WO2014090923A1 - Process for producing 3-hydroxytyrosol from 3,4-dihydroxyphenylacetic acid|employing a carboxylic acid reductase - Google Patents

Process for producing 3-hydroxytyrosol from 3,4-dihydroxyphenylacetic acid|employing a carboxylic acid reductase Download PDF

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WO2014090923A1
WO2014090923A1 PCT/EP2013/076327 EP2013076327W WO2014090923A1 WO 2014090923 A1 WO2014090923 A1 WO 2014090923A1 EP 2013076327 W EP2013076327 W EP 2013076327W WO 2014090923 A1 WO2014090923 A1 WO 2014090923A1
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seq
reaction
sequence
car
sequence seq
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Christiaan RIJKSEN
Karen Robins
Antonio Victor OSORIO LOZADA
Kamila NAPORA-WIJATA
Margit WINKLER
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Lonza AG
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Lonza AG
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • C12Y102/0103—Aryl-aldehyde dehydrogenase (NADP+) (1.2.1.30)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/08—Transferases for other substituted phosphate groups (2.7.8)
    • C12Y207/08007—Holo-[acyl-carrier-protein] synthase (2.7.8.7)

Definitions

  • the invention relates to the preparation of 3-hydroxytyrosol (3-HT) from 3,4- dihydroxyphenylacetic acid (DOPAC) using a carboxylic acid reductase CAR.
  • 3-HT is a potent antioxidant found in olive leafs and olive oil, and is present in high abundance in olive mill waste waters. 3-HT has been associated with lower mortality and incidence of cancer in Mediterranean regions and has been attributed cardio-protective properties. It is used as a food additive. There has been therefore an increased interest in the manufacturing and commercialization of 3-HT as nutritional supplement.
  • E. coli BL21-CodonPlus (DE2)-RP/pPV2.85 that expresses car, npt and gdh, for the reduction of vanillic acid to vanillin.
  • AADH and CAR are used synonymously in this specification for an enzyme of the enzyme class EC 1.2.1.30.
  • ACPS and PPTase are used synonymously in this specification for an enzyme of the enzyme class EC 2.7.8.7.
  • the enzyme class EC is defined by the NC-IUBMB, which is accessible in the internet via http://www.chem.qmul.ac.uk/iubmb/enzyme/, and is also compiled e.g. in the database BRENDA.
  • sequences have the same residues at the same positions in an alignment, often expressed as a percentage identity of the amino acid residues, based on the total number of amino acid residues of the amino acid sequence. If the two sequences, which are being compared, have a different number of amino acid residues, the percentage is based on the number of the shorter amino acid sequence.
  • homologs biological components (genes, proteins, structures) are called homologs.
  • homolog is used interchangeably with the term variant, meaning rather a certain degree of identity between two sequences without implying a common ancestor. Therefore within the meaning of the invention, the term variant of a sequence means a sequence with a certain degree of identity to the sequence, if not otherwise stated.
  • sequence identity, similarity and positives values provided herein refer to the value obtained using the BLAST suite of programs using default parameters, a description is found in Altschul et al., J. Mol. Biol., 1990, 215, 403-410 and in "The NCBI Handbook" (Internet), editors J. McEntyre and J. Ostell, Bethesda (MD): National Center for Biotechnology Information (US); 2002-, and using the program version BLAST 2.2.27+, if not otherwise stated.
  • sequence can mean a nucleotide sequence, which constitutes a polynucleotide, or an amino acid sequence, which constitutes a peptide. Nucleotide sequences are found e.g. under the respective accession no. in the EMBL Nucleotide Sequence Database of the EMBL- EBI, or in the Nucleotide database of the NCBI; and amino acid sequences are found e.g. under the respective accession no. in the UniProt database of the EMBL-EBI, or in the Protein database of the NCBI; if not otherwise stated.
  • Heterologous nucleic acid sequence or “nucleic acid sequence heterologous to a host” means a nucleic acid sequence which encodes e. g. an expression product such as a polypeptide that is foreign to the host ("heterologous expression” or “heterologous product”) e.g. a nucleic acid sequence originating from a donor different from the host or a chemically synthesized nucleic acid sequence which encodes e. g. an expression product such as a polypeptide that is foreign to the host.
  • heterologous protein means a protein that is foreign to the host.
  • Form to the host can mean, that the nucleic acid or protein originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition or genomic locus by deliberate human intervention.
  • a heterologous nucleic acid sequence as referred herein encompasses also nucleic acids, which are codon optimized for the host according to the codon usage of the host.
  • Exogenous DNA refers to any deoxyribonucleic acid that originates outside of the organism of concern or study.
  • host refers not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, plant, amphibian, or mammalian cells.
  • a “vector”, “vector expressible in a host” or “expression vector” are used interchangeably and is a polynucleic acid construct that is used in transfection or transformation of a host cell and into which a polynucleotide can be inserted, it is generated recombinantly or synthetically, with a series of specified polynucleic acid elements that permit transcription of a particular nucleic acid sequence in a host cell.
  • this vector includes a transcriptional unit comprising a particular nucleic acid sequence to be transcribed operably linked to a promoter.
  • Vectors are often replicons.
  • a vector expressible in a host can be e. g. an autonomously or self-replicating plasmid, a cosmid, a phage, a virus or a retrovirus.
  • nucleic acid incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
  • the nucleic acid may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
  • Bacteria In molecular biology, the process by which cells take up exogenous DNA from the outside is called “transformation”. Bacteria need to be in a certain physiological state to successfully take up exogenous DNA, which is described as one of "competence”. Some bacteria are naturally competent, but usually only for a brief time at a certain stage of their growth cycle. Bacteria can also be made competent through a variety of chemical treatments including exposure to calcium ions, or a mixture of polyethylene glycol and dimethylsulfoxide, which make the cell membrane more permeable, leading to the uptake of the exogenous DNA. Another treatment method is the utilization of electricity as the membrane-permeabilizing agent (electroporation or electro transformation). Finally, liposome-mediated transformation can be used. In this method, DNA is coated with lipid. Fusion of this lipid and the membrane lipid can occur, facilitating the entry of DNA.
  • Transduction is usually used for the process of injection of foreign DNA by a virus into the host bacterium.
  • Transfection is the process of deliberately introducing nucleic acids into cells.
  • the term is often used for the introduction of foreign DNA into eukaryotic cells, notably for non- viral methods.
  • Competence is the ability of a cell to take up extracellular DNA from its environment.
  • the terms “transformation”, “transformed” or “introducing a nucleic acid into a host cell” are used interchangeably and denote any process wherein an extracellular nucleic acid like a vector, with or without accompanying material, enters a host cell.
  • the term “cell transformed” or “transformed cell” means the cell or its progeny into which the extracellular nucleic acid has been introduced and thus harbors the extracellular nucleic acid.
  • the nucleic acid might be introduced into the cell so that the nucleic acid is replicable either as a chromosomal integrant or as an extra chromosomal element. Transformation of appropriate host cells with e. g.
  • an expression vector can be accomplished by well-known methods such as microinjection, electroporation, particle bombardment or by chemical methods such as Calcium phosphate-mediated transformation, described e. g. in Maniatis et al. 1982, Molecular Cloning, A laboratory Manual, Cold Spring Harbor Laboratory, in Ausubel et al. 1994, Current protocols in molecular biology, John Wiley and Sons, or in "Molecular Cloning: A laboratory Manual, Third edition (3 volume set)", Cold Spring Harbor Laboratory Press, 2001.
  • nucleic acid "nucleic acid sequence” or “polynucleotide” are used interchangeably and together with “isolated and purified nucleic acid or nucleic acid sequence or polynucleotide", as referred in the present invention, might be DNA, RNA, or DNA/RNA hybrid. Unless otherwise indicated, the terms include reference to the specified sequence as well as the complementary sequence thereof. In case the nucleic acid sequence is located on a vector it is usually DNA.
  • DNA which is referred to herein can be any polydeoxynuclotide sequence, including, e.g.
  • double-stranded DNA single-stranded DNA, double-stranded DNA wherein one or both strands are composed of two or more fragments, double-stranded DNA wherein one or both strands have an uninterrupted phosphodiester backbone, DNA containing one or more single- stranded portion(s) and one or more double-stranded portion(s), double-stranded DNA wherein the DNA strands are fully complementary, double-stranded DNA wherein the DNA strands are only partially complementary, circular DNA, covalently- closed DNA, linear DNA, covalently cross-linked DNA, cDNA, chemically synthesized DNA, semi-synthetic DNA, biosynthetic DNA, naturally-isolated DNA, enzyme-digested DNA, sheared DNA, labeled DNA, such as radio labeled DNA and fluorochrome-labeled DNA, DNA containing one or more non-naturally occurring species of nucleic acid.
  • DNA sequences can be synthesized by standard chemical techniques, for example, the phosphotriester
  • RNA which is referred to herein can be e.g. single-stranded RNA, cRNA, double- stranded RNA, double stranded RNA wherein one or both strands are composed of two or more fragments, double-stranded RNA wherein one or both strands have an uninterrupted phosphodiester backbone, RNA containing one or more single-stranded portion(s) and one or more double-stranded portion(s), double-stranded RNA wherein the RNA strands are fully complementary, double-stranded RNA wherein the RNA strands are only partially complementary, covalently cross-linked RNA, enzyme digested RNA, sheared RNA, mRNA, chemically-synthesized RNA, semi-synthetic RNA, biosynthetic RNA, naturally isolated RNA, labeled RNA, such as radiolabeled RNA and fluorochrome-labeled RNA, RNA containing one or more non-naturally occurring species of nu
  • DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein.
  • variants or variant of a sequence is meant a nucleic acid sequence that vary from the reference sequence by nucleic acid substitutions, preferably by conservative nucleic acid substitution, whereby one or more nucleic acids are substituted by another with
  • Variants encompass as well degenerated sequences, sequences with deletions and insertions, as long as such modified sequences exhibit the same function (functionally equivalent) as the reference sequence.
  • polypeptide peptide
  • protein protein
  • polypeptidic peptidic
  • peptidic amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • isolated and purified nucleic acid sequence refers to the state in which the nucleic acid sequence will be free or substantially free of material with which they are naturally associated such as other nucleic acids with which they are found in their natural environment, or the environment in which they are prepared (e. g. cell culture) when such preparation is by recombinant technology practiced in vitro or in vivo.
  • the "origin of replication” (also called the replication origin) is a particular sequence in a genome at which replication is initiated. This can either involve the replication of DNA in living organisms such as prokaryotes and eukaryotes, or that of DNA or RNA in viruses, such as double-stranded RNA viruses. DNA replication may proceed from this point
  • Promoter refers to a nucleic acid sequence that regulates expression of a transcriptional unit.
  • a “promoter region” is a regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. Within the promoter region will be found a transcription initiation site (conveniently defined by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase such as the putative - 35 region and the Pribnow box.
  • Signal sequence refers to a nucleic acid sequence which encodes a short amino acid sequence (i.e., signal peptide) present at the NH2 -terminus of certain proteins that are normally exported by cells to non-cytoplasmic locations (e.g., secretion) or to be membrane components. Signal peptides direct the transport of proteins from the cytoplasm to non-cytoplasmic locations.
  • Translation initiation region is a signal region which promotes translation initiation and which functions as the ribosome binding site such as the Shine Dalgarno sequence.
  • Terminator “Terminator”, “transcription terminator” and “transcription termination region” are used interchangeably and indicate a section of genetic sequence that marks the end of gene or operon on genomic DNA for transcription. It causes RNA polymerase to terminate transcription.
  • the transcription termination region is usually part of a transcriptional unit and increases the stability of the mRNA.
  • Transcriptional unit refers to a nucleic acid sequence that is normally transcribed into a single RNA molecule.
  • the transcriptional unit might contain one gene (monocistronic) or two (dicistronic) or more genes (polycistronic) that code for functionally related polypeptide molecules.
  • a nucleic acid sequence is "operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a signal sequence is operably linked to DNA for a protein if it is expressed as a preprotein that participates in the secretion of the protein; a promoter is operably linked to a coding sequence if it affects the transcription of the sequence; or a translation initiation region such as a ribosome binding site is operably linked to a nucleic acid sequence encoding e. g. a polypeptide if it is positioned so as to facilitate translation of the polypeptide.
  • Linking can be accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • Subject of the invention is a method for the preparation of 3-hydroxytyrosol by a reaction, the reaction comprises an enzymatic reduction of 3,4-dihydroxyphenyl acetic acid, wherein the enzymatic reduction is done using two enzymes, a carboxylic acid reductase CAR and a 4'-phosphopantetheinyl transferase PPTase;
  • CAR comprises each of the three amino acid sequences SEQ ID NO: 1, SEQ ID NO: 3 and
  • PPTase comprises the sequence SEQ ID NO: 12 or comprises the sequence SEQ ID NO: 14, each with 70 to 100 % identity respectively.
  • SEQ ID NO: 1 to 6 and 10 are derived from SEQ ID 7.
  • SEQ ID NO: 1 position 90 to 535 of SEQ ID NO: 7
  • SEQ ID NO: 2 position 90 to 544 of SEQ ID NO: 7
  • SEQ ID NO: 3 position 661 to 693 of SEQ ID NO: 7
  • SEQ ID NO: 4 position 658 to 704 of SEQ ID NO: 7
  • SEQ ID NO: 10 position 655 to 730 of SEQ ID NO: 7
  • SEQ ID NO: 5 position 789 to 1080 of SEQ ID NO: 7
  • SEQ ID NO: 6 position 750 to 1094 of SEQ ID NO: 7
  • SEQ ID NO: 7 UniProt database of the EMBL-EBI, EMBL accession no. Q6RKB1, amino acid sequence of CAR from Nocardia sp. Strain NRRL 5646
  • SEQ ID NO: 8 EMBL Nucleotide Sequence Database of the EMBL-EBI, EMBL accession no. AY495697, nucleotide sequence of CAR gene of Nocardia iowensis, registered as "Nocardia sp. NRRL 5646 ATP/NADPH-dependent carboxylic acid reductase (car)" and translating in sequence of SEQ ID NO: 7
  • SEQ ID NO: 9 this is a SEQ ID NO: 8, which is codon optimized for E. coli SEQ ID NO: 11 : Nucleotide database of the NCBI, Escherichia coli BL21 (DE3), complete genome, accession no. AM946981.2 region 569939 to 570568, nucleotide sequence of a EcPPTase gene in its complementary form
  • SEQ ID NO: 12 Protein database of the NCBI, phosphopantetheinyl transferase, subunit of enterobactin synthase multi enzyme complex [Escherichia coli BL21 (DE3)], accession no. CAQ31055.1, amino acid sequence of a EcPPTase
  • SEQ ID NO: 13 Nucleotide database of the NCBI, Corynebacterium glutamicum ATCC 13032, NC 003450, region 2634745 to 2635152, nucleotide sequence of a CgPPTase gene in its complementary form
  • SEQ ID NO: 14 Protein database of the NCBI, 4'-phosphopantetheinyl transferase
  • SEQ ID NO: 15 Gene sequence of plasmid pJexpress-404-9-CAR
  • SEQ ID NO: 16 Gene sequence of plasmid pJexpress-401-11 -EcPPTase
  • SEQ ID NO: 1 and SEQ ID NO: 2 represent the adenylating domain of CAR, where AMP is bound.
  • SEQ ID NO: 3 represent the phosphopantetheinyl binding site of CAR.
  • SEQ ID NO: 4 and SEQ ID NO: 10 represent the thioester reductase domain of CAR, where NADPH is bound.
  • CAR is classified in the literature to be an enzyme that catalyzes the reaction, which enzymes of the enzyme class EC 1.2.99.6 or of the enzyme class EC 1.2.1.30 catalyze.
  • enzyme class EC 1.2.1.30 is assigned to enzymes, that have the accepted name "aryl-aldehyde dehydrogenase (NADP+)".
  • enzyme class EC 1.2.99.6 is assigned to enzymes, that have the accepted name: carboxylate reductase. They catalyze the reaction:
  • aldehyde + acceptor + H20 a carboxylate + reduced acceptor
  • aldehyde: (acceptor) oxidoreductase a carboxylate + reduced acceptor
  • enzyme class EC 2.7.8.7 is assigned to enzymes, that have the accepted name "holo-[acyl-carrier- protein] synthase".
  • CoA-[4'-phosphopantetheine] + apo-[acyl-carrier protein] adenosine 3',5'-bisphosphate + holo-[acyl-carrier protein]
  • Other names are acyl carrier protein holo protein (holo-ACP) synthetase; holo-ACP synthetase; coenzyme A: fatty acid synthetase apo enzyme 4'-phosphopantetheine transferase; holo synthase; acyl carrier protein synthetase; holo-ACP synthase; PPTase; AcpS; ACPS; acyl carrier protein synthase; P-pant transferase; CoA: apo-[acyl-carrier-protein]
  • Systematic name is "CoA-[4'-phosphopantetheine] :apo-[acyl-carrier protein] 4'- pantetheinephosphotransferase”.
  • the CAS registry number is 37278-30-1.
  • CAR and PPTase are polypeptides.
  • the scope of CAR and of PPTase within the meaning of the invention comprises “functional equivalents".
  • functional equivalents of CAR and of PPTase are polypeptides which differ from CAR and of PPTase but which still possess the desired biological activity such as substrate specificity, for example.
  • “functional equivalents” mean, for example, enzymes which reduce DOPAC to 3-HT and which have at least 50%, preferably at least 60%, more preferably at least 75%, even more preferably at least 90%, of the activity of an enzyme having the amino acid sequence listed in SEQ ID NO 7 or 12 respectively.
  • Functional equivalents are also preferably stable between pH 4 to 10. They preferably possess a pH optimum between pH 5 and 8.
  • Functional equivalents thus comprise e.g. the mutants obtainable by one or more amino acid additions, amino acid substitutions, amino acid deletions and/or or amino acid inversions, it being possible for said alterations to occur in any sequence position, as long as they result in a mutant having the property profile of the invention.
  • Functional equivalence also exists, in particular, when the reactivity patterns of the mutant and the unaltered enzyme with the amino acid sequence SEQ ID NO: 7 or 12 respectively, agree qualitatively, i.e. DOPAC is reduced to 3-HT at different rates.
  • precursors are natural or synthetic precursors of the polypeptides with the desired reduction activity.
  • salts means both salts of carboxyl groups and/or of amino groups of the polypeptides of the invention.
  • Salts of carboxyl groups can be prepared in a manner known per se and comprise inorganic salts such as sodium, calcium, ammonium, iron and zinc salts as well as salts with organic bases, for example amines, such as triethanolamine, arginine, lysine, piperidine and the like.
  • Salts of amino groups are for example salts with mineral acids such as hydrochloric acid or sulfuric acid and salts with organic acids such as acetic acid and oxalic acid.
  • “Functional derivatives" of polypeptides of the invention are covalently modified polypeptides of the invention, and may be prepared with the aid of known techniques e.g. by modification of functional side groups of the polypeptide such as amino side groups or hydroxy side groups, or by modification of the N-terminus or C-terminus. Modification is done preferably with protecting groups, which are commonly used in polypeptide chemistry and which are known to the skilled person.
  • Functional derivatives of this kind comprise, for example, aliphatic esters of carboxylic acid groups, amides of carboxylic acid groups, which amides are obtainable by reacting with ammonia or with a primary or secondary amine; N- acyl derivatives of free amino groups, which derivatives are prepared by reacting the free amino groups with acyl groups; or O-acyl derivatives of free hydroxyl groups, which derivatives are prepared by reacting the free hydroxyl groups with acyl groups.
  • Functional equivalents naturally also comprise polypeptides which are available from other organisms than E. coli and also naturally occurring variants.
  • areas of homologous sequence regions can be established by sequence comparison and equivalent enzymes can be determined on the basis of the specific guidelines of the invention.
  • polypeptide sequences or functional equivalents derived therefrom and at least one further heterologous sequence which is functionally different therefrom and is covalently linked, preferably N -terminally or C-terminally (i.e. without any substantial reciprocal functional impairment of the fusion protein moieties).
  • heterologous sequences are signal peptides or enzymes, for example.
  • “Functional equivalents” which are comprised in the invention are variants of the specifically disclosed sequences. Said variants comprise an amino acid sequence with 70 to 100% identity, preferably with 80 to 100% identity, more preferably with 90 to 100%) identity, even more preferably with 95 to 100%> identity, especially with 100%> identity, with the amino acid sequence with which it is compared.
  • variant of a sequence means preferably a sequence with a certain degree of identity to the sequence.
  • functional equivalents comprise proteins of the above described type in deglycosylated or glycosylated form and also modified forms which can be obtained by altering the glycosylation pattern.
  • Variants of the proteins or polypeptides of the invention may be generated by mutagenesis, for example by point mutation or truncation of the protein.
  • Variants of the proteins of the invention may be identified by screening combinatorial libraries of mutants such as truncation mutants.
  • a variegated library of protein variants may be generated by combinatorial mutagenesis at the nucleic acid level, for example by enzymatically ligating a mixture of synthetic oligonucleotides.
  • a degenerate gene sequence maybe synthesized chemically in a DNA synthesizer and the synthetic gene may then be ligated into a suitable expression vector.
  • Using a degenerate set of genes makes it possible to prepare all the sequences in a mixture which encode the desired set of potential protein sequences.
  • a plurality of techniques for screening gene products of combinatorial libraries which have been prepared by point mutations or truncation and for screening cDNA libraries for gene products having a selected property are known in the prior art. These techniques can be adapted for rapidly screening the gene libraries which have been generated by combinatorial mutagenesis of variants of the invention.
  • the most frequently employed techniques for screening large gene libraries which are subject to high-throughput analysis comprise cloning the gene library into replicable expression vectors, transforming the appropriate cells with the resulting vector library and expressing the combinatorial genes under conditions under which detection of the desired activity facilitates isolation of the vector which encodes the gene whose product was detected.
  • REM Recursive ensemble mutagenesis
  • CAR is of enzyme class EC 1.2.1.30;
  • PPTase is of enzyme class EC 2.7.8.7.
  • PPTase is a EcPPTase or a CgPPTase
  • CAR is of enzyme class EC 1.2.1.30 and PPTase is of enzyme class EC 2.7.8.7.
  • CAR is of enzyme class EC 1.2.1.30 and PPTase is a EcPPTase or a CgPPTase; more preferably, is a PPTase of E. coli.
  • CAR comprises each of the three amino acid sequences SEQ ID NO: 1
  • PPTase comprises the sequence SEQ ID NO: 12 or comprises the sequence SEQ ID NO:
  • each sequence is comprised with 80 to 100 % identity, preferably with 90 to 100 % identity, more preferably with 95 to 100 % identity, even more preferably with 100 % identity.
  • CAR comprises each of the three amino acid sequences SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6;
  • PPTase comprises the sequence SEQ ID NO: 12 or comprises the sequence SEQ ID NO:
  • each sequence is comprised with 70 to 100% identity, preferably with 80 to 100% identity, more preferably with 90 to 100%> identity, even more preferably with 95 to 100%> identity, especially with 100% identity.
  • CAR comprises each of the three amino acid
  • PPTase comprises the sequence SEQ ID NO: 12 or comprises the sequence SEQ ID NO:
  • CAR comprises the CAR from Nocardia sp. Strain NRRL 5646; and PPTase comprises the sequence SEQ ID NO: 12 or comprises the sequence SEQ ID NO:
  • each of the sequences SEQ ID NO: 12 and SEQ ID NO: 14 is comprised with 70 to 100% identity, preferably with 80 to 100% identity, more preferably with 90 to 100% identity, even more preferably with 95 to 100% identity, especially with 100% identity.
  • CAR comprises the sequence SEQ ID NO: 7;
  • PPTase comprises the sequence SEQ ID NO: 12 or comprises the sequence SEQ ID NO:
  • each sequence is comprised with 70 to 100%) identity, preferably with 80 to 100%) identity, more preferably with 90 to 100%> identity, even more preferably with 95 to 100%> identity, especially with 100%> identity;
  • CAR has the sequence SEQ ID NO: 7;
  • PPTase has the sequence SEQ ID NO: 12 or has the sequence SEQ ID NO: 14;
  • sequence SEQ ID NO: 12 is the preferred embodiment for PPTase.
  • CAR and PPTase are used in the reaction
  • the cell free extract containing the enzyme or both enzymes can be e.g. in form of a powdered protein composition, in form of a solution or suspension containing the enzyme or in form of a purified enzyme. It is possible to use for the process of the invention growing cells. It is also possible to use resting or disrupted cells. Disrupted cells mean, for example, cells which have been made permeable by way of treatment with solvents for example, or cells which have been broken up by way of treatment with enzymes, by way of mechanical treatment (e.g. French press or ultrasonication) or by way of another method.
  • the crude extracts obtained in this manner are advantageously suitable for the process of the invention. It is also possible to use purified or partially purified enzymes for the process. Immobilized microorganisms or enzymes which may advantageously be applied in the reaction are likewise suitable.
  • microorganism that expresses or expressed one of the enzymes or both, is also called host organism or host cell.
  • CAR and PPTase are used in the reaction in form of a microorganism, that expresses both enzymes, or in form of two mircoorganisms, one microorganism expressing the one enzyme and the other microorganism expressing the other enzyme, or in form of a cell free extract containing the enzymes;
  • microorganism even more preferably in form of a microorganism, that expresses both enzymes, or in form of two mircoorganisms, one mircroorganism expressing the one enzyme and the other mircroorganism expressing the other enzyme.
  • a suitable microorganism used as host is in principle any prokaryotic or eukaryotic organism.
  • microorganisms such as bacteria, fungi or yeasts are used as host organisms.
  • Preferred eukaryotic microorganisms are Saccharomyces or Pichia, more preferably
  • Preferred prokaryotic microorganisms used as hosts are gram-positive or gram-negative bacteria, preferably selected from the group of families consisting of Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Streptomycetaceae and Nocardiaceae, more preferably selected from the group consisting of Escherichia, Bacillus, Lactobacillus, Pseudomonas, Streptomyces, Nocardia, Burkholderia, Salmonella, Agrobacterium and Rhodococcus; even more preferably the microorganism is Escherichia coli.
  • further advantageous bacteria can be found in the group of the alpha-proteobacteria, beta-proteobacteria or gamma- proteobacteria.
  • E. coli is used preferably in form of the strains selected from the group consisting of BL21, TGI, W311 0, DH1, XL 1-Blue and Origami, which are commercially available or can be obtained via the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany).
  • ECOLI-1 is E. coli BL21, more preferably E. coli BL21 (DE3).
  • the organisms used in the process of the invention are, depending on the host organism, grown or cultured in a manner known to the skilled worker.
  • Microorganisms are usually grown in a liquid medium which comprises a carbon source, usually in the form of sugars, a nitrogen source, usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as iron salts, manganese salts, magnesium salts and, if appropriate, vitamins, at temperatures of between 0° C. and 100° C, preferably between 10° C and 60 °C, while being aerated.
  • the pH of the nutrient liquid may kept at a fixed value, i.e. it is not necessary but preferable to be regulated during cultivation.
  • the cultivation may be carried out batch wise, semibatchwise or continuously.
  • Nutrients may be introduced at the beginning of the fermentation or be fed in subsequently in a semicontinuous or continuous manner.
  • DOPAC may be added directly to the culture or, advantageously, after cultivation.
  • the enzymes can be used according to the methods described in the examples or can be used for the reaction as a crude extract.
  • Host cells are usually cultured in conventional media as known in the art such as complex media like LB broth, "nutrient yeast broth medium", minimal media or a glycerol containing medium as described by Kortz et al., J. Biotechnol., 1995, 39, 59-65 , or a mineral salt media as described by Kulla et al., Arch. Microbiol., 1983, 135, 1-7.
  • complex media like LB broth, "nutrient yeast broth medium", minimal media or a glycerol containing medium as described by Kortz et al., J. Biotechnol., 1995, 39, 59-65 , or a mineral salt media as described by Kulla et al., Arch. Microbiol., 1983, 135, 1-7.
  • LB broth and LB medium are used interchangeable. Glucose can be added to the medium for cultivation.
  • induction is done using IPTG.
  • the vector used in the invention is preferably an autonomously or self-replicating plasmid, a cosmid, a phage, a virus or a retrovirus.
  • a wide variety of host/vector combinations may be employed in expressing the nucleic acid sequences encoding CAR and/or PPTase
  • Useful expression vectors may consist of segments of
  • Suitable vectors include vectors with specific host range such as vectors specific for e. g. E. coli as well as vectors with broad-host-range such as vectors useful for Gram-negative bacteria. "Low-copy”, “medium-copy” as well as “high copy” plasmids can be used.
  • Useful vectors for e.g. expression in E. coli are: pQE70, pQE60 und pQE-9 (QIAGEN, Inc.); pBluescriptVectors, PhagescriptVectors, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene Cloning Systems, Inc.); ptrc99a, pKK223-3, pKK233-3, pOR540, pRIT5 (Pharmacia Biotech, Inc.); pLG338, pACYC184, pBR322, pUC18, pUC19,pKC30, pRep4, pACYC177, pACYC184, pRSFlOlO and pBW22 (Wilms et al., Biotechnology and Bioengineering, 2001, 73, 95-103) or derivatives thereof such as plasmid pBW22-Fab-H or plasmid
  • Preferred vectors to be used in the method of the present inventions are autonomously or self- replicating plasmids, more preferred are vectors with specific host range such as vectors specific for e. g. E. coli.
  • pJexpress vectors are used.
  • one of the enzymes CAR and PPTase or both are used as in form of a microorganism, that expresses both enzymes, or in form of two mircoorganisms, one mircroorganism expressing the one enzyme and the other mircroorganism expressing the other enzyme, the respective microorganism is used in the reaction in form of its cells with an optical density of from 0.1 to 300, more preferably from 0.5 to 300, even more preferably from 1 to 300, especially from 5 to 300, more especially from 5 to 250, even more especially from 10 to 250, in particular from 10 to 200, more in particular from 50 to 200.
  • the amount of each of the enzymes CAR and PPTase in the reaction is from 0.001 to 100 mg/mL, more preferably from 0.005 to 100 mg/mL, even more preferably from 0.01 to 100 mg/mL, especially from 0.05 to 100 mg/mL, more especially from 0.1 to 30 mg/mL, even more especially from 0.5 to 30 mg/mL;
  • the amount of each of the enzymes CAR and PPTase in the reaction, when used as in form of an isolated enzyme, is from 0.001 to 100 mg/mL, more preferably from 0.005 to 100 mg/mL, even more preferably from 0.01 to 100 mg/mL, especially from 0.05 to 100 mg/mL, more especially from 0.1 to 30 mg/mL, even more especially from 0.5 to 30 mg/mL.
  • the molar ratio of CAR : DOPAC in the reaction is from 1 50000 to 1 : 1 ; preferably from 1 : 10000 to 1 : 5, more preferably from 1 : 5000 to 1 : 2'
  • the molar ratio of PPTase : CAR in the reaction is from 1 : 1000 to 1 : 1 , more preferably from 1 : 500 to 1 : 2.
  • DOPAC is used in the reaction in a concentration of from 0.1 to 100 mM, more preferably of from 1 to 100 mM, even more preferably of from 1 to 50 mM, especially of from 5 to 50 mM.
  • the reduction of DOPAC to 3-HT proceeds chemically in two steps: in the first reduction step the DOPAC is reduced to the corresponding aldehyde, the compound of formula (1),
  • enzyme SECREDSTEP is present in the reaction in addition to the two enzymes CAR and PPTase; enzyme SECREDSTEP is an enzyme which catalyzes the reduction of compound of formual (1) to 3-HT.
  • Enzyme SECREDSTEP can also be named in the literature as being an alcohol
  • dehydrogenase a aldo-keto reductase, a keto reductase, an aldehyde reductase or aldehyde hydrogenase.
  • Enzyme SECREDSTEP is preferably classified in the literature to be an enzyme that catalyzes the reaction, which enzymes of the enzyme class EC 1.1.1 catalyze.
  • enzyme class EC 1.1.1 is assigned to enzymes called "EC 1 Oxidoreductases - EC 1.1 Acting on the CH-OH group of donors - EC 1.1.1 With NAD+ or NADP+ as acceptor".
  • enzyme SECREDSTEP is an enzyme classified in the literature to be an enzyme that catalyzes the reaction, which enzymes of enzyme classes EC 1.1.1.1 or EC 1.1.1.2 catalyze.
  • enzyme class EC 1.1.1.1 is assigned to enzymes, that have the accepted name "alcohol
  • Systematic name is "alcohol:NAD + oxidoreductase”.
  • the CAS registry number is 9031-72-5.
  • enzyme class EC 1.1.1.2 is assigned to enzymes, that have the accepted name "alcohol dehydrogenase (NADP + )”.
  • NADPH 2 NADP-alcohol dehydrogenase
  • NADP- aldehyde reductase NADP-dependent aldehyde reductase
  • NADPH-aldehyde reductase NADP-dependent aldehyde reductase
  • nonspecific succinic semialdehyde reductase ALR 1 ;
  • Systematic name is " alcohol :NADP + oxidoreductase”.
  • the CAS registry number is 9028-12-0.
  • enzyme SECREDSTEP is an endogenous enzyme of the micororganism that expresses or expressed CAR or PPTase or both.
  • enzyme SECREDSTEP is an endogenous enzyme of the micororganism that expresses or expressed CAR or PPTase or both.
  • enzyme SECREDSTEP is used in the reaction in the form of a
  • the respective microorganism is used in the reaction in the form of its cells with an optical density of from 0.1 to 300, more preferably from 0.5 to 300, even more preferably from 1 to 300, especially from 5 to 300, more especially from 5 to 250, even more especially from 10 to 250, in particular from 10 to 200, more in particular from 50 to 200.
  • the amount of enzyme SECREDSTEP in the reaction is from 0.001 to 100 mg/mL, more preferably from 0.005 to 100 mg/mL, even more preferably from 0.01 to 100 mg/mL, especially from 0.05 to 100 mg/mL, more escpecially from 0.1 to 30 mg/mL, even more especially from 0.5 to 30 mg/mL;
  • the amount of enzyme SECREDSTEP in the reaction when used as in form of an isolated enzyme, is from 0.001 to 100 mg/mL, more preferably from 0.005 to 100 mg/mL, even more preferably from 0.01 to 100 mg/mL, especially from 0.05 to 100 mg/mL, more especially from 0.1 to 30 mg/mL, even more especially from 0.5 to 30 mg/mL.
  • the molar ratio of enzyme SECREDSTEP : DOPAC in the reaction is from 1 : 50000 to 1 : 1; preferably from 1 : 10000 to 1 : 5.
  • the reaction is done in an aqueous reaction medium.
  • the reaction medium is a medium conventionally used for a cell culture.
  • reaction is done in the presence of a buffer (REAC).
  • RRC buffer
  • Buffer is a buffer commonly used in enzyme catalyzed reactions.
  • buffer is selected from the group consisting of phosphate buffer, HEPES (a buffer based on 4-2-hydroxyethyl-l-piperazineethanesulfonic acid), TES (a buffer based on 2- ⁇ [tris(hydroxymethyl)methyl]amino ⁇ ethanesulfonic acid), MOPS (a buffer based on 3-(N-morpholino)propanesulfonic acid), PIPES (a buffer based on piperazine-
  • HEPES a buffer based on 4-2-hydroxyethyl-l-piperazineethanesulfonic acid
  • TES a buffer based on 2- ⁇ [tris(hydroxymethyl)methyl]amino ⁇ ethanesulfonic acid
  • MOPS a buffer based on 3-(N-morpholino)propanesulfonic acid
  • PIPES a buffer based on piperazine-
  • buffer is phosphate buffer, MES or mixtures thereof.
  • a phosphate buffer is preferably a sodium or potassium phosphate buffer.
  • buffer is sodium or potassium phosphate buffer or MES
  • buffer (REAC) is MES.
  • the concentration of buffer (REAC) in the reaction mixture is from 0.005 to 0.5 M, more preferably from 0.02 to 0.2 M.
  • the enzymes are used in the reaction in form of a microorganism, that expresses both enzymes, or in form of two mircoorganisms, one mircroorganism expressing the one enzyme and the other mircroorganism expressing the other enzyme;
  • reaction medium is buffer (REAC).
  • the reaction is started in the presence of buffer (REAC) with a pH of from 5 to 8.5, more preferably of from 5.5 to 8.5, even more preferably of from 5.5 to 8.0, especially of from 5.5 to 7.6.
  • buffer RRC
  • the reaction is done in the presence of buffer (REAC) with at a pH of from 5 to 8.5, more preferably of from 5.5 to 8.5, even more preferably of from 5.5 to 8.0, especially of from 5.5 to 7.6.
  • RRC buffer
  • the reaction is done in the presence of DTT. DTT is preferably added to the reaction mixture.
  • the amount of DTT is preferably from 0.01 to 20 mM, more preferably from 0.1 to 10 mM, even more preferably from 0.4 to 10 mM.
  • the reaction is done in the presence of EDTA.
  • EDTA is preferably added to the reaction mixture.
  • the amount of EDTA is preferably from 0.01 to 10 mM, more preferably from 0.1 to 5 mM, even more preferably from 0.4 to 5 mM.
  • reaction is started in the presence of buffer (REAC) with a pH of from 5 to 8.5, and
  • the reaction is done in the presence of DTT, the amount of DTT is from 0.01 to 20 mM, and
  • the reaction is done in the presence of EDTA, the amount of EDTA is from 0.01 to 10 mM;
  • the pH is from 5.5 to 8.5
  • the amount of DTT is from 0.1 to 10 mM
  • the amount of EDTA is from 0.1 to 5 mM; even more especially, the pH is from 5.5 to 8.0,
  • the amount of DTT is from 0.4 to 10 mM
  • the amount of EDTA is from 0.4 to 5 mM
  • the pH is from 5.5 to 7.6,
  • the amount of DTT is from 0.4 to 10 mM
  • the amount of EDTA is from 0.4 to 5 mM.
  • the reaction is done at a reaction temperature of from 4 to 45 °C, more preferably of from 10 to 45 °C, even more preferably of from 15 to 40 °C, especially of from 19 to 39 °C, more especially of from 20 to 38 °C.
  • the reaction time is from 10 min to 96 h, more preferably from 20 min to 48 h.
  • the reaction is done under aerobic conditions.
  • the reaction medium is stirred, either by shaking the reaction tube or by a stirrer.
  • shaking is done at 500 to 1500 rpm, preferably at 750 to 1250 rpm.
  • stirring is done at 50 to 500 rpm.
  • the reaction is done in the presence of a cofactor
  • the cofactor is selected from the group consisting of NADPH and NADH.
  • cofactor either endogenous cofactor is used or the cofactor is added to the reaction mixture or both.
  • the cofactor when the cofactor is added, the cofactor is added to provide for a concentration of from 0.05 to 500 mM, more preferably of from 0.05 to 100 mM, even more preferably of from 0.1 to 50 mM.
  • the cofactor when the cofactor is added, the cofactor is added to provide for a 2 to 10 fold molar amount based on the molar amount of DOPAC.
  • endogenous cofactor is used.
  • the cofactor can be regenerated.
  • the reaction is (i) done in the presence of an enzyme ENZREGENCOF, that regenerates the cofactor, and a respective substrate REGENSUB, that is used by ENZREGENCOF to regenerate the cofactor; or
  • reaction is done using electrical current via redox electrodes for the regeneration of the cofactor
  • substrate is a substrate of the citric acid cycle or glucose.
  • reaction is
  • the citric acid cycle is also known as tricarboxylic acid cycle (TCA cycle).
  • ENZREGENCOF is selected from the group consisting of glucose 1 - dehydrogenase (GDH), formate dehydrogenase (FDH), phosphonate dehydrogenase,
  • NAD(P) oxidase NAD(P) oxidase, alcohol dehydrogenase (ADH) and mixture thereof;
  • REGENSUB is glucose, formate, phosphonate, hydrogen peroxide or an alcohol as
  • ENZREGENCOF is GDH with REGENSUB being glucose or
  • ENZREGENCOF is FDH with REGENSUB being formate or ENZREGENCOF is a mixture of GDH and FDH with REGENSUB being glucose and formate;
  • ENZREGENCOF is GDH with REGENSUB being glucose.
  • ENZREGENCOF and REGENSUB are commercially available, e.g. from Sigma Aldrich, or ENZREGENCOF can be cloned according to known procedures from literature.
  • a redox mediator is preferably a viologen or a mixture of viologens.
  • Viologens are bipyridinium derivatives of 4,4'-bipyridyl, preferred viologens are selected from the consisting of methyl viologen with CAS 1910-42-5 and benzyl viologen with CAS 1102-19- 8.
  • a substrate is preferably selected from the group consisting of glucose citrate, cis-aconitate, D-isocitrate, alpha-ketoglutarate, succinate, fumarate, malate, oxaloacetate and mixtures thereof; more preferably glucose, citrate and mixtures thereof.
  • substrate is present in a concentration of from 0.001 to 0.5 M, more preferably of from 0.005 to 0.1 M.
  • substrate is present in a 1 to 3 fold molar amount based on the molar amount of DOPAC.
  • substrate SUBSCIT
  • substrate SUBSCIT
  • An alcohol as substrate for ADH can be any substrate that is a known substrate for AHD, preferably an alcohol as substrate for ADH is selected from the group consisting of C2-8 alcohol; more preferably ethanol, propanol or butanol.
  • GDH is preferably an enzyme of the enzyme class EC 1.1.1.47.
  • enzyme class EC 1.1.1.47 is assigned to enzymes that have the accepted name "glucose 1 -dehydrogenase”.
  • FDH is preferably an enzyme of the enzyme class EC 1.2.1.2.
  • enzyme class EC 1.2.1.2 is assigned to enzymes, that have the accepted name "formate dehydrogenase”.
  • formate + NAD + C0 2 + NADH
  • formate-NAD oxidoreductase FDH I; FDH II; N-FDH
  • formic hydrogen- lyase formate hydrogenlyase; hydrogenlyase
  • NAD-linked formate dehydrogenase NAD-dependent formate dehydrogenase
  • formate dehydrogenase NAD
  • NAD-formate dehydrogenase formate benzyl-viologenoxidoreductase
  • Systematic name is "formate: NAD + oxidoreductase”.
  • the CAS registry number is 9028-85-7.
  • phosphonate dehydrogenase is preferably an enzyme of the enzyme class EC 1.20.1.1.
  • enzyme class EC 1.20.1.1 is assigned to enzymes, that have the accepted name "phosphonate dehydrogenase”.
  • NAD phosphite oxidoreductase
  • phosphite dehydrogenase phosphite dehydrogenase
  • NAD(P) oxidase is preferably an enzyme of the enzyme class EC 1.6.3.1.
  • enzyme class EC 1.6.3.1 is assigned to enzymes, that have the accepted name "NAD(P)H oxidase".
  • THOX2 ThOX
  • dual oxidase pl38tox
  • thyroid NADPH oxidase thyroid oxidase
  • thyroid oxidase 2 thyroid oxidase 2
  • NADPH oxidase Other names are "THOX2; ThOX; dual oxidase; pl38tox; thyroid NADPH oxidase; thyroid oxidase; thyroid oxidase 2; NADPH oxidase”.
  • NAD(P)H oxygenoxidoreductase
  • the CAS registry number is 77106-92-4.
  • ADH is preferably an enzyme of the enzyme class EC 1.1.1.1 or of the enzyme class EC 1.1.1.2.
  • the amount of ENZREGENCOF is from 0.001 to 100 mg/mL, more preferably from 0.005 to 100 mg/mL, even more preferably from 0.005 to 30 mg/mL, especially from 0.005 to 20 mg/mL; more especially from 0.005 to 15 mg/mL.
  • the molar ratio of ENZREGENCOF : DOPAC is from 1 : 50000 to 1 : 1 ; preferably from 1 : 10000 to 1 : 1.
  • the total optical density of the combined microorganisms in the reaction is preferably of from 0.1 to 300, more preferably from 0.5 to 300, even more preferably from 1 to 300, especially from 5 to 300, more especially from 5 to 250, even more especially from 10 to 250, in particular from 10 to 200, more in particular from 50 to 200.
  • the molar amount of REGENSUB is from 1 to 10 fold, more preferably from 1 to 6 fold, based on the molar amount of DOPAC.
  • ENZREGENCOF is used as isolated enzyme, or to clone ENZREGENCOF separately from CAR and PPTase into a different microorganism, which then is present in the reaction, or to clone ENZREGENCOF together with one of the enzymes CAR or PPTase or with both enzymes CAR and PPTase in the same microorganism, even on the same plasmid in the same microorganism.
  • the reaction is done in the presence of a kation KAT, KAT is selected from the group consisting of Mg 2+ , Zn 2+ and mixtures thereof.
  • KAT is present in form of a sulfate, hydrogen sulfate, chloride or acetate of KAT.
  • KAT is Mg 2+ ;
  • KAT is present in form of a sulfate, hydrogen sulfate, chloride or acetate of Mg 2+ or as a mixture thereof;
  • KAT as added to the reaction mixture.
  • KAT is used in a concentration of from 0.01 to 50 mM, more preferably of from 0.1 to 30 mM, even more preferably 0.5 to 30 mM, especially 0.5 to 20 mM.
  • the reaction is started in the presence of buffer (REAC) with a pH of from 5 to 8.5, and the reaction is done in the presence of DTT, the amount of DTT is from 0.01 to 20 mM, and
  • the reaction is done in the presence of EDTA, the amount of EDTA is from 0.01 to 10 mM, and
  • KAT is used in a concentration of from 0.01 to 50 mM
  • the pH is from 5.5 to 8.5
  • the amount of DTT is from 0.1 to 10 mM
  • the amount of EDTA is from 0.1 to 5 mM
  • KAT is used in a concentration of from 0.01 to 50 mM
  • the pH is from 5.5 to 8.0
  • the amount of DTT is from 0.4 to 10 mM
  • the amount of EDTA is from 0.4 to 5 mM
  • KAT is used in a concentration of from 0.1 to 30 mM
  • the pH is from 5.5 to 7.6,
  • the amount of DTT is from 0.4 to 10 mM
  • the amount of EDTA is from 0.4 to 5 mM
  • KAT is used in a concentration of from 0.5 to 30 mM
  • the reaction is done with resting cells, which are frozen and are thawed for the reaction, these cells should be frozen for a time period of up to one month. To have the cells for a longer period of time in the frozen state might reduce their activity. Generally, this was observed in the disclosed experiments.
  • the reaction can be monitored, e.g. by HPLC, GC or by NMR.
  • 3-HT can be isolated from the reaction mixture by standard methods known to the skilled person.
  • the cells are separated by centrifugation or filtration.
  • the product is preferably isolated by extraction with a solvent, preferably after centrifugation or filtration, from the mother liquor, and can be purified by flash column chromatography or crystallization or both.
  • extraction is done with a solvent EXTR selected from the group consisting of toluene, dichloromethane, cyclohexane, methylcyclohexane, ethyl acetate and methyl tert- butyl ether, preferably ethyl acetate.
  • Flash column chromatography is preferably done using a column packed with silicagel G 60, the solvent system for flash chromatography is preferably methyl tert-butyl ether : n-heptane 1 :1 v/v.
  • Crystallization is preferably done from a solvent CRYST selected from the group consisting of petroleum : ether, 3.5 : 1 v/v; the ether is preferably methyl tert-butyl ether.
  • Buffer B 50 mM MES, 10 mM MgCl 2 , 1 mM EDTA, 1 mM DTT, 10% v/v glycerol, pH
  • Buffer C 50 mM MES, 10 mM MgCl 2 , 1 mM EDTA, 1 mM DTT, pH 6.0 adjusted with
  • Buffer D 50 mM MES, pH 6.0 adjusted with NaOH
  • Centrifugation parameter can also be specified with the g value (gravitational constant)
  • Electrocompetent cells E. coliBL21 (DE3) Gold Agilent Technologies, Inc., CA 95051
  • 3-HT can be purchased from Sigma-Aldrich, www.sigmaaldrich.com
  • IPTG isopropyl beta-D-l-thiogalactopyranoside, Biosynth, 9422 Staad, Switzerland, www.biosynth.com
  • KPi Phosphate buffer containing different ratio of equalmolar KH2PO4 and
  • LB-plates Luria broth medium also called LB -Lennox- Agar (20g/L) containing agarose
  • LB medium Luria broth medium also called LB-Lennox (20g/L: lOg/L Tryptone, 5g/L
  • Tris tris-(hydroxymethyl)-aminomethane (IUPAC name: 2-Amino-2- hydroxymethyl-propane-l,3-diol), Carl ROTH, 76185 Düsseldorf, Germany, www.carlroth.com
  • Tryptone Tryptone is the assortment of peptides formed by the digestion of casein by the protease trypsin, supplier: Oxoid Limited, Hampshire, United Kingdom, www.oxoid.com, a subsidiary of Thermo Fisher Scientific,
  • U 1 enzyme unit (U) 1 ⁇ min -1 , that is 1 U converts 1 ⁇ of substrate per minute
  • MS e.g. Agilent G1956B, equipped with Atmospheric pressure
  • MS electrospray ionization, positive mode (MS) recording for scan (90 to 450 m/z) and SIMs (155 m/z and 153 m/z for alcohol and aldehyde, resp.) and negative mode (for acid)
  • the concentration a calibration was done with known concentration of the respective substance to be detected; the % value is the analytical yield of product (product is 3-HT if not otherwise stated) based on the molar amount of substrate used; if not otherwise stated.
  • This pellet does not contain any of the DNA.
  • the supernatant should contain approximately 2 ⁇ g plasmid DNA (ca. 20 ng ⁇ L).
  • the isolated DNA can subsequently be transformed, cut with restriction enzymes, or sequenced without further purification.
  • Running Buffer NuPAGE® MOPS SDS Running Buffer (for Bis-Tris Gels only)
  • Power Supplier PowerEase® 500 Power Supply (Invitrogen)
  • Electrophoresis cell XCellSureLock® Mini-Cell
  • Electrophoresis was run for 45 min at 200 V.
  • the cell pellets were dispersed in buffer. Typically, per gram of wet cell paste, 10 mL of buffer was used. The dispersion was placed into an aluminium beaker that was placed on a stirred water-ice bath. A Branson Sonifier ® 250 analog ultrasonic cell disruptor equipped with a 1/2" tapped Disruptor horn was used for 6 min set to 80% duty cycle and to output control 8.
  • the gene with sequence SEQ ID NO: 9 and the gene with sequence SEQ ID NO: 11 were ordered from DNA2.0 and were delivered in form of plasmid pJexpress-404-9-CAR with SEQ ID NO: 15, carrying the gene with SEQ ID NO: 9 for CAR, and of plasmid pJexpress- 401-11-EcPPTase with SEQ ID NO: 16, carrying the gene for PPTase with SEQ ID NO: 11, on GFC filters.
  • FIG 1 shows pJexpress-404-9-CAR
  • T5 promoter T5 promoter sequence was described in Gentz et al., J.
  • FIG 2 shows pJexpress-401-11 -EcPPTase.
  • T5 promoter T5 promoter sequence was described in Gentz et al., J. Bacterid
  • lacl lacl gene which expresses the repressor protein that binds to the lac operator Plasmid DNA was retrieved according to the protocol of DNA2.0 delivered with the plasmids (see above under Methods).
  • E. coli BL21 (DE3) Gold 60 ⁇ . of chemically competent cells E. coli BL21 (DE3) Gold were transformed with the plasmids separately, using 100 ng of pJexpress-404-9-CAR to give strain CE1 and 75 ng of pJexpress-401-11-EcPPTase to give strain PEl respectively.
  • the transformation was done as follows: The two mixtures of cells and plasmid were incubated on ice for 30 min, heat shocked for 30 s at 42°C and cooled down on ice for 2 min. 250 ⁇ of pre-warmed SOC medium was added, incubated 1 h at 37°C.
  • the strains were selected as follows:
  • Plasmid DNA was isolated from the transformed cells CE1 and PEl according to the Purification Protocol of GeneJETTM Plasmid Miniprep Kit of Fermentas.
  • Plasmid DNA isolated from PEl and CE1 was sequenced by LGC genomics using Primers 1 to 6 for SEQ ID NO: 9 and Primers 1 and 2 for SEQ ID NO: 11 and confirmed SEQ ID NO: 9 and SEQ ID NO: 11 respectively.
  • Primer 1 is called pTF5 by DNA2.0
  • Primer 2 is called pTR by DNA2.0.
  • electroporation cuvettes were placed in a MicroPulserElectroporator411BR (Bio-Rad, www.bio-rad.com) and electroporation with following conditions 25 mFD, 200 W, and 2.5 kV (time constant ca. 5 msec) was started. Afterwards, the cells were transferred to a 1.5 mL tube, 600 ⁇ of SOC medium was added and the cells were incubated on a Thermomixer comfort (Eppendorf) at 37°C and 600 rpm for 1 h.
  • Thermomixer comfort Eppendorf
  • the strains were selected as follows:
  • FIG 3 shows overexpression of CAR, expected molecular masses are 128 kDa for CAR and 23 kDa for EcPPTase.
  • the NuPage® electrophoresis was done with a concentration of ca. 1 mg/mL of total protein and the amount of CAR was estimated by visual inspection of the strength of the bands. This results in a approximate concentration of CAR of ca. 8 ⁇ in the enzymatic reduction reactions described in the following examples.
  • FIG 3 shows, that whereas the over expression of CAR enzyme is evident in CFE in lane 2, EcPPTase is not visible in overexpressed amounts. However, the presence of sufficient
  • reaction buffer was used to adjust the final volume to 500 ⁇
  • reaction mixture composition was studied under the following conditions: CPE1 resting cell sample aliquots, prepared according to Example 1, were thawed and centrifuged. The supernatant was discarded and the cell pellet washed twice with buffer B. The cell pellets were dispersed in 50 mM MES buffer pH 6.0 containing variations of components as specified in Table3. 0.5 M NADPH in water and 100 mM of DOPAC in water were added to give final concentrations of 20 mM and 10 mM respectively, in a total volume of 500 ⁇ . The thus prepared samples in Eppendorf vessels were incubated in an Eppendorf Thermomixer at 28°C and 1000 rpm for 4 h with open lid. The samples were refilled with water to 500 ⁇ , centrifuged for 10 min at 28°C and the supernatants were filtered through plate filters (0.2 ⁇ , polypropylene). The filtrates were analyzed with Method A.
  • Relative % means that the % value of Ex 3-1 was set to 100 % and all other % values were normalized to this value.
  • “Relative DOPAC and 3-HT recovery [%]” means the value of the sum of substrate and product concentration of Ex 3-16 was set to 100 % and all other values were normalized to this value
  • the reaction temperature was studied under the following conditions: CPE1 resting cell sample aliquots, which can be prepared according to Example 1 , were thawed and centrifuged. The supernatant was discarded and the cell pellet washed twice with buffer B. The cell pellets were dispersed in buffer B, and 0.5 M NADPH in water and 100 mM of
  • DOPAC in water were added to give final concentrations of 20 mM and 10 mM respectively, in a total volume of 500 ⁇ .
  • the samples were incubated in five separate Eppendorf
  • Relative % means that the % value of Ex 4-3 was set to 100 % and all other % values were normalized to this value.
  • CPEl resting cell samples were prepared according to Example l .
  • the tested reaction times are given in table 5.
  • CPEl fresh cells were used, which were prepared according to example 1 , wherein the cells were harvested as described in example 1 by centrifugation at 2831 g at 4°C for 10 min after the protein expression phase, resulting in a cell pellet of fresh cells. These fresh cell pellets were washed with buffer B and then dispersed in buffer B.
  • Relative % means that the % value of Ex 6-8 was set to 100 % and all other % values were normalized to this value. Table 6 CPEl CPE2
  • the FDH was used in form of a cell free extract of a E. coli Rosetta containing the plasmid pRSF:FDH from Candida boidinii as described in Madje et al., Microb. Cell Fact., 2012, 11 :7, with the described 18°C during the induction phase which is reported by Madje to provide FHD in an amount of 28 % of the total soluble protein in the extract.
  • FDH and formate were added to give final concentrations as listed in Table 8.
  • citrate was used, with and without extra NADP+ in final concentrations as listed in Table 8.
  • the DNA of SEQ ID NO: 13 was amplified from genomic DNA of Corynebacterium glutamicum DSM 20300, ATCC 13032 including the introduction of restriction sites Ncol and Hindlll using Phusion ® High-Fidelity DNA polymerase (Finnzymes) and primers of SEQ ID NO: 23 and of SEQ ID NO: 24.
  • a single colony of Corynebacterium glutamicum was picked with a sterile tooth pick into 50 ⁇ of distilled water. After incubation at 95°C for 10 min and centrifugation for 5 min at room temperature and 13 200 rpm, 5 ⁇ of the supernatant was used as template DNA for the PCR reaction.
  • the PCR reaction was thermally cycled: 98°C for 30 s, followed by 20 cycles of 98°C for 10 s, 50°C for 20 s, and 72°C for 15 s, then a final incubation of 72°C for 7 min.
  • the PCR products were gel purified with the QIAquick® Gel Extraction Kit (QIAGEN).
  • the thus obtained PCR product was double digested with Ncol and Hindlll restriction enzymes (Fermentas) in the presence of lx Tango buffer (Fermentas) and column purified according to QIAquick® PCR purification protocol (QIAGEN).
  • the gene was ligated into the pEHISTEV vector (Huanting et al. Protein Expression and Purification 2009, 63, 102-111) which was also digested with Ncol and Hindlll. The ligation was carried out overnight at 16°C in the presence of T4 ligase
  • ligation product was desalted for 40 min on MF-MilliporeTM membrane filters (0.025 ⁇ filter code VSWP) (Millipore), transformed into electrocompetent E. coli TOP10 F' cells and plated out on a LB-plate supplemented with 50 ⁇ g Kan/mL.
  • the resulting plasmid called pEHISTEV- 14-CgPPTase, was harvested by GeneJETTM Plasmid Miniprep Kit (Fermentas) and the sequence was confirmed by LGC Genomics.
  • CE1, CPE1, PE1, PE2 and CPE2 CFEs of resting cell samples were obtained as follows: For each strain, 20 mL of LB medium containing 100 mg/L Amp or 50 mg/L Kan or both respectively, was inoculated with 5 ⁇ of a respective glycerol stock sample of each strain respectively, prepared according to example 1 and example 9 respectively.
  • the protein expression phase proceeded for 4 h at 37°C and 120 rpm for strains CE1, PE1, CPE1 and CPE2, and overnight at 25°C and 120 rpm for strain PE2. Subsequently, cells were harvested by centrifugation at 2831 g at 4°C for 10 min. The cell pellets were washed twice with Buffer B and centrifuged again at 2831 g at 4°C for 10 min. The supernatant was removed and the cell pellets were dispersed in Buffer B, sonicated, ultracentrifuged at
  • Thawed CFEs were mixed in Eppendorf vessels according to the combinations described in Table 10, 0.5 M NADPH in water, 250 mM ATP in water and 100 mM of DOPAC in water were added to give final concentrations of 20 mM, 10 mM and 10 mM respectively, in a total volume of 500 ⁇ .
  • DOE 'design of experiments' method
  • PPCO beaker PPCO beaker
  • gauze was covered with gauze and incubated on a magnetic stirrer at 28°C and 100 rpm stirring speed. 200 ⁇ ⁇ samples were withdrawn periodically (see column “Time” in Table 12), centrifuged for 10 min at room temperature and the supernatants were filtered through a plate filter (30 kDa cut off, modified polyethersulfone). The filtrates were analyzed with Method A.
  • the reaction was stopped by centrifugation at 15344 g.
  • the pellet was first washed with 25 ml of buffer D by disperging and centrifugation, and then additionally two times with 10 ml of buffer D.
  • the pH of the combined supernatants was adjusted to pH 2.0 with concentrated HC1 and the compounds were extracted with ethyl acetate four times.
  • the combined extracts (175 mL) were dried with sodium sulfate and the solvent was removed at 135 mbar at 40°C.
  • the residue was purified by silica gel chromatography using 15 g of silica gel and dichloromethane : MeOH of 15:1 with 0.15 % of triethylamine as the eluent.

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Abstract

The invention relates to the preparation of 3-hydroxytyrosol (3-HT) from 3,4-dihydroxyphenylacetic acid (DOPAC) using a carboxylic acid reductase CAR.

Description

PROCESS FOR PRODUCING 3-HYDROXYTYROSOL FROM
3,4-DIHYDROXYPHENYLACETIC ACID|EMPLOYING A CARBOXYLIC ACID REDUCTASE
The invention relates to the preparation of 3-hydroxytyrosol (3-HT) from 3,4- dihydroxyphenylacetic acid (DOPAC) using a carboxylic acid reductase CAR.
BACKGROUND OF THE INVENTION
3-HT is a potent antioxidant found in olive leafs and olive oil, and is present in high abundance in olive mill waste waters. 3-HT has been associated with lower mortality and incidence of cancer in Mediterranean regions and has been attributed cardio-protective properties. It is used as a food additive. There has been therefore an increased interest in the manufacturing and commercialization of 3-HT as nutritional supplement.
For the reduction of carboxylic acids by chemical means, the acid has usually to be converted into a more reactive derivative, and strong reducing agents are required. There is a need to find an environmentally friendly, efficient and cost effective process in an aqueous system.
US 5795759 B and Li et al., Journal of Bacteriology, 1997, 179, 3482-3487, disclose purification, characterization and properties of a CAR from Nocardia sp. Strain NRRL 5646 of the enzyme class EC 1.2.1.30. A number of carboxylic acids as substrates were tested for the ability of the CAR to reduce them to their corresponding aldehydes in in vitro experiments. CAR does not reduce an aldehyde to an alcohol, whereas intact whole cells of Nocardia strain NRRL 5646 reduce carboxylic acids first to the aldehyde and then to the corresponding alcohol. US7056714B and He et al., Applied and Environmental Microbiology, 2004, 70, 1974-1881, disclose the CAR from Nocardia sp. Strain NRRL 5646 with its gene and amino acid sequence. The DNA sequence is listed at the Gen-Bank-EMBL database under accession code AY495697.
In vitro reduction of benzoate, vanillic acid and ferulic acid with the recombinant CAR resulted in the corresponding aldehydes. In vivo biotransformations of the same substrates with recombinant E. coli B21 strains resulted in the smooth conversion of carboxylic acids to aldehydes and subsequently partly also to alcohols. Reduction of aldehydes formed by CAR to alcohols by an endogenous E. coli ADH similar to that observed in Nocardia is relatively slow. Biochemical engineering approaches with the recombinant organism might be exploited to diminish this unwanted side reaction.
Venkitasubramaman et al., The Journal of Biological Chemistry, 2007, 282, 478-485, disclose that the CAR from Nocardia sp. Strain NRRL 5646 requires post-translational
phosphopantetheinylation by a PPTase in order to be transformed from an apo- to a holo- enzyme, with only the latter showing the CAR activity.
Venkitasubramaman et al., Enzyme and Microbial Technology, 2008, 42, 130-137, disclose the use of recombinant E. coli BL21-CodonPlus (DE2)-RP/pPV2.85 that expresses car, npt and gdh, for the reduction of vanillic acid to vanillin.
SUMMARY OF THE INVENTION
There was a need for finding a suitable system for the preparation of 3-HT from DOPAC by biotransformation.
The data of the in vitro experiments of Li et al., Journal of Bacteriology, 1997, 179,
3482-3487, in table 2, shows the conversion for the following substrates in relation to the conversion of benzoate, with the conversion of benzoate set to 100 % relative activity:
Relative Activity
Benzoic acid 100%
3- hydroxylbenzoic acid 77%
4- hydroxylbenzoic acid 6%>
phenylacetic acid 43%>
This means, that especially a hydroxy residue in 4 position reduces the rate of conversion considerably. The skilled person would expect, that the relative activity for DOPAC as substrate would be (0.77 x 0.06 x 0.43) = 0.02, i.e. 2 %>, which is rather low.
The teaching of the prior art disclosing the application of CAR of Nocardia sp. is directed to the application of CAR for the reduction of carboxylic acids to their corresponding aldehydes. The subsequent reduction, i.e. the reduction of the aldehyde to its corresponding alcohol, is seen as an unwanted side reaction, for which He et al., Applied and Environmental Microbiology, 2004, 70, 1974-1881, on page 1880 right column end of third last paragraph, suggest expressly the possibility of exploitation of biochemical engineering approaches with the recombinant organism in order to diminish this unwanted side reaction. Venkitasubramanian et al., Enzyme and Microbial Technology, 2008, 42, 130-137, disclose on page 134 end of first paragraph in left column, that the mass balance for the reduction by whole, resting cells of respective E. coli constructs of vanillic acid to vanillin by exogenous CAR and subsequently to vanillyl alcohol by an endogenous E. coli ADH was essentially 100 %, as it was for the reduction of benzoic acid to benzyl alcohol, but ferulic acid was never completely reduced with mixtures of the starting acid, coniferyl aldehyde and coniferyl alcohol obtained at all times. This suggests that the carboxy residue should be directly connected with the phenyl ring, and not via intermediate carbon atoms. Further it is assumed on page 136 in the right column middle of first paragraph that the rate of reduction of vanillic acid by holo-Car is much faster than the rate of reduction of the aldehyde vanillin by endogenous ADH.
The skilled person, in an attempt to find a suitable biotransformation system for DOPAC to 3-HT, would be dissuaded by the data presented in the prior art to use the disclosed CAR of Nocardia sp. in E. coli for this task: firstly, one would expect to have a low conversion rate in the first reduction step due to the substitution pattern of DOPAC, secondly, one would expect to end with a product mixture of the substrate, the acid, the intermediate, the aldehyde, and the desired product, the alcohol. Also the conversion rate of the endogenous ADH of E. coli was assumed to be lower relative to the conversion rate of CAR for the reduction of vanillic acid to vanillin.
Unexpectedly, against this teaching of prior art, a biotransformation system for the reduction of DOPAC to 3-HT based on a CAR of the enzyme class EC 1.2.1.30 was found, that is not only able to reduced DOPAC to 3-HT, but in all experiments it was not even possible to detect the intermediate aldehyde. This high efficiency not only for the first reduction step from the carboxylate to the aldehyde, but also for the second reduction step from the aldehyde to the alcohol, was unexpected especially in view of the disclosure of the prior art.
DETAILED DESCRIPTION OF THE INVENTION
Abbreviations AADH aryl-aldehyde dehydrogenase (NADP+), EC 1.2.1.30
ACPS holo-[acyl-carrier-protein] synthase, EC 2.7.8.7
acpS gene coding for CgPPTase
ADH alcohol dehydrogenase
BLAST Basic Local Alignment Search Tool of NCBI,
http://blast.ncbi.nlm.nih.gov/Blast.cgi
BRENDA database compiling enzymes and enzyme classes, accessible under
http://www.brenda-enzymes.org/, published by Department of Biomformatics and Biochemistry, Institute for Biochemistry and Biotechnology Technische Universitat Braunschweig, Langer Kamp 19B, 38106 Braunschweig, Germany
CAR carboxylic acid reductase
car gene coding for CAR
CgPPTase 4'-phosphopantetheinyl transferase of Corynebacterium glutamicum
DNA2.0 DNA2.0, Inc., Menlo Park, USA, www.dna20.com
DOPAC 3,4-dihydroxyphenylacetic acid
DTT 1,4-Dithiothreitol
EDTA Ethylene diamine tetra acetic acid
E. coli Escherichia coli
EcPPTase 4'-phosphopantetheinyl transferase of E. coli
EMBL European Molecular Biology Laboratory, Heidelberg, Germany,
http://www.embl.de/
EMBL-EBI European Molecular Biology Laboratory - European Biomformatics Institute,
Hinxton, UK, http://www.ebi.ac.uk/
entD gene coding for EcPPTase
FDH formate dehydrogenase
fdh gene coding for fdh
GDH glucose 1 -dehydrogenase (DSM Innovative Synthesis BV, Geleen, The
Netherlands, www.dsm.com)
gdh gene coding for GDH
3-HT 3-hydroxytyrosol
IUBMB International Union of Biochemistry and Molecular Biology,
http ://www.iubmb. org/
NCBI National Center for Biotechnology Information, Bethesda, USA,
http://www.ncbi.nlm.nih.gov/ NC-IUBMB Nomenclature Committee of the International Union of Biochemistry and
Molecular Biology, http://www.chem.qmul.ac.uk/iupac/jcbn/
Novagene® Merck Milipore, Merck KGaA, Darmstadt, Deutschland,
www.merckmillipore . de
NPT Nocardia sp. phosphopantetheinyl transferase
npt gene coding for NPT
OD Optical Density
PPTase 4'-phosphopantetheinyl transferase
U Unit is abbreviated with "U" for the purpose of this invention, if not otherwise stated
AADH and CAR are used synonymously in this specification for an enzyme of the enzyme class EC 1.2.1.30.
ACPS and PPTase are used synonymously in this specification for an enzyme of the enzyme class EC 2.7.8.7.
The enzyme class EC is defined by the NC-IUBMB, which is accessible in the internet via http://www.chem.qmul.ac.uk/iubmb/enzyme/, and is also compiled e.g. in the database BRENDA.
The terms "alignment, "conserved substitution", "identity", "homology" and "similarity" are used as defined in BLAST:
alignment The process or result of matching up the nucleotide or amino acid residues of two or more biological sequences to achieve maximal levels of identity and, in the case of amino acid sequences, conservation, for the purpose of assessing the degree of similarity and the possibility of homology.
conserved substitution A change at a specific position of an amino acid or, less commonly, DNA sequence that preserves the physico-chemical properties of the original residue or achieves a positive score in the governing scoring matrix.
identity or sequence identity The extent to which two (nucleotide or amino acid)
sequences have the same residues at the same positions in an alignment, often expressed as a percentage identity of the amino acid residues, based on the total number of amino acid residues of the amino acid sequence. If the two sequences, which are being compared, have a different number of amino acid residues, the percentage is based on the number of the shorter amino acid sequence.
homology Similarity attributed to descent from a common ancestor. Homologous
biological components (genes, proteins, structures) are called homologs. Often the term homolog is used interchangeably with the term variant, meaning rather a certain degree of identity between two sequences without implying a common ancestor. Therefore within the meaning of the invention, the term variant of a sequence means a sequence with a certain degree of identity to the sequence, if not otherwise stated.
similarity The extent to which nucleotide or protein sequences are related. Similarity between two sequences can be expressed as percent sequence identity and/or percent positive substitutions. Unless otherwise stated, sequence identity, similarity and positives values provided herein refer to the value obtained using the BLAST suite of programs using default parameters, a description is found in Altschul et al., J. Mol. Biol., 1990, 215, 403-410 and in "The NCBI Handbook" (Internet), editors J. McEntyre and J. Ostell, Bethesda (MD): National Center for Biotechnology Information (US); 2002-, and using the program version BLAST 2.2.27+, if not otherwise stated.
The term "sequence" can mean a nucleotide sequence, which constitutes a polynucleotide, or an amino acid sequence, which constitutes a peptide. Nucleotide sequences are found e.g. under the respective accession no. in the EMBL Nucleotide Sequence Database of the EMBL- EBI, or in the Nucleotide database of the NCBI; and amino acid sequences are found e.g. under the respective accession no. in the UniProt database of the EMBL-EBI, or in the Protein database of the NCBI; if not otherwise stated.
"Heterologous nucleic acid sequence" or "nucleic acid sequence heterologous to a host" means a nucleic acid sequence which encodes e. g. an expression product such as a polypeptide that is foreign to the host ("heterologous expression" or "heterologous product") e.g. a nucleic acid sequence originating from a donor different from the host or a chemically synthesized nucleic acid sequence which encodes e. g. an expression product such as a polypeptide that is foreign to the host. In a similar way, "heterologous protein" means a protein that is foreign to the host.
"Foreign to the host" can mean, that the nucleic acid or protein originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition or genomic locus by deliberate human intervention.
A heterologous nucleic acid sequence as referred herein encompasses also nucleic acids, which are codon optimized for the host according to the codon usage of the host.
Exogenous DNA refers to any deoxyribonucleic acid that originates outside of the organism of concern or study.
The terms "host", "host cell" and "recombinant host cell" are used interchangeably herein to indicate a cell, which contains or shall contain a vector or an isolated nucleic acid sequence, and supports the replication and/or expression of the vector or of the nucleic acid. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, plant, amphibian, or mammalian cells.
A "vector", "vector expressible in a host" or "expression vector" are used interchangeably and is a polynucleic acid construct that is used in transfection or transformation of a host cell and into which a polynucleotide can be inserted, it is generated recombinantly or synthetically, with a series of specified polynucleic acid elements that permit transcription of a particular nucleic acid sequence in a host cell. Typically, this vector includes a transcriptional unit comprising a particular nucleic acid sequence to be transcribed operably linked to a promoter. Vectors are often replicons. A vector expressible in a host can be e. g. an autonomously or self-replicating plasmid, a cosmid, a phage, a virus or a retrovirus.
The term "introduced" in the context of inserting a nucleic acid into a cell, means
"transfection" or "transformation" or "transduction" and includes reference to the
incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
In molecular biology, the process by which cells take up exogenous DNA from the outside is called "transformation". Bacteria need to be in a certain physiological state to successfully take up exogenous DNA, which is described as one of "competence". Some bacteria are naturally competent, but usually only for a brief time at a certain stage of their growth cycle. Bacteria can also be made competent through a variety of chemical treatments including exposure to calcium ions, or a mixture of polyethylene glycol and dimethylsulfoxide, which make the cell membrane more permeable, leading to the uptake of the exogenous DNA. Another treatment method is the utilization of electricity as the membrane-permeabilizing agent (electroporation or electro transformation). Finally, liposome-mediated transformation can be used. In this method, DNA is coated with lipid. Fusion of this lipid and the membrane lipid can occur, facilitating the entry of DNA.
"Transduction" is usually used for the process of injection of foreign DNA by a virus into the host bacterium.
"Transfection" is the process of deliberately introducing nucleic acids into cells. The term is often used for the introduction of foreign DNA into eukaryotic cells, notably for non- viral methods.
In microbiology, genetics, cell biology and molecular biology, "competence" is the ability of a cell to take up extracellular DNA from its environment.
Within the meaning of the invention and if not otherwise stated, the terms "transformation", "transformed" or "introducing a nucleic acid into a host cell" are used interchangeably and denote any process wherein an extracellular nucleic acid like a vector, with or without accompanying material, enters a host cell. The term "cell transformed" or "transformed cell" means the cell or its progeny into which the extracellular nucleic acid has been introduced and thus harbors the extracellular nucleic acid. The nucleic acid might be introduced into the cell so that the nucleic acid is replicable either as a chromosomal integrant or as an extra chromosomal element. Transformation of appropriate host cells with e. g. an expression vector can be accomplished by well-known methods such as microinjection, electroporation, particle bombardment or by chemical methods such as Calcium phosphate-mediated transformation, described e. g. in Maniatis et al. 1982, Molecular Cloning, A laboratory Manual, Cold Spring Harbor Laboratory, in Ausubel et al. 1994, Current protocols in molecular biology, John Wiley and Sons, or in "Molecular Cloning: A laboratory Manual, Third edition (3 volume set)", Cold Spring Harbor Laboratory Press, 2001.
"Nucleic acid", "nucleic acid sequence" or "polynucleotide" are used interchangeably and together with "isolated and purified nucleic acid or nucleic acid sequence or polynucleotide", as referred in the present invention, might be DNA, RNA, or DNA/RNA hybrid. Unless otherwise indicated, the terms include reference to the specified sequence as well as the complementary sequence thereof. In case the nucleic acid sequence is located on a vector it is usually DNA. DNA which is referred to herein can be any polydeoxynuclotide sequence, including, e.g. double-stranded DNA, single-stranded DNA, double-stranded DNA wherein one or both strands are composed of two or more fragments, double-stranded DNA wherein one or both strands have an uninterrupted phosphodiester backbone, DNA containing one or more single- stranded portion(s) and one or more double-stranded portion(s), double-stranded DNA wherein the DNA strands are fully complementary, double-stranded DNA wherein the DNA strands are only partially complementary, circular DNA, covalently- closed DNA, linear DNA, covalently cross-linked DNA, cDNA, chemically synthesized DNA, semi-synthetic DNA, biosynthetic DNA, naturally-isolated DNA, enzyme-digested DNA, sheared DNA, labeled DNA, such as radio labeled DNA and fluorochrome-labeled DNA, DNA containing one or more non-naturally occurring species of nucleic acid. DNA sequences can be synthesized by standard chemical techniques, for example, the phosphotriester method or via automated synthesis methods and PCR methods. The purified and isolated DNA sequence may also be produced by enzymatic techniques.
RNA which is referred to herein can be e.g. single-stranded RNA, cRNA, double- stranded RNA, double stranded RNA wherein one or both strands are composed of two or more fragments, double-stranded RNA wherein one or both strands have an uninterrupted phosphodiester backbone, RNA containing one or more single-stranded portion(s) and one or more double-stranded portion(s), double-stranded RNA wherein the RNA strands are fully complementary, double-stranded RNA wherein the RNA strands are only partially complementary, covalently cross-linked RNA, enzyme digested RNA, sheared RNA, mRNA, chemically-synthesized RNA, semi-synthetic RNA, biosynthetic RNA, naturally isolated RNA, labeled RNA, such as radiolabeled RNA and fluorochrome-labeled RNA, RNA containing one or more non-naturally occurring species of nucleic acid. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. With "variants" or "variants of a sequence" is meant a nucleic acid sequence that vary from the reference sequence by nucleic acid substitutions, preferably by conservative nucleic acid substitution, whereby one or more nucleic acids are substituted by another with
same characteristics. Variants encompass as well degenerated sequences, sequences with deletions and insertions, as long as such modified sequences exhibit the same function (functionally equivalent) as the reference sequence.
The terms "polypeptide", "peptide", "protein", "polypeptidic" and "peptidic" are used interchangeably herein to refer to a polymer of amino acid residues connected to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
The term "isolated and purified nucleic acid sequence" refers to the state in which the nucleic acid sequence will be free or substantially free of material with which they are naturally associated such as other nucleic acids with which they are found in their natural environment, or the environment in which they are prepared (e. g. cell culture) when such preparation is by recombinant technology practiced in vitro or in vivo. The "origin of replication" (also called the replication origin) is a particular sequence in a genome at which replication is initiated. This can either involve the replication of DNA in living organisms such as prokaryotes and eukaryotes, or that of DNA or RNA in viruses, such as double-stranded RNA viruses. DNA replication may proceed from this point
bidirectionally or unidirectionally.
"Promoter" as used herein refers to a nucleic acid sequence that regulates expression of a transcriptional unit. A "promoter region" is a regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. Within the promoter region will be found a transcription initiation site (conveniently defined by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase such as the putative - 35 region and the Pribnow box. "Signal sequence" or "signal peptide sequence" refers to a nucleic acid sequence which encodes a short amino acid sequence (i.e., signal peptide) present at the NH2 -terminus of certain proteins that are normally exported by cells to non-cytoplasmic locations (e.g., secretion) or to be membrane components. Signal peptides direct the transport of proteins from the cytoplasm to non-cytoplasmic locations.
"Translation initiation region" is a signal region which promotes translation initiation and which functions as the ribosome binding site such as the Shine Dalgarno sequence.
"Terminator", "transcription terminator" and "transcription termination region" are used interchangeably and indicate a section of genetic sequence that marks the end of gene or operon on genomic DNA for transcription. It causes RNA polymerase to terminate transcription. The transcription termination region is usually part of a transcriptional unit and increases the stability of the mRNA. "Transcriptional unit" as used herein refers to a nucleic acid sequence that is normally transcribed into a single RNA molecule. The transcriptional unit might contain one gene (monocistronic) or two (dicistronic) or more genes (polycistronic) that code for functionally related polypeptide molecules. A nucleic acid sequence is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a signal sequence is operably linked to DNA for a protein if it is expressed as a preprotein that participates in the secretion of the protein; a promoter is operably linked to a coding sequence if it affects the transcription of the sequence; or a translation initiation region such as a ribosome binding site is operably linked to a nucleic acid sequence encoding e. g. a polypeptide if it is positioned so as to facilitate translation of the polypeptide. Linking can be accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. Subject of the invention is a method for the preparation of 3-hydroxytyrosol by a reaction, the reaction comprises an enzymatic reduction of 3,4-dihydroxyphenyl acetic acid, wherein the enzymatic reduction is done using two enzymes, a carboxylic acid reductase CAR and a 4'-phosphopantetheinyl transferase PPTase;
wherein
CAR comprises each of the three amino acid sequences SEQ ID NO: 1, SEQ ID NO: 3 and
SEQ ID NO: 5 with 70 to 100% identity;
PPTase comprises the sequence SEQ ID NO: 12 or comprises the sequence SEQ ID NO: 14, each with 70 to 100 % identity respectively.
List of Sequences
SEQ ID NO: 1 to 6 and 10 are derived from SEQ ID 7.
SEQ ID NO: 1 : position 90 to 535 of SEQ ID NO: 7
SEQ ID NO: 2: position 90 to 544 of SEQ ID NO: 7
SEQ ID NO: 3: position 661 to 693 of SEQ ID NO: 7
SEQ ID NO: 4: position 658 to 704 of SEQ ID NO: 7
SEQ ID NO: 10: position 655 to 730 of SEQ ID NO: 7
SEQ ID NO: 5: position 789 to 1080 of SEQ ID NO: 7
SEQ ID NO: 6: position 750 to 1094 of SEQ ID NO: 7
SEQ ID NO: 7: UniProt database of the EMBL-EBI, EMBL accession no. Q6RKB1, amino acid sequence of CAR from Nocardia sp. Strain NRRL 5646
SEQ ID NO: 8: EMBL Nucleotide Sequence Database of the EMBL-EBI, EMBL accession no. AY495697, nucleotide sequence of CAR gene of Nocardia iowensis, registered as "Nocardia sp. NRRL 5646 ATP/NADPH-dependent carboxylic acid reductase (car)" and translating in sequence of SEQ ID NO: 7
SEQ ID NO: 9: this is a SEQ ID NO: 8, which is codon optimized for E. coli SEQ ID NO: 11 : Nucleotide database of the NCBI, Escherichia coli BL21 (DE3), complete genome, accession no. AM946981.2 region 569939 to 570568, nucleotide sequence of a EcPPTase gene in its complementary form
SEQ ID NO: 12: Protein database of the NCBI, phosphopantetheinyl transferase, subunit of enterobactin synthase multi enzyme complex [Escherichia coli BL21 (DE3)], accession no. CAQ31055.1, amino acid sequence of a EcPPTase
SEQ ID NO: 13: Nucleotide database of the NCBI, Corynebacterium glutamicum ATCC 13032, NC 003450, region 2634745 to 2635152, nucleotide sequence of a CgPPTase gene in its complementary form
SEQ ID NO: 14: Protein database of the NCBI, 4'-phosphopantetheinyl transferase
[Corynebacterium glutamicum ATCC 13032], NCBI Reference Sequence: NP 601692, amino acid sequence of a CgPPTase
SEQ ID NO: 15: Gene sequence of plasmid pJexpress-404-9-CAR
SEQ ID NO: 16: Gene sequence of plasmid pJexpress-401-11 -EcPPTase
Primers:
SEQ ID NO: 17: Primer 1
SEQ ID NO: 18: Primer 2
SEQ ID NO: 19: Primer 3
SEQ ID NO: 20: Primer 4
SEQ ID NO: 21 : Primer 5
SEQ ID NO: 22: Primer 6
SEQ ID NO: 23: Primer 7
SEQ ID NO: 24: Primer 8
SEQ ID NO: 1 and SEQ ID NO: 2 represent the adenylating domain of CAR, where AMP is bound.
SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 10 represent the phosphopantetheinyl binding site of CAR. SEQ ID NO: 5 and SEQ ID NO: 6 represent the thioester reductase domain of CAR, where NADPH is bound.
CAR is classified in the literature to be an enzyme that catalyzes the reaction, which enzymes of the enzyme class EC 1.2.99.6 or of the enzyme class EC 1.2.1.30 catalyze.
According to IUBMB Enzyme Nomenclature and for the purpose of this invention, enzyme class EC 1.2.1.30 is assigned to enzymes, that have the accepted name "aryl-aldehyde dehydrogenase (NADP+)".
They catalyze the reaction:
"an aromatic aldehyde + NADP+ + AMP + diphosphate + H20 =
an aromatic acid + NADPH + H+ + ATP"
Other names are "aromatic acid reductase" and "aryl-aldehyde dehydrogenase (NADP)". Systematic name is "aryl-aldehyde: NADP+ oxidoreductase (ATP-forming)". The CAS registry number is 9074-94-6.
According to IUBMB Enzyme Nomenclature and for the purpose of this invention, enzyme class EC 1.2.99.6 is assigned to enzymes, that have the accepted name: carboxylate reductase. They catalyze the reaction:
"an aldehyde + acceptor + H20 = a carboxylate + reduced acceptor" Other name is "aldehyde: (acceptor) oxidoreductase".
Systematic name is "aldehyde: acceptor oxidoreductase".
IUBMB enzyme Nomenclature gives a comments: "A tungsten protein. Methyl viologen can act as acceptor. In the reverse direction, non-activated acids are reduced by reduced viologens to aldehydes, but not to the corresponding alcohols." The CAS registry number ir 125008-36-
PPTase catalyzes the reaction, which enzymes of the enzyme class EC 2.7.8.7. catalyze. According to IUBMB Enzyme Nomenclature and for the purpose of this invention, enzyme class EC 2.7.8.7 is assigned to enzymes, that have the accepted name "holo-[acyl-carrier- protein] synthase".
They catalyze the reaction:
"CoA-[4'-phosphopantetheine] + apo-[acyl-carrier protein] = adenosine 3',5'-bisphosphate + holo-[acyl-carrier protein]" Other names are acyl carrier protein holo protein (holo-ACP) synthetase; holo-ACP synthetase; coenzyme A: fatty acid synthetase apo enzyme 4'-phosphopantetheine transferase; holo synthase; acyl carrier protein synthetase; holo-ACP synthase; PPTase; AcpS; ACPS; acyl carrier protein synthase; P-pant transferase; CoA: apo-[acyl-carrier-protein]
pantetheinephosphotransferase.
Systematic name is "CoA-[4'-phosphopantetheine] :apo-[acyl-carrier protein] 4'- pantetheinephosphotransferase". The CAS registry number is 37278-30-1.
CAR and PPTase are polypeptides. The scope of CAR and of PPTase within the meaning of the invention comprises "functional equivalents". Within the context of the present invention, functional equivalents of CAR and of PPTase are polypeptides which differ from CAR and of PPTase but which still possess the desired biological activity such as substrate specificity, for example. Thus, "functional equivalents" mean, for example, enzymes which reduce DOPAC to 3-HT and which have at least 50%, preferably at least 60%, more preferably at least 75%, even more preferably at least 90%, of the activity of an enzyme having the amino acid sequence listed in SEQ ID NO 7 or 12 respectively. Functional equivalents are also preferably stable between pH 4 to 10. They preferably possess a pH optimum between pH 5 and 8. They preferably possess a temperature optimum in the range from 20° C to 80° C. Functional equivalents thus comprise e.g. the mutants obtainable by one or more amino acid additions, amino acid substitutions, amino acid deletions and/or or amino acid inversions, it being possible for said alterations to occur in any sequence position, as long as they result in a mutant having the property profile of the invention. Functional equivalence also exists, in particular, when the reactivity patterns of the mutant and the unaltered enzyme with the amino acid sequence SEQ ID NO: 7 or 12 respectively, agree qualitatively, i.e. DOPAC is reduced to 3-HT at different rates.
Functional equivalents in the above sense are also "precursors" of the polypeptides described and also "functional derivatives" and "salts" of said polypeptides.
In this context, "precursors" are natural or synthetic precursors of the polypeptides with the desired reduction activity.
The expression "salts" means both salts of carboxyl groups and/or of amino groups of the polypeptides of the invention. Salts of carboxyl groups can be prepared in a manner known per se and comprise inorganic salts such as sodium, calcium, ammonium, iron and zinc salts as well as salts with organic bases, for example amines, such as triethanolamine, arginine, lysine, piperidine and the like. Salts of amino groups are for example salts with mineral acids such as hydrochloric acid or sulfuric acid and salts with organic acids such as acetic acid and oxalic acid.
"Functional derivatives" of polypeptides of the invention are covalently modified polypeptides of the invention, and may be prepared with the aid of known techniques e.g. by modification of functional side groups of the polypeptide such as amino side groups or hydroxy side groups, or by modification of the N-terminus or C-terminus. Modification is done preferably with protecting groups, which are commonly used in polypeptide chemistry and which are known to the skilled person. Functional derivatives of this kind comprise, for example, aliphatic esters of carboxylic acid groups, amides of carboxylic acid groups, which amides are obtainable by reacting with ammonia or with a primary or secondary amine; N- acyl derivatives of free amino groups, which derivatives are prepared by reacting the free amino groups with acyl groups; or O-acyl derivatives of free hydroxyl groups, which derivatives are prepared by reacting the free hydroxyl groups with acyl groups.
Functional equivalents naturally also comprise polypeptides which are available from other organisms than E. coli and also naturally occurring variants. For example, areas of homologous sequence regions can be established by sequence comparison and equivalent enzymes can be determined on the basis of the specific guidelines of the invention.
Functional equivalents are moreover fusion proteins which contain any of the
abovementioned polypeptide sequences or functional equivalents derived therefrom and at least one further heterologous sequence which is functionally different therefrom and is covalently linked, preferably N -terminally or C-terminally (i.e. without any substantial reciprocal functional impairment of the fusion protein moieties). Non limiting examples of such heterologous sequences are signal peptides or enzymes, for example.
"Functional equivalents" which are comprised in the invention are variants of the specifically disclosed sequences. Said variants comprise an amino acid sequence with 70 to 100% identity, preferably with 80 to 100% identity, more preferably with 90 to 100%) identity, even more preferably with 95 to 100%> identity, especially with 100%> identity, with the amino acid sequence with which it is compared.
Therefore within the meaning of the invention, the term variant of a sequence means preferably a sequence with a certain degree of identity to the sequence.
In the case of a possible protein glycosylation, functional equivalents comprise proteins of the above described type in deglycosylated or glycosylated form and also modified forms which can be obtained by altering the glycosylation pattern. Variants of the proteins or polypeptides of the invention may be generated by mutagenesis, for example by point mutation or truncation of the protein.
Variants of the proteins of the invention may be identified by screening combinatorial libraries of mutants such as truncation mutants. For example, a variegated library of protein variants may be generated by combinatorial mutagenesis at the nucleic acid level, for example by enzymatically ligating a mixture of synthetic oligonucleotides.
There are a large number of methods which may be used for preparing libraries of potential variants from a degenerate oligonucleotide sequence. A degenerate gene sequence maybe synthesized chemically in a DNA synthesizer and the synthetic gene may then be ligated into a suitable expression vector. Using a degenerate set of genes makes it possible to prepare all the sequences in a mixture which encode the desired set of potential protein sequences.
Processes for synthesizing degenerate oligonucleotides are known to the skilled person (e.g. Narang et al., Tetrahedron, 1983 39, 3-22; Itakura et al., Annu. Rev. Biochem., 1984, 53, 323- 356; Itakura et al., Science, 1984, 198, 1056-1063; Ike et al., Nucleic Acids Res., 1983, 11, 477-488).
A plurality of techniques for screening gene products of combinatorial libraries which have been prepared by point mutations or truncation and for screening cDNA libraries for gene products having a selected property are known in the prior art. These techniques can be adapted for rapidly screening the gene libraries which have been generated by combinatorial mutagenesis of variants of the invention. The most frequently employed techniques for screening large gene libraries which are subject to high-throughput analysis comprise cloning the gene library into replicable expression vectors, transforming the appropriate cells with the resulting vector library and expressing the combinatorial genes under conditions under which detection of the desired activity facilitates isolation of the vector which encodes the gene whose product was detected. Recursive ensemble mutagenesis (REM), a technique which increases the frequency of functional mutants in the libraries, may be used in combination with the screening tests in order to identify variants (Arkin et al., Proc. Acad. Natl. Sci., 1992, 89, 7811-7815; Delgrave et al., Protein Engineering, 1993, 6, 327-331 ; in this case the term homolog is used interchangeably with the term variant).
Preferably, CAR is of enzyme class EC 1.2.1.30;
Preferably, PPTase is of enzyme class EC 2.7.8.7.
In another preferred embodiment, PPTase is a EcPPTase or a CgPPTase;
more preferably, is a PPTase of E. coli. More preferably, CAR is of enzyme class EC 1.2.1.30 and PPTase is of enzyme class EC 2.7.8.7.
In another more preferred embodiment, CAR is of enzyme class EC 1.2.1.30 and PPTase is a EcPPTase or a CgPPTase; more preferably, is a PPTase of E. coli.
In an especial embodiment, CAR comprises each of the three amino acid sequences SEQ ID
NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5;
and PPTase comprises the sequence SEQ ID NO: 12 or comprises the sequence SEQ ID NO:
14;
each sequence is comprised with 80 to 100 % identity, preferably with 90 to 100 % identity, more preferably with 95 to 100 % identity, even more preferably with 100 % identity.
In a more especial embodiment, CAR comprises each of the three amino acid sequences SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6;
and PPTase comprises the sequence SEQ ID NO: 12 or comprises the sequence SEQ ID NO:
14;
each sequence is comprised with 70 to 100% identity, preferably with 80 to 100% identity, more preferably with 90 to 100%> identity, even more preferably with 95 to 100%> identity, especially with 100% identity.
In an even more especial embodiment, CAR comprises each of the three amino acid
sequences SEQ ID NO: 2, SEQ ID NO: 10 and SEQ ID NO: 6;
and PPTase comprises the sequence SEQ ID NO: 12 or comprises the sequence SEQ ID NO:
14;
each sequence is comprised with 70 to 100% identity, preferably with 80 to 100% identity, more preferably with 90 to 100%> identity, even more preferably with 95 to 100%> identity, especially with 100%> identity. In a particular embodiment, CAR comprises the CAR from Nocardia sp. Strain NRRL 5646; and PPTase comprises the sequence SEQ ID NO: 12 or comprises the sequence SEQ ID NO:
14;
the CAR from Nocardia sp. Strain NRRL 5646 and each of the sequences SEQ ID NO: 12 and SEQ ID NO: 14 is comprised with 70 to 100% identity, preferably with 80 to 100% identity, more preferably with 90 to 100% identity, even more preferably with 95 to 100% identity, especially with 100% identity.
In a more particular embodiment, CAR comprises the sequence SEQ ID NO: 7;
and PPTase comprises the sequence SEQ ID NO: 12 or comprises the sequence SEQ ID NO:
14;
each sequence is comprised with 70 to 100%) identity, preferably with 80 to 100%) identity, more preferably with 90 to 100%> identity, even more preferably with 95 to 100%> identity, especially with 100%> identity;
In an even more particular embodiment, CAR has the sequence SEQ ID NO: 7;
and PPTase has the sequence SEQ ID NO: 12 or has the sequence SEQ ID NO: 14;
each sequence with 70 to 100% identity, preferably with 80 to 100%) identity, more preferably with 90 to 100%) identity, even more preferably with 95 to 100% identity, especially with 100% identity.
In each of the above embodiments, the sequence SEQ ID NO: 12 is the preferred embodiment for PPTase.
Preferably, CAR and PPTase are used in the reaction
• in form of a cell free extract of a microorganism containing one of the enzymes or both, e.g. in form of a protein extract of a microorganism, that expressed one of the enzymes or both;
• in form of a microorganism, that expresses or expressed both enzymes, or in form of two mircoorganisms, one microorganism expressing the one enzyme and the other microorganism expressing the other enzyme; e.g. in form of a cell suspension of such microorganism;
• in form of parts of a microorganism, that expressed one of the enzymes or both, e.g. in form of cell debris of such microorganism; or
• as a combination of these embodiments.
The cell free extract containing the enzyme or both enzymes can be e.g. in form of a powdered protein composition, in form of a solution or suspension containing the enzyme or in form of a purified enzyme. It is possible to use for the process of the invention growing cells. It is also possible to use resting or disrupted cells. Disrupted cells mean, for example, cells which have been made permeable by way of treatment with solvents for example, or cells which have been broken up by way of treatment with enzymes, by way of mechanical treatment (e.g. French press or ultrasonication) or by way of another method. The crude extracts obtained in this manner are advantageously suitable for the process of the invention. It is also possible to use purified or partially purified enzymes for the process. Immobilized microorganisms or enzymes which may advantageously be applied in the reaction are likewise suitable.
The microorganism, that expresses or expressed one of the enzymes or both, is also called host organism or host cell.
More preferably, CAR and PPTase are used in the reaction in form of a microorganism, that expresses both enzymes, or in form of two mircoorganisms, one microorganism expressing the one enzyme and the other microorganism expressing the other enzyme, or in form of a cell free extract containing the enzymes;
even more preferably in form of a microorganism, that expresses both enzymes, or in form of two mircoorganisms, one mircroorganism expressing the one enzyme and the other mircroorganism expressing the other enzyme.
It was also unexpected that the two enzymes CAR and PPTase can be maintained stably in one microorganism in form of two plasmids each carrying the gene of one of the eynzymes in spite of the fact that two identical origins of replications are present in this case.
It is possible to prepare recombinant microorganisms which are, for example, transformed with a vector carrying a gene or genes encoding for one of the enzymes or for both, and which are expressing one the enzymes or both. In this connection, familiar cloning and transfection methods known to the skilled worker, such as, for example, coprecipitation, protoplast fusion, electroporation, retroviral transfection and the like, are preferably used in order to cause said nucleic acids to be expressed in the particular expression system. Suitable systems are described, for example, in Current Protocols in Molecular Biology, F. Ausubel et al., Eds., Wiley Interscience, New York 1997, or Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd edition, Cold Spring 10 Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, or in "Molecular Cloning: A laboratory Manual, Third edition (3 volume set)", Cold Spring Harbor Laboratory Press, 2001.
A suitable microorganism used as host is in principle any prokaryotic or eukaryotic organism. Advantageously, microorganisms such as bacteria, fungi or yeasts are used as host organisms.
Preferred eukaryotic microorganisms are Saccharomyces or Pichia, more preferably
Saccharomyces cerevisiae or Pichia pastoris. Preferred prokaryotic microorganisms used as hosts are gram-positive or gram-negative bacteria, preferably selected from the group of families consisting of Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Streptomycetaceae and Nocardiaceae, more preferably selected from the group consisting of Escherichia, Bacillus, Lactobacillus, Pseudomonas, Streptomyces, Nocardia, Burkholderia, Salmonella, Agrobacterium and Rhodococcus; even more preferably the microorganism is Escherichia coli. In addition, further advantageous bacteria can be found in the group of the alpha-proteobacteria, beta-proteobacteria or gamma- proteobacteria.
E. coli is used preferably in form of the strains selected from the group consisting of BL21, TGI, W311 0, DH1, XL 1-Blue and Origami, which are commercially available or can be obtained via the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany).
Preferably, ECOLI-1 is E. coli BL21, more preferably E. coli BL21 (DE3). The organisms used in the process of the invention are, depending on the host organism, grown or cultured in a manner known to the skilled worker. Microorganisms are usually grown in a liquid medium which comprises a carbon source, usually in the form of sugars, a nitrogen source, usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as iron salts, manganese salts, magnesium salts and, if appropriate, vitamins, at temperatures of between 0° C. and 100° C, preferably between 10° C and 60 °C, while being aerated. In this connection, the pH of the nutrient liquid may kept at a fixed value, i.e. it is not necessary but preferable to be regulated during cultivation. The cultivation may be carried out batch wise, semibatchwise or continuously. Nutrients may be introduced at the beginning of the fermentation or be fed in subsequently in a semicontinuous or continuous manner.
DOPAC may be added directly to the culture or, advantageously, after cultivation. The enzymes can be used according to the methods described in the examples or can be used for the reaction as a crude extract.
As cell culture system continuous or discontinuous culture such as batch culture or fed batch culture can be applied in culture tubes, shake flasks or bacterial fermenters. Host cells are usually cultured in conventional media as known in the art such as complex media like LB broth, "nutrient yeast broth medium", minimal media or a glycerol containing medium as described by Kortz et al., J. Biotechnol., 1995, 39, 59-65 , or a mineral salt media as described by Kulla et al., Arch. Microbiol., 1983, 135, 1-7.
LB broth and LB medium are used interchangeable. Glucose can be added to the medium for cultivation.
Methods for induction, protein expression and harvesting of cells are known to the skilled person, and are described for example in "Molecular Cloning: A laboratory Manual, Third edition (3 volume set)", Cold Spring Harbor Laboratory Press, 2001.
Preferably, induction is done using IPTG.
The vector used in the invention is preferably an autonomously or self-replicating plasmid, a cosmid, a phage, a virus or a retrovirus. A wide variety of host/vector combinations may be employed in expressing the nucleic acid sequences encoding CAR and/or PPTase
respectively. Useful expression vectors, for example, may consist of segments of
chromosomal, non-chromosomal and/or synthetic nucleic acid sequences. Suitable vectors include vectors with specific host range such as vectors specific for e. g. E. coli as well as vectors with broad-host-range such as vectors useful for Gram-negative bacteria. "Low-copy", "medium-copy" as well as "high copy" plasmids can be used.
Useful vectors for e.g. expression in E. coli are: pQE70, pQE60 und pQE-9 (QIAGEN, Inc.); pBluescriptVectors, PhagescriptVectors, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene Cloning Systems, Inc.); ptrc99a, pKK223-3, pKK233-3, pOR540, pRIT5 (Pharmacia Biotech, Inc.); pLG338, pACYC184, pBR322, pUC18, pUC19,pKC30, pRep4, pACYC177, pACYC184, pRSFlOlO and pBW22 (Wilms et al., Biotechnology and Bioengineering, 2001, 73, 95-103) or derivatives thereof such as plasmid pBW22-Fab-H or plasmid pAKL 14, pET Vectors (Novagene®) and pJexpress vectors (DNA2.0). Further useful plasmids are well known to the person skilled in the art and are described e.g. in "Cloning Vectors", Eds.
Pouwels P. H. et al. Elsevier, Amsterdam-New York-Oxford, 1985.
Preferred vectors to be used in the method of the present inventions are autonomously or self- replicating plasmids, more preferred are vectors with specific host range such as vectors specific for e. g. E. coli.
Preferably, pJexpress vectors are used. Preferably, when one of the enzymes CAR and PPTase or both are used as in form of a microorganism, that expresses both enzymes, or in form of two mircoorganisms, one mircroorganism expressing the one enzyme and the other mircroorganism expressing the other enzyme, the respective microorganism is used in the reaction in form of its cells with an optical density of from 0.1 to 300, more preferably from 0.5 to 300, even more preferably from 1 to 300, especially from 5 to 300, more especially from 5 to 250, even more especially from 10 to 250, in particular from 10 to 200, more in particular from 50 to 200.
In another preferred embodiment, the amount of each of the enzymes CAR and PPTase in the reaction is from 0.001 to 100 mg/mL, more preferably from 0.005 to 100 mg/mL, even more preferably from 0.01 to 100 mg/mL, especially from 0.05 to 100 mg/mL, more especially from 0.1 to 30 mg/mL, even more especially from 0.5 to 30 mg/mL;
more preferably, the amount of each of the enzymes CAR and PPTase in the reaction, when used as in form of an isolated enzyme, is from 0.001 to 100 mg/mL, more preferably from 0.005 to 100 mg/mL, even more preferably from 0.01 to 100 mg/mL, especially from 0.05 to 100 mg/mL, more especially from 0.1 to 30 mg/mL, even more especially from 0.5 to 30 mg/mL.
In another preferred embodiment, the molar ratio of CAR : DOPAC in the reaction is from 1 50000 to 1 : 1 ; preferably from 1 : 10000 to 1 : 5, more preferably from 1 : 5000 to 1 : 2'
Preferably, the molar ratio of PPTase : CAR in the reaction is from 1 : 1000 to 1 : 1 , more preferably from 1 : 500 to 1 : 2. Preferably, DOPAC is used in the reaction in a concentration of from 0.1 to 100 mM, more preferably of from 1 to 100 mM, even more preferably of from 1 to 50 mM, especially of from 5 to 50 mM. The reduction of DOPAC to 3-HT proceeds chemically in two steps: in the first reduction step the DOPAC is reduced to the corresponding aldehyde, the compound of formula (1),
Figure imgf000025_0001
in the second reduction step compound of formula (1) is reduced to 3-HT.
Preferably an enzyme SECREDSTEP is present in the reaction in addition to the two enzymes CAR and PPTase; enzyme SECREDSTEP is an enzyme which catalyzes the reduction of compound of formual (1) to 3-HT.
Enzyme SECREDSTEP can also be named in the literature as being an alcohol
dehydrogenase, a aldo-keto reductase, a keto reductase, an aldehyde reductase or aldehyde hydrogenase.
Enzyme SECREDSTEP is preferably classified in the literature to be an enzyme that catalyzes the reaction, which enzymes of the enzyme class EC 1.1.1 catalyze.
According to IUBMB Enzyme Nomenclature and for the purpose of this invention, enzyme class EC 1.1.1 is assigned to enzymes called "EC 1 Oxidoreductases - EC 1.1 Acting on the CH-OH group of donors - EC 1.1.1 With NAD+ or NADP+ as acceptor".
More preferably, enzyme SECREDSTEP is an enzyme classified in the literature to be an enzyme that catalyzes the reaction, which enzymes of enzyme classes EC 1.1.1.1 or EC 1.1.1.2 catalyze.
According to IUBMB Enzyme Nomenclature and for the purpose of this invention, enzyme class EC 1.1.1.1 is assigned to enzymes, that have the accepted name "alcohol
dehydrogenase".
They catalyze the reaction:
"(1) a primary alcohol + NAD+ = an aldehyde + NADH + H+ (2) a secondary alcohol + NAD+ = a ketone + NADH + H+" Other names are "aldehyde reductase; ADH; alcohol dehydrogenase (NAD); aliphatic alcohol dehydrogenase; ethanol dehydrogenase; NAD-dependent alcohol dehydrogenase; NAD- specific aromatic alcohol dehydrogenase; NADH-alcohol dehydrogenase; NADH-aldehyde dehydrogenase; primary alcohol dehydrogenase; yeast alcohol dehydrogenase".
Systematic name is "alcohol:NAD+ oxidoreductase". The CAS registry number is 9031-72-5.
According to IUBMB Enzyme Nomenclature and for the purpose of this invention, enzyme class EC 1.1.1.2 is assigned to enzymes, that have the accepted name "alcohol dehydrogenase (NADP+)".
They catalyze the reaction:
"an alcohol + NADP+ = an aldehyde + NADPH + H+"
Other names are "aldehyde reductase (NADPH2); NADP-alcohol dehydrogenase; NADP- aldehyde reductase; NADP-dependent aldehyde reductase; NADPH-aldehyde reductase; NADPH-dependent aldehyde reductase; nonspecific succinic semialdehyde reductase; ALR 1 ;
\ow-Km aldehyde reductase; high-^m aldehyde reductase; alcohol dehydrogenase (NADP)".
Systematic name is " alcohol :NADP+ oxidoreductase". The CAS registry number is 9028-12-0.
It is possible to use enzyme SECREDSTEP in the reaction
· as an isolated enzyme; or
• to express enzyme SECREDSTEP separately from CAR and PPTase into a different microorganism, which is present in the reaction; or
• to express enzyme SECREDSTEP together with one of the enzymes CAR or PPTase or with both enzymes CAR and PPTase in the same microorganism, even on the same plasmid in the same microorganism; or
• enzyme SECREDSTEP is an endogenous enzyme of the micororganism that expresses or expressed CAR or PPTase or both.
Preferably, enzyme SECREDSTEP is an endogenous enzyme of the micororganism that expresses or expressed CAR or PPTase or both.
Preferably, when enzyme SECREDSTEP is used in the reaction in the form of a
microorganism, that expresses enzyme SECREDSTEP, the respective microorganism is used in the reaction in the form of its cells with an optical density of from 0.1 to 300, more preferably from 0.5 to 300, even more preferably from 1 to 300, especially from 5 to 300, more especially from 5 to 250, even more especially from 10 to 250, in particular from 10 to 200, more in particular from 50 to 200. In another preferred embodiment, the amount of enzyme SECREDSTEP in the reaction is from 0.001 to 100 mg/mL, more preferably from 0.005 to 100 mg/mL, even more preferably from 0.01 to 100 mg/mL, especially from 0.05 to 100 mg/mL, more escpecially from 0.1 to 30 mg/mL, even more especially from 0.5 to 30 mg/mL;
more preferably, the amount of enzyme SECREDSTEP in the reaction, when used as in form of an isolated enzyme, is from 0.001 to 100 mg/mL, more preferably from 0.005 to 100 mg/mL, even more preferably from 0.01 to 100 mg/mL, especially from 0.05 to 100 mg/mL, more especially from 0.1 to 30 mg/mL, even more especially from 0.5 to 30 mg/mL. In another preferred embodiment, the molar ratio of enzyme SECREDSTEP : DOPAC in the reaction is from 1 : 50000 to 1 : 1; preferably from 1 : 10000 to 1 : 5.
Preferably, the reaction is done in an aqueous reaction medium.
Preferably, the reaction medium is a medium conventionally used for a cell culture.
More preferably, the reaction is done in the presence of a buffer (REAC).
Buffer (REAC) is a buffer commonly used in enzyme catalyzed reactions.
Preferably, buffer (REAC) is selected from the group consisting of phosphate buffer, HEPES (a buffer based on 4-2-hydroxyethyl-l-piperazineethanesulfonic acid), TES (a buffer based on 2- {[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid), MOPS (a buffer based on 3-(N-morpholino)propanesulfonic acid), PIPES (a buffer based on piperazine-
N,N'-bis(2-ethanesulfonic acid), MES (a buffer based on 2-(N-morpholino)ethanesulfonic acid) and mixtures thereof;
more preferably, buffer (REAC) is phosphate buffer, MES or mixtures thereof.
A phosphate buffer is preferably a sodium or potassium phosphate buffer.
Even more, buffer (REAC) is sodium or potassium phosphate buffer or MES;
especially, buffer (REAC) is MES.
Preferably, the concentration of buffer (REAC) in the reaction mixture is from 0.005 to 0.5 M, more preferably from 0.02 to 0.2 M. Even more preferably, the enzymes are used in the reaction in form of a microorganism, that expresses both enzymes, or in form of two mircoorganisms, one mircroorganism expressing the one enzyme and the other mircroorganism expressing the other enzyme;
and the reaction medium is buffer (REAC).
Preferably, the reaction is started in the presence of buffer (REAC) with a pH of from 5 to 8.5, more preferably of from 5.5 to 8.5, even more preferably of from 5.5 to 8.0, especially of from 5.5 to 7.6.
In another preferred embodiment, the reaction is done in the presence of buffer (REAC) with at a pH of from 5 to 8.5, more preferably of from 5.5 to 8.5, even more preferably of from 5.5 to 8.0, especially of from 5.5 to 7.6. Preferably, the reaction is done in the presence of DTT. DTT is preferably added to the reaction mixture.
The amount of DTT is preferably from 0.01 to 20 mM, more preferably from 0.1 to 10 mM, even more preferably from 0.4 to 10 mM. Preferably, the reaction is done in the presence of EDTA. EDTA is preferably added to the reaction mixture.
The amount of EDTA is preferably from 0.01 to 10 mM, more preferably from 0.1 to 5 mM, even more preferably from 0.4 to 5 mM.
Especially, the reaction is started in the presence of buffer (REAC) with a pH of from 5 to 8.5, and
the reaction is done in the presence of DTT, the amount of DTT is from 0.01 to 20 mM, and
the reaction is done in the presence of EDTA, the amount of EDTA is from 0.01 to 10 mM;
more especially, the pH is from 5.5 to 8.5,
the amount of DTT is from 0.1 to 10 mM, and
the amount of EDTA is from 0.1 to 5 mM; even more especially, the pH is from 5.5 to 8.0,
the amount of DTT is from 0.4 to 10 mM, and
the amount of EDTA is from 0.4 to 5 mM;
in particular, the pH is from 5.5 to 7.6,
the amount of DTT is from 0.4 to 10 mM, and
the amount of EDTA is from 0.4 to 5 mM.
Preferably, the reaction is done at a reaction temperature of from 4 to 45 °C, more preferably of from 10 to 45 °C, even more preferably of from 15 to 40 °C, especially of from 19 to 39 °C, more especially of from 20 to 38 °C.
Preferably, the reaction time is from 10 min to 96 h, more preferably from 20 min to 48 h.
Preferably, the reaction is done under aerobic conditions.
Preferably, the reaction medium is stirred, either by shaking the reaction tube or by a stirrer. Preferably in case of shaking a small reaction vessel such as an eppendorf reaction tube, shaking is done at 500 to 1500 rpm, preferably at 750 to 1250 rpm.
Preferably in case of the use of a stirrer, stirring is done at 50 to 500 rpm.
Preferably, the reaction is done in the presence of a cofactor, the cofactor is selected from the group consisting of NADPH and NADH.
As cofactor either endogenous cofactor is used or the cofactor is added to the reaction mixture or both.
Preferably, when the cofactor is added, the cofactor is added to provide for a concentration of from 0.05 to 500 mM, more preferably of from 0.05 to 100 mM, even more preferably of from 0.1 to 50 mM.
In another preferred embodiment, when the cofactor is added, the cofactor is added to provide for a 2 to 10 fold molar amount based on the molar amount of DOPAC.
Preferably, endogenous cofactor is used.
The cofactor can be regenerated. For this purpose, preferably the reaction is (i) done in the presence of an enzyme ENZREGENCOF, that regenerates the cofactor, and a respective substrate REGENSUB, that is used by ENZREGENCOF to regenerate the cofactor; or
(ii) the reaction is done using electrical current via redox electrodes for the regeneration of the cofactor; or
(iii) the reaction is done in the presence of a redox mediator for the regeneration of the
cofactor; or
(iv) done in the presence of a substrate (SUBSCIT), substrate (SUBSCIT) is a substrate of the citric acid cycle or glucose.
More preferably, the reaction is
(i) done in the presence of an enzyme ENZREGENCOF, that regenerates the cofactor, and a respective substrate REGENSUB, that is used by ENZREGENCOF to regenerate the cofactor; or
(iv) done in the presence of a substrate (SUBSCIT).
The citric acid cycle is also known as tricarboxylic acid cycle (TCA cycle).
Preferably ENZREGENCOF is selected from the group consisting of glucose 1 - dehydrogenase (GDH), formate dehydrogenase (FDH), phosphonate dehydrogenase,
NAD(P) oxidase, alcohol dehydrogenase (ADH) and mixture thereof;
and REGENSUB is glucose, formate, phosphonate, hydrogen peroxide or an alcohol as
substrate for the respective ENZREGENCOF;
more preferably, ENZREGENCOF is GDH with REGENSUB being glucose or
ENZREGENCOF is FDH with REGENSUB being formate or ENZREGENCOF is a mixture of GDH and FDH with REGENSUB being glucose and formate;
even more preferably, ENZREGENCOF is GDH with REGENSUB being glucose.
ENZREGENCOF and REGENSUB are commercially available, e.g. from Sigma Aldrich, or ENZREGENCOF can be cloned according to known procedures from literature.
A redox mediator is preferably a viologen or a mixture of viologens. Viologens are bipyridinium derivatives of 4,4'-bipyridyl, preferred viologens are selected from the consisting of methyl viologen with CAS 1910-42-5 and benzyl viologen with CAS 1102-19- 8.
A substrate (SUBSCIT) is preferably selected from the group consisting of glucose citrate, cis-aconitate, D-isocitrate, alpha-ketoglutarate, succinate, fumarate, malate, oxaloacetate and mixtures thereof; more preferably glucose, citrate and mixtures thereof.
Preferably, substrate (SUBSCIT) is present in a concentration of from 0.001 to 0.5 M, more preferably of from 0.005 to 0.1 M.
In another preferred embodiment, substrate (SUBSCIT) is present in a 1 to 3 fold molar amount based on the molar amount of DOPAC.
Preferably, substrate (SUBSCIT) is added to the reaction mixture.
An alcohol as substrate for ADH can be any substrate that is a known substrate for AHD, preferably an alcohol as substrate for ADH is selected from the group consisting of C2-8 alcohol; more preferably ethanol, propanol or butanol.
For the purpose of this invention, GDH is preferably an enzyme of the enzyme class EC 1.1.1.47.
According to IUBMB Enzyme Nomenclature, enzyme class EC 1.1.1.47 is assigned to enzymes that have the accepted name "glucose 1 -dehydrogenase".
They catalyze the reaction:
"beta-D-glucose + NAD(P)+ = D-glucono-l,5-lactone + NAD(P)H + H+"
Other names are "D-glucose dehydrogenase (NAD(P)); hexose phosphate dehydrogenase". Systematic name is "beta-D-glucose: NAD(P)+ 1-oxidoreductase". The CAS registry number is 9028-53-9.
For the purpose of this invention, FDH is preferably an enzyme of the enzyme class EC 1.2.1.2.
According to IUBMB Enzyme Nomenclature, enzyme class EC 1.2.1.2 is assigned to enzymes, that have the accepted name "formate dehydrogenase".
They catalyze the reaction:
"formate + NAD+ = C02 + NADH" Other names are "formate-NAD oxidoreductase; FDH I; FDH II; N-FDH; formic hydrogen- lyase; formate hydrogenlyase; hydrogenlyase; NAD-linked formate dehydrogenase; NAD- dependent formate dehydrogenase; formate dehydrogenase (NAD); NAD-formate dehydrogenase; formate benzyl-viologenoxidoreductase; formic acid dehydrogenase".
Systematic name is "formate: NAD+ oxidoreductase". The CAS registry number is 9028-85-7.
For the purpose of this invention, phosphonate dehydrogenase is preferably an enzyme of the enzyme class EC 1.20.1.1.
According to IUBMB Enzyme Nomenclature, enzyme class EC 1.20.1.1 is assigned to enzymes, that have the accepted name "phosphonate dehydrogenase".
They catalyze the reaction:
"phosphonate + NAD+ + H20 = phosphate + NADH + H+"
Other names are "NAD: phosphite oxidoreductase; phosphite dehydrogenase".
Systematic name is "phosphonate: NAD+ oxidoreductase". The CAS registry number is 9031- 35-0.
For the purpose of this invention, NAD(P) oxidase is preferably an enzyme of the enzyme class EC 1.6.3.1.
According to IUBMB Enzyme Nomenclature, enzyme class EC 1.6.3.1 is assigned to enzymes, that have the accepted name "NAD(P)H oxidase".
They catalyze the reaction:
"NAD(P)H + H+ + 02 = NAD(P)+ + H202"
Other names are "THOX2; ThOX; dual oxidase; pl38tox; thyroid NADPH oxidase; thyroid oxidase; thyroid oxidase 2; NADPH oxidase".
Systematic name is "NAD(P)H: oxygenoxidoreductase". The CAS registry number is 77106-92-4.
For the purpose of this invention, ADH is preferably an enzyme of the enzyme class EC 1.1.1.1 or of the enzyme class EC 1.1.1.2.
Preferably, the amount of ENZREGENCOF is from 0.001 to 100 mg/mL, more preferably from 0.005 to 100 mg/mL, even more preferably from 0.005 to 30 mg/mL, especially from 0.005 to 20 mg/mL; more especially from 0.005 to 15 mg/mL. In another preferred embodiment, the molar ratio of ENZREGENCOF : DOPAC is from 1 : 50000 to 1 : 1 ; preferably from 1 : 10000 to 1 : 1.
Since the enzymes CAR, PPTase, optionally enzyme ENZREGENCOF and optionally enzyme SECREDSTEP can be used in the reaction either expressed in one, in two, in three or even in four microorganisms, the total optical density of the combined microorganisms in the reaction is preferably of from 0.1 to 300, more preferably from 0.5 to 300, even more preferably from 1 to 300, especially from 5 to 300, more especially from 5 to 250, even more especially from 10 to 250, in particular from 10 to 200, more in particular from 50 to 200.
Preferably, the molar amount of REGENSUB is from 1 to 10 fold, more preferably from 1 to 6 fold, based on the molar amount of DOPAC.
It is possible to use ENZREGENCOF as isolated enzyme, or to clone ENZREGENCOF separately from CAR and PPTase into a different microorganism, which then is present in the reaction, or to clone ENZREGENCOF together with one of the enzymes CAR or PPTase or with both enzymes CAR and PPTase in the same microorganism, even on the same plasmid in the same microorganism. Preferably, the reaction is done in the presence of a kation KAT, KAT is selected from the group consisting of Mg2+, Zn2+ and mixtures thereof.
Preferably, KAT is present in form of a sulfate, hydrogen sulfate, chloride or acetate of KAT. Preferably, KAT is Mg2+;
more preferably, KAT is present in form of a sulfate, hydrogen sulfate, chloride or acetate of Mg2+ or as a mixture thereof;
even more preferably in form of MgS04 or MgC^;
especially in form of MgCl2.
Preferably, KAT as added to the reaction mixture.
Preferably, KAT is used in a concentration of from 0.01 to 50 mM, more preferably of from 0.1 to 30 mM, even more preferably 0.5 to 30 mM, especially 0.5 to 20 mM.
In particular, the reaction is started in the presence of buffer (REAC) with a pH of from 5 to 8.5, and the reaction is done in the presence of DTT, the amount of DTT is from 0.01 to 20 mM, and
the reaction is done in the presence of EDTA, the amount of EDTA is from 0.01 to 10 mM, and
KAT is used in a concentration of from 0.01 to 50 mM;
more in particular, the pH is from 5.5 to 8.5,
the amount of DTT is from 0.1 to 10 mM, and
the amount of EDTA is from 0.1 to 5 mM, and
KAT is used in a concentration of from 0.01 to 50 mM;
even more in particular, the pH is from 5.5 to 8.0,
the amount of DTT is from 0.4 to 10 mM, and
the amount of EDTA is from 0.4 to 5 mM, and
KAT is used in a concentration of from 0.1 to 30 mM;
specifically, the pH is from 5.5 to 7.6,
the amount of DTT is from 0.4 to 10 mM, and
the amount of EDTA is from 0.4 to 5 mM, and
KAT is used in a concentration of from 0.5 to 30 mM;
with KAT as defined above, also with any of its preferred embodiments. Preferably, if the reaction is done with resting cells, which are frozen and are thawed for the reaction, these cells should be frozen for a time period of up to one month. To have the cells for a longer period of time in the frozen state might reduce their activity. Generally, this was observed in the disclosed experiments. The reaction can be monitored, e.g. by HPLC, GC or by NMR.
After the reaction, 3-HT can be isolated from the reaction mixture by standard methods known to the skilled person. Preferably, the cells are separated by centrifugation or filtration. The product is preferably isolated by extraction with a solvent, preferably after centrifugation or filtration, from the mother liquor, and can be purified by flash column chromatography or crystallization or both. Preferably, extraction is done with a solvent EXTR selected from the group consisting of toluene, dichloromethane, cyclohexane, methylcyclohexane, ethyl acetate and methyl tert- butyl ether, preferably ethyl acetate. Flash column chromatography is preferably done using a column packed with silicagel G 60, the solvent system for flash chromatography is preferably methyl tert-butyl ether : n-heptane 1 :1 v/v.
Crystallization is preferably done from a solvent CRYST selected from the group consisting of petroleum : ether, 3.5 : 1 v/v; the ether is preferably methyl tert-butyl ether.
The foregoing description will be more fully understood with reference to the following Examples. Such examples, are, however, exemplary of methods of practicing the present invention and are not intended to limit the scope of the invention. Especially techniques of genetic engineering, such as cloning, transformation and sequencing, also sequence verification after cloning, can be carried out in accordance with procedures described in "Molecular Cloning: A laboratory Manual, Third edition (3 volume set)", Cold Spring Harbor Laboratory Press, 2001.
Examples
Materials, equipment, sources, further abbreviations
Amp Ampicillin, Sigma Aldrich, www.sigmaaldrich.com
ACN Acetonitrile, J. T. Baker, Aventor Performance Materials B.V., The
Netherlands.
ATP Adenosine-5'-triphosphate, Roche, Basel, Switzerland, www.roche.com
Bacto Yeast Extract Becton, Dickinson and Company, NJ USA 07417, www.bd.com Buffer A 50 mM Tris, 10 mM MgCl2, 1 mM EDTA, 1 mM DTT, 10% v/v glycerol, pH
7.5 adjusted with HC1
Buffer B 50 mM MES, 10 mM MgCl2, 1 mM EDTA, 1 mM DTT, 10% v/v glycerol, pH
6.0 adjusted with NaOH
Buffer C 50 mM MES, 10 mM MgCl2, 1 mM EDTA, 1 mM DTT, pH 6.0 adjusted with
NaOH
Buffer D 50 mM MES, pH 6.0 adjusted with NaOH
CD cell debris - debris of cells and possibly remaining unbroken cells which result from sonication and ultracentrifugation
Centrifuges: Small: (e.g. for Eppendorf tubes) Centrifuge 5415R (Eppendorf)
Medium: (e.g. for Falcon tubes) Centrifuge 581 OR (Eppendorf) Big: (e.g. for volumes of more than 50 mL) Avanti centrifuge J-20
(Beckman Coulter, www.beckmancoulter.com)
Ultracentrifuge: Optima LE80K (Beckman Coulter,
www.beckmancoulter.com)
Centrifugation parameter can also be specified with the g value (gravitational constant)
CFE cell free extract
Chemically competent cells E. coliBL21 (DE3) Gold Biocompare, South San Francisco,
CA 94080, USA, www.biocompare.com
CoA Coenzyme A trilithium salt, Sigma Aldrich, www.sigmaaldrich.com
DOPAC Sigma-Aldrich, www.sigmaaldrich.com
DTT Carl ROTH, 76185 Karlsruhe, Germany, www.carlroth.com
EDTA Carl ROTH,76185 Karlsruhe, Germany, www.carlroth.com
Electrocompetent cells E. coliBL21 (DE3) Gold Agilent Technologies, Inc., CA 95051
United States, www.genomics.agilent.com Fermentas FERMENTAS GMBH, St. Leon-Rot, Germany, a subsidiary of Thermo Fisher
Scientific Inc., www.fermentas.com
Finnzymes part of Thermo Scientific Molecular Biology, www.thermoscientificbio.com Glucose monohydrate Carl ROTH, 76185 Karlsruhe, Germany, www.carlroth.com Glycerol Carl ROTH, 76185 Karlsruhe, Germany, www.carlroth.com
3-HT can be purchased from Sigma-Aldrich, www.sigmaaldrich.com
Invitrogen Invitrogen Corporation, www.invitrogen.com, a subsidiary of Life
Technologies Corporation, www.lifetechnologies.com
IPTG isopropyl beta-D-l-thiogalactopyranoside, Biosynth, 9422 Staad, Switzerland, www.biosynth.com
pJexpress IP-Free™ bacterial expression vectors of DNA2.0, information reproduced in this specification is provided on the internet homepage of DNA2.0 and is reproduced with permission of DNA2.0
pJexpress 401 plasmid of DNA2.0 with kanamycin resistance maker and T5 promoter,
information is provided on the internet homepage of DNA2.0
pJexpress 404 plasmid of DNA2.0 with ampicillin resistance maker and T5 promoter,
information is provided on the internet homepage of DNA2.0
Kan Kanamycin, Carl ROTH, 76185 Karlsruhe, Germany, www.carlroth.com
KH2PO4 Potassium dihydrogen phosphate, Carl ROTH, 76185 Karlsruhe, Germany, www.carlroth.com
K2HPO4 di-Potassium hydrogen phosphate, Carl ROTH, 76185 Karlsruhe, Germany, www.carlroth.com
KPi Phosphate buffer, containing different ratio of equalmolar KH2PO4 and
K2HP04; depending on desired pH, Carl ROTH, 76185 Karlsruhe, Germany, www.carlroth.com
LB-plates Luria broth medium (also called LB -Lennox- Agar) (20g/L) containing agarose
(15g/L) and optionally the appropriate antibiotic, Carl ROTH, 76185
Karlsruhe, Germany, www.carlroth.com
LB medium Luria broth medium (also called LB-Lennox) (20g/L: lOg/L Tryptone, 5g/L
Yeast extract, 5 g/L NaCl) , Carl ROTH, 76185 Karlsruhe, Germany, www.carlroth.com
LGC Genomics LGC Genomics GmbH, Berlin, Germany, www.lgcgroup.com
MES 2-(N-morpholino)ethanesulfonic acid, Carl ROTH, 76185 Karlsruhe, Germany, www.carlroth.com MES buffer 50 mM MES, pH adjusted with 1 M NaOH
MgCl2 Magnesium chloride, Carl ROTH, 76185 Karlsruhe, Germany,
www.carlroth.com
MgS04 Magnesium sulfate, Carl ROTH, 76185 Karlsruhe, Germany,
www.carlroth.com
MWD multi wavelength detector
NAD+ Nicotinamide adenine dinucleotide, oxidized form, Roche Diagnostics,
www.roche.com
NADH Nicotinamide adenine dinucleotide, Roche Diagnostics, www.roche.com NADP+ Nicotinamide adenine dinucleotide phosphate, oxidized form, Roche
Diagnostics, www.roche.com
NADPH Nicotinamide adenine dinucleotide phosphate, Roche Diagnostics,
www.roche.com
NaOH Sodium hydroxide, Carl ROTH, 76185 Karlsruhe, Germany,
www.carlroth.com
PageRuler Prestained Protein Ladder Fermentas#SM0671 , published by Thermo
Scientific under "CERTIFICATE OF ANALYSIS PageRuler™ Prestained Protein Ladder#SM0671 " in the internet at www.fermentas.com
SOC medium Super Optimal broth with Catabolite repression medium: 2% w/v Tryptone,
0.5% w/v Yeast Extract, 10 mM NaCl, 2.5 mM KC1, 10 mM MgCl2 , 10 mM
MgS04, 20 mM Glucose
Tris tris-(hydroxymethyl)-aminomethane (IUPAC name: 2-Amino-2- hydroxymethyl-propane-l,3-diol), Carl ROTH, 76185 Karlsruhe, Germany, www.carlroth.com
Tryptone Tryptone is the assortment of peptides formed by the digestion of casein by the protease trypsin, supplier: Oxoid Limited, Hampshire, United Kingdom, www.oxoid.com, a subsidiary of Thermo Fisher Scientific,
www.thermofisher.com,
U 1 enzyme unit (U) = 1 μιηοΐ min-1, that is 1 U converts 1 μιηοΐ of substrate per minute
Methods
OD Optical Density measured at 600 nm in cuvettes with a pathlength of 1 cm HPLC Method A
HPLC Agilent Technologies 1200 Series equipped with G1379B degasser,G1312B
Binary Pump SL, G1367C HiP-ALS SL Autosampler including well plate handler, G1314C VWD SL UV detector, G1316B TCC SL column oven and G1956B MSD Mass selective detector.
Injection volume 10μL
Column Chromolith Performance RP-18 end capped 100-4.6 operated at 30°C
Flow 1.2 mL/min, split flow to 0.8 mL/min between UV and MS detector
Solvent 95% formic acid 0.1 %; 5% ACN
Detection either MS: e.g. Agilent G1956B, equipped with Atmospheric pressure
electrospray ionization, positive mode (MS) recording for scan (90 to 450 m/z) and SIMs (155 m/z and 153 m/z for alcohol and aldehyde, resp.) and negative mode (for acid)
and/or UV: e.g. Agilent 1100 equipped with MWD recording 230 or 254 nm Evaluation: Results are given in the examples in concentration and/or as % value. To
determine the concentration, a calibration was done with known concentration of the respective substance to be detected; the % value is the analytical yield of product (product is 3-HT if not otherwise stated) based on the molar amount of substrate used; if not otherwise stated.
In some experiments more than the theoretical possible yield of 3-HT was detected and is reported in the results. This stems from the refilling after reaction of the Eppendorf tubes to a volume of 500 μΕ, which was done visually using a reference mark for the volume on the Eppendorf tubes, and introduced a respective inaccuracy.
Protocol of DNA2.0 delivered with the plasmids for retrieval of plasmid DNA
Instructions for Use
Accessing plasmid DNA from GFC Filter
• Remove filter from plastic bag and place on a sterile and clean surface.
· Add 100 μΕ of 10 mM Tris-HCI, pH 7.5 directly to the center of the filter.
• Incubate at room temperature for 2 min.
• Puncture the bottom of a 0.6 mL tube using a syringe.
• Place filter in the 0.6 mL tube and place the 0.6 mL tube in a 1.5 mL tube. • Place the 1.5 mL tube (now containing punctured 0.6 mL tube with filter) in tabletop centrifuge.
• Spin 1 min at full speed. The DNA containing liquid will transfer from the filter in the 0.6 mL tube to the 1.5 mL tube.
• Discard the 0.6 mL tube with filter. The 1.5 mL tube now contains ca. 90 μΐ^ buffer and DNA.
• Carefully remove supernatant. There may be a small pellet consisting of filter debris.
This pellet does not contain any of the DNA. The supernatant should contain approximately 2 μg plasmid DNA (ca. 20 ng^L).
The isolated DNA can subsequently be transformed, cut with restriction enzymes, or sequenced without further purification.
NuPAGE® electrophoresis (Invitrogen)
NuPAGE® electrophoresis was performed to check the overexpression of the target proteins as follows:
Standard: 5 μΐ PageRuler™Prestained Protein Ladder (Fermentas)
Samples: 15 μΐ of cell free extract (CFE) or cell debris (CD) with protein concentration 2 mg/mL + 5 μΐ of NuPAGE® LDS Loading Buffer (4X) (Invitrogen), boiled at 99°C for 5 min on Thermomixer (Eppendorf), spun on Centrifuge 5415R (Eppendorf), loaded on the gel.
Gel: 4 to 12 % NuPAGE® Bis-Tris Gel (Invitrogen)
Running Buffer: NuPAGE® MOPS SDS Running Buffer (for Bis-Tris Gels only)
(20X)
Power Supplier: PowerEase® 500 Power Supply (Invitrogen)
Electrophoresis cell: XCellSureLock® Mini-Cell
Electrophoresis was run for 45 min at 200 V.
Staining: The gel was washed 3 times for 5min with distilled water, stained for
1 h with SimplyBlue™ SafeStain (Invitrogen) and destained overnight in distilled water under shaking on PMR-30 Platform Rocker (Grant Bio, subsidiary of Keison, www.keison.co.uk). After that the gel was scanned.
Purification Protocol of GeneJET™ Plasmid Miniprep Kit of Fermentas published by Thermo Scientific under "PRODUCTINFORMATION Thermo Scientific GeneJET Plasmid Miniprep Kit#K0502, #K0503"in the internet at
www.thermoscientific.com/fermentas, © 2011 Thermo Fisher Scientific Inc. Sonication
Sonication is done as follows, if not otherwise stated:
The cell pellets were dispersed in buffer. Typically, per gram of wet cell paste, 10 mL of buffer was used. The dispersion was placed into an aluminium beaker that was placed on a stirred water-ice bath. A Branson Sonifier® 250 analog ultrasonic cell disruptor equipped with a 1/2" tapped Disruptor horn was used for 6 min set to 80% duty cycle and to output control 8.
Example 1 - Strains and cultivation
The gene with sequence SEQ ID NO: 9 and the gene with sequence SEQ ID NO: 11 were ordered from DNA2.0 and were delivered in form of plasmid pJexpress-404-9-CAR with SEQ ID NO: 15, carrying the gene with SEQ ID NO: 9 for CAR, and of plasmid pJexpress- 401-11-EcPPTase with SEQ ID NO: 16, carrying the gene for PPTase with SEQ ID NO: 11, on GFC filters.
FIG 1 shows pJexpress-404-9-CAR
Legend of FIG 1 :
pUC ori origin of replication of the plasmid pUC (p short for "plasmid",
"UC" short for "University of California"), which is one of the most common origins of replication that is used in plasmids for genetic engineering
rpn txn terminator terminator of the rpn txn gene
Primer 1 to 6 as defined under List of Sequences
bla txn terminator terminator of the bla txn gene
T5 promoter T5 promoter sequence was described in Gentz et al., J.
Bacterid. 1985, 164, 70-77. Silencing of the promoter prior to IPTG induction is achieved using symmetrical lac operators (Sadler et al., Proc. Natl. Acad. Sci. USA 1983, 80, 6785-6789) spaced around the promoter to maximize cooperativity (Oehler et al., EMBO J., 1994, 13, 3348-3355). This operator pair ensures significantly tighter repression than regular lac operators. Overlapping T5 promoter/lac operator has been
described (Lanzer et al., Proc. Natl. Acad. Sci. USA, 1988, 85, 8973-8977)
CAR of SEQ ID NO: 9 gene with SEQ ID NO: 9 for CAR
rrnBl B2 Tl txn terminator terminator of the rrnBl B2 Tl txn gene
amp gene for ampicillin resistance
lacl lacl gene, which expresses the repressor protein that binds to the lac operator FIG 2 shows pJexpress-401-11 -EcPPTase.
Legend of FIG 2:
pUC ori origin of replication of the plasmid pUC (p short for "plasmid",
"UC" short for "University of California"), which is one of the most common origin of replication that is used in plasmids for genetic engineering
rpn txn terminator terminator of the rpn txn gene
Primer 1 and 2 as defined under List of Sequences
bla txn terminator terminator of the bla txn gene
T5 promoter T5 promoter sequence was described in Gentz et al., J. Bacterid
1985, 164, 70ff. Silencing of the promoter prior to IPTG induction is achieved using symmetrical lac operators (Sadler et al, Proc. Natl. Acad. Sci. USA 1983, 80, 6785-6789) spaced around the promoter to maximize cooperativity (Oehler et al, EMBO J 1994, 13, 3348-3355). This operator pair ensures significantly tighter repression than regular lac operators.
Overlapping T5 promoter/lac operator has been described (Lanzer et al, Proc. Natl. Acad. Sci. USA, 1988, 85, 8973-8977) EcPPTase of SEQ ID NO: 11 gene with SEQ ID NO: 11 for EcPPTase
rrnBl B2 Tl txn terminator terminator of the rrnBl B2 Tl txn gene
kan gene for kanamycin resistance
lacl lacl gene, which expresses the repressor protein that binds to the lac operator Plasmid DNA was retrieved according to the protocol of DNA2.0 delivered with the plasmids (see above under Methods).
60 μΐ. of chemically competent cells E. coli BL21 (DE3) Gold were transformed with the plasmids separately, using 100 ng of pJexpress-404-9-CAR to give strain CE1 and 75 ng of pJexpress-401-11-EcPPTase to give strain PEl respectively. The transformation was done as follows: The two mixtures of cells and plasmid were incubated on ice for 30 min, heat shocked for 30 s at 42°C and cooled down on ice for 2 min. 250 μΕ of pre-warmed SOC medium was added, incubated 1 h at 37°C.
The strains were selected as follows:
100 μΕ from each transformation was streaked on LB-plates, supplemented with 100 μg/mL of Amp or with 50 μg/mL Kan respectively, and incubated overnight at 37°C.
Plasmid DNA was isolated from the transformed cells CE1 and PEl according to the Purification Protocol of GeneJET™ Plasmid Miniprep Kit of Fermentas.
Plasmid DNA isolated from PEl and CE1 was sequenced by LGC genomics using Primers 1 to 6 for SEQ ID NO: 9 and Primers 1 and 2 for SEQ ID NO: 11 and confirmed SEQ ID NO: 9 and SEQ ID NO: 11 respectively.
Primer 1 is called pTF5 by DNA2.0, Primer 2 is called pTR by DNA2.0.
Strains CPE1 and CPE2 were obtained by simultaneous transformation with two plasmids of electrocompetent cells as follows:
For transformation, ice cooled electroporation cuvettes 2 mm (Cell Projects Ltd,
www.cellprojects.com) were filled with 80 μΕ of electrocompetent cells E. coli BL21 (DE3) Gold for CPE1 and with 1.13 μg of pJexpress-404-9-CAR and 0.11 μg of pJexpress-401-11- EcPPTase; and with 80 μΕ of electrocompetent cells E. coli BL21 (DE3) Gold for CPE2 and with 0.3 μg of pJexpress-404-9-CAR and 0.03 μg of pEHISTEV-14-CgPPTase prepared according to example 9 respectively. After 2 min incubation on ice, electroporation cuvettes were placed in a MicroPulserElectroporator411BR (Bio-Rad, www.bio-rad.com) and electroporation with following conditions 25 mFD, 200 W, and 2.5 kV (time constant ca. 5 msec) was started. Afterwards, the cells were transferred to a 1.5 mL tube, 600 μΕ of SOC medium was added and the cells were incubated on a Thermomixer comfort (Eppendorf) at 37°C and 600 rpm for 1 h.
The strains were selected as follows:
Next, 100 μΕ of cell suspension was streaked on LB-plate supplemented with 100 μg/mL Amp and 50 μg/mL Kan and incubated overnight at 37°C.
Preparation of glycerol stock samples:
20 mL of LB medium containing 100 mg/L Amp and/or 50 mg/L Kan respectively was inoculated with a single colony of one the strains CEl, PEl, CPEl and CPE2 respectively. Cultivation was carried out overnight at 37°C and 120 rpm. Samples of 500 μΕ were mixed with 250 μΕ LB medium and 250 μΕ aqueous glycerol 50% v/v and shock-frozen in liquid nitrogen and stored at -80°C to provide so called glycerol stock samples. The strains were cultivated as follows:
20 mL of LB medium containing 100 mg/L Amp and/or 50 mg/L Kan respectively was inoculated with 5 μΕ of a glycerol stock sample of one the strains CEl, PEl, CPEl and CPE2 respectively. Cultivation was carried out overnight at 37°C and 120 rpm. Subsequently, 500 mL LB medium, containing 100 mg/L Amp and/or 50 mg/L Kan respectively, in sterile 2L baffled flask were inoculated with pre-culture in such an amount in order to obtain an OD of ca. 0.1. Incubation for 1.5 to 2 h at 37°C and 120 rpm delivered cultures with an OD between 0.4 and 0.6, which were induced with 1 mM IPTG (0.5mL of 1M aqueous solution). The protein expression phase proceeded for 4 h at 37°C and 120 rpm. Subsequently, cells were harvested by centrifugation at 2831 g, 4°C for 10 min. The cell pellet was washed twice with Buffer A, and centrifuged again at 2831 g, 4°C for 10 min. The supernatant was removed and the cell pellet was dispersed in 50% glycerol and shock-frozen as 1 mL aliquots in liquid nitrogen. These resting cell samples were stored at -20°C.
Protein expression was visualized for CEl, PEl and CPEl with NuPage® electrophoresis. FIG 3 shows overexpression of CAR, expected molecular masses are 128 kDa for CAR and 23 kDa for EcPPTase.
The NuPage® electrophoresis was done with a concentration of ca. 1 mg/mL of total protein and the amount of CAR was estimated by visual inspection of the strength of the bands. This results in a approximate concentration of CAR of ca. 8 μΜ in the enzymatic reduction reactions described in the following examples.
FIG 3 shows, that whereas the over expression of CAR enzyme is evident in CFE in lane 2, EcPPTase is not visible in overexpressed amounts. However, the presence of sufficient
EcPPTase is evident because CPEl cells are active for carboxylate reduction whereas the CEl strain is inactive.
Legend of FIG 3:
Lane No
1 PageRuler™ Prestained Protein Ladder
2 CFE of CPEl
3 CD of CPEl Example 2
With the resting cell samples, prepared according to Example 1 and stored at -20°C, several reaction conditions were tested, details are given in table 2. For this purpose, the 1 niL aliquots were thawed and centrifuged. The supernatant was discarded and the cell pellet washed twice with reaction buffer, the reaction buffer was 50 mM KPi buffer of pH 7.5. After the second centrifugation and buffer removal, in case of addition of CoA, the cell pellets were dispersed in the reaction buffer and CoA was added and the cells were preincubated for 1 h at 28°C and at 1000 rpm prior to addition of the other ingredients . In case of no addition of CoA, the cell pellets were dispersed in the reaction buffer and the other ingredients were added without said preincubation.
The other ingredients were added according to table 2 to have the stated concentrations in the reaction mixture.
Reactions were done with the following parameters:
• with whole cells of strain CPEl,
· addition of MgCl2 for a concentration of 20 mM,
• addition of DOPAC for a concentration of 20 mM,
• reaction buffer was used to adjust the final volume to 500 μΕ
• the pH after addition of all ingredients was not controlled,
• incubation over night in Eppendorf tubes at 28 °C with open or closed lids. After incubation, the reaction mixtures were filtered through syringe filters (size 0.2 μιη, nylon) and analyzed by HPLC Method A. Results are summarized in Table 2.
Figure imgf000046_0001
Example 3 - various reaction parameters
The effect of reaction mixture composition was studied under the following conditions: CPE1 resting cell sample aliquots, prepared according to Example 1, were thawed and centrifuged. The supernatant was discarded and the cell pellet washed twice with buffer B. The cell pellets were dispersed in 50 mM MES buffer pH 6.0 containing variations of components as specified in Table3. 0.5 M NADPH in water and 100 mM of DOPAC in water were added to give final concentrations of 20 mM and 10 mM respectively, in a total volume of 500 μΕ. The thus prepared samples in Eppendorf vessels were incubated in an Eppendorf Thermomixer at 28°C and 1000 rpm for 4 h with open lid. The samples were refilled with water to 500 μΕ, centrifuged for 10 min at 28°C and the supernatants were filtered through plate filters (0.2 μιη, polypropylene). The filtrates were analyzed with Method A.
"Relative %" means that the % value of Ex 3-1 was set to 100 % and all other % values were normalized to this value. "Relative DOPAC and 3-HT recovery [%]" means the value of the sum of substrate and product concentration of Ex 3-16 was set to 100 % and all other values were normalized to this value
Figure imgf000047_0001
Example 4- temperature of reaction
The reaction temperature was studied under the following conditions: CPE1 resting cell sample aliquots, which can be prepared according to Example 1 , were thawed and centrifuged. The supernatant was discarded and the cell pellet washed twice with buffer B. The cell pellets were dispersed in buffer B, and 0.5 M NADPH in water and 100 mM of
DOPAC in water were added to give final concentrations of 20 mM and 10 mM respectively, in a total volume of 500 μΕ. The samples were incubated in five separate Eppendorf
Thermomixers at different temperatures at 1000 rpm with open lid as outlined in table 4. After 16 h, the samples were refilled with water to 500 μΕ, centrifuged for 10 min at 28°C and the supematants were filtered through a plate filter (0.2 μηι, polypropylene). The filtrates were analyzed with Method A. Table 4 shows the influence of the reaction temperature on the reaction.
"Relative %" means that the % value of Ex 4-3 was set to 100 % and all other % values were normalized to this value.
Figure imgf000048_0001
Example 5 - time of reaction
The influence of the reaction time was studied under the following conditions: CPEl resting cell samples were prepared according to Example l .The tested reaction times are given in table 5. For the reaction times of 16 h to 24 h, CPEl fresh cells were used, which were prepared according to example 1 , wherein the cells were harvested as described in example 1 by centrifugation at 2831 g at 4°C for 10 min after the protein expression phase, resulting in a cell pellet of fresh cells. These fresh cell pellets were washed with buffer B and then dispersed in buffer B.
For the reaction times of 30 min to 8 h, CPEl resting cell sample aliquots, prepared according to Example 1 , were thawed and centrifuged. The supernatant was discarded and the cell pellet washed twice with buffer B. These cell pellets were dispersed in buffer B.
To any of these cell pellets dispersed in buffer B, 0.5 M NADPH in water and 100 mM of DOPAC in water were added to give final concentrations of 20 mM and 10 mM respectively, in a total volume of 500 μΕ.
The thus prepared samples in Eppendorf vessels were incubated in an Eppendorf
Thermomixer at 28°C and 1000 rpm with open lid. At the reaction times indicated in Table5, the samples were refilled with water to 500 μΕ, centrifuged for 10 min at 28°C and the supematants were filtered through a plate filter (0.2 μιη polypropylene). The filtrates were analyzed with Method A. Table 5 shows the influence of the reaction time of the reaction. Table 5
Example Reaction time DOPAC 3-HT
[h] [mM] [mM]
5-1 0.5 7.85 0.35
5-2 1 8.71 0.69
5-3 2 5.79 2.54
5-4 4 3.14 6.95
5-5 6 1.37 10.04
5-6 8 0 9.78
5-7 16 0 11.29
5-8 18 0 9.47
5-9 20 0 9.73
5-10 22 0 11.19
5-11 24 0 10.79
Example 6-influence of cofactors on reaction
The influence of different cofactors and cofactor concentrations on the reaction of DOPAC to 3-HT was studied under the following conditions: CPEl and CPE2 resting cell sample aliquots respectively, prepared according to Example 1 , were thawed and centrifuged. The supernatant was discarded and the cell pellet washed twice with buffer B in case of CPEl, with buffer C in case of CPE2. These cell pellets were dispersed in buffer B in case of CPEl, with buffer C in case of CPE2.
The cofactors listed in Table 6 were added in form of an aqueous solution to give final concentrations as listed in Table 6. Subsequently, 100 mM of DOPAC in water was added to give a final concentration of 10 mM in a total volume of 500 μΕ. The thus prepared samples in Eppendorf vessels were incubated in an Eppendorf Thermomixer at 28°C and 1000 rpm with open lid. After 4 h they were centrifuged for 10 min at 28°C and the supernatants were filtered through a plate filter (0.2 μιη, polypropylene). The filtrates were analyzed with
Method A. Table 6 shows the influence of the cofactor composition and concentration, only one example was done with CPE2.
"Relative %" means that the % value of Ex 6-8 was set to 100 % and all other % values were normalized to this value. Table 6 CPEl CPE2
NADPH NADP+ NADH Example Relative Example Relative
[mM] [mM] [mM] % %
0 0 0 6-1-CPEl 3 — —
10 0 0 6-2-CPEl 37 — —
20 0 0 6-3-CPEl 83 6-3-CPE2 55
10 0 10 6-4-CPEl 91 — —
5 0 5 6-5-CPEl 22 — —
0 10 10 6-6-CPEl 7 — —
0 0 10 6-7-CPEl 18 — —
0 0 20 6-8-CPEl 100 — —
0 5 5 6-9-CPEl 5 — —
Example 7-cofactor recycling using GDH and glucose
Recycling of NAD(P)H was studied under the following conditions: CPEl resting cell sample aliquots, prepared according to Example 1 but with the difference that the protein expression phase after induction was 16 to 18 h instead of 4 h, were used. After this protein expression phase, cells were harvested by centrifugation at 2831 g, 4°C for 10 min. The supernatant was discarded and the cell pellet washed twice with buffer C. These cell pellets were dispersed in buffer C.
GDH and glucose were added to give final concentrations as listed in Table 7. Subsequently, 100 mM DOPAC in water was added to give a final concentration of 10 mM in a total volume of 500 μΕ. The thus prepared samples in Eppendorf vessels were incubated in an Eppendorf Thermomixer at 28°C and 1000 rpm with open lid. After 5 h, the samples were centrifuged for 10 min at 28°C and the supernatants were filtered through a plate filter (0.2 μιη polypropylene). The filtrates were analyzed with Method A. Table 7 shows the influence of cofactor recycling with the system GDH and glucose
1 mg/mL GDH corresponds to 2.7 U for NADP+ reduction (1 U catalyzes the conversion of 1 μιηοΐ NADP+ per min). "Relative %" means that the % value of Ex 7-5 was set to 100 % and all other % values were normalized to this value. Table 7
Example GDH glucose Relative
[U] |mM ] %
7-1 0.027 10 72
7-2 0.135 10 70
7-3 0.27 10 97
7-4 0.27 20 97
7-5 0.27 50 100
7-6 2.7 10 83
7-7 13.5 10 43
7-8 27 10 61
7-9 27 20 60
7-10 27 50 57
Example 8-cofactor recycling using FDH and formate or citrate
Recycling of NAD(P)H was studied under the following conditions: CPEl and CPE2 resting cell sample aliquots respectively, prepared according to Example 1 , were thawed and centrifuged. The supernatant was discarded and the cell pellet washed twice with buffer C. The cell pellets were dispersed in buffer C.
The FDH was used in form of a cell free extract of a E. coli Rosetta containing the plasmid pRSF:FDH from Candida boidinii as described in Madje et al., Microb. Cell Fact., 2012, 11 :7, with the described 18°C during the induction phase which is reported by Madje to provide FHD in an amount of 28 % of the total soluble protein in the extract. FDH and formate were added to give final concentrations as listed in Table 8. Alternatively, citrate was used, with and without extra NADP+ in final concentrations as listed in Table 8.
Subsequently, 100 mM DOPAC in water was added to give a final concentration of 10 mM in a total volume of 500 μΕ. The thus prepared samples in Eppendorf vessels were incubated in an Eppendorf Thermomixer at 28°C and 1000 rpm with open lid. After 5 h, the samples were centrifuged for 10 min at 28°C and the supernatants were filtered through a plate filter (0.2 μιη polypropylene). The filtrates were analyzed with Method A.
Table 8 gives the respective values and results. 1.08 mg/mL CFE containing FDH
corresponds to 0.27 Units (1 U catalyzes the conversion of 1 μιηοΐ NAD+ per min). Table 8 CPE1 CPE2
FDH formate Citrate NADP+ Example % Example % [U] |mM ] |mM] |mM ]
0.27 10 — — 8-1-CPEl 65 8-1-CPE2 36
— — 10 — 8-2-CPEl 26 — —
— — 10 1 8-3-CPEl 67 8-3-CPE2 46
— — 50 — 8-4-CPEl 77 — —
Example 9 - strain PE2
Preparation of pEHISTEV- 14-CgPPTase
The DNA of SEQ ID NO: 13 was amplified from genomic DNA of Corynebacterium glutamicum DSM 20300, ATCC 13032 including the introduction of restriction sites Ncol and Hindlll using Phusion® High-Fidelity DNA polymerase (Finnzymes) and primers of SEQ ID NO: 23 and of SEQ ID NO: 24. A single colony of Corynebacterium glutamicum was picked with a sterile tooth pick into 50 μΕ of distilled water. After incubation at 95°C for 10 min and centrifugation for 5 min at room temperature and 13 200 rpm, 5 μΕ of the supernatant was used as template DNA for the PCR reaction. The PCR reaction was thermally cycled: 98°C for 30 s, followed by 20 cycles of 98°C for 10 s, 50°C for 20 s, and 72°C for 15 s, then a final incubation of 72°C for 7 min. The PCR products were gel purified with the QIAquick® Gel Extraction Kit (QIAGEN). The thus obtained PCR product was double digested with Ncol and Hindlll restriction enzymes (Fermentas) in the presence of lx Tango buffer (Fermentas) and column purified according to QIAquick® PCR purification protocol (QIAGEN). The gene was ligated into the pEHISTEV vector (Huanting et al. Protein Expression and Purification 2009, 63, 102-111) which was also digested with Ncol and Hindlll. The ligation was carried out overnight at 16°C in the presence of T4 ligase
(Fermentas) and T4 ligation buffer (Fermentas). The ligation product was desalted for 40 min on MF-Millipore™ membrane filters (0.025μιη filter code VSWP) (Millipore), transformed into electrocompetent E. coli TOP10 F' cells and plated out on a LB-plate supplemented with 50 μg Kan/mL. The resulting plasmid, called pEHISTEV- 14-CgPPTase, was harvested by GeneJET™ Plasmid Miniprep Kit (Fermentas) and the sequence was confirmed by LGC Genomics.
Transformation with pEHISTEV- 14-CgPPTase to provide PE2 60 μΐ. of chemically competent cells E. coli BL21 (DE3) Gold were transformed with 100 ng of pEHISTEV-14-CgPPTase.The transformation, selection, using Kan only for selection, and glycerol stock sample preparation were done as described in Example 1 to provide strain PE2 in form of glycerol stock samples.
Example 10 - 3-HT preparation using CFEs
CE1, CPE1, PE1, PE2 and CPE2 CFEs of resting cell samples were obtained as follows: For each strain, 20 mL of LB medium containing 100 mg/L Amp or 50 mg/L Kan or both respectively, was inoculated with 5 μΕ of a respective glycerol stock sample of each strain respectively, prepared according to example 1 and example 9 respectively.
Cultivation was carried out overnight at 37°C and 120 rpm. Subsequently, 500 mL LB medium, containing 100 mg/L Amp or 50 mg/L Kan or both respectively, in sterile 2 L baffled flask were inoculated with pre-culture in such an amount in order to obtain an OD of ca. 0.1. Incubation for 1.5 to 2 h at 37°C and 120 rpm delivered cultures with an OD between 0.4 and 0.6, which were induced with 1 mM IPTG (0.5 mL of 1M aqueous solution). The protein expression phase proceeded for 4 h at 37°C and 120 rpm for strains CE1, PE1, CPE1 and CPE2, and overnight at 25°C and 120 rpm for strain PE2. Subsequently, cells were harvested by centrifugation at 2831 g at 4°C for 10 min. The cell pellets were washed twice with Buffer B and centrifuged again at 2831 g at 4°C for 10 min. The supernatant was removed and the cell pellets were dispersed in Buffer B, sonicated, ultracentrifuged at
107527.5 g and 4°C for 45 min. The supernatants representing the CFEs were shock frozen in liquid nitrogen and stored at -20°C.
Thawed CFEs, volumes as given in table 10, were mixed in Eppendorf vessels according to the combinations described in Table 10, 0.5 M NADPH in water, 250 mM ATP in water and 100 mM of DOPAC in water were added to give final concentrations of 20 mM, 10 mM and 10 mM respectively, in a total volume of 500 μΕ.
The thus prepared samples were incubated in an Eppendorf Thermomixer at 28°C and 1000 rpm with closed lid. After overnight incubation, the samples were centrifuged for 10 min at 28°C and the supernatants were filtered through a plate filter (0.2 μιη polypropylene). The filtrates were analyzed with Method A. Table 10 shows the influence of the CFE composition on the reaction. Table 10
Example CPE1 CE1 PE1 PE2 CPE2 %
[μί] [μί]
10-1 200 — — — — 3.7
10-2 — 200 — — — 3.1
10-3 — 200 200 — — 5.9
10-4 — 200 — 200 — 16.4
10-5 — — — — 200 6.0
Example 11-reaction condition optimization using citrate and glucose
In order to optimize the reaction conditions, the 'design of experiments' method (DOE) was used.
CPE1 resting cell sample aliquots respectively, prepared according to Example 1, were thawed and centrifuged. The supernatant was discarded and the cell pellet was washed twice with buffer D. The cell pellets were dispersed in buffer D. Stock solutions of sodium citrate and glucose in buffer D at pH 6.0 were added to give final concentrations as specified in Table 11. The samples were incubated in 1.5 ml reaction tubes for lh at 28°C and 1000 rpm with open lids.
After that, DTT, EDTA and magnesium chloride, each dissolved in buffer D at pH 6.0, were added to give final concentrations as specified in Table 11. Subsequently, 300 mM DOPAC in buffer D at pH 6.0 was added to give final concentrations as specified in Table 11 in a total volume of 500 μΕ. The thus prepared samples in Eppendorf vessels were incubated in an Eppendorf Thermomixer at 28°C and 1000 rpm with open lid. After 21 h, the samples were centrifuged for 10 min at 28°C and the supernatants were filtered through a plate filter (0.2 μιη polypropylene). The filtrates were analyzed with Method A.
Table 11 gives selected values and results.
Table 11
Example DOPAC citrate Glucose DTT EDTA MgCl2 3-HT
[mM] [mM] [mM] [mM] [mM] [mM] [mM]
11-1 30 55 55 5.5 5.5 5.5 22.7
11-2 10 10 10 1 1 1 6.8
11-3 10 10 10 10 1 10 6.7
11-4 10 10 100 1 10 10 9.2
11-5 10 10 100 10 10 1 8.6
11-6 10 100 10 1 10 10 7.2
11-7 10 100 10 10 10 1 5.6
11-8 10 100 100 1 1 1 10.4
11-9 10 100 100 10 1 10 10.0
11-10 50 10 10 1 10 1 1.0
11-11 50 10 10 10 10 10 0.8
11-12 50 10 100 1 1 10 2.7
11-13 50 10 100 10 1 1 14.2
11-14 50 100 10 1 1 10 4.6
11-15 50 100 10 10 1 1 3.9
11-16 50 100 100 1 10 1 3.4
11-17 50 100 100 10 10 10 2.0
11-18 30 100 10 5 4.2 1.1 11.2
11-19 30 67 58 1 5.5 4.9 28.3
Example 12: Reaction on Preparative Scale
120 mL of LB medium containing 100 mg/L Amp and 50 mg/L Kan, respectively, was inoculated with a single colony of CPE1 grown on LB-plates containing 100 mg/L Amp and 50 mg/L Kan, respectively; this single colony was prepared according to example 1 described after the sentence "The strains were selected as follows:" and before the sentence "Preparation of glycerol stock samples:".
Cultivation was carried out overnight at 37°C and 120 rpm. Subsequently, 6 times 430 mL LB medium, containing 100 mg/L Amp and 50 mg/L Kan respectively, in sterile 2 L baffled flask were inoculated with pre-culture in such an amount in order to obtain an OD of ca. 0.1.
Incubation for 1.5 to 2 h at 37°C and 110 rpm delivered cultures with an OD between 0.4 and 0.6, which were induced with 1 mM IPTG (0.45mL of 1 M aqueous solution). The protein expression phase proceeded for 4 h at 37°C and 110 rpm. Subsequently, cells were harvested by centrifugation at 2831 g, 4°C for 10 min. The cell pellet was washed twice with buffer C, and centrifuged again at 2831 g, 4°C for 10 min. The supernatant was removed and the cell pellet was dispersed in 50 % glycerol and shock-frozen as 40 mL aliquots in liquid nitrogen. These resting cell samples were stored at -80°C. One aliquot of CPE1 resting cells was thawed and centrifuged. The supernatant was discarded and the cell pellet washed twice with buffer C. A stirring bar was added and the cell pellet (3 g wet weight) was dispersed in buffer D in a 500 mL polypropylene (PPCO) beaker. Stock solutions of sodium citrate and glucose in buffer D at pH 6.0 were added to give final concentrations (based on the total reaction volume or 100 mL) of 10 mM and 100 mM, respectively. The sample was incubated for lh at 28°C and 100 rpm in the open PPCO beaker on a magnetic stirrer.
After that, DTT, EDTA and magnesium chloride, each dissolved in buffer D at pH 6.0, were added to give final concentrations of 1 mM, 10 mM and 10 mM, respectively. Subsequently, 300 mM DOPAC in buffer D at pH 6.0 was added to give a final concentration of 10 mM in a total volume of 100 mL. The thus prepared suspension in the PPCO beaker (called "first
PPCO beaker") was covered with gauze and incubated on a magnetic stirrer at 28°C and 100 rpm stirring speed. 200 μΐ^ samples were withdrawn periodically (see column "Time" in Table 12), centrifuged for 10 min at room temperature and the supernatants were filtered through a plate filter (30 kDa cut off, modified polyethersulfone). The filtrates were analyzed with Method A.
After 23 h and 45 h, new portions of resting cells (3.0 g and 2.9 g wet cell weight) were prepared as follows: One aliquot of CPE1 resting cells, prepared according to this example, was thawed on ice, diluted with buffer C and centrifuged. The supernatant was discarded and the cell pellet was washed two more times with buffer C. The cell pellet was dispersed in buffer D in a PPCO beaker. Stock solutions of sodium citrate (1.5 mL, 0.67 M) and glucose (10 mL, 1 M), both in buffer D at pH 6.0, were added to the cell pellet. The sample was incubated for lh at 28°C and 90 rpm in the open vessel in a rotary shaker. The mixture was added to the first PPCO beaker with the ongoing DOPAC reduction reaction and the stirring speed was increased to 200 rpm after the first addition of fresh cells. The second addition was carried out likewise.
After 144 h, the reaction was stopped by centrifugation at 15344 g. The pellet was first washed with 25 ml of buffer D by disperging and centrifugation, and then additionally two times with 10 ml of buffer D. The pH of the combined supernatants was adjusted to pH 2.0 with concentrated HC1 and the compounds were extracted with ethyl acetate four times. The combined extracts (175 mL) were dried with sodium sulfate and the solvent was removed at 135 mbar at 40°C. The residue was purified by silica gel chromatography using 15 g of silica gel and dichloromethane : MeOH of 15:1 with 0.15 % of triethylamine as the eluent. The product containing fractions were pooled and the solvent was removed under reduced pressure to give 29.2 mg of a yellowish oil. Product purity was confirmed with Method A to be > 99 % and product identity was confirmed by comparison of chromatographic behavior and by ¾ and 13C NMR against standard 3-HT.
Table 12
Entry Time [h] Bioconversion [%] 3-HT |mM]
12-1 3 0.9 0.1
12-2 20 11.3 1.2
12-3 23 13.0 1.3
12-4 45 19.7 1.8
12-5 92 32.5 2.7
12-6 95 33.6 3.0
12-7 116 42.7 4.4
12-8 144 38.7 3.8
Sequence Listing Free Text
<210> 9
<223> Nocardia sp. NRRL 5646 ATP/NADPH-dependent carboxylic acid reductase (car) , EMBL Nucleotide Sequence Database accession no. AY495697, codon optimized for E.coli
<210> 15
<223> Gene sequence of plasmid pJexpress-404- 9-CAR <210> 16
<223> Gene sequence of plasmid pJexpress-401-ll-EcPPTase
<210> 17
<223> Primer 1
<210> 18
<223> Primer 2
<210> 19
<223> Primer 3
<210> 20
<223> Primer 4 <210> 21
<223> Primer 5
<210> 22
<223> Primer 6
<210> 23
<223> Primer 7
<210> 24
<223> Primer 8

Claims

Claims
1. Method for the preparation of 3-hydroxytyrosol by a reaction, the reaction comprises an enzymatic reduction of 3,4-dihydroxyphenyl acetic acid,
wherein the enzymatic reduction is done using two enzymes, a carboxylic acid reductase CAR and a 4'-phosphopantetheinyl transferase PPTase;
wherein
CAR comprises each of the three amino acid sequences SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5 with 70 to 100% identity;
PPTase comprises the sequence SEQ ID NO: 12 or comprises the sequence SEQ ID NO: 14, each sequence is comprised with 70 to 100 % identity.
2. Method according to claim 1, wherein CAR comprises each of the three amino acid sequences SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6;
and PPTase comprises the sequence SEQ ID NO: 12 or comprises the sequence SEQ ID NO:
14;
each sequence is comprised with 70 to 100% identity.
3. Method according to claim 1, wherein CAR comprises each of the three amino acid sequences SEQ ID NO: 2, SEQ ID NO: 10 and SEQ ID NO: 6;
and PPTase comprises the sequence SEQ ID NO: 12 or comprises the sequence SEQ ID NO:
14;
each sequence is comprised with 70 to 100%> identity.
4. Method according to claim 1, wherein CAR comprises the CAR from Nocardia sp. Strain NRRL 5646;
and PPTase comprises the sequence SEQ ID NO: 12 or comprises the sequence SEQ ID NO:
14;
the CAR from Nocardia sp. Strain NRRL 5646 and each of the sequences SEQ ID NO: 12 and SEQ ID NO : 14 is comprised with 70 to 100% identity.
5. Method according to claim 1, wherein CAR comprises the sequence SEQ ID NO: 7; and PPTase comprises the sequence SEQ ID NO: 12 or comprises the sequence SEQ ID NO:
14, each sequence is comprised with 70 to 100% identity.
6. Method according to claim 1, wherein CAR has the sequence SEQ ID NO: 7;
and PPTase has the sequence SEQ ID NO: 12 or has the sequence SEQ ID NO: 14;
each sequence with 70 to 100% identity.
7. Method according to claim 1, wherein CAR has the sequence SEQ ID NO: 7;
and PPTase has the sequence SEQ ID NO: 12 or has the sequence SEQ ID NO: 14;
each sequence with 80 to 100% identity.
8. Method according to claim 1, wherein CAR has the sequence SEQ ID NO: 7;
and PPTase has the sequence SEQ ID NO: 12 or has the sequence SEQ ID NO: 14;
each sequence with 90 to 100%> identity.
9. Method according to claim 1, wherein CAR has the sequence SEQ ID NO: 7;
and PPTase has the sequence SEQ ID NO: 12 or has the sequence SEQ ID NO: 14;
each sequence with 95 to 100% identity.
10. Method according to one or more of claims 1 to 9, wherein the sequence SEQ ID NO: 12 is the embodiment for PPTase.
11. Method according to one or more of claims 1 to 10, wherein CAR and PPTase are used in the reaction
• in form of a cell free extract of a microorganism containing one of the enzymes or both;
• in form of a microorganism, that expresses or expressed both enzymes, or in form of two mircoorganisms, one microorganism expressing the one enzyme and the other microorganism expressing the other enzyme;
• in form of parts of a microorganism, that expressed one of the enzymes or both; or · as a combination of these embodiments.
12. Method according to one or more of claims 1 to 11 , wherein CAR and PPTase are used in the reaction in form of a microorganism, that expresses both enzymes, or in form of two mircoorganisms, one microorganism expressing the one enzyme and the other microorganism expressing the other enzyme, or in form of a cell free extract containing the enzymes.
13. Method according to claim 11 or 12, wherein the microorganism is selected from the group of families consisting of Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae,
Streptomycetaceae and Nocardiaceae.
14. Method according to one or more of claims 11 to 13, wherein the mircoroganism is selected from the group consisting of Escherichia, Bacillus, Lactobacillus, Pseudomonas, Streptomyces, Nocardia, Burkholderia, Salmonella, Agrobacterium and Rhodococcus.
15. Method according to one or more of claims 11 to 13, wherein the microorganism is Escherichia coli.
16. Method according to one or more of claims 1 to 15, wherein the reaction is done in the presence of a buffer (REAC);
buffer (REAC) is selected from the group consisting of phosphate buffer, a buffer based on 4- 2-hydroxyethyl-l-piperazineethanesulfonic acid, a buffer based on 2- {[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid, a buffer based on 3-(N- morpholino)propanesulfonic acid, a buffer based on piperazine-N,N'-bis(2-ethanesulfonic acid, a buffer based on 2-(N-morpholino)ethanesulfonic acid and mixtures thereof.
17. Method according to claim 16, wherein buffer (REAC) is phosphate buffer, 2-(N- morpholino)ethanesulfonic acid or mixtures thereof.
18. Method according to claim 16 or 17, wherein the reaction is started in the presence of buffer (REAC) with a pH of from 5 to 8.5.
19. Method according to one or more of claims 1 to 18, wherein the reaction is done
presence of DTT.
20. Method according to one or more of claims 1 to 19, wherein the reaction is done in the presence of EDTA.
21. Method according to one or more of claims 1 to 20, wherein the reaction is done in the presence of a cofactor, the cofactor is selected from the group consisting of NADPH and NADH.
22. Method according to one or more of claims 1 to 21, wherein the reaction is
(i) done in the presence of an enzyme ENZREGENCOF, that regenerates the cofactor, and a respective substrate REGENSUB, that is used by ENZREGENCOF to regenerate the cofactor; or
(ii) the reaction is done using electrical current via redox electrodes for the regeneration of the cofactor; or
(iii) the reaction is done in the presence of a redox mediator for the regeneration of the
cofactor; or
(iv) done in the presence of a substrate (SUBSCIT), substrate (SUBSCIT) is a substrate of the citric acid cycle or glucose.
23. Method according to claim 22, wherein the reaction is
(i) done in the presence of an enzyme ENZREGENCOF, that regenerates the cofactor, and a respective substrate REGENSUB, that is used by ENZREGENCOF to regenerate the cofactor; or
(iv) done in the presence of a substrate (SUBSCIT).
24. Method according to claim 22 or 23, wherein ENZREGENCOF is selected from the group consisting of glucose 1 -dehydrogenase, formate dehydrogenase, phosphonate dehydrogenase, NAD(P) oxidase, alcohol dehydrogenase and mixture thereof;
and REGENSUB is glucose, formate, phosphonate, hydrogen peroxide or an alcohol as
substrate for the respective ENZREGENCOF.
25. Method according to one or more of claims 22 to 24, wherein ENZREGENCOF is GDH with REGENSUB being glucose or ENZREGENCOF is FDH with REGENSUB being formate or ENZREGENCOF is a mixture of GDH and FDH with REGENSUB being glucose and formate.
26. Method according to one or more of claims 1 to 25, wherein the reaction is done in the pprreesseennccee ooff aa kkaattiion KAT, KAT is selected from the group consitsing of Mg2+, Zn2+ and mixtures thereof.
PCT/EP2013/076327 2012-12-13 2013-12-12 Process for producing 3-hydroxytyrosol from 3,4-dihydroxyphenylacetic acid|employing a carboxylic acid reductase Ceased WO2014090923A1 (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998040472A2 (en) * 1997-03-10 1998-09-17 University Of Iowa Research Foundation Carboxylic acid reductase, and methods of using same

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998040472A2 (en) * 1997-03-10 1998-09-17 University Of Iowa Research Foundation Carboxylic acid reductase, and methods of using same

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HE, A. ET AL.: "Nocardia sp. Carboxylic Acid Reductase: Cloning, Expression, and Characterization of a New Aldehyde Oxidoreductase Family", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 70, no. 3, March 2004 (2004-03-01), pages 1874 - 1881, XP002483657 *
VENKITASUBRAMANIAN, P. ET AL.: "Aldehyde oxidoreductase as a biocatalyst: Reductions of vanillic acid", ENZYME AND MICROBIAL TECHNOLOGY, vol. 42, no. 2, 12 December 2007 (2007-12-12), pages 130 - 137, XP022387256 *
XU, C.L. & SIM, M.K.: "REDUCTION OF DIHYDROXYPHENYLACETIC ACID BY A NOVEL ENZYME IN THE RAT BRAIN", BIOCHEMICAL PHARMACOLOGY, vol. 50, no. 9, 26 October 1995 (1995-10-26), pages 1333 - 1337, XP002711205 *

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