Monooxygenase mutant for the biosynthesis of 2,6-bis(hydroxymethyl)pyridine and method for preparing 2,6-bis(hydroxymethyl)pyridine using said monooxygenase mutant

Abstract

The present invention relates to an enzymatic method for the preparation of 2,6-bis(hydroxymethyl)pyridine starting from 2,6-lutidine using a mutant xylene monooxygenase enzyme (designated ppXMO) comprising the xylM and xylA subunits from Pseudomonas putida, said mutant enzyme having an amino acid exchange at position 116 of the amino acid sequence of the XylM component. The key to the invention is the replacement of methionine (M) at this position with an amino acid selected from the group consisting of asparagine (N), lysine (K), arginine (R) and glycine (G), which surprisingly results in the direct methylhydroxylation of 6-methyl-2-pyridinemethanol, thereby improving the overall process yield, producing fewer by-products, avoiding toxic reaction intermediates and minimizing the involvement of endogenous reductases and the need for NADPH and its regeneration. Other enzymes related to XylM of P. putida that have the same amino acid exchange in the highly conserved region around position 116 or its equivalent show similar improved properties.

Description

[Technical field]

[0001] The present invention belongs to the field of biochemistry, more precisely to genetic engineering for preparing enzymes and mutant enzymes for various applications. The present invention also belongs to the field of organic chemistry. The present invention relates to a monooxygenase mutant for the biosynthesis of 2,6-bis(hydroxymethyl)pyridine (formula I) and a method for preparing 2,6-bis(hydroxymethyl)pyridine using said monooxygenase mutant. [Background technology]

[0002] 2,6-Bis(hydroxymethyl)pyridine (Formula I) is a compound that can be used as a versatile intermediate in the preparation of other complex products. The hydroxyl groups can be converted to other functional groups, such as aldehyde groups, halogenated hydrocarbons, and amino groups, which are then used in the preparation of useful compounds. In addition, 2,6-bis(hydroxymethyl)pyridine can also be used in the synthesis of macrocyclic compounds by substitution at the 2- and 6-positions. One example is picrene, an azamacrocyclic framework that incorporates an aromatic pyridine moiety into a 12-membered macrocyclic unit.

[0003] The compounds of formula I can be synthesized from the readily available starting material 2,6-lutidine II by oxidation to the respective dicarboxylic acid using KMnO4, conversion to the respective ester, and finally reduction of the ester group to the alcohol (Journal of Dispersion Science and Technology 2006,27,p.15-21). The cited references do not state the yield of the conversion of the three steps. Moreover, this synthesis method is laborious since it requires the isolation of several intermediates with all three steps and purification.

[0004] Patent application CN105646334A discloses the above synthetic approach by omitting the ester conversion step, i.e., the dicarboxylic acid is first isolated and then directly converted to the bis-alcohol. The Chinese patent application reports that the total yield of this two-step process is 64%, which is a moderate yield for such a short synthesis.

[0005] Egorov et al. reported in 1985 that they found that 2,6-dimethylpyridine could be hydroxylated to 2-methyl-6-hydroxymethylpyridine by suspensions of certain non-growing cells (Prikladnaya Biokhimiya i Mikrobiologiya,21(3),pp.349-353). Small amounts of 2,6-bis(hydroxymethyl)pyridine were found to be formed only by Sporotrichum sulfurescens ATCC7159. It was suggested that the insertion of the first hydroxyl group increases the polarity of the substrate, which prevents the oxidation of the second methyl group. The document discloses that the yield cannot be significantly increased by increasing the time of the conversion reaction.

[0006] It is desirable to develop a selective process for producing 2,6-bis(hydroxymethyl)pyridine (Formula I) from 2,6-lutidine (Formula II) in high yield without the need for isolating intermediates, which is cost-effective from an industrial production perspective and is also more sustainable.

[0007] It is a further object of the present invention to provide an enzyme variant that improves the efficiency of producing 2,6-bis(hydroxymethyl)pyridine from 2,6-lutidine.

[0008] Cutting-edge technology Recently filed patent application PCT / EP2021 / 068920, which has not been published as of the time of writing, discloses a process for converting 2,6-lutidine II to 2,6-bis(hydroxymethyl)pyridine I, [ka] Here, the conversion is carried out in the presence of an enzyme and the conversion can proceed via the formation of 6-methyl-2-hydroxypyridine (formula III). [ka]

[0009] The enzyme used in this process is an oxidoreductase, preferably an NAD(P)H-dependent oxidoreductase, which uses molecular oxygen to oxidize 2,6-lutidine II. Possible oxidoreductases according to this document are: - the xylene monooxygenase enzyme (called ppXMO) encoded by the xylM and xylA genes of Pseudomonas putida (Arthrobacter siderocapsulatus), or - Alteromonas macleodii, or Tepidiphilus succinatimandens, or Novosphingobium kunmingense, or Hyphomonas oceanitis, or Sphingobium sp. 32-64-5, or Halioxenophilus aromaticivorans XylMA-like enzymes, or -It is a XylMA-like enzyme with over 70% sequence identity at the amino acid level.

[0010] Although this solution process is cost-effective from an industrial manufacturing standpoint, the above reaction pathway is not optimal for several reasons, including: - the highly toxic nature of the intermediates formed, 6-methyl-2-pyridinecarbaldehyde and 6-(hydroxymethyl)pyridine-2-carbaldehyde, the formation of 6-methyl-2-pyridinecarboxylic acid as a by-product, - at least one endogenous enzyme is involved; and - High requirement for both NADH and NADPH cofactors.

[0011] These factors have a negative impact on the overall process yield, and therefore, resolving these shortcomings is a technical challenge. Summary of the Invention

[0012] The present invention discloses an enzymatic process for preparing a compound of formula I starting from 2,6-lutidine (formula II). The process disclosed herein comprises one step, which involves the presence of an enzyme capable of carrying out the dual oxidation in a selective manner. [ka]

[0013] To address the shortcomings of the method using the wild-type xylene monooxygenase enzyme ppXMO, comprising the XylM and XylA subunits from Pseudomonas putida (Arthrobacter siderocapsulatus), genetic engineering has been used to generate mutant enzymes, which have an amino acid exchange at position 116 of the amino acid sequence of XylM. The key point of the present invention is that the methionine (M) at position 116 in the highly conserved region is replaced with any different amino acid, preferably an amino acid selected from the group consisting of asparagine (N), lysine (K), arginine (R), and glycine (G), which surprisingly leads to the direct methylhydroxylation of 6-methyl-2-pyridinemethanol (Formula III). It has been observed that the mutant enzyme: -Improve overall process yield by at least 50%; - Improve the end product profile (which means fewer by-products are produced), - facilitating process control during pre-synthesis (in bioreactors); -reducing the toxicity of reaction intermediates; - Minimizing / eliminating the need for endogenous reductase involvement, -Minimize / eliminate the need for NADPH and its regeneration.

[0014] Other enzymes related to the XylM component of P. putida (A. siderocapsulatus) with the same amino acid exchange within the highly conserved region around position 116 show similar improved properties and are therefore suitable for preparing compounds of formula I starting from 2,6-lutidine. In some XylM-related enzymes, such as XylM from Halioxenophilus aromaticivorans (GenBank: BBB44451.1), the equivalent position for methionine is 134, whereas in the mutant ntnMO (GenBank: AAC38359.1) derived from the ntnMO wild type, the equivalent position is 116, with a tryptophan (W) instead of methionine. To ensure the retention of enzyme activity and the effect of the described mutations, a 50% homology at the amino acid level with the XylM component of P. putida is required.

[0015] The bis-OH products from the process of the present invention may be used to prepare the respective bis-LG compounds, most commonly 2,6-bis(chloromethyl)pyridine, 2,6-bis(bromomethyl)pyridine, 2,6-bis(mesyloxymethyl)pyridine, and 2,6-bis(tosyloxymethyl)pyridine.

[0016] Furthermore, the above-described method and the enzymes used may be used in methods for preparing other compounds, diagnostic complexes or other products. In a possible embodiment, the method according to the invention may be a step in a method for preparing a pyridine-based tetraazaheterocycle, preferably picrene (3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene). Also, the product of the method according to the invention may be used in a method for preparing a pyridine-based tetraazaheterocycle, preferably picrene.

[0017] Furthermore, the method according to the invention and / or the method for preparing a pyridine-based tetraazaheterocycle, preferably picrene, and / or the product obtained therefrom may be used in methods for preparing various diagnostic conjugates or other compounds. In the most preferred embodiment, the method according to the invention or its product is used to prepare gadopiclenol (C), a contrast agent used in magnetic resonance imaging (MRI) to detect and visualize lesions associated with abnormal angiogenesis in the central nervous system and body. 35 H 54 GdNO 15 , INN, trade name Elucirem). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] definition The following terms have the respective meanings set forth below for purposes of this application, including the claims appended hereto. When reference is made herein to general terms such as enzymes, solvents, etc., it is understood that one of skill in the art can make appropriate selections of such reagents from those provided in the definitions below and additional reagents described subsequently herein or found in references in the art.

[0019] As used herein, the term "enzymatic process" or "enzymatic method" refers to a process or method that uses an enzyme or a microorganism.

[0020] The term "microbial cell" refers to a wild-type microbial cell, a wild-type microbial cell, or a genetically modified unicellular microorganism, also called a genetically modified organism, that serves as a host for producing functional entities (enzymes) involved in enzymatic processes. The terms host and cell are used interchangeably throughout the present invention.

[0021] The term "recombinant cell" indicates that the microbial cell further comprises heterologous DNA encoding enzymatic functions, either supplied genomically integrated or in the form of plasmid DNA.

[0022] The term "feed rate" refers to the amount of a substance (such as glucose, glycerol, lutidine, or any other nutrient, cofactor, etc.) added to a reaction medium during the course of an enzymatic process per unit time and unit volume.

[0023] The term "reaction medium" refers to any growth medium used to carry out a process involving an enzyme. This medium can have starting materials, enzymes (alone or as part of a cell), and products and by-products. Typically, the reaction medium is an aqueous solvent.

[0024] The term "cofactor regeneration system" refers to an enzyme or set of enzymes that uses a biocompatible substrate, such as a standard carbon source (glucose, glycerol), alcohol, or organic acid (e.g., formate), to reduce a biological cofactor, preferably NAD+ to NADH, NADP+ to NADPH, or more preferably NAD+ to NADH.

[0025] The term "formate" refers to the anion produced by the respective salt, such as sodium formate.

[0026] The term "nutrient" refers to organic or inorganic molecules that serve as sources of carbon (e.g., glucose, glycerol), nitrogen (e.g., ammonia, amino acids), phosphorus (phosphate, phytate), and micronutrients (metal ions, vitamins).

[0027] The enzymes used in the present invention are derived from microbial genomes.

[0028] The genes may be codon optimized and prepared synthetically or isolated (e.g., by PCR) from the respective host. For example, they may be cloned (e.g., using restriction enzymes and DNA ligase) into a suitable expression vector or integrated into the genome of a recombinant host to generate a genetically modified host cell.

[0029] Furthermore, in the processes and claims herein, the pronoun "a", when used to refer to a reagent, e.g., "base", "solvent", etc., is intended to mean "at least one" and, therefore, should be understood to include mixtures of reagents as well as single reagents, where appropriate.

[0030] Detailed Description of the Invention The present invention discloses an enzymatic method for preparing a compound of 2,6-bis(hydroxymethyl)pyridine (formula I). ​​Using wild-type ppXMO from P. putida (A. siderocapsulatus) having the amino acid sequence of SEQ ID NO: 1 below, the compound of formula I can be obtained starting from the readily available formula II in the presence of the enzyme in high yield and without the formation of significant amounts of by-products (side products).

[0031] SEQ ID NO:1: MDTLRYYLIPVVTACGLIGFYYGGYWVWLGAATFPALMVLDVILPKDFSARKVSPFFADLTQYLQLPLMIGLYGLLVFGVENGRIELSEPLQV AGCILSLAWLSGVPTLPVSHELMHRRHWLPRKMAQLLAMFYGDPNRDIAHVNTHHLY LDTPLDSDTPYRGQTIYSFVISATVGSVKDAIKIEAETLRRKGQSPWNLSNKTYQYVALLLALPGLVSYLGGPALGLVTIASMIIAKGIVEGFNYFQHYGLVRDLDQPI LLHHAWNHMGTIVRPLGCEITNHINHHIDGYTRFYELRPEKEAPQMPSLFVCFLLGLIPPLWFALIAKPKLRDWDQRYATPGERELAMAANKKAGWPLWCESELGRVASI

[0032] The inventors have surprisingly found that a mutant ppXMO enzyme from P. putida (A. siderocapsulatus), prepared by genetic engineering and carrying an amino acid exchange at position 116 of the amino acid sequence of the XylM component, is capable of direct methyl hydroxylation of 6-methyl-2-pyridinemethanol III. The methionine (M) at position 116 in the wild-type XylM component is replaced by any amino acid different from M, preferably an amino acid selected from the group consisting of asparagine (N), lysine (K), arginine (R) and glycine (G), the preferred choice being glycine (G). The present invention therefore relates to the mutant enzyme as well as to any polynucleic acid encoding the mutant enzyme having the following amino acid sequence:

[0033] SEQ ID NO:2: MDTLRYYLIPVVTACGLIGFYYGGYWVWLGAATFPALMVLDVILPKDFSARKVSPFFADLTQYLQLPLMIGLYGLLVFGVENGRIELSEPLQV AGCILSLAWLSGVPTLPVSHELGHRRHWLPRKMAQLLAMFYGDPNRDIAHVNTHHLY LDTPLDSDTPYRGQTIYSFVISATVGSVKDAIKIEAETLRRKGQSPWNLSNKTYQYVALLLALPGLVSYLGGPALGLVTIASMIIAKGIVEGFNYFQHYGLVRDLDQPI LLHHAWNHMGTIVRPLGCEITNHINHHIDGYTRFYELRPEKEAPQMPSLFVCFLLGLIPPLWFALIAKPKLRDWDQRYATPGERELAMAANKKAGWPLWCESELGRVASI

[0034] SEQ ID NO:3: MDTLRYYLIPVVTACGLIGFYYGGYWVWLGAATFPALMVLDVILPKDFSARKVSPFFADLTQYLQLPLMIGLYGLLVFGVENGRIELSEPLQV AGCILSLAWLSGVPTLPVSHELNHRRHWLPRKMAQLLAMFYGDPNRDIAHVNTHHLYLDTPLDSDTPYRGQTIYSFVISATVGSVKDAIKIEAETLRRKGQSPWNLSNKTYQYVALLLALPGLVSYLGGPALGLVTIASMIIAKGIVEGFNYFQHYGLVRDLDQPI LLHHAWNHMGTIVRPLGCEITNHINHHIDGYTRFYELRPEKEAPQMPSLFVCFLLGLIPPLWFALIAKPKLRDWDQRYATPGERELAMAANKKAGWPLWCESELGRVASI

[0035] SEQ ID NO:4: MDTLRYYLIPVVTACGLIGFYYGGYWVWLGAATFPALMVLDVILPKDFSARKVSPFFADLTQYLQLPLMIGLYGLLVFGVENGRIELSEPLQV AGCILSLAWLSGVPTLPVSHELKHRRHWLPRKMAQLLAMFYGDPNRDIAHVNTHHLY LDTPLDSDTPYRGQTIYSFVISATVGSVKDAIKIEAETLRRKGQSPWNLSNKTYQYVALLLALPGLVSYLGGPALGLVTIASMIIAKGIVEGFNYFQHYGLVRDLDQPI LLHHAWNHMGTIVRPLGCEITNHINHHIDGYTRFYELRPEKEAPQMPSLFVCFLLGLIPPLWFALIAKPKLRDWDQRYATPGERELAMAANKKAGWPLWCESELGRVASI

[0036] SEQ ID NO:5: MDTLRYYLIPVVTACGLIGFYYGGYWVWLGAATFPALMVLDVILPKDFSARKVSPFFADLTQYLQLPLMIGLYGLLVFGVENGRIELSEPLQV AGCILSLAWLSGVPTLPVSHELRHRRHWLPRKMAQLLAMFYGDPNRDIAHVNTHHLY LDTPLDSDTPYRGQTIYSFVISATVGSVKDAIKIEAETLRRKGQSPWNLSNKTYQYVALLLALPGLVSYLGGPALGLVTIASMIIAKGIVEGFNYFQHYGLVRDLDQPI LLHHAWNHMGTIVRPLGCEITNHINHHIDGYTRFYELRPEKEAPQMPSLFVCFLLGLIPPLWFALIAKPKLRDWDQRYATPGERELAMAANKKAGWPLWCESELGRVASI

[0037] The underlined portions of the sequence above indicate highly conserved regions, and the substitution of M at position 116 results in a significant change in the enzyme properties compared to the wild type. In some ppXMO-related enzymes, such as hoXMO, the equivalent position for methionine is 134, whereas in ntnMO the equivalent position is 116, with a tryptophan (W) instead of a methionine. The effects of the mutations are the same or very similar. Mutant enzymes have been observed to: - improving the overall yield of the pre-synthesis process (especially in bioreactors), typically improving yield by at least 50%; - Improve the end product profile (which means fewer by-products are produced), - facilitating process control during pre-synthesis (in bioreactors); - reducing the problem of toxicity of reaction intermediates, which is a direct consequence of the mutant enzyme being able to simultaneously convert compounds of formula I and III (in contrast to the wild type, which must first deplete compound II before starting the conversion of compound III); -Minimizing the need for endogenous enzyme involvement, -Minimize / eliminate the need for NADPH and its regeneration.

[0038] The less favorable and favorable mutations at position 116 can be further improved when combined with other mutations in XylMA.

[0039] Other enzymes related to ppXMO from P. putida (A. siderocapsulatus) that have the same amino acid exchange of methionine in a highly conserved region (equivalent to position 116 in the XylM component) show similar improved properties and are suitable for use in the preparation of compounds of formula I starting from 2,6-lutidine. More preferably, the oxidoreductase is a xylene monooxygenase enzyme encoded by the xylM and xylA genes of P. putida (A. siderocapsulatus), or a XylMA-like enzyme of Alteromonas macleodii, or Tepidiphilus succinatimandens, or Novosphingobium kunmingense, or Hyphomonas oceanitis, or Sphingobium sp. 32-64-5, or Halioxenophilus aromaticivorans, or a XylM-like enzyme having greater than 50% sequence identity at the amino acid level with the entire XylM sequence. More preferably, the oxidoreductase is the xylene monooxygenase enzyme ppXMO encoded by the xylM and xylA genes of Pseudomonas putida. Alternatively, a preferred option is the XylMA-like enzyme from H. aromaticivorans having the amino acid sequence of SEQ ID NO: 6:

[0040] SEQ ID NO:6: MDTIRYYLIPLVSACGALGFYYGGDWVWLGAATFPSLMILDVLLPRDYEERKVSPFFADLTQYLQLPLMIAMYGFLIFGVREGRIDLGEPVQFLGSILSLAWLSGVPTLPVSHELMHRRHWLPRRMAQLLATFYGDPNRDIAHVNTHHLELDTPLDSDTPFRGQTMYSFVVSATVGSVMDA AKIEAETLRRKGKSPWHLSNKMYQYVMLLIALPGVVTYFGGAESGLVTIISMLIAKAIVEGFNYFQHYGLVREIGHPILLHHAWNHMGMIVRPLGCEITNHINHHLDGYTRFYYLHPEKEAPQMPSLFLCFLLGLVPPLWENLVAKPKLKDWDLQYATPGERKLAMEANKNAGWPQWIPEAA

[0041] The process for converting 2,6-lutidine II to 2,6-bis(hydroxymethyl)pyridine I is carried out using the mutant enzymes described above. [ka] The disclosed enzymes can be used in the disclosed methods according to techniques well known to those of skill in the art. The enzymes may be used as part of the cell that produces them (whole cell catalysts) or in vitro, where the enzymes are available and used in the reaction medium under appropriate reaction conditions.

[0042] In a preferred embodiment, the enzyme according to the invention is expressed in a microbial host, which may then be referred to as a recombinant microbial host. The recombinant host may be further tailored by genetic engineering. Preferred microbial hosts are Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Pseudomonas putida, Rhodobacter sphaeroides, Streptomyces spp, Propionibacterium shermanii, Ketogulonigenium vulgare, Acinetobacter baylyi, and Halomonas bluephagenesis. More preferred is Escherichia coli.

[0043] Those skilled in the art are familiar with techniques for expressing specific enzymes in microbial hosts, which are exemplified in relevant texts such as "Methods in Enzymology" (Book series, Elsevier, ISSN 0076-6879) or "Molecular Cloning" (ISBN 978-1-936113-42-2).

[0044] When a wild-type enzyme is used, the enzymatic process proceeds at least in part through the formation of 6-methyl-2-hydroxypyridine III. When the enzyme is a xylene monooxygenase enzyme, the enzymatic conversion of the compound of formula II to the compound of formula I proceeds through the formation of the compound of formula IV in addition to the compound of formula III. [ka]

[0045] It is beneficial to keep 2,6-lutidine II at a suitable feed rate to maintain the balance between the various conversions occurring within the enzymatic process.The feed rate does not need to be constant, as long as it is adjusted according to the following embodiment.The feed rate also needs to be at an appropriate level so as not to reach growth-inhibiting levels of 2,6-lutidine II.

[0046] In a preferred embodiment, the supply rate of 2,6-lutidine II in the reaction medium is adjusted so that the concentration of 2,6-lutidine II in the reaction medium does not exceed 1 g / L, preferably 0.1 g / L, more preferably 0.02 g / L.

[0047] In another preferred embodiment, the feed rate of 2,6-lutidine II in the reaction medium is adjusted so that the concentration of 2,6-lutidine II does not fall below a value of 10 mg / L, preferably 0.1 mg / L, more preferably 0.01 mg / L.

[0048] The method of the present invention is carried out in an aqueous medium, which may be water or deionized water, which may further include buffers and nutrients.

[0049] The weight of biomass used in the process may be adjusted according to the general knowledge of the person skilled in the art.

[0050] The temperature of the reaction medium may be such that the enzyme retains its enzymatic activity, and is preferably maintained at 25-37°C, and most preferably at 28-32°C.

[0051] The pH may be such that the enzyme and recombinant strain retain their enzymatic activity, preferably the pH is 6.0 to 8.0, more preferably 6.5 to 7.5, and even more preferably 7.0±0.1.

[0052] The dissolved oxygen tension (DOT) must be maintained above 0%. As the recombinant cells grow and biomass accumulates in the bioreactor, the DOT decreases, and decreases even more drastically when the substrate 2,6-lutidine II is added. Therefore, it is important to maintain the DOT above 0%, or better, above 3-5%, for the biocatalytic reaction to occur. The DOT can be controlled by the energy input used to mix the aqueous medium velocity, the rate at which the bioreactor is aerated, or by supplementing the supplied air with oxygen.

[0053] The supply rate of carbon sources such as glucose and glycerol can be adjusted according to the general knowledge of those skilled in the art.

[0054] The reaction time may vary depending on the amount of enzyme and its specific activity. Furthermore, it may be further adjusted by the temperature or other conditions of the enzyme reaction that are familiar to those skilled in the art. Typical reaction times range from 1 hour to 72 hours.

[0055] In another embodiment, the process of converting 2,6-lutidine II to 2,6-bis(hydroxymethyl)pyridine I optionally involves the presence of a dehydrogenase. The conversion may be carried out directly in the microbial cell without further engineering of a housekeeping dehydrogenase. In yet another embodiment, the microbial cell further synthesizes a dehydrogenase from another microbial cell. In yet another embodiment, one or more housekeeping dehydrogenases are inactivated or engineered.

[0056] In a preferred embodiment, the microbial cell further synthesizes a dehydrogenase enzyme from another microbial cell, and one or more housekeeping dehydrogenases are inactivated or engineered.

[0057] The enzyme which catalyzes the oxidative conversion of the methyl group of 2,6-lutidine II to the respective hydroxymethyl group of 2,6-bis(hydroxymethyl)pyridine I and is used in this embodiment is according to the previous embodiment.

[0058] To the extent that one arrives at a particular combination of enzymes, their expression in the same microbial host is a technique well known to those skilled in the art, references of which are provided above.

[0059] In a preferred embodiment, the dehydrogenase is NAD(P)H-dependent or NADH-dependent, preferably NADH-dependent.

[0060] In another preferred embodiment, the dehydrogenase catalyzes the reduction of 6-methylpyridine-2-carboxaldehyde IV to 6-methyl-2-hydroxypyridine III or the reduction of 6-(hydroxymethyl)-2-pyridinecarbaldehyde V to 2,6-bis(hydroxymethyl)pyridine I. Preferably, the dehydrogenase catalyzes both the reduction of 6-methylpyridine-2-carboxaldehyde IV to 6-methyl-2-hydroxypyridine III and the reduction of 6-(hydroxymethyl)-2-pyridinecarbaldehyde V to 2,6-bis(hydroxymethyl)pyridine I. [ka]

[0061] In another preferred embodiment, the dehydrogenase is selected from the list: AKR from Kluyveromyces lactis, XylB from Acinetobacter baylyi ADP1, and AFPDH from Candida maris.

[0062] In another embodiment, a process is provided for converting 2,6-lutidine II to 2,6-bis(hydroxymethyl)pyridine I, the conversion being carried out in the presence of an enzyme that catalyzes the oxidative conversion of the methyl groups of 2,6-lutidine II to the respective hydroxymethyl groups of 2,6-bis(hydroxymethyl)pyridine I, and additionally in the presence of a cofactor regenerating system.

[0063] The enzyme which catalyzes the oxidative conversion of the methyl group of 2,6-lutidine II to the respective hydroxymethyl group of 2,6-bis(hydroxymethyl)pyridine I and is used in this embodiment is according to the previous embodiment.

[0064] As disclosed in the previous embodiment, the conversion may be carried out directly in the microbial cells without further manipulation of the housekeeping dehydrogenases.

[0065] In yet another embodiment, the microbial cell further synthesizes a dehydrogenase enzyme from another microbial cell.

[0066] In yet another embodiment, one or more housekeeping dehydrogenases are inactivated or engineered.

[0067] In a preferred embodiment, the microbial cell further synthesizes a dehydrogenase enzyme from another microbial cell, and one or more housekeeping dehydrogenases are inactivated or engineered.

[0068] The dehydrogenase used in this embodiment is according to the above-mentioned embodiment.

[0069] The cofactor may be NAD(P)H or NADH and the regenerating system is an NAD(P)H or NADH regenerating system. Preferably, the regenerating system is an NADH regenerating system.

[0070] The regeneration system is preferably co-expressed in the same microbial host that expresses the enzymes that catalyze the oxidative conversion. In a more preferred embodiment, the same microbial host also co-expresses the dehydrogenases, as described in the previous embodiment.

[0071] Cofactors are non-proteinaceous compounds that play an important role in many enzyme-catalyzed biochemical reactions. Cofactors function to transfer chemical groups between enzymes. Nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) and the reduced forms of the molecules (NADH and NADPH, respectively) are biological cofactors that play a central role in cellular metabolism, functioning as electron transfer agents. The oxidized forms NAD+ and NADP+ function as electron acceptors and are reduced during the process. On the other hand, NADH and NADPH act as reducing agents and are oxidized during the process. Most of the enzymes that mediate oxidation or reduction reactions depend on cofactors such as NADPH or NADH. Cofactor regeneration systems are used to ensure that the cofactors involved in a given bioprocess are not depleted and / or to reduce the overall cost of the process.

[0072] In a preferred embodiment, the NADH regeneration system is a formate dehydrogenase regeneration system.

[0073] In another preferred embodiment, the NADH regeneration system is a formate dehydrogenase-based system, more preferably a cytosolic dehydrogenase that is not sensitive to oxygen.

[0074] In another preferred embodiment, the NADH regenerating system active towards NAD+ species is composed of a metal-independent formate dehydrogenase of bacterial or fungal origin.

[0075] Preferably, the metal-independent formate dehydrogenase active towards NAD+ species is derived from Candida tropicalis or Mycobacterium vaccae formate dehydrogenase (FDH).

[0076] In a preferred embodiment, formate is provided to the process, as defined in any of the previous embodiments, to regenerate NADH consumed by the enzyme, dehydrogenase, or both, that catalyzes the oxidative conversion of the methyl group of 2,6-lutidine II to the respective hydroxymethyl group of 2,6-bis(hydroxymethyl)pyridine I. Preferably, formate is provided to the process to regenerate NADH consumed by the oxidoreductase, dehydrogenase, or both.

[0077] In a preferred embodiment, the feed rate of formate in the reaction medium is adjusted so that the concentration of formate does not exceed a value of 150 mM, preferably 100 mM, more preferably 50 mM.

[0078] In another preferred embodiment, the feed rate of formate in the reaction medium is adjusted so that the concentration of formate in the reaction medium does not fall below a value of 50 mM, preferably 25 mM, more preferably 5 mM.

[0079] In a more preferred embodiment, the supply rate of formate in the reaction medium is adjusted so that the concentration of 2,6-lutidine II in the reaction medium does not exceed a value of 150 mM and does not fall below a value of 50 mM, preferably 25 mM, more preferably 5 mM.

[0080] In another preferred embodiment, the feed rate of formate in the reaction medium is adjusted so that the concentration of formate in the reaction medium does not exceed a value of 100 mM and does not fall below a value of 50 mM, preferably 25 mM, more preferably 5 mM.

[0081] In another preferred embodiment, the feed rate of formate in the reaction medium is adjusted so that the concentration of formate in the reaction medium does not exceed a value of 50 mM and does not fall below a value of 50 mM, preferably 25 mM, more preferably 5 mM. [Brief description of the drawings]

[0082] The invention will be further explained on the basis of the examples and figures showing:

[0083] [Figure 1] Figure 1. Time course of 2,6-bis(hydroxymethyl)pyridine formation from 2,6-lutidine catalyzed by wild-type monooxygenase XylMA from P. putida (squares) and mutant XylMA with M replaced by G at position 116 (triangles). B. Whole-cell biotransformation of 1 g / L 2,6-lutidine II to 2,6-bis(hydroxymethyl)pyridine I by XMO wild-type (WT) and mutants with M at position 116 replacement. For simplicity, only the concentration of target product I is plotted over the course of the biotransformation. [Diagram 2] Figure 2. HPLC-UV chromatograms resulting from overnight laboratory-scale biotransformation using wild-type monooxygenase XylMA from P. putida (left) and mutant XylMA with an M to G exchange at position 116 (right). The peak area corresponds to the by-product 6-methyl-2-pyridine carboxylic acid V. [Diagram 3] Figure 3. Reaction scheme for the conversion of 2,6-lutidine II to 2,6-bis(hydroxymethyl)pyridine I. Wild-type XylMA (top) and mutant XylMA (bottom), unlike the wild-type, efficiently catalyze the reaction via the direct hydroxylation of 6-methyl-2-hydroxypyridine III. [Figure 4] Figure 4. Accumulation of intermediates and products in the preparative synthesis of 2,6-bis(hydroxymethyl)pyridine I by XylMA wild type (left) and XylMA mutants (right). [Figure 5-1] FIG. 5 Multiple sequence alignment showing the highly conserved features of the region around M116 in XylM from P. putida and sequences with over 50% homology to the protein. [Figure 5-2] FIG. 5 Multiple sequence alignment showing the highly conserved features of the region around M116 in XylM from P. putida and sequences with over 50% homology to the protein. [Figure 6]Figure 6 HPLC chromatograms of overnight conversion of 0.5 g / L of compound III by two related proteins (GenBank: BBB44451.1 (hoXMO wild type) and GenBank: AAC38359.1 (ntnMA wild type)) and two side directed mutagenesis mutants harboring a mutation at position 116. [Figure 7] Fig. 7 Phylogenetic relationships among different XylM and XylM-like enzymes and their enzymatic activities Example 1

[0084] Example 1: Genetic engineering of wild-type XylM protein Wild-type amino acid sequence >sp|P21395|XYLM_PSEPU Xylene monooxygenase subunit 1 OS=Pseudomonas putida OX=303 GN=xylM PE=3 SV=1

[0085] MDTLRYYLIPVVTACGLIGFYYGGYWVWLGAATFPALMVLDVILPKDFSARKVSPFFADLTQYLQLPLMIGLYGLLVFGVENGRIELSEPLQVAGCILSLAWLSGVPTLPVSHELMHRRHWLPRKMAQLLAMFYGDPNRDIAHVNTHHLYLDTPLDSDTPYRGQTIYSFVISATVGSVKDAIKI EAETLRRKGQSPWNLSNKTYQYVALLLALPGLVSYLGGPALGLVTIASMIIAKGIVEGFNYFQHYGLVRDLDQPILLHHAWNHMGTIVRPLGCEITNHINHHIDGYTRFYELRPEKEAPQMPSLFVCFLLGLIPPLWFALIAKPKLRDWDQRYATPGERELAMAANKKAGWPLWCESELGRVASI https: / / www.uniprot.org / uniprot / P21395

[0086] Manipulation of the gene sequence of SEQ ID NO:1 with the aim of introducing random mutations was carried out using standard techniques of random mutagenesis, i.e. error-prone PCR amplification of the xylM gene using a mutagenic DNA polymerase. The resulting PCR product was then inserted into pBR322 origin of replication, the kan gene encoding the kanamycin resistance protein, and the inducible P gene for XylMA induction with dicyclopropylketone (DCPK) suitable for protein expression. alkS The mutants were cloned into a promoter-containing vector backbone and transformed into an expression strain by electroporation. The presence of the random mutations was confirmed by DNA sequencing. A library of over 50,000 unique mutants was generated.

[0087] Screening for target activity was performed using MALDI-MS. This protocol allowed the measurement of 384 samples (one 384-well microtiter plate) in approximately 20 minutes, thereby allowing the screening of over 20,000 clones from the library. Mutants from wells showing the highest signal corresponding to the product of interest were screened again in 96-well plates, product formation was quantified by HPLC-UV, and mutations contained in the gene sequence were identified by DNA sequencing.

[0088] Example 2: Conversion of lutidine by recombinant E. coli expressing XylMA protein in shake flasks The polynucleotide sequences of the xylM and xylA genes of Pseudomonas putida (Arthrobacter siderocapsulatus), encoding the multicomponent xylene monooxygenase XylMA, were cloned into pBR322 replication origin, the kan gene encoding the kanamycin resistance protein, and the inducible P gene for XylMA induction by dicyclopropyl ketone (DCPK). alkSThe promoter was cloned into a plasmid containing the promoter, transformed into the E. coli BL21 host by electroporation, and plated on LB agar plates supplemented with kanamycin. After overnight incubation at 37°C, single colonies were picked and grown in 4 mL of LB growth medium at 37°C for 12–14 h on a shaker at 200 rpm. The following day, the overnight culture in LB was inoculated into the main culture in minimal medium containing 4.5 g / L KH2PO4, 6.3 g / L Na2HPO4, 2.3 g / L (NH4)2SO4, 1.9 g / L NH4Cl, 1 g / L citric acid, 20 mg / L thiamine, 10 g / L glycerol, 55 mg / L CaCl2, 240 mg / L MgSO4, 1× trace elements (0.5 mg / L CaCl2·2H2O, 0.18 mg / L ZnSO4·7H2O, 0.1 mg / L MnSO4·H2O, 20.1 mg / L Na2-EDTA, 16.7 mg / L FeCl3·6H2O, 0.16 mg / L CuSO4·5H2O), and 50 mg / L kanamycin (adjusted to pH 7 with NH4OH). The starting optical density (OD600) of 20 mL main cultures in 100 mL shake flasks was adjusted to 0.05 and the flasks were incubated at 37°C and 200 rpm until the OD reached 0.6-0.8, then 0.025% DCPK was added and the cultures were further incubated at 30°C and 200 rpm for an additional hour or until the OD reached 1. At the target OD, various sub-growth inhibitory concentrations of 2,6 lutidine II were added to the cells and the cultures were further incubated until complete substrate conversion was achieved and cell growth ceased for at least 2 hours. Reaction progress was monitored and quantified at 270 nm using a RP-HPLC equipped with a C18 column and specific activity ranges of 0.3-0.6 g / gCDW / h were calculated for the individual reactions catalyzed by whole cells. The results showed the production of 1.25 g / L of hydroxylated products (93% 2,6-bis(hydroxymethyl)pyridine I, 5-7% 6-methyl-2-pyridinecarboxylic acid V).

[0089] FIG. 1 shows the progress of the lab-scale reaction and the rate of accumulation of the target product when the reaction is catalyzed by wild-type monooxygenase (squares in FIG. 1a and triangles in FIG. 1b) versus different monooxygenase mutants. FIG. 1a shows the results for the mutant with an amino acid exchange from M to G, where the desired reaction product, 2,6-bis(hydroxymethyl)pyridine, begins to accumulate sooner and reaches a maximum concentration sooner (about 2 hours earlier). FIG. 1b shows a comparison between the individual preferred mutant monooxygenases, with higher reaction rates and final yields for the mutants with amino acid sequences of SEQ ID NO:2 and SEQ ID NO:3. The other two mutants with amino acid sequences of SEQ ID NO:4 and SEQ ID NO:5 have lower yields and lower reaction rates, but the amount of toxic by-products is still greatly reduced compared to the wild-type, and therefore these mutants are still preferred over the wild-type.

[0090] Figure 2 shows the difference in the final product profile, i.e., HPLC-UV chromatograms produced by the wild-type monooxygenase (left) and the mutant monooxygenase with an M to G amino acid exchange (and thus the sequence of SEQ ID NO: 2) (right). The chromatograms were obtained from overnight shake flask biotransformation as described above. The peak area of ​​the peak corresponding to the by-product 6-methyl-2-pyridine carboxylic acid V is much smaller in the product profile obtained with the mutant enzyme, indicating superior catalysis, improved efficiency, and lower toxicity.

[0091] FIG. 3 shows the difference in the reaction scheme for the conversion of 2,6-lutidine II to 2,6-bis(hydroxymethyl)pyridine I by XylMA wild-type (top) and the XylMA mutant (sequence number 2, bottom), where, unlike the wild-type, the XylMA mutant efficiently catalyzes the reaction via direct hydroxylation of 6-methyl-2-hydroxypyridine III.

[0092] Example 3: Conversion of 2,6-lutidine by recombinant E. coli expressing XylMA protein in a bioreactor The microbial strain, medium, and growth conditions up to inoculation of the main culture are the same as in Example 1. However, in this example, the main culture is prepared in a bioreactor where parameters such as temperature, pH, dissolved oxygen tension, mixing, and glucose availability can be controlled, which allows for batch fermentation. The pH fluctuations are maintained by appropriate addition of ammonium hydroxide or sulfuric acid controlled by a pH stat. In the batch phase of the fermentation, 1 L of growth medium (same as in Example 1) is inoculated at a starting OD600 value of 0.025 and the cells are grown at 30°C for 12-13 hours or until the cells have completely consumed the initially provided carbon source (e.g., glucose or glycerol), as indicated by a sudden rise in dissolved oxygen in the bioreactor. At this stage, the fed-batch phase of fermentation was started by adding the appropriate glucose from a 500 g / L glucose stock supplemented with 1x trace elements, 1x kanamycin, and 240 mg / L MgSC4 at an appropriate feed rate and incubated for 0.31 h until an OD600 of 35 was reached when 0.05% DCPK was added. -1 A growth rate of 0.025 h was maintained. One hour after induction with DCPK, 2,6-lutidine II was added to the bioreactor (feed rate: 0.1 mL / L broth / min) and the reaction was allowed to proceed for 14–18 h. Once the first feed amount was converted to 2,6-bis(hydroxymethyl)pyridine I, a second addition of 2,6-lutidine II could be made and the reaction was continued until conversion was complete or for 0.025 h. -1 The reaction was allowed to proceed as long as the growth rate of the larger cells was maintained. Up to 20 g / L of total product (90% 2,6-bis(hydroxymethyl)pyridine I, 10% 6-methyl-2-pyridinecarboxylic acid V) could be produced within 20 h of biotransformation.

[0093] FIG. 4 shows the accumulation of intermediates and products in the preliminary synthesis of 2,6-bis(hydroxymethyl)pyridine I by XylMA wild type (left) and XylMA mutant (right). The formation of the main product, by-products, and intermediates formed during the biotransformation of 2,6-lutidine II catalyzed by wild type monooxygenase and monooxygenase mutant M116G is shown separately. The preliminary reaction catalyzed by mutant M116G yielded about 30% more product under non-optimized bioprocess conditions. With optimization of bioprocess conditions and use of mutant M116G, it is predicted to be more than 50%. Furthermore, the average rate of target product formation increases from 0.8 g / L / h (monooxygenase wild type) to 1 g / L / h (monooxygenase M116G mutant), and compounds II and III are converted simultaneously by the mutant, rather than hierarchically. Finally, the formation of by-products is significantly reduced in the mutant (from about 5% (wild type) to less than 1% (mutant M116G)).

[0094] Example 4: Conversion of 2,6-lutidine by recombinant E. coli expressing XylMA-like proteins Introduction of an exchange at the functionally equivalent position of M116 in P. putida has a similar effect on biotransformation efficiency. We have shown this using a xylM-like gene from H. aromaticivorans. H. aromaticivorans mutants carrying a mutation at the equivalent position to M116 showed improved reaction rates, higher yields, and simultaneous (but not hierarchical) conversion of compounds of formula II and formula III.

[0095] Figure 5 shows a multiple sequence alignment showing that the region around M116 of XylM in P. putida (highlighted by a box in the figure) is highly conserved among related sequences with sequences with more than 50% identity to XylM in P. putida. To show that the replacement of methionine or equivalent at position 116 causes the same effect in related enzymes, two mutant enzymes were prepared by targeted mutagenesis and expressed in an E. coli host with reduced aromatic aldehyde reduction capacity (E. coli RARE). HPLC chromatograms of overnight, i.e., 1020 min, reactions of the conversion of 0.5 g / L of compound III by two related proteins (GenBank: BBB44451.1 (hoXMOWT) and GenBank: AAC38359.1 (ntnMOWT)) and two site-directed mutagenesis mutants with mutations at position 116 are shown. In the aforementioned E. coli host, the desired product I can only be formed by direct hydroxylation of the free methyl group of compound III catalyzed by the tested enzymes (two WT and two mutants). In both cases, as in the case of mutant XylM from P. putida, the amino acid exchange in the mutant enzymes significantly improves the formation of the desired product. In the figure, only the peaks corresponding to the substrate (grey arrow) and the desired product (black arrow) are shown. The additional peaks corresponding to the peroxidation products (aldehydes, acids) are not explicitly highlighted. While both mutants show an increase in the desired product, the amount of reactant is significantly reduced.

[0096] Figures 6 and 7 show the results for various XylM-like enzymes with at least 50% homology to the P. putida enzyme. As shown in Figure 6, HPLC chromatograms of overnight conversion of 0.5 g / L of compound III by two related proteins (GenBank: BBB44451.1 (hoXMO wild type) and GenBank: AAC38359.1 (ntnMA wild type)) and two position-induced mutagenesis variants with mutations at position 116 showed improved product yields and fewer by-products. Similarly, Figure 7 shows data for many more XylM-like enzymes, namely from the following species: Hyphomonas oceanitis, Sphingobium sp., Novosphingobium kunmingense, Pseudomonas TW3, Halioxenophilus aromaticivorans, Alteromonas macleodii, and Tepidiphilus succinatimandens, all of which show similar enzymatic activity to XylM from P. putida. Since the amino acid sequence homology is at least 50%, these results indicate that this is sufficiently homologous to retain enzymatic activity and show yield improvement due to the above mutations.

[0097] These results confirm the importance and effectiveness of the amino acid exchange at position 116 or an equivalent position where methionine or tryptophan resides in the wild-type enzyme.

[0098] Sequence Listing SEQ ID NO:1: MDTLRYYLIPVVTACGLIGFYYGGYWVWLGAATFPALMVLDVILPKDFSARKVSPFFADLTQYLQLPLMIGLYGLLVFGVENGRIELSEPLQVAGCILSLAWLSGVPTLPVSHELMHRRHWLPRKMAQLLAMFYGDPNRDIAHVNTHHLYLDTPLDSDTPYRGQTIYSFVISATVGSVKDAIKI EAETLRRKGQSPWNLSNKTYQYVALLLALPGLVSYLGGPALGLVTIASMIIAKGIVEGFNYFQHYGLVRDLDQPILLHHAWNHMGTIVRPLGCEITNHINHHIDGYTRFYELRPEKEAPQMPSLFVCFLLGLIPPLWFALIAKPKLRDWDQRYATPGERELAMAANKKAGWPLWCESELGRVASI

[0099] SEQ ID NO:2: MDTLRYYLIPVVTACGLIGFYYGGYWVWLGAATFPALMVLDVILPKDFSARKVSPFFADLTQYLQLPLMIGLYGLLVFGVENGRIELSEPLQVAGCILSLAWLSGVPTLPVSHELGHRRHWLPRKMAQLLAMFYGDPNRDIAHVNTHHLYLDTPLDSDTPYRGQTIYSFVISATVGSVKDAIKI EAETLRRKGQSPWNLSNKTYQYVALLLALPGLVSYLGGPALGLVTIASMIIAKGIVEGFNYFQHYGLVRDLDQPILLHHAWNHMGTIVRPLGCEITNHINHHIDGYTRFYELRPEKEAPQMPSLFVCFLLGLIPPLWFALIAKPKLRDWDQRYATPGERELAMAANKKAGWPLWCESELGRVASI

[0100] SEQ ID NO:3: MDTLRYYLIPVVTACGLIGFYYGGYWVWLGAATFPALMVLDVILPKDFSARKVSPFFADLTQYLQLPLMIGLYGLLVFGVENGRIELSEPLQVAGCILSLAWLSGVPTLPVSHELNHRRHWLPRKMAQLLAMFYGDPNRDIAHVNTHHLYLDTPLDSDTPYRGQTIYSFVISATVGSVKDAIKI EAETLRRKGQSPWNLSNKTYQYVALLLALPGLVSYLGGPALGLVTIASMIIAKGIVEGFNYFQHYGLVRDLDQPILLHHAWNHMGTIVRPLGCEITNHINHHIDGYTRFYELRPEKEAPQMPSLFVCFLLGLIPPLWFALIAKPKLRDWDQRYATPGERELAMAANKKAGWPLWCESELGRVASI

[0101] SEQ ID NO:4: MDTLRYYLIPVVTACGLIGFYYGGYWVWLGAATFPALMVLDVILPKDFSARKVSPFFADLTQYLQLPLMIGLYGLLVFGVENGRIELSEPLQVAGCILSLAWLSGVPTLPVSHELKHRRHWLPRKMAQLLAMFYGDPNRDIAHVNTHHLYLDTPLDSDTPYRGQTIYSFVISATVGSVKDAIKI EAETLRRKGQSPWNLSNKTYQYVALLLALPGLVSYLGGPALGLVTIASMIIAKGIVEGFNYFQHYGLVRDLDQPILLHHAWNHMGTIVRPLGCEITNHINHHIDGYTRFYELRPEKEAPQMPSLFVCFLLGLIPPLWFALIAKPKLRDWDQRYATPGERELAMAANKKAGWPLWCESELGRVASI

[0102] SEQ ID NO:5: MDTLRYYLIPVVTACGLIGFYYGGYWVWLGAATFPALMVLDVILPKDFSARKVSPFFADLTQYLQLPLMIGLYGLLVFGVENGRIELSEPLQVAGCILSLAWLSGVPTLPVSHELRHRRHWLPRKMAQLLAMFYGDPNRDIAHVNTHHLYLDTPLDSDTPYRGQTIYSFVISATVGSVKDAIKI EAETLRRKGQSPWNLSNKTYQYVALLLALPGLVSYLGGPALGLVTIASMIIAKGIVEGFNYFQHYGLVRDLDQPILLHHAWNHMGTIVRPLGCEITNHINHHIDGYTRFYELRPEKEAPQMPSLFVCFLLGLIPPLWFALIAKPKLRDWDQRYATPGERELAMAANKKAGWPLWCESELGRVASI

[0103] SEQ ID NO:6: MDTIRYYLIPLVSACGALGFYYGGDWVWLGAATFPSLMILDVLLPRDYEERKVSPFFADLTQYLQLPLMIAMYGFLIFGVREGRIDLGEPVQFLGSILSLAWLSGVPTLPVSHELMHRRHWLPRRMAQLLATFYGDPNRDIAHVNTHHLELDTPLDSDTPFRGQTMYSFVVSATVGSVMDA AKIEAETLRRKGKSPWHLSNKMYQYVMLLIALPGVVTYFGGAESGLVTIISMLIAKAIVEGFNYFQHYGLVREIGHPILLHHAWNHMGMIVRPLGCEITNHINHHLDGYTRFYYLHPEKEAPQMPSLFLCFLLGLVPPLWENLVAKPKLKDWDLQYATPGERKLAMEANKNAGWPQWIPEAA

Claims

1. 1 or an enzyme having at least 50% homology with said sequence at the amino acid level, said homology ensuring the enzymatic activity of said enzyme, said protein having a mutation at position 116 or an equivalent position, said mutation being a replacement of methionine (M) or tryptophan (W) by a different amino acid.

2. 2. The enzyme according to claim 1, wherein the M or W at position 116 or an equivalent position is replaced by an amino acid selected from the group consisting of G, N, R or K, preferably G.

3. 3. An enzyme according to claim 1 or 2, having further mutations and / or deletions.

4. The enzyme is the XylMA enzyme of Pseudomonas putida, or - Alteromonas macleodii, or Tepidiphilus succinatimandens, or Novosphingobium cummingense, or Hyphomonas oceanitis, or Sphingobium sp. 32-64-5, or Halioxenophilus aromaticivorans or - a XylMA-like enzyme having a sequence identity of more than 50% with SEQ ID NO:1 at the amino acid level The enzyme according to any one of claims 1 to 3.

5. A nucleic acid encoding the enzyme according to any one of claims 1 to 4.

6. An expression vector comprising the nucleic acid of claim 5.

7. A host cell comprising an expression vector that expresses the nucleic acid and / or enzyme according to any one of claims 1 to 6.

8. The host cell according to claim 7, wherein the host cell is a microbial cell, preferably a bacterial cell.

9. The host cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Pseudomonas putida, Rhodobacter sphaeroides, Streptomyces spp, Propionibacterium shermanii, Ketogulonigenium vulgare, and the like.

9. The host cell of claim 8, which is a cell of Acinetobacter vulgare, Acinetobacter baylyi, Halomonas bruephagenesis, and most preferably a cell of Escherichia coli.

10. Use of an enzyme, a nucleic acid and / or a host cell according to any one of claims 1 to 9 in a process for converting 2,6-lutidine II to 2,6-bis(hydroxymethyl)pyridine I.

11. A process for converting 2,6-lutidine II to 2,6-bis(hydroxymethyl)pyridine I, comprising the steps of: 【Chemistry 1】 A process, characterized in that the conversion is carried out in the presence of an enzyme and an enzyme or a host cell according to any one of claims 1 to 10 is used.

12. 12. The process according to claim 11, wherein the feed rate of 2,6-lutidine II in the reaction medium is adjusted so that the concentration of 2,6-lutidine II in the reaction medium does not exceed a value of 1 g / L, preferably 0.1 g / L, more preferably 0.02 g / L, and the feed rate of 2,6-lutidine II in the reaction medium is adjusted so that the concentration of 2,6-lutidine II in the reaction medium does not fall below a value of 10 mg / L, preferably 0.1 mg / L, more preferably 0.01 mg / L.

13. The process according to any one of claims 8 to 12, wherein a dehydrogenase is used, said dehydrogenase being preferably co-expressed in a microbial host.

14. 14. The process according to claim 13, wherein the dehydrogenase is NADH-dependent, NADP-dependent, NADPH-dependent or GDH-dependent, preferably selected from the list of AKR from Kluyveromyces lactis, XylB from Acinetobacter baylyi ADP1 and AFPDH from Candida maris.

15. 15. The process according to claim 13 or 14, wherein an NADH regeneration system, an NADP regeneration system, an NADPH regeneration system or a GDH regeneration system is co-expressed in the microbial host, the NADH regeneration system being preferably a formate dehydrogenase-based system, the NADH regeneration system being preferably composed of a metal-independent formate dehydrogenase active on NAD+ species of bacterial or fungal origin.

16. 16. The process according to any one of claims 12 to 15, wherein the feed rate of formate is adjusted so that the concentration of formate in the reaction medium does not exceed a value of 150 mM, preferably 100 mM, more preferably 50 mM, and wherein the feed rate of formate is adjusted so that the concentration of formate in the reaction medium does not fall below a value of 50 mM, preferably 25 mM, more preferably 5 mM.

17. Use of the product of the process according to any one of claims 1 to 16 in the preparation of other compounds, diagnostic conjugates, most preferably 2-[3,9-bis[1-carboxylato-4-(2,3-dihydroxypropylamino)-4-oxobutyl]-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-trien-6-yl]-5-(2,3-dihydroxypropylamino)-5-oxopentanoate; gadolinium(3+), 2,6-bis(chloromethyl)pyridine, 2,6-bis(bromomethyl)pyridine, 2,6-bis(mesyloxymethyl)pyridine and 2,6-bis(tosyloxymethyl)pyridine.

18. 17. A process for preparing a pyridine-based tetraazaheterocycle, preferably 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene, i.e. picrene, comprising the method and / or product of the method according to any one of claims 1 to 16.

19. A process for preparing various diagnostic conjugates or other compounds comprising the method and / or product of the method of any one of claims 1 to 16.

20. 20. The process according to claim 19, which is the preparation of 2-[3,9-bis[1-carboxylato-4-(2,3-dihydroxypropylamino)-4-oxobutyl]-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-trien-6-yl]-5-(2,3-dihydroxypropylamino)-5-oxopentanoate; gadolinium(3+), i.e. gadopiclenol.

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