes i) to iv) in an aqueous medium, b) activation of the whole cell catalyst with an inducer, the inducer leading to the expression of genes i) to iv). , and c) contacting the whole-cell catalyst with a substance comprising at least one compound of formula (I), wherein the at least one compound of formula (I) is reacted with the enzymes defined in (a) to form a compound of formula (V).

According to the invention, there is no significant further metabolization of the compound of formula (V), ie no significant further conversion of the compound of formula (V) by the whole-cell catalyst. When selecting the whole-cell catalyst, the person skilled in the art can evaluate whether potentially disruptive metabolic pathways are present in the cell, for example disruptive genes in the tyrosine metabolism (in particular homoprotocatechuate degradation) or in the Phenylalanine metabolism (ring-opening mechanisms in dihydroxylated aromatic structures). In the case of potentially disruptive metabolic pathways, the possibility of inactivating such disruptive metabolic pathways by means of genetic engineering methods (negative mutant) or via targeted culture management (e.g. substrate-mediated inhibition by glucose or omission of media components which potentially have an inducing effect on such disruptive metabolic pathways) are known to the person skilled in the art or derivable. Advantageously, there is no change of substituents on the benzene ring in the process according to the invention.

In embodiments, the method according to the invention takes place with the sequence of steps a), b) and c), steps b) and c) taking place simultaneously or step c) taking place after step b). The process according to the invention preferably takes place with the sequence of steps a), b) and c), with steps b) and c) taking place simultaneously.

The inventive method has the advantage that through the use of whole-cell catalysts, which express a phenolic acid decarboxylase, a monooxygenase comprising an oxygenase subunit and / or a reductase subunit, a styrene isomerase and an alcohol dehydrogenase, all enzymes simultaneously for the biocatalytic Synthesis of 2-phenylethanols, preferably 3-hydroxytyrosol. With the process according to the invention, it is advantageously possible to obtain compounds of the formula (V) or 2-phenylethanols, in particular 3-hydroxytyrosol, in a sustainable manner. Yields of at least 95% were also advantageously achieved with the process according to the invention.

The compound according to formula (I) is preferably a trans compound.

In embodiments, at least one of Ri and R2 is a hydroxy group.

In embodiments, the compound of formula (I) is selected from caffeic acid (3-(3,4-dihydroxyphenyl)propenoic acid), cinnamic acid (3-phenylpropenoic acid), 3-hydroxycinnamic acid, p-coumaric acid (4-hydroxycinnamic acid), 3-methoxycinnamic acid and ferulic acid (4-hydroxy-3-methoxycinnamic acid). The compound according to formula (I) is preferably caffeic acid.

In embodiments, the compound of formula (V) is selected from 3-hydroxytyrosol (2-(3,4-dihydroxyphenyl)ethanol), 2-(3-hydroxyphenyl)ethanol, 2-(4-hydroxyphenyl)ethanol, 2-(3- methoxyphenyl)ethanol and 2-(4-hydroxy-3-methoxy-phenyl)ethanol (homovanillyl alcohol). The compound of formula (V) is preferably 3-hydroxytyrosol.

In embodiments, Ri and R2 are each a hydroxy group.

The term "whole cell catalyst" means a catalytically active cell. In embodiments, the whole cell catalyst is an authentic microorganism (ie natural or unmodified microorganism) or a genetically modified microorganism.

A “genetically modified microorganism” is understood to mean a microorganism with at least one mutation, deletion or substitution in the genome or a microorganism in which further genes would be made available via additionally introduced vectors. The combination of artificial changes in the genome and introduction by means of vectors is also included here.

In embodiments, the genetically engineered microorganism is a knockout mutant or a deletion mutant of an authentic microorganism (i.e., an authentic microorganism with at least one gene knockout to inactivate a particular gene) (negative mutant) and/or an insertion mutant. The term "insertion mutation" is understood to mean the introduction of at least one gene by genome insertion or by introduction of expression vectors.

In preferred embodiments, the whole cell catalyst is a recombinant bacterial cell.

In embodiments, the bacterial cell is selected from Escherichia, Arthrobacter, Bacillus, Rhodococcus, Pseudomonas, Sphingobium, Sphingopyxis, Corynebacterium, Marinobacterium, Variovorax and Gordonia, preferably from Escherichia coli, Rhodococcus opacus 1 CP, Rhodococcus species ST-5, Pseudomonas fluorescens ST, Corynebacterium species AC-5, Pseudomonas putida CA-3, Pseudomonas putida 312 and Variovorax paradoxus EPS.

In preferred embodiments, the bacterial cell is Escherichia coli, in particular a derivative of strain BL21 or strain B.

In preferred embodiments, the whole cell catalyst is Escherichia coli with insertion mutation.

The nucleotide sequences of genes i.) to iv.) include authentic and/or artificial reading frames. An artificial reading frame is preferably adapted to the "codon usage" of the host organism via gene synthesis.

In principle, suitable genes i.) to iv.) by methods known per se, such as by polymerase chain reaction (PCR) using short, synthetic nucleotide sequences (primers) starting from the DNA of conceivable donor organisms, amplified and then isolated. The primers used are generally produced using known gene sequences due to existing homologies with genes i.) to iv.). Alternatively, the genes can also be constructed using gene synthesis, which allows additional adaptations of the genes. Ideally, the vector for cloning an amplified gene is of low molecular mass and has selectable genes to result in a readily recognizable phenotype in a cell, allowing for easy selection of vector-containing and vector-free host cells. In order to obtain a high yield of DNA and corresponding gene products, the expression vector should have at least one strong promoter and/or regulatory sequence. Alternatively, promoters are introduced in front of the genes by means of a PCR (via the primers) or by means of gene synthesis. In addition, a ribosomal binding site (RBS) is expediently introduced in front of each gene by means of a vector, PCR or gene synthesis for optimal translation. An origin of replication is also important for replication of the vector. For example, pET vector systems based on antibiotic selection, among others, are suitable.

The methods for cultivating microorganisms are known to those skilled in the art, with the microorganisms being cultured continuously or discontinuously in a batch process (batch cultivation) or in a fed-batch (feed process) or repeated fed-batch process (repetitive feed process) for the purpose of cultivation or biocatalytic conversion of a substance comprising a compound according to formula (I) are cultivated.

In embodiments, the cultivation takes place in shake flasks or in a fermenter. The cultivation preferably takes place under physiological conditions at a temperature in the range from 10° C. to 50° C., preferably in the range from 20° C. to 40° C., particularly preferably in the range from 23° C. to 37° C., with the pH Value of the aqueous component is preferably in the range from 5.8 to 8.5, particularly preferably in the range from 6.5 to 8.0.

In embodiments, the whole-cell catalyst is provided with a low bacterial density, preferably with an ODeoo in the range from 0.3 to 1.7, particularly preferably with an ODeoo in the range from 0.5 to 1.1. In step b) and at the beginning of step c), the whole-cell catalyst expediently has a low bacterial density, preferably with an ODeoo in the range from 0.3 to 1.7, particularly preferably with an ODeoo in the range from 0.5 to 1.1 , before. The term “ODeoo” is a measure of the bacterial density, which is measured using UV/Vis spectroscopy or a photometer at a wavelength of 600 nm. In embodiments, the at least one whole-cell catalyst is provided in step a) by inoculating a main culture using a preculture with an ODfeoo in the range from 0.7 to 5, so that the main culture has an ODeoo of at most 0.1, and then growing the main culture up to to an ODeoo in the range from 0.3 to 1.7, particularly preferably up to an ODeoo in the range from 0.5 to 1.1, at which the induction (step b) is carried out and the conversion (step c) is started.

In embodiments, the genes i.) to iv.) are naturally present in the whole-cell catalyst according to the invention or are introduced by genetic engineering methods.

A phenolic acid decarboxylase is an enzyme in bacteria that catalyzes the cleavage of a carboxy group (-COOH) from organic acids, particularly phenolic acids.

In embodiments, the whole cell catalyst comprises a phenolic acid decarboxylase capable of converting compound (I) to compound (II)

where R ¹ is -H or -OH and where R ² is -H, -OH or -OCH3.

According to the invention, the whole-cell catalyst has at least one gene coding for an enzyme comprising an oxygenase subunit and/or a reductase subunit, preferably a styrene monooxygenase or indole monooxygenase. Advantageously, styrene monooxygenase and indole monooxygenase have a substrate spectrum which enables high conversion of the compounds according to the invention.

An oxygenase is an enzyme in bacteria that incorporates at least one oxygen atom into an organic compound, forming at least one new hydroxy, carboxy, or epoxy group. Preferably the oxygenase is a monooxygenase, i. H. an enzyme that incorporates an oxygen atom into an organic compound.

A styrene monooxygenase is an enzyme in bacteria that chemically reacts styrene with flavin adenine dinucleotide (FADH2) according to the reaction:

styrene + FADH2 + O2 «-> 2-phenyloxirane + FAD + H2O as the first step of the aerobic styrene degradation pathway (ie in the presence of oxygen) in bacteria to the intermediate 2-phenyloxirane (styrene oxide). FAD is regenerated by the reductase subunit of styrene monooxygenase using NADH.

An indole monooxygenase is an enzyme in bacteria that chemically reacts indole with flavin adenine dinucleotide (FADH2) according to the reaction:

Indole + FADH2 + O2 «-> indole oxide + FAD + H2O catalyzed. FAD is regenerated by the reductase subunit of indole monooxygenase using NADH.

A reductase is an enzyme which catalyses the reduction of an organic compound, in particular FAD, with the simultaneous oxidation of a co-substrate, in particular NADH.

In embodiments, the whole cell catalyst comprises at least one enzyme having an oxygenase subunit and/or a reductase subunit capable of converting compound (II) to compound (III)

where R ¹ is -H or -OH and where R ² is -H, -OH or -OCH3.

In embodiments, the enzyme comprising an oxygenase subunit and/or a reductase subunit is a fusion protein.

A styrene oxide isomerase is an enzyme in bacteria that catalyzes the chemical conversion of unsubstituted or substituted styrene oxides to unsubstituted or substituted phenylacetaldehydes.

In embodiments, the whole cell catalyst comprises a styrene oxide isomerase capable of converting compound (III) to compound (IV)

where R ¹ is -H or -OH and where R ² is -H, -OH or -OCH3.

An alcohol dehydrogenase is an enzyme that catalyzes the chemical conversion of alcohols into aldehydes or ketones and the reverse reaction. The enzyme is dependent on cofactors. Cofactors NADH, NADPH or cytochrome C are preferred. Alcohol dehydrogenases often have a broad substrate spectrum, which is why a larger number of alcohol dehydrogenases can be used in the method according to the invention. Furthermore, advantageously, several alcohol dehydrogenases occur naturally in bacterial cells, which is why authentic alcohol dehydrogenases can be used in some whole-cell catalysts.

In embodiments, the whole cell catalyst comprises an alcohol dehydrogenase capable of converting compound (IV) to compound (V)

where R ¹ is -H or -OH and where R ² is -H, -OH or -OCH3.

The corresponding gene and amino acid sequences for the enzymes mentioned (phenolic acid decarboxylase, oxygenase of a monooxygenase, reductase of a monooxygenase, styrene oxide isomerase and alcohol dehydrogenase) are well known to the person skilled in the art or can be found in known databases (e.g. NCBI, RCSB PDB, UniProt, PDB Europe) can be taken.

In embodiments, the whole cell catalyst comprises i. At least two genes coding for an enzyme phenolic acid decarboxylase and/or ii. at least two genes coding for an enzyme comprising an oxygenase subunit and/or a reductase subunit, preferably a styrene monooxygenase or indole monooxygenase, and/or iii. at least two genes coding for an enzyme styrene oxide isomerase, and/or iv. at least two genes coding for an enzyme alcohol dehydrogenase, it being possible for the genes coding for an enzyme to be identical or different in each case.

Advantageously, two different genes for an enzyme allow an increase in the substrate spectrum. Furthermore, advantageously, two different genes for an enzyme allow a variation of the expression and thus an optimization of the running time of the method according to the invention.

In embodiments, the whole cell catalyst comprises i. an enzyme phenolic acid decarboxylase capable of compound (I)

_(N) to implement, and ii. at least one enzyme having an oxygenase subunit and/or a reductase subunit capable of compound (II)

to compound (III)

implement, and iii. an enzyme styrene oxide isomerase capable of compound (III)

(IV) implement, and iv. an enzyme alcohol dehydrogenase capable of compound (IV)

where R ¹ is -H or -OH and where R ² is -H, -OH or -OCH3.

In embodiments, the whole-cell catalyst comprises a phenolic acid decarboxylase having an amino acid sequence with at least 90% sequence identity with a sequence selected from SEQ ID no. 1 to SEQ ID No. 3, preferably with at least 95% sequence identity with a Sequence selected from SEQ ID No. 1 to SEQ ID No. 3, particularly preferably with at least

98% sequence identity with a sequence selected from SEQ ID no. 1 to SEQ ID No. 3.

In embodiments, the whole cell catalyst comprises a phenolic acid decarboxylase having an amino acid sequence selected from SEQ ID no. 1 to SEQ ID No. 3.

In further embodiments, the whole-cell catalyst comprises an oxygenase subunit having an amino acid sequence with at least 90% sequence identity with a sequence selected from SEQ ID no. 4 to SEQ ID No. 9, preferably with at least 95% sequence identity with a sequence selected from SEQ ID no. 4 to SEQ ID No. 9, particularly preferably with at least 98% sequence identity with a sequence selected from SEQ ID no. 4 to SEQ ID No. 9.

In embodiments, the whole cell catalyst comprises an oxygenase subunit having an amino acid sequence selected from SEQ ID no. 4 to SEQ ID No. 9.

In preferred embodiments, the whole-cell catalyst comprises an oxygenase subunit having an amino acid sequence with at least 90% sequence identity with a sequence selected from SEQ ID no. 4, SEQ ID No. 5, SEQ ID No. 8 or SEQ ID no. 9, preferably with at least 95% sequence identity with a sequence selected from SEQ ID no. 4, SEQ ID No. 5, SEQ ID No. 8 or SEQ ID no. 9, particularly preferably with at least 98% sequence identity with a sequence selected from SEQ ID no. 4, SEQ ID No. 5, SEQ ID No. 8 or SEQ ID no. 9.

In embodiments, the whole cell catalyst comprises an oxygenase subunit having an amino acid sequence selected from SEQ ID no. 4, SEQ ID No. 5, SEQ ID No. 8 or SEQ ID no. 9. Advantageously, a whole-cell catalyst with an oxygenase subunit with an amino acid sequence selected from SEQ ID no. 4, SEQ ID No. 5, SEQ ID No. 8 or SEQ ID no. 9 shows a higher conversion for hydroxylated, especially 3,4-dihydroxylated, compounds.

In further embodiments, the whole cell catalyst comprises a reductase subunit having an amino acid sequence with at least 90% sequence identity with a sequence selected from SEQ ID no. 10 to SEQ ID No. 15, preferably with at least 95% sequence identity with a sequence selected from SEQ ID no. 10 to SEQ ID No. 15, particularly preferably with at least 98% sequence identity with a sequence selected from SEQ ID no. 10 to SEQ ID No. 15 In embodiments, the whole cell catalyst comprises a reductase subunit having an amino acid sequence selected from SEQ ID no. 10 to SEQ ID No. 15

In further embodiments, the whole-cell catalyst comprises an oxygenase subunit having an amino acid sequence with at least 90% sequence identity with a sequence selected from SEQ ID no. 4 to SEQ ID No. 9, preferably SEQ ID no. 4, SEQ ID No. 5, SEQ ID No. 8 or SEQ ID no. 9; and/or a reductase subunit having an amino acid sequence with at least 90% sequence identity with a sequence selected from SEQ ID no. 10 to SEQ ID No. 15

Advantageously, there is no inhibition of the enzyme comprising an oxygenase subunit with an amino acid sequence with at least 90% sequence identity with a sequence selected from SEQ ID no. 4, SEQ ID No. 5, SEQ ID No. 8 or SEQ ID no. 9 and/or a reductase subunit having an amino acid sequence with at least 90% sequence identity with a sequence selected from SEQ ID no. 10 to SEQ ID No. 15 by hydroxylated, in particular 3,4-dihydroxylated, intermediates which can occur in the process of the invention.

In further embodiments, the whole-cell catalyst comprises a styrene oxide isomerase having an amino acid sequence with at least 90% sequence identity with SEQ ID no. 16, SEQ ID NO. 17 or SEQ ID no. 18, preferably with at least 95% sequence identity with SEQ ID no. 16, SEQ ID NO. 17 or SEQ ID no. 18, particularly preferably with at least 98% sequence identity with SEQ ID no. 16, SEQ ID NO. 17 or SEQ ID no. 18

In embodiments, the whole cell catalyst comprises a styrene oxide isomerase having an amino acid sequence selected from SEQ ID no. 16, SEQ ID NO. 17 or SEQ ID no. 18

In preferred embodiments, the whole cell catalyst comprises i. a phenolic acid decarboxylase having an amino acid sequence with at least 90% sequence identity with a sequence selected from SEQ ID no. 1 to SEQ ID No. 3, and ii. an oxygenase subunit having an amino acid sequence with at least 90% sequence identity with a sequence selected from SEQ ID no. 4 to SEQ ID No. 9, and iii. a reductase subunit having an amino acid sequence with at least 90% sequence identity with a sequence selected from SEQ ID no. 10 to SEQ ID No. 15, and iv. a styrene oxide isomerase having an amino acid sequence with at least 90% sequence identity with SEQ ID no. 16, SEQ ID NO. 17 or SEQ ID no. 18

In embodiments, the whole cell catalyst comprises an oxygenase subunit having an amino acid sequence with at least 90% sequence identity with a sequence selected from SEQ ID no. 4 to SEQ ID No. 9 and a reductase subunit having an amino acid sequence with at least 90% sequence identity with a sequence selected from SEQ ID no. 10 to SEQ ID No. 13.

In alternative embodiments, the whole cell catalyst comprises an oxygenase subunit having an amino acid sequence with at least 90% sequence identity with a sequence selected from SEQ ID no. 4 to SEQ ID No. 9 and a reductase subunit having an amino acid sequence with at least 90% sequence identity with SEQ ID no. 14 or SEQ ID no. 15

In alternative embodiments, the whole-cell catalyst comprises a reductase subunit having an amino acid sequence with at least 90% sequence identity with SEQ ID no. 14 or SEQ ID no. 15

In embodiments, the alcohol dehydrogenase is naturally occurring in the whole cell catalyst.

The term "promoter" means a nucleotide sequence on the DNA that enables the regulated expression of a gene.

The genes coding for an enzyme phenolic acid decarboxylase, an enzyme comprising an oxygenase subunit and/or a reductase subunit, a styrene oxide isomerase and an alcohol dehydrogenase are functionally under the control of at least one regulatable promoter, with the promoters being identical or interrelated can be different.

In embodiments, the whole-cell catalyst is activated in a signal-dependent manner by contacting the whole-cell catalyst with an inductor, also activator, the induction the expression of the genes is signal-dependently induced and the whole-cell catalyst is converted into its active form. The inducers preferably activate the regulatable promoter by interacting directly with a regulatable promoter or by binding to a repressor protein which then detaches from the promoter. By contacting the whole-cell catalyst with an inducer, the above-mentioned enzymes are formed in the whole-cell catalyst and are thus available for the biocatalytic conversion of a substrate of the formula (I).

In embodiments, the at least one promoter for the regulatable expression of genes i) to iv) is a common promoter.

In alternative embodiments, genes i) to iv) are each functionally under the control of a regulatable promoter.

In embodiments, the regulatable promoters differ from one another, such that the promoters can be activated in a primary signal-specific manner. The presence of different inducers and/or activators in the whole-cell catalyst can thus advantageously lead to the expression of selected genes, as a result of which the stress for a recombinant cell is minimized.

The corresponding nucleic acid sequences for promoters, e.g. B. T7 promoter in pET16 expression systems that can be used for a method according to the invention are known to the person skilled in the art or can be found in known databases (eg EPD, TRED, MPromDB). Advantageously, promoters introduced artificially into organisms and naturally occurring promoters can be used.

The term “inductor” is understood to mean a mostly low-molecular compound which switches on the genes required for the method according to the invention or inactivates an existing repressor so that the genetic information of the genes can be converted into proteins or enzymes.

In embodiments, the inducer is selected from isopropyl-β-D-thiogalactopyranoside (IPTG), tryptophan, or sugars such as arabinose or lactose.

In further embodiments, the inducer is a substrate inducer when using a corresponding promoter. A substrate inducer is preferably glucose, an aromatic and/or aliphatic alcohol, styrene or a derivative thereof. In preferred embodiments, the inducer is isopropylbβ-D-thiogalactopyranoside (IPTG).

The contacting of the whole-cell catalysts with an inducer preferably takes place in a concentration of the inducer based on the total volume of the aqueous component of between 10 pM and 1500 pM, particularly preferably between 100 pM and 1200 pM, very particularly preferably between 500 pM and 1000 pM. The inductor is preferably added once.

In embodiments, at least step) takes place in a single-phase aqueous system or in a two-phase system. A "two-phase system" is understood to mean a mixture of two solvents which are immiscible. In embodiments, water or an aqueous medium is used as the first phase and a polar, organic solvent or an ionic liquid which is substantially immiscible with water is used as the second phase, the compound of the formula (I), the intermediates (II ) to (IV) and/or the product (V) preferably accumulates in the organic phase. The organic phase advantageously protects the cells which are in the aqueous phase from the harmful effects of greater concentrations of the intermediates (II) to (IV) and from a high concentration of the product (V).

In embodiments, substantially water-immiscible polar organic solvents are ethyl acetate, n-octanol, n-decanol, or solvents with a comparable polarity. The solvents mentioned are expediently suitable for the compounds of the formulas (I) to (V).

By contacting the whole-cell catalyst with a substance comprising a compound of the formula (I), a biocatalytic conversion to a corresponding reaction product of the formula (V) takes place within a whole-cell catalyst. In embodiments, the compound of formula (I) is resorbed by the whole cell catalyst, i. H. absorbed into the cell and biocatalytically converted by the enzymes of the whole-cell catalyst.

The whole-cell catalyst is preferably brought into contact with a compound according to formula (I) by direct addition of the substance to the culture medium as a solution and/or solid. When added directly to the whole-cell biocatalyst, an organic phase can also be used as a substrate reservoir.

Expediently, by varying the process control with regard to the form of addition of the substance comprising a compound of formula (I), an adaptation to the cell density and the used organism, whereby an optimal biocatalytic conversion of compounds of formula (I) and the longest possible process time is made possible.

In embodiments, the substance comprising at least one compound according to formula (I) is a pure substance, a plant component or a plant extract. Plant waste, for example potato peelings or coffee residues from roasting plants, can also be used advantageously in the method according to the invention. Advantageously, the use of a plant component or a plant extract comprising a compound according to formula (I) uses renewable raw materials and thus provides a sustainable process

In preferred embodiments, the compound of formula (I) is caffeic acid. Caffeic acid is expediently obtained from various plants or by enzymatic synthesis from other plant constituents, such as chlorogenic acid. Suitable plants for obtaining chlorogenic acid and/or caffeic acid include mugwort, stinging nettle, yarrow, coffee, potatoes or goutweed.

The substance comprising a compound of formula (I) is preferably mixed with a total concentration of the compound of formula (I) in the range of 5 mM and 30 mM, more preferably in the range of 10 mM and 25 mM, most preferably in the range of 15 mM and 20 mM used in a biocatalytic conversion and can be tracked continuously or discontinuously.

In embodiments, use is made of portion sizes in the range of 3mM to 8mM with batch addition or in the range of 1mM to 2mM per hour with continuous addition. A combination of discontinuous addition at the beginning with an initial portion size in the range from 5 mM to 8 mM and then after 2 hours to 7 hours of continuous addition with a portion size in the range from 1.2 mM to 1.8 mM per hour is particularly suitable .

The term "aqueous medium" is understood to mean a liquid comprising water. The aqueous component must suitably meet the requirements of the whole-cell catalyst.

Compositions of aqueous components, in particular culture media, for various microorganisms are known to those skilled in the art and are described, for example, by Panke et al. described (Panke et al. 1999). Other additives (e.g. carbon sources, Nitrogen sources, metal salts) can be added to the aqueous component in the form of a one-time batch or supplied in a suitable manner during the cultivation. To control the pH of the aqueous component, buffer compounds such as e.g. B. hydrogen phosphate salts or TRIS; and basic compounds such as B. sodium hydroxide, potassium hydroxide or ammonia; or acidic compounds such as B. phosphoric acid or sulfuric acid; used in an appropriate manner.

In embodiments, phosphate-buffered media are used in the method according to the invention (Panke et al. 1999), with glucose being used as the carbon source. Glucose is preferably initially used for culturing at a concentration in the range from 10 mM to 15 mM, based on the culture volume which contains the whole-cell catalyst.

In further embodiments, before, at the same time as or after the activation of the whole-cell catalyst with an inducer in step b), glucose with a total concentration in the range of 40 mM to 60 mM is added in addition to the initial portion, preferably by means of continuous addition in the range of 2 mM to 4mM per hour. The glucose can be added as a solution or as a solid.

To control foaming, antifoams such as B. fatty acid polyglycol esters or n-octanol; be used. To maintain the stability of plasmids, the aqueous component can include antibiotics such. B. chloramphenicol, ampicillin or kanamycin; to be added. In embodiments, bacterial cells with partially inactivated metabolic pathways (e.g. auxotrophic mutants) are used to maintain plasmid stability, with an (expression) vector containing at least one gene defined in i.) to iv.), e.g. Genes to complete incomplete metabolic pathways are included. Bacterial cells are preferably used in the method according to the invention which contain at least one antibiotic resistance gene on an (expression) vector containing at least one gene defined in i.) to iv.).

In embodiments, the method according to the invention takes place under aerobic conditions. In order to maintain aerobic conditions, oxygen or oxygen-containing gas mixtures, such as. As air, entered into the aqueous component.

In embodiments, step c) takes place in a period of time in the range from 0 h to 2 h, preferably 0 h to 1 h, particularly preferably 0 h to 0.5 h after step b). Surprisingly, the highest conversion was obtained when inductor and substrate were added at the same time. In comparison In biotechnological processes, a standard pre-induction of a whole-cell catalyst with an inductor takes place for a few hours, in particular 6 h to 14 h, before the actual addition of the substrate to start the conversion.

In further embodiments, the method according to the invention comprises at least one further step, the at least one further step being isolation of the compound of the formula (V) after steps a) to c). The purification of the compound of the formula (V), obtained by means of the process according to the invention, is advantageously simpler than in the case of extraction by extraction, since fewer structurally similar ingredients have to be separated off. Furthermore, the reaction product of the formula (V) is advantageously secreted by the whole-cell catalyst into the aqueous component, which promotes the isolation of at least one reaction product of the formula (V) from the biomass and the aqueous component.

The reaction product of the formula (V) is preferably isolated stepwise from the biomass and the aqueous component, the biomass being separated off from the aqueous component containing a reaction product of the formula (V) in a first step by centrifugation or filtration.

In embodiments, the compound of formula (V) is isolated by extraction of the aqueous component with an organic solvent selected from ethyl acetate, n-octanol, n-decanol or an aromatic and/or aliphatic solvent with comparable polarity.

In embodiments, the organic solvents mentioned are used in a single-phase, aqueous system for extraction after conversion of a compound of the formula (V). In alternative embodiments, the organic solvents mentioned serve in a two-phase system in the form of a second phase in addition to the aqueous component as a reservoir for a compound of formula (I) and/or for separating off a compound of formula (V).

In addition to extraction with organic solvents, solid phase extraction is also suitable for product separation. Possible solid-phase materials include commercially available hydrophobic copolymers consisting of monomers of styrene, styrene derivatives, benzene, vinylbenzenes and/or vinylpyrrolidone, which, among other things, can also be polar modified or can contain groups with ion exchange properties. The target product is expediently eluted from these materials with methanol or ethanol and/or by varying the pH.

In embodiments, the purification of the compound of formula (V) is followed by distillation after the liquid extraction or solid-phase extraction, the organic solvent preferably being separated off. In embodiments, the organic solvent is removed by evaporation at a pressure in the range from 0.1 mbar to 1000 mbar, preferably in the range from 0.1 mbar to 750 mbar, particularly preferably in the range from 1 mbar to 400 mbar. The product obtained can then be taken up in water and/or purified using preparative high-performance liquid chromatography. Depending on the work-up, the separated organic solvent can be used again for the next extraction or elution.

Another aspect of the invention relates to a whole cell catalyst comprising: i. a gene coding for an enzyme phenolic acid decarboxylase and ii. at least one gene coding for an enzyme comprising an oxygenase subunit and/or a reductase subunit, preferably a styrene monooxygenase or indole monooxygenase, and iii. a gene coding for an enzyme styrene oxide isomerase, and iv. a gene coding for an enzyme alcohol dehydrogenase, and at least one promoter for the controllable expression of genes i) to iv).

In embodiments, the whole cell catalyst is a recombinant microorganism, preferably a recombinant bacterial cell.

In preferred embodiments, the whole cell catalyst is Escherichia coli with insertion mutation. The term "insertion mutation" is understood to mean the introduction of at least one gene by genome insertion or by introduction of expression vectors. The nucleotide sequences of genes i.) to iv.) include authentic and/or artificial reading frames. An artificial reading frame is preferably adapted to the "codon usage" of the host organism via gene synthesis.

where R ¹ is -H or -OH and where R ² is -H, -OH or -OCH3.

According to the invention, the whole-cell catalyst has at least one gene coding for an enzyme comprising an oxygenase subunit and/or a reductase subunit, preferably a monooxygenase, particularly preferably a styrene monooxygenase or an indole monooxygenase.

where R ¹ is -H or -OH and where R ² is -H, -OH or -OCH3.

In embodiments, the enzyme comprising an oxygenase subunit and/or a reductase subunit is a fusion protein, preferably according to SEQ ID no. 14 or SEQ ID no. 15

where R ¹ is -H or -OH and where R ² is -H, -OH or -OCH3.

where R ¹ is -H or -OH and where R ² is -H, -OH or -OCH3.

The corresponding gene and amino acid sequences for the enzymes mentioned (phenolic acid decarboxylase, oxygenase, reductase, styrene oxide isomerase and alcohol dehydrogenase) are well known to those skilled in the art or can be found in known databases (e.g. NCBI, RCSB PDB, UniProt, PDB Europe). will.

In embodiments, the whole cell catalyst comprises i. At least two genes coding for an enzyme phenolic acid decarboxylase and/or ii. at least two genes coding for an enzyme comprising an oxygenase subunit and/or a reductase subunit, preferably a styrene monooxygenase or indole monooxygenase, and/or iii. at least two genes coding for an enzyme styrene oxide isomerase, and/or iv. at least two genes coding for an enzyme alcohol dehydrogenase, it being possible for the genes coding for an enzyme to be identical or different in each case. In embodiments, the whole cell catalyst comprises i. an enzyme phenolic acid decarboxylase capable of compound (I)

implement, and ii. at least one enzyme having an oxygenase subunit and/or a reductase subunit capable of compound (II)

to compound (III)

implement, and iii. an enzyme styrene oxide isomerase capable of compound (III)

to connection (IV)

implement, and iv. an enzyme alcohol dehydrogenase capable of compound (IV)

where R ¹ is -H or -OH and where R ² is -H, -OH or -OCH3.

In embodiments, the whole-cell catalyst comprises a phenolic acid decarboxylase having an amino acid sequence with at least 90% sequence identity with a sequence selected from SEQ ID no. 1 to SEQ ID No. 3, preferably with at least 95% sequence identity with a sequence selected from SEQ ID no. 1 to SEQ ID No. 3, particularly preferably with at least 98% sequence identity with a sequence selected from SEQ ID no. 1 to SEQ ID No. 3.

In further embodiments, the whole-cell catalyst comprises an oxygenase subunit having an amino acid sequence with at least 90% sequence identity with a sequence selected from SEQ ID no. 4 to SEQ ID No. 9, preferably with at least 95% sequence identity with a sequence selected from SEQ ID no. 4 to SEQ ID No. 9, particularly preferably with at least 98% sequence identity with a sequence selected from SEQ ID no. 4 to SEQ ID No. 9. In embodiments, the whole cell catalyst comprises an oxygenase subunit having an amino acid sequence selected from SEQ ID no. 4 to SEQ ID No. 9.

In further embodiments, the whole-cell catalyst comprises a reductase subunit having an amino acid sequence with at least 90% sequence identity with a sequence selected from SEQ ID no. 10 to SEQ ID No. 15, preferably with at least 95% sequence identity with a sequence selected from SEQ ID no. 10 to SEQ ID No. 15, particularly preferably with at least 98% sequence identity with a sequence selected from SEQ ID no. 10 to SEQ ID No. 15

In embodiments, the whole cell catalyst comprises a reductase subunit having an amino acid sequence selected from SEQ ID no. 10 to SEQ ID No. 15

In embodiments, the whole cell catalyst comprises a styrene oxide isomerase having an amino acid sequence selected from SEQ ID no. 16, SEQ ID NO. V or SEQ ID No. 18

In embodiments, the regulatable promoters differ from one another, such that the promoters can be activated in a primary signal-specific manner.

The corresponding nucleic acid sequences for promoters, e.g. B. T7 promoter in pET16 expression systems that can be used for a method according to the invention are known to the person skilled in the art or can be found in known databases (eg EPD, TRED, MPromDB). Advantageously, promoters introduced artificially into organisms and natural promoters can be used. The invention also includes the use of a whole-cell catalyst according to the invention for the biocatalytic synthesis of a compound according to formula (V),

by the biocatalytic conversion of a substance comprising at least one compound according to formula (I)

wherein R ¹ is -H or -OH, wherein R ² is -H, -OH or -OCH3.

For the realization of the invention, it is also expedient to combine the above-described embodiments and features of the claims.

The invention will be explained in more detail below with reference to some exemplary embodiments and associated figures. The exemplary embodiments are intended to describe the invention without restricting it.

It show the

1 shows the influence of the pre-induction period (step b) before addition of the substrate caffeic acid (step c) on the formation of 3-hydroxytyrosol.

2 shows the course of the conversion of caffeic acid into 3-hydroxytyrosol with a whole-cell catalyst according to the invention in the fermenter. Example 1 Whole-cell catalyst according to the invention

In a first embodiment, the BL21 variant of Escherichia coli is used, which has its own alcohol dehydrogenase. Escherichia coli T7Express lysY/lq or Escherichia coli BL21 (DE3)pLysS are particularly suitable. The whole-cell catalyst according to the invention comprises SEQ ID no. 1 , SEQ ID No. 9, SEQ ID No. 15 with SEQ ID no. 17 (catalyst A) or SEQ ID no. 1 , SEQ ID No. 8, SEQ ID No. 14 with SEQ ID no. 17 (catalyst B) or SEQ ID no. 1 , SEQ ID No. 5, SEQ ID No. 12 with SEQ ID no. 17 (catalyst C) or SEQ ID no. 1 , SEQ ID No. 4, SEQ ID No. 10 with SEQ ID no. 17 (catalyst D) or SEQ ID no. 1 , SEQ ID No. 15 with SEQ ID no. 17 (Catalyst E). The genes are equipped with a ribosomal binding site (RBS) upstream of each gene and subjected to an IPTG-sensitive promoter one after the other, in particular by introduction into the multiple cloning site (MCS) of pET or pRSF vectors or into a vector with several MCSs, especially pCOLA Duet. All genes can be introduced into the cell via one vector or alternatively via two vectors.

In the case of catalysts A to D, all genes mentioned are introduced into strain T7Express lysY/lq via a single pET vector. Catalyst E, on the other hand, corresponds to strain BL21(DE3)pLysS, with the relevant genes being provided by means of the pCOLA Duet vector and pET vector.

Example 2: Conversion of caffeic acid into 3-hydroxytyrosol on a 50 ml scale

Catalysts A to D (see Example 1) are used on a 50 ml scale for the conversion of caffeic acid. The pre-cultivation starting from cryocultures takes place first in test tubes with 4 ml LB medium, which additionally contains 100 pg/ml ampicillin, at 37° C. and 120 rpm overnight. Then the main culture, consisting of 50 ml M9* medium (according to Panke et al. 1999) with 100 pg/ml ampicillin, is inoculated by introducing 3.5 ml preculture. These cultures are grown at 37° C. and 120 rpm to a cell density of ODsoo=0.6 and then induced with 1 mM IPTG. At the same time, a concentration of 8 mM is set in the main cultures with solid caffeic acid. Further cultivation takes place at 120 rpm and 30°C. After about 6 hours, caffeic acid is added again at a concentration of 4 mM. After 12 h, samples are taken to demonstrate the functionality of the whole-cell catalysts by removing 100 to 250 μl of the main culture, stopping with methanol, centrifuging to separate the cells and analyzing the clear supernatant removed using high-performance liquid chromatography. With catalyst A, 7.4 mM product could be produced under these conditions after 12 h, which corresponded to a yield of 62%. Catalysts B and C revealed 8.7 mM product after 12 h and a yield of ca. 73%. Catalyst D achieved a product concentration of 10.3 mM and a yield of 86%.

Example 3: Conversion of ferulic acid and caffeic acid with catalyst E

Cultivation of the biomass of catalyst E (see Example 1) in the preculture and in the main culture up to induction with 1 mM IPTG is carried out according to Example 2. Either 3 mM caffeic acid or 3 mM ferulic acid are added once in parallel with the induction. The cultures are then incubated at 30° C. and 120 rpm. After 24 hours, product formation is quantified as described in Example 2.

If ferulic acid was used as starting material, 1.5 mM homovanillyl alcohol could be detected after 24 h, which corresponds to a yield of 50%. In the case of the cultures that were incubated with caffeic acid, 1.6 mM 3-hydroxytyrosol could be detected, which corresponds to a yield of 53%.

Example 4 Influence of the induction time on the conversion of caffeic acid

For catalyst D (see Example 1), the influence of a pre-induction before the addition of the starting material was investigated. The pre-cultivation and main cultivation takes place as described in Example 2, but a portion of 8 mM caffeic acid is added only once. Depending on the batch, the caffeic acid is added after 0 h, 0.5 h, 1 h, 1.5 h, 2 h, 4 h, 6 h or 12 h after the addition of the inductor IPTG. Samples are taken and analyzed as described in Example 2. The results of the conversions after 21 hours are shown in FIG.

A significant decrease in the product quantities that can be achieved is clearly recognizable if the caffeic acid is only added 1 h after the induction. Consequently, addition over a period of 0 to 2 hours, preferably 0 to 0.5 hours, is useful according to the invention. On the other hand, longer, otherwise usual pre-induction times would lead to extremely low amounts of 3-hydroxytyrosol in this case. Example 5 Conversion of caffeic acid into 3-hydroxytyrosol with biocatalyst A on a scale of

9.25 I in the fermenter

Catalyst A (see Example 1) is selected for this embodiment. The cultivation conditions correspond to example 2. However, the cultivation of the preculture only takes place over a period of 6 h. Thereafter, 200 ml each of LB medium, which additionally contains 100 g/ml ampicillin, is inoculated twice with the contents of a preculture. Cultivation then takes place for 14 h at 37° C. and 120 rpm. The contents of the two flasks are mixed and 250 ml of this preculture are used to inoculate a fermenter. This contains 9 l of an M9* medium preheated to 37° C. (according to Panke et al., 1999) with 100 pg/ml ampicillin and 12.7 mM glucose. The agitation speed and oxygen supply of the main culture is adjusted so that the pC>2 value is 50 to 90%. Just before induction, the temperature is lowered to 30°C. The induction with 1 mM IPTG takes place at a cell density of the main culture of ODeoo=0.6. At the same time, caffeic acid is added to a final concentration of 8 mM. Further cultivation takes place at 30°C, with the stirring speed and air entry being adjusted to the needs of the culture so that the pO2 value is kept between 30 and 85%. 1.5 hours after the induction, starting from a liquid stock solution, glucose is automatically added at a concentration of 2.4 mM per hour. A total of 50 to 51 mM glucose is introduced. After 6 hours, another 6 mM caffeic acid and after 14 hours 3 mM caffeic acid are added to the fermenter by adding solid substrate. The conversion was complete 24 hours after the induction. Samples were taken during the conversion and analyzed as described under example 2.

A total of 14.6 mM 3-hydroxytyrosol could be formed from 15 mM caffeic acid, which corresponds to a yield of 97%. This still corresponds to a product quantity of 2.3 g/l. Based on the total volume, about 21 g of product could be realized within 25 hours.

Example 6 Conversion of caffeic acid into 3-hydroxytyrosol with biocatalyst D on a scale of 9.25 l in the fermenter

Catalyst D (see Example 1) was used for this example. The pre-cultivation is carried out according to example 5. The contents of the two flasks are mixed and 250 ml of this pre-culture for inoculation of a fermenter with 9 l of an M9* medium preheated to 37° C. (according to Panke et al., 1999) with 100 pg/ml Ampicillin and 12.7 mM glucose used. The agitation speed and oxygen supply of the main culture is adjusted in such a way that the pCh Value is 50 to 90%. Just before induction, the temperature is lowered to 30°C. The induction takes place at a cell density of ODeoo=0.6 with 1 mM IPTG. At the same time, caffeic acid is added to a final concentration of 8 mM. Further cultivation takes place at 30°C, with the stirring speed and air entry being adjusted to the needs of the culture so that the pC>2 value is in the range between 40 and 75%. 1.75 hours after the induction, starting from a liquid stock solution, glucose is automatically added at a concentration of 2.4 mM per hour. A total of 40 to 41 mM glucose is added. In addition, 5.75 h after the induction, caffeic acid solution is automatically introduced into the fermenter at 1.2 mM per hour (a total of 12 mM in addition to the initial portion of 8 mM). The conversion is complete no later than 20 h after the induction. Samples were taken during the conversion and analyzed as in Example 2. The course of the conversion is shown in FIG.

A total of 19.4 mM 3-hydroxytyrosol were formed by using 20 mM caffeic acid, which corresponds to a yield of 97%. This also corresponds to a product quantity of almost 3 g/l. Based on the total volume, 27.7 g of product were obtained within 20 h.

Non-patent literature cited

Bedoya LM, Beltran M, Obregon-Calderon P, Garcia-Perez J, de la Torre HE, Gonzalez N, Perez-Olmeda M, Aunon D, Capa L, Gomez-Acebo E, et al. (2016) Hydroxytyrosol: A new class of microbicide displaying broad anti-HIV-1 activity. AIDS 30:2767-2776.

Bellumori M, Cecchi L, Innocenti M, Clodoveo ML, Corbo F, Mulinacci N (2019) The EFSA health claim on olive oil polyphenols: acid hydrolysis validation and total hydroxytyrosol and tyrosol determination in Italian virgin olive oils. Molecules 24(11):2179.

Fernandez-Bolanos J, Rodriguez G, Rodriguez R, Heredia A, Guillen R, Jimenez A (2002) Production in large quantities of highly purified hydroxytyrosol from liquid-solid waste of two-phase olive oil processing or “Alperujo”. J Agric. Food Chem. 50: 6804-6811.

Hu T, He X-W, Jiang J-G, Xu X-L (2014) Hydroxytyrosol and its potential therapeutic effects. J Agric. Food Chem. 62:1449-1455.

Panke S, Meyer A, Huber CM, Witholt B, Wubbolts MG (1999) An alkane-responsive expression system for the production of fine chemicals. appl. environment microbiol. 65: 2324-2332.

Tafesh A, Najami N, Jadoun J, Halahlih F, Riepl H, Azaizeh H (2011) Synergistic antibacterial effects of polyphenolic compounds from olive mill wastewater. Evidence-Based Complementary and Alternative Medicine: eCAM, 431021.

Claims

patent claims

1. Process for the Biocatalytic Synthesis of a Compound of Formula (V)

wherein R ¹ is -H or -OH, wherein R ² is -H, -OH or -OCH3, comprising the following steps: a) providing at least one whole cell catalyst comprising i. a gene coding for an enzyme phenolic acid decarboxylase and ii. at least one gene coding for an enzyme comprising an oxygenase subunit and/or a reductase subunit and iii. a gene coding for an enzyme styrene oxide isomerase, and iv. a gene coding for an enzyme alcohol dehydrogenase, and at least one promoter for the controllable expression of genes i) to iv) in an aqueous medium, b) activation of the whole cell catalyst with an inducer, the inducer leading to the expression of genes i) to iv). , and c) contacting the whole-cell catalyst with a substance comprising at least one compound of formula (I), wherein the at least one compound of formula (I) is reacted with the enzymes defined in (a) to form a compound of formula (V). ears according to claim 1, characterized in that the whole cell catalyst i. an enzyme phenolic acid decarboxylase capable of compound (I)

to compound (III)

implement, and iii. an enzyme styrene oxide isomerase capable of compound (III)

to connection (IV)

implement, and iv. an enzyme alcohol dehydrogenase capable of compound (IV)

(V) wherein R ¹ is -H or -OH, wherein R ² is -H, -OH or -OCH3. The method according to claim 1 or 2, characterized in that the whole cell catalyst i. a phenolic acid decarboxylase having an amino acid sequence with at least 90% sequence identity with a sequence selected from SEQ ID no. 1 to SEQ ID No. 3, and/or ii. an oxygenase subunit having an amino acid sequence with at least 90% sequence identity with a sequence selected from SEQ ID no. 4 to SEQ ID No. 9, and/or iii. a reductase subunit having an amino acid sequence with at least 90% sequence identity with a sequence selected from SEQ ID no. 10 to SEQ ID No. 15, and/or iv. a styrene oxide isomerase having an amino acid sequence with at least 90% sequence identity with SEQ ID no. 16, SEQ ID NO. 17 or SEQ ID no. 18 includes. Method according to one of Claims 1 to 3, characterized in that the whole-cell catalyst contains an oxygenase subunit with an amino acid sequence with at least 90% sequence identity with a sequence selected from SEQ ID no. 4 to SEQ ID No. 9 and a reductase subunit having an amino acid sequence with at least 90% sequence identity with a sequence selected from SEQ ID no. 10 to SEQ ID No. 13, or an oxygenase subunit having an amino acid sequence with at least 90% sequence identity with a sequence selected from SEQ ID no. 4 to SEQ ID No. 9 and a reductase subunit having an amino acid sequence with at least 90% sequence identity with SEQ ID no. 14 or SEQ ID no. 15, or a reductase subunit having an amino acid sequence with at least 90% sequence identity with SEQ ID no. 14 or SEQ ID no. 15 includes. Process according to one of Claims 1 to 4, characterized in that the whole-cell catalyst is a recombinant microorganism. Process according to any one of Claims 1 to 5, characterized in that the inducer is isopropyl-β-D-thiogalactopyranoside (IPTG). Process according to one of Claims 1 to 6, characterized in that at least step e) takes place in a single-phase aqueous system or in a two-phase system. Method according to one of Claims 1 to 7, characterized in that the substance comprising at least one compound according to formula (I) is a pure substance, a plant component or plant extract. Method according to one of Claims 1 to 8, characterized in that step c) takes place in a period in the range from 0 h to 2 h after step b). Whole cell catalyst comprising: i. a gene coding for an enzyme phenolic acid decarboxylase and ii. at least one gene coding for an enzyme comprising an oxygenase subunit and/or a reductase subunit and iii. a gene coding for an enzyme styrene oxide isomerase, and iv. a gene coding for an enzyme alcohol dehydrogenase, and at least one promoter for the controllable expression of genes i) to iv). A cell catalyst according to claim 10 comprising i. an enzyme phenolic acid decarboxylase capable of compound (I)

implement, and iii. an enzyme styrene oxide isomerase capable of compound (III)

(V) wherein R ¹ is -H or -OH, wherein R ² is -H, -OH or -OCH3. nz cell catalyst according to claim 10 or 11 comprising i. a phenolic acid decarboxylase having an amino acid sequence with at least 90% sequence identity with a sequence selected from SEQ ID no. 1 to SEQ ID No. 3, and/or ii. an oxygenase subunit having an amino acid sequence with at least 90% sequence identity with a sequence selected from SEQ ID no. 4 to SEQ ID No. 9, and/or iii. a reductase subunit having an amino acid sequence with at least 90% sequence identity with a sequence selected from SEQ ID no. 10 to SEQ ID No. 15, and/or iv. a styrene oxide isomerase having an amino acid sequence with at least 90% sequence identity with SEQ ID no. 16, SEQ ID NO. 17 or SEQ ID no. 18. whole-cell catalyst according to any one of claims 10 to 12 comprising an oxygenase subunit having an amino acid sequence with at least 90% sequence identity with a sequence selected from SEQ ID no. 4 to SEQ ID No. 9 and a reductase subunit having an amino acid sequence with at least 90% sequence identity with a sequence selected from SEQ ID no. 10 to SEQ ID No. 13, or an oxygenase subunit having an amino acid sequence with at least 90% sequence identity with a sequence selected from SEQ ID no. 4 to SEQ ID No. 9 and a reductase subunit having an amino acid sequence with at least 90% sequence identity with SEQ ID no. 14 or SEQ ID no. 15, or a reductase subunit having an amino acid sequence with at least 90% sequence identity with SEQ ID no. 14 or SEQ ID no. 15. whole-cell catalyst according to any one of claims 10 to 13, characterized in that the whole-cell catalyst is a recombinant microorganism. Use of a whole-cell catalyst according to any one of claims 10 to 14 for the biocatalytic synthesis of a compound of formula (V),

wherein R ¹ is -H or -OH, wherein R ² is -H, -OH or -OCH3.