DE102014218121A1 - Process for the enzymatic preparation of 2-hydroxycarboxylic acids - Google Patents

Abstract

The present invention relates to a process for the enzymatic preparation of 2-hydroxycarboxylic acids. To prepare 2-hydroxycarboxylic acids, formyl-CoA and a carbonyl compound selected from ketone or aldehyde are incubated in an aqueous reaction solution. The solution contains and / or produces an enzyme system which has at least one 2-hydroxycarbonyl-CoA synthase to form a 2-hydroxycarbonyl-CoA ester. About the corresponding 2-hydroxycarbonyl-CoA ester, the 2-hydroxycarboxylic acid is obtained as the free acid or in the form of their salts.

Description

The present invention relates to a process for the enzymatic preparation of 2-hydroxycarboxylic acids.

2-hydroxycarboxylic acids, especially the 2-hydroxy-2-alkylcarboxylic acids and the resulting after dehydration unsaturated 2-alkylcarboxylic acids, namely the C ₄ acids 2-hydroxyisobutyric acid and methacrylic acid and their alkyl or especially methyl esters are important building blocks for the synthesis a variety of chemical products, such as. B. of polymers and coatings.

Since industrial production is currently only carried out by non-enzymatic processes and requires a great deal of energy and considerable environmental pollution, biotechnological processes are required that make it possible to produce 2-hydroxycarboxylic acids in a more resource-efficient and environmentally friendly manner.

The biotechnological production of 2-hydroxy-2-methylcarboxylic acids via 3-hydroxycarboxylic acids based on the cobalamin-dependent 3-hydroxycarbonyl- or 2-hydroxy found in the bacterial strains Aquincola tertiaricarbonis HCM-10 (DSM 18028) and Kyrpidia tusciae DSM 2912 Although 2-methylcarbonyl-CoA mutase activities are already known ( DE 102006017760 A1 . WO 2009/156214 A1 . DE 102012216904 A1 . WO 2014/044674 A2 ), but these acyl-CoA mutase-dependent methods have a number of disadvantages. Thus, the metabolic pathway for the synthesis of the (R) and (S) -3-hydroxycarbonyl-CoA esters required for the mutase reaction, in particular of (R) -3-hydroxycarbonyl-CoA and poly- (R) -3- hydroxyalkanoates, although well established biotechnologically, in the isomerization to the 2-hydroxy-2-methylcarbonyl-CoA ester by the known 3-hydroxycarbonyl-CoA mutases but either the (R) - or (S) -enantiomers of the 3- Hydroxycarbonyl-CoA ester preferred. This stereospecificity must therefore be taken into account in the selection of the 3-hydroxycarbonyl-CoA ester-producing metabolism and thus limits the range of application of the respective mutase enzymes. Conversion into the other enantiomers is only possible by supplementing with corresponding racemic enzymatic activities. A major disadvantage of the mutase-based biotechnological processes for the preparation of 2-hydroxy-2-methylcarboxylic acids is their cobalamin dependence. This reaction is very sensitive to oxygen and other radicals, so that the co-factor attached to the mutase is easily inactivated. In order to maintain a high enzyme activity, so in addition to the mutase additionally appropriate protection or repair systems must be present. In addition, the required cobalamin needs to be prepared by the microorganism in a very extensive synthesis route consuming and in large quantities or added as vitamin B12 in the aqueous reaction solution and then absorbed by the microorganism, which in turn the presence of appropriate transport systems in the cell wall or cell membrane and also other enzymatic steps for the conversion of the vitamin into the active cofactor and the loading of the mutase enzymes. This cobalamin dependence thus increases the complexity and thus the susceptibility and the cost of these known biotechnological processes.

The object of the present invention is therefore to find biotechnological methods which avoid one or more disadvantages of the prior art. In particular, it is an object of the invention to provide enzyme systems which are capable of effecting the preparation of 2-hydroxycarbonyl-CoA esters from simple starting compounds from which the 2-hydroxycarboxylic acids can be further prepared, the disadvantage being the cobalamin dependence when using mutases, should be avoided.

Surprisingly, it has been found that 2-hydroxycarbonyl-CoA synthase activity enzyme systems can produce 2-hydroxycarbonyl-CoA esters from carbonyl compounds and formyl-coenzyme A (formyl-CoA), which are then converted to corresponding 2-hydroxycarboxylic acids can.

The present invention solves the problem by an enzymatic process for the preparation of 2-hydroxycarboxylic acids, wherein formyl-CoA and a carbonyl compound selected from ketone or aldehyde are incubated in an aqueous reaction solution in the presence of an enzyme system having at least one 2-hydroxycarbonyl-CoA synthase. The 2-hydroxycarbonyl-CoA synthase enzyme system is capable of forming a 2-hydroxycarbonyl-CoA ester. After incubation, the correspondingly converted 2-hydroxycarboxylic acid is obtained via the 2-hydroxycarbonyl-CoA ester as free acid or in the form of its salts.

Unless the context clearly indicates otherwise, the use of singular forms or plural forms always includes both the multiple and the singular.

The term "2-hydroxycarboxylic acids" denotes hydroxycarboxylic acids having the general structure of the formula (I): (R ₁ ) (R ₂ ) C (OH) COOH (I), wherein R ₁ and R _{2 may be the} same or different and are independently selected from H and organic radicals which have at least the size of a methyl group and stand for a straight-chain or branched aliphatic and also for an aromatic radical. In these organic radicals, carbon and hydrogen atoms may also be replaced, e.g. By O, S or N. Preferably, they are alkyl, aralkyl or aryl groups in saturated or unsaturated, isomeric or non-isomeric, straight-chain or branched-chain or cyclic form, preferably having 1 to 20 carbon atoms, wherein R ₁ and R ₂ may also be substituted by the above radicals, wherein said 2-hydroxycarboxylic acid may be present as free acid or in lactone form or in salt form with an organic base or an inorganic cation, as well as stereoisomers in D-, L- and DL-form when R ₁ and R _{2 are} not identical.

The produced 2-hydroxycarboxylic acid may, for. Example, be an alkylhydroxycarboxylic acid or 2-hydroxy-2-alkylcarboxylic acid, preferably selected from the group consisting of 2-hydroxyacetic acid (glycolic acid), 2-hydroxypropanoic acid (lactic acid), 2-methyl-2-hydroxypropanoic acid (2-hydroxyisobutyric acid), 2 -Hydroxybutanoic acid, 2-methyl-2-hydroxybutanoic acid, 2-ethyl-2-hydroxybutanoic acid, 2-hydroxypentanoic acid, 2-hydroxyhexanoic acid, 2-hydroxyheptanoic acid, 2-hydroxyoctanoic acid, 2-hydroxynonanoic acid, 2-hydroxydecanoic acid, 2-hydroxyundecanoic acid, 2-hydroxydodecanoic acid (alpha-hydroxylauric acid), 2-hydroxytetradecanoic acid (alpha-hydroxymyristic acid), 2-hydroxyhexadecanoic acid (alpha-hydroxypalmitic acid), 2-hydroxyoctadecanoic acid (alpha-hydroxystearic acid), 2-hydroxyeicosanoic acid (alpha-hydroxyarachidonic acid), 2-hydroxytetraeicosanoic acid (cerebronic acid) and 2 -Hydroxytetraeicosenoic acid (alpha-hydroxynvonic acid).

The 2-hydroxycarboxylic acid may furthermore be an aralkyl or aryl-2-hydroxycarboxylic acid, preferably selected from the group consisting of 2-phenyl-2-hydroxyacetic acid (mandelic acid), 2,2-diphenyl-2-hydroxyacetic acid (benzilic acid), 2-hydroxycarboxylic acid, Phenyl-2-methyl-2-hydroxyacetic acid (atrolactic acid), 2- (4'-hydroxyphenyl) -2-hydroxyacetic acid, 2- (4'-chlorophenyl) -2-hydroxyacetic acid, 2- (3'-hydroxy-4'-methyl) methoxyphenyl) -2-hydroxyacetic acid, 2- (4'-hydroxy-3-methoxyphenyl) -2-hydroxyacetic acid, and 2- (3 ', 4'-dihydroxyphenyl) -2-hydroxyacetic acid.

The 2-hydroxycarboxylic acid may also be a polyhydroxycarboxylic acid or hydroxypolycarboxylic acid, preferably selected from the group consisting of 2,3-dihydroxypropanoic acid (glyceric acid), 2,3,4-trihydroxybutanoic acid (isomers, erythronic acid, threonic acid), 2,3,4, 5-Tetrahydroxypentansure (isomers; ribonic acid, arabic acid, xylonic acid, lyxonic acid), 2,3,4,5,6-pentahydroxyhexanoic acid (isomers, allonic acid, altronic acid, gluconic acid, mannonic acid, gulonic acid, idonic acid, galactonic acid, talc acid), 2,3, 4,5,6,7-hexahydroxyheptanoic acid (isomers; glucoheptonic acid, galactoheptonic acid, etc.) and hydroxypropane-1,3-diacid (tartronic acid), 2-hydroxybutane-1,4-diacid (malic acid), 2,3-dihydroxybutane-1 , 4-diacid (tartaric acid), 2-hydroxy-2-carboxypentane-1,5-diacid (citric acid), 2,3,4,5-tetrahydroxyhexane-1,6-diacid (isomers, saccharic acid, mucic acid, etc.).

2-Hydroxycarboxylic acids present in salt or lactone form are preferably selected from the group consisting of gluconolactone, galactonolactone, glucuronlactone, galacturonlactone, gulonlactone, ribonolactone, saccharinic acid lactone, pantoyl lactone, glucoheptonlactone, mannonlactone and galactoheptonlactone.

The carbonyl compound used in the process according to the invention for the preparation of the 2-hydroxycarboxylic acid is selected from ketone or aldehyde, from which then the corresponding acid results. Preferably, a ketone compound is used in the process according to the invention as the carbonyl compound.

Ketones and aldehydes share the -C = O group, also called the carbonyl group. In this functional group, the tetravalent carbon atom is linked to an oxygen atom via a double bond. The carbonyl compounds have the general formula R ₁ R ₂ C = O, where R ₁ and R _{2 have} the abovementioned meanings. When using an aldehyde, one of the radicals R ₁ or R _{2 is} always a hydrogen atom.

In the context of the present invention, the term "2-hydroxycarbonyl-CoA synthase-containing enzyme system" is understood to mean a catalytic system comprising one or more constituents or components which is at least capable of forming a 2-hydroxycarbonyl from the starting materials formyl-CoA and carbonyl compound -CoA ester to form or produce. In this case, the enzyme system having 2-hydroxycarbonyl-CoA synthase may be an isolated enzyme, an isolated enzyme system and / or a microorganism or a crude extract thereof, which has or produces the enzyme system containing at least the 2-hydroxycarbonyl synthase.

As the 2-hydroxycarbonyl-CoA synthase-containing enzyme system, a microorganism or cell-free crude extract comprising or producing the enzyme system is preferably used in the method of the present invention.

"Microorganism" in the sense of the method according to the invention are microscopic single or multi-line living beings, which are usually not recognizable as single beings with the naked eye. It may be both prokaryotic organisms such. As bacteria and archaea, as well as eukaryotic organisms such as fungi, z. As yeasts, act. Microorganisms suitable for the present invention are in particular those bacteria, archaea, yeasts and fungi which are deposited as bacterial, archaeal, yeast or fungal strains in the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ), Braunschweig, Germany ,

Microorganisms suitable according to the invention belong to those genera which are subject to " https://www.dsmz.de/de/kataloge/katalog-mikroorganismen.html "As archaea, bacteria and yeasts / fungi are listed. Suitable microorganisms may also be obtained from other sources, e.g. From other strain collections such as the American Type Culture Collection (ATCC), USA.

Preferred microorganisms which can be used in the process according to the invention z. B. Actinomycetospora chiangmaiensis, Aquincola tertiaricarbonis, Escherichia coli, Cupriavidus necator, Azohydramonas lata, Pseudomonas oleovorans, Methylosinus spp., Methylocyctis trichosporium, Methylobacter spp., Methylobacterium organophilum, Methylobacterium extorquens, Methylobacterium rhodesianum, Ideonella spp., Rhodococcus ruber, Bacillus subtilis, Bacillus mycoides, Bacillus thuringiensis, Bacillus macerans, Bacillus laterosporus, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus brevis, Bacillus sphaericus, Bacillus circulans, Bacillus cereus, Bacillus megaterium, Streptomyces coelicolor, Streptomyces fradiae, Streptomyces parvus, Nocardia corallina, Corynebacterium spp. , Candida spp., Pichia spp., Saccharomyces spp., Yarrowia spp. Particularly preferred are Actinomycetospora chiangmaiensis, Aquincola tertiaricarbonis, Escherichia coli, Cupriavidus necator, Methylobacterium extorquens or Bacillus methanolicus.

The reaction conditions (pH, ion concentration, oxygen / carbon dioxide requirement, trace elements, temperatures and the like) are of course chosen so that the microorganisms are capable of optimal conversion of ketones / aldehydes and formyl-CoA to 2-hydroxycarboxylic acids. Under these process conditions, the enzyme system in the natural micro-milieu, ie within the cell, a higher stability and effectiveness than an isolated enzyme. In addition, cell proliferation and thus an increase in enzyme concentration may be possible under suitable conditions. Thus, enzymatic conversion by means of microorganisms may be a significant advantage in terms of reliability, automation and simplicity, as well as quality and yield of the end product of the process.

For the inventive enzymatic reaction of ketones / aldehydes in the desired 2-hydroxycarboxylic acids, it is also possible to introduce the 2-hydroxycarbonyl-CoA synthase in purified, enriched and / or isolated form in the reaction solution, wherein the enzymes z. B. of natural origin. Furthermore, the enzymes may be recombinantly produced enzymes from a genetically engineered organism.

The enzymes are used in the novel process according to the invention as catalysts both in the form of intact microbial cells and in the form of permeabilized microbial cells. Other uses exist in the form of components (one or more) of microbial cell extracts, but also in partially purified or purified form. The enzymatic catalysts may be immobilized or attached to a dissolved or undissolved carrier material.

In a preferred embodiment, certain cell compartments or parts thereof are separated or pooled, that is, carbohydrate structures, lipids or proteins and / or peptides and nucleic acids capable of positively or negatively affecting the 2-hydroxycarbonyl-CoA synthase moiety may be combined or separated. In order to use such influence consciously, from the microorganisms z. B. expert crude extracts prepared, which may be centrifuged to perform an inventive reaction with the sediment or the supernatant can. Accordingly, the enzyme system comprising 2-hydroxycarbonyl-CoA synthase may, for example, also be a cell-free crude extract of a microorganism which comprises or produces this enzyme system. Such a "raw extract" obtained from a microorganism refers to an extract of one or more substances or components which are dissolved out of this microorganism, withdrawn or isolated. Processes and production or production of such crude extracts from microorganisms are described in the prior art and known to the person skilled in the art. For example, such crude extracts can be obtained by disrupting microorganisms by means of ultrasound, enzymatic methods, by a possibly repeated combination of shock freezing and thawing, autolysis, flow homogenizer, French press, ball mill, and / or combinations thereof. These crude extracts have essentially no living, intact or functioning microorganisms.

The 2-hydroxycarbonyl-CoA synthase-containing enzyme system is capable in the process according to the invention at least for the formation of a 2-hydroxycarbonyl-CoA ester. The enzyme system has a catalyzing activity and condenses formyl-CoA with the carbonyl compound via a C-C ligation to form the 2-hydroxycarbonyl-CoA ester. Naturally, depending on the carbonyl compound used, a corresponding 2-hydroxycarbonyl-CoA ester is formed.

The enzyme system comprising 2-hydroxycarbonyl-CoA synthase preferably comprises thiamine diphosphate as coenzyme. "Thiamine diphosphate", or thiamine pyrophosphate, is a phosphate ester of thiamine, which is also known as vitamin B1. The addition of thiamine diphosphate in the process according to the invention surprisingly leads to a more than 20-fold increase in the enzyme activity over a mixture without thiamine diphosphate addition.

The process according to the invention is particularly preferably suitable for the preparation of 2-hydroxy-2-methyl or 2-hydroxy-2-alkylcarboxylic acids, where an alkylmethyl or a dialkyl ketone reacts with the formyl-CoA. The process according to the invention is particularly preferably suitable for the preparation of 2-hydroxyisobutyric acid (2-hydroxy-2-methylpropanoic acid), 2-hydroxy-2-methylbutyric acid or 2-hydroxy-2-ethylbutyric acid. For example, if the carbonyl compound is acetone (dimethyl ketone), the enzyme system having 2-hydroxycarbonyl-CoA synthase catalyses e.g. For example, the formation of a 2-hydroxyisobutyryl CoA ester. If butanone (ethyl methyl ketone) is used as the carbonyl compound, a 2-hydroxy-2-methylbutyryl-CoA ester is formed in the process according to the invention. If 3-pentanone (diethyl ketone) is used as the carbonyl compound, a 2-hydroxy-2-ethylbutyryl-CoA ester is formed.

Is the carbonyl compound an aldehyde z. Propanal, the 2-hydroxycarbonyl-CoA synthase enzyme system catalyzes an (R) - and / or (S) -2-hydroxybutyryl-CoA ester.

It has been found that an enzyme which can be used according to the invention is preferably an enzyme having the amino acid sequence SEQ ID NO 2, as well as at least 49% homologs, preferably at least 75%, particularly preferably at least 95%, very particularly preferably at least 99% homologs. These enzymes are particularly capable of reacting ketones with formyl-CoA.

Further preferred homologous enzymes to SEQ ID NO 2 have SEQ ID NO 3 to 20, these enzymes having the same properties. The enzymes are present as monomer or oligomer.

Preferably, these enzymes have a binding site for thiamine diphosphate, as z. B. for thiamine diphosphate-dependent enzymes is described ( Widmann et al. 2010, BMC Biochem. 11: 9 ), and use this as a cofactor during the invention catalyzed synthase reaction.

Both nucleic acids for the production of the enzymes and the enzymes themselves are present as naturally occurring biocatalysts in microorganism strains or they can be isolated from natural sources. Microorganisms of the genus Actinomycetospora, z. B. Actinomycetospora chiangmaiensis DSM 45062; Pseudonocardia, Rubrobacter, Skermanella, Nisaea, e.g. B. Nisaea denitrificans DSM 18348; Roseivivax, Bradyrhizobium, Xanthobacter, Azoarcus, Methylibium, e.g. B. methylibium petroleiphilum, Aquincola, preferably A. tertiaricarbonis DSM 18028 (= HCM-10), 18260 (= L108) and 18512 (= 110) are particularly preferred.

The enzymes can also be synthesized by known methods. The nucleic acid molecule on which the enzyme of SEQ ID NO 2 is based has SEQ ID NO 1.

"Homologs", "sequence homology" or "homologous sequences" means a sequence identity to said nucleic acid / amino acid sequences of at least 49%, preferably at least 75%, more preferably at least 95%, most preferably at least 99% at the nucleic acid or amino acid level ,

For the purposes of the present invention, sequence homology can be objectively determined by various methods. Such methods or methods for determining relationships between two or more sequences (eg identity, similarity and / or homology) are described in the prior art and known to those skilled in the art, which is capable of providing a suitable method without undue burden select. For example, such methods may include manual matching, computer-assisted sequence matching, and combinations thereof. Various, usually computer-implemented, algorithms for performing sequence alignment are well known or can be made by those skilled in the art. The degree of homology or identity of one amino acid sequence or nucleotide sequence to another can be determined using the following algorithm BLAST by Karlin and Altschul ( Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993 ) be determined. Programs like BLASTN and BLASTX, which were developed based on this algorithm ( Altschul et al. (1990) J. Mol. Biol. 215: 403-410 ) can also be used. Specific techniques for such analysis are known in the art (see http://www.ncbi.nim.nih.gov. ).

In the process according to the invention, the conversion of the 2-hydroxycarbonyl-CoA ester into the corresponding 2-hydroxycarboxylic acid can be carried out by means of further enzymes which are added or are part of the enzyme system.

In addition come z. As enzymes having an esterase activity, in question, by means of which the resulting CoA ester is split into the corresponding acid and CoA. Examples of suitable enzymes with esterase activity are thioesterases (eg EC 3.1.2.2, EC 3.1.2.11 and EC 3.1.2.20) such as eg. B. TesB from E. coli; Acetoacetyl-CoA-hydrolase / thioesterase, e.g. B. TEII _srf from Bacillus subtilis or YbgC from Haemophilus influenza.

In a variant of the process, the resulting CoA esters can also be prepared first by a Phosphotransacylase, z. B. Phosphotransbutyrylase (EC 2.3.1.19) from Clostridium acetobutylicum, be transesterified into a corresponding phosphate ester and then to form ATP from ADP by a carboxylic acid kinase, eg. B. butyrate kinase (EC 2.7.2.7) from Clostridium acetobutylicum are converted into the free carboxylic acid, as z. B. for the production of 3-hydroxybutyric acid via 3-hydroxybutyryl-CoA has been described ( Tseng et al. 2009, Appl. Environ. Microbiol. 75: 3137-3145 ).

In a further variant, the 2-hydroxycarbonyl-CoA esters or the free 2-hydroxycarboxylic acids can also be enzymatically transesterified or esterified with alcohols, which may require the recovery of the products z. B. facilitated by extraction or needed for further processing. Suitable enzymes include z. B. to the carboxylic acid O-methyltransferases (EC 2.1.1.15), which can transfer a methyl group of S-adenosyl-L-methionine to the carboxylic acid, so that the corresponding methyl ester is formed. A direct transesterification of the CoA ester with an alcohol can, for. B. by alcohol O-acyltransferase (EC 2.3.1.75) take place. Furthermore, the free carboxylic acids z. B. esterified by carboxylesterases (EC 3.1.1.1) with alcohols.

The conversion of the 2-hydroxycarbonyl-CoA ester into the corresponding 2-hydroxycarboxylic acid and optionally a derivatization, for. As the conversion into alkyl esters, can also be non-enzymatic.

So z. B. after digestion of the microbial cells or directly in a cell-free reaction solution of the CoA esters according to the invention, such as any other thioester compound are hydrolyzed by the addition of inorganic acids or bases. At the same time, transesterification, for example with methanol, may be catalyzed at the same time, so that corresponding methyl or general alkyl esters are formed. Preferably, a dehydration of the 2-hydroxycarboxylic acids can be carried out, so that z. B. in the case of 2-hydroxyisobutyric finally the important basic chemical methyl methacrylic acid is formed.

Preferably, the 2-hydroxycarboxylic acid is released into the medium and can be recovered as acid or in the form of its salts. Process and recovery or isolation of the 2-hydroxycarboxylic acids Media are described in the art and known in the art. Such methods include, for example, concentration, ion exchange, distillation, electrodialysis, extraction and crystallization.

Furthermore, it is possible to use in the process enzymatic activities which are already capable of product formation of formyl-CoA and / or the carbonyl compound or control these. Thus, additionally at least one further enzyme for the production of the starting compounds formyl-CoA and / or carbonyl compound can be contained or added in the reaction solution. Both inorganic and organic carbon sources can be used for the synthesis of formyl-CoA and / or ketones or aldehydes. Examples of organic carbon sources are, for. As glucose, methane and reduced C1 compounds such as methanol, formaldehyde or formic acid. Suitable inorganic carbon sources may, for. As carbon monoxide or carbon dioxide.

Suitable enzymes that can be used for the production of formyl-CoA are mentioned in the examples and are z. Acetyl-CoA: oxalate CoA transferases (EC 2.8.3.19), oxalyl-CoA decarboxylases (EC 4.1.1.8) and / or acetyl-CoA synthetase (EC 6.2.1.1). Furthermore, suitable CoA-dependent glyoxylic acid dehydrogenases (EC 1.2.1.17), z. B. from Methylobacterium extorquens AM1. However, a synthesis of formyl-CoA can not only z. B. via oxalyl-CoA, but z. B. also directly from formic acid, ATP and CoA by acyl-CoA synthetases such. B. the acetyl-CoA synthetase from Pyrobaculum aerophilum ( Bräsen et al. 2005, FEBS Lett. 579: 477-482 ) respectively.

Suitable enzymes for the corresponding production of carbonyl compounds from corresponding metabolic precursors (see examples) are e.g. Acetoacetyl-CoA-hydrolase / thioesterase (EC 3.1.2.11), e.g. B. TEII _srf from Bacillus subtilis or YbgC from Haemophilus influenza, and / or 3-oxocarboxylic decarboxylases, e.g. B. acetoacetate decarboxylase (EC 4.1.1.4) from Clostridium acetobutylicum.

Alternatively or additionally, formyl CoA and / or carbonyl compound may be added to the aqueous reaction solution. From these externally added starting materials then the 2-hydroxycarbonyl-CoA esters can be prepared by means of the 2-hydroxycarbonyl-CoA synthase having enzyme system.

In one embodiment of the invention, the 2-hydroxycarbonyl-CoA synthase-containing enzyme system is capable of producing formyl CoA and / or the carbonyl compound from a carbon source present in the aqueous reaction solution, from the formyl CoA and the carbonyl compound the formation of a 2-hydroxylcarbonyl -CoA ester to catalyze and convert the formed 2-hydroxylcarbonyl-CoA ester into the corresponding 2-hydroxycarboxylic acid.

The microorganisms, which naturally do not comprise 2-hydroxycarbonyl-CoA synthase and / or the enzymes for the production of formyl-CoA and / or carbonyl compounds and / or the enzymes for the conversion of the 2-hydroxycarbonyl-CoA-esters, can be manipulated expertly, so that they are suitable for the process according to the invention for the preparation of 2-hydroxycarboxylic acids.

Mutations can be generated in the nucleic acid molecules used according to the invention by means of molecular biological techniques known per se, which enables the desired enzymes with analogous or similar properties to be synthesized which can be used according to the invention. Mutations can z. B. deletion mutations that lead to shortened enzymes. By other molecular mechanisms such. As insertions, duplications, transpositions, gene fusion, nucleotide exchange or gene transfer between different strains of microorganisms can also be produced modified enzymes with similar or analogous properties. The identification and isolation of such nucleic acid molecules can be accomplished using the nucleic acid molecules or portions thereof. The molecules hybridizing with the nucleic acid molecules also include fragments, derivatives and allelic variants of the nucleic acid molecules described above which encode an enzyme useful in the invention. By fragments are meant parts of the nucleic acid molecules that are long enough to encode the described enzyme. Derivative is understood as meaning sequences of these molecules which differ from the sequences of the above-described nucleic acid molecules at one or more positions, but have a high degree of homology to these sequences.

For example, the microorganisms can be further transformed with one or more nucleic acid sequences which, in addition to enzymes for forming the 2-hydroxycarbonyl-CoA ester, enable the enzymes to produce formyl-CoA and / or carbonyl compounds and / or the enzymes to convert the 2-hydroxycarbonyl-CoA esters. Hydroxylcarbonyl-CoA esters in the corresponding 2-hydroxycarboxylic acids and / or optionally other enzymes for derivatization, such as. B. to esterify the 2-hydroxycarboxylic acids with alcohols to produce.

In addition, the invention encompasses the use of enzymes in the process of the invention comprising the 2-hydroxycarbonyl-CoA synthases as monomer or oligomer as described above, fused to additional proteins selected from the group consisting of other thiamine and thiamine diphosphate-binding Proteins, CoA synthetases, esterases, dehydrogenases, decarboxylases and the like. a. available.

Included here are variants in which the further proteins are fused with only one subunit and the proteins with at least 49% homology, preferably at least 75%, more preferably at least 95%, or at least 99% homology to these sequences.

In the following the invention will be explained in more detail by means of exemplary embodiments.

embodiments

General methods

Homology comparisons of nucleotide and amino acid sequences were carried out with the program BLAST (BLASTN or BLASTP) with the general basic settings (matrix BLOSUM62) ( Altschul et al. 1990, Journal of Molecular Biology 215: 403-410 ). The BLAST program can be obtained from the National Center for Biotechnology Information (NCBI) and other sources.

For the screening of microorganisms with 2-hydroxycarbonyl-CoA synthase activity, for the cultivation of relevant microorganisms and for the degradation experiments with microorganisms and 2-hydroxy-2-alkylcarboxylic acids, media based on a basal medium (Table 1) and a modified ISP-2 medium (Table 2) used. The mixture was incubated with shaking or stirring in the dark at the temperatures, pH values and concentrations of 2-hydroxycarboxylic acids mentioned in the Examples.

To check the vitamin B12 independence of the degradation of 2-hydroxycarboxylic acids, a mixture with basal medium without vitamin B12 and a batch with vitamin B12 (0.05 mg / L) was incubated in each case. Table 1: Basal medium (mg / l) NH ₄ Cl 761.4 KH ₂ PO ₄ 340.3 K ₂ HPO ₄ 435.5 CaCl ₂ × 6H ₂ O 5.5 MgSO ₄ × 7H ₂ O 71.2 ZnSO ₄ × 7H ₂ O 0.44 MnSO ₄ × H ₂ O 0.615 CuSO ₄ × 5H ₂ O 0,785 Na ₂ MoO ₄ × 2H ₂ O 0.252 FeSO ₄ × 7H ₂ O 14.96 biotin 0.02 folic acid 0.02 Pyridoxine HCl 0.1 thiamine 0.05 riboflavin 0.05 nicotinic acid 0.05 DL-calcium pantothenate 0.05 p-aminobenzoic acid 0.05 lipoic acid 0.05 Table 2: Modified ISP-2 medium (mg / L) yeast extract 4000 malt extract 10000 glucose 4000 inositol 500 KH ₂ PO ₄ 340.3 KH ₂ PO ₄ 435.5 biotin 0.02 folic acid 0.02 Pyridoxine HCl 0.1 thiamine 0.05 riboflavin 0.05 nicotinic acid 0.05 lipoic acid 0.05 p-aminobenzoic acid 0.05 DL-calcium pantothenate 0.05

Stock cultures of microorganisms used are stored in 20% glycerol solution in liquid nitrogen.

For the enzyme assay for determining the 2-hydroxycarbonyl-CoA synthase activity in crude protein extracts or enzyme preparations, reaction mixtures (Table 3) were incubated with formyl-CoA and the carbonyl compounds mentioned in the examples as substrates at the temperatures indicated in the examples. Table 3 MgCl 1 mm potassium phosphate 50 mM thiamine 0.05mM carbonyl 1000 ppm Formyl-CoA 1.3 mM PH value 7.2

To test a thiamine diphosphate dependence in the formation of the 2-hydroxycarbonyl-CoA ester from formyl-CoA and a carbonyl compound, an enzyme assay mixture with thiamine diphosphate according to Tab. 3 and an approach without thiamine diphosphate were respectively performed.

The formyl-CoA used in the enzyme assay for the determination of 2-hydroxycarbonyl-CoA synthase activity was prepared according to the relevant method from formic acid after reaction with acetic anhydride to the mixed anhydride, activation with thiophenol and subsequent transesterification with coenzyme A (CoA), such as they z. In Jonsson et al. 2004 ( Jonsson et al. 2004, Kinetic and mechanistic characterization of the formyl-CoA transferase from Oxalobacter formigenes. J. Biol. Chem. 279: 36003-36012 ) and regarding transesterification in detail in Sly and Stadtman 1963 ( Sly and Stadtman 1963, Formats metabolism: 1. Formyl coenzyme A, to intermediate in the formate-dependent decomposition of acetyl phosphates in Clostridium kluyveri. J. Biol. Chem. 238: 2632-2638 ) is described.

Standards for the 2-hydroxycarbonyl-CoA esters formed in the enzyme assay for determining the 2-hydroxycarbonyl-CoA synthase activity were prepared from the free acids by the relevant method via activation with thiophenol and subsequent transesterification with coenzyme A (CoA), as they are z. In Padmakumar et al. 1993 ( Padmakumar et al. 1993, A rapid method for the synthesis of methylamlonylcoenzymes A and other CoA esters. Anal. Biochem. 214: 318-320 ) is described.

The 2-hydroxycarbonyl-CoA esters formed in the enzyme assay for determining the 2-hydroxycarbonyl-CoA synthase activity and the formyl-CoA used were determined by means of HPLC. The mobile phase (rate 0.8 ml / min) used was the mobile phase consisting of 10 mM tetrabutylammonium hydrogensulfate and 100 mM sodium phosphate having a pH of 4.5 and an acetonitrile content of 14.5% by volume (mobile phase A) and 21, respectively. 6% by volume (solvent B). Solvent A was used to separate free CoA and formyl-CoA and, with mobile phase B, the 2-hydroxycarbonyl-CoA esters on Saute Nucleosil 100-5 C18 (Macherey-Nagel). Detection was at 260 nm using a photometric detector.

Non-esterified organic acids, which served or were formed as substrate when incubated with whole cells, were also determined by HPLC. The mobile phase (0.5 mL / min) was an eluent consisting of 10 mN sulfuric acid in water. Thus, the acids were separated on the column Nucleogel Ion 300 OA (Macherey-Nagel). Detection was performed using a refractive index detector.

Volatiles such as aldehydes and ketones (eg, propanal, acetone, 2-butanone and 3-pentanone) were determined by GC analysis. For this purpose, headspace GC vials were incubated with culture samples for 20 min at 70 ° C, gas samples taken and injected into the GC system. The substances were separated on an Optima Delta 3 column (60 m, 0.32 mm, 0.35 μm, Macherey-Nagel) at 40 or 50 ° C. Detection was carried out using a Fl detector.

The protein concentration of the crude protein extracts and the enzyme preparations was determined using the Bradford reagent (Merck) according to the manufacturer's protocol. Bovine serum albumin served as standard.

Example 1:

Screening for microbial degradation of 2-hydroxycarboxylic acids

Various enrichment cultures and microbial pure cultures were tested to determine whether they can degrade 2-hydroxyisobutyric acid, 2-hydroxy-2-methylbutyric acid and / or 2-hydroxy-2-ethylbutyric acid (starting concentration in each case 0.5 g / l) in liquid culture. The bacterial strain Actinomycetospora chiangmaiensis DSM 45062 surprisingly showed the sought-after ability. By incubation of the strain DSM 45062 at 30 ° C. and pH 7.0 in a medium which consisted of 19 volumes of basal medium (Table 1) and 1 volume proportion of modified ISP-2 medium (Table 2), all tested Hydroxycarboxylic degraded.

Surprisingly, it was also found that strain DSM 45062 2-hydroxy-2-alkylcarboxylic acids not such. B. for strain L108 described ( Yaneva et al. 2012, J. Biol. Chem. 287: 15502-15511 ) degrades via 3-hydroxybutyric acid or generally via 3-hydroxycarboxylic acids, but that ketones are formed during the degradation. Thus, during incubation with 2-hydroxyisobutyric acid, acetone was temporarily accumulated, with 2-hydroxy-2-methylbutyric acid corresponding to 2-butanone and then 3-pentanone with 2-hydroxy-2-ethylbutyric acid. This degradation was vitamin B12-independent.

By homology comparison (BLASTP program) using the sequence of the methylmalonyl-CoA mutase from Propionibacterium freudenreichii subsp. shermanii NCIB 9885 (GenBank: CAA33090), all cobalamin-dependent acyl-CoA mutases or the genes coding for them were identified in the published genome of the strain DSM 45062 (see, eg, GenBank). In total, only 3 regions with mutase genes were found. The first region (GenBank: WP_018333124) clearly encodes a methylmalonyl CoA mutase, as the sequence of the corresponding gene product is in great agreement with the enzyme from Propionibacterium freudenreichii subsp. shermanii NCIB 9885 shows (71% identical residues) and also has the characteristic of this enzyme amino acid residues in the active center, such. As described by Cracan and Banerjee 2012 ( Cracan and Banerjee 2012, Biochemistry 51: 6039-6046 ). Accordingly, the second region (GenBank: WP_018335024) likewise clearly codes for an ethylmalonyl-CoA mutase, as described, for example, in US Pat. B. characterized for Rhodobacter sphaeroides ( Erb et al. 2008, J. Biol. Chem. 283: 32283-32293 ). The third region (GenBank: WP_018333903) clearly encodes an isobutyryl-CoA mutase fused to the MeaB-like chaperone meal, as has recently been described for other bacterial strains ( Cracan et al. 2010, J. Biol. Chem. 285: 655-666 ).

Thus, strain DSM 45062 does not have 2-hydroxyisobutyryl-CoA mutase, so surprisingly, the observed degradation of 2-hydroxycarboxylic acids occurs cobalamin and acyl-CoA mutase independent.

Example 2:

Testing for 2-hydroxycarbonyl-CoA synthase Aktviät in cell-free crude extracts of strain DSM 45062

For testing a 2-hydroxycarbonyl-CoA synthase activity, strain DSM 45062 was initially incubated at 30 ° C. and pH 7.0 in a medium consisting of 3 volumes of basal medium (Table 1) and 1 volume of modified ISP-2 medium ( Tab. 2), in the presence of 500 mg / l of 2-hydroxyisobutyric acid. After induction of 2-hydroxyisobutyric acid degradation, the cells were harvested by filtration and centrifugation (8,000 xg, 4 ° C, 10 min), suspended in phosphate buffer (50 mM potassium phosphate, pH 7.2), and digested (30 min at one frequency of 30 Hz, MM 400, Retsch GmbH, with glass beads 212-300 μm, Sigma-Aldrich). Glass beads and cell debris were separated by centrifugation (18,000 x g, 4 ° C, 20 min). The crude extract thus obtained was used directly for the enzyme assay.

In this crude extract surprisingly a 2-hydroxycarbonyl-CoA synthase activity could be detected in an enzyme assay according to Tab. 3 at a protein concentration of 325 mg / l. It was with formyl-CoA and acetone as substrates at an incubation temperature of 30 ° C for the formation of 2-hydroxyisobutyryl-CoA a specific enzyme activity of 3440 nmol / l / min and 10.6 nmol / min / mg total protein in Presence of thiamine diphosphate achieved. Surprisingly, the addition of thiamine diphosphate resulted in a 22-fold increase in enzyme activity over a non-thiamine diphosphate addition ( ).

The incubation according to Tab. 3 with the crude extract and acetone but with formic acid or Na formate (1.5 mM) instead of formyl-CoA did not lead to the formation of 2-hydroxyisobutyryl-CoA.

Example 3:

Synthesis of 2-hydroxyisobutyric acid with transformation of an E. coli strain with the enzymes E1 to E5:

E1 to E5 are enzymes for the microbial synthesis of 2-hydroxyisobutyric acid from oxalate and acetone using the 2-hydroxycarbonyl-CpA synthase having SEQ ID NO 2
E1: acetyl-CoA: oxalate CoA transferase YfdE from E. coli (eg GenBank: AAC75430, GI: 87082093);
E2: oxalyl-CoA decarboxylase YfdU from Methylobacterium extorquens AM1 (GenBank: ACS38885, GI: 240007659, MexAM1_META1p0990) or YfdU from E. coli (eg GenBank: NP_416874, GI: 16130305);
E3: 2-hydroxycarbonyl-CoA synthase from strain DSM 45062 (SEQ ID NO 2)
E4: thioesterase TesB from E. coli (eg GenBank: YP_488744, GI: 388476558);
E5: Acetyl-CoA synthetase from E. coli (eg GenBank: AAC77039, GI: 1790505).

shows metabolism and the stoichiometries for the synthesis of 2-hydroxyisobutyric acid from oxalate and acetone on the example of an E. coli strain after additional expression of the enzymes E1 to E5

Example 4

Synthesis of 2-hydroxyisobutyric acid with transformation of a Methylobacterium extorquens AM1 strain with the following enzymes E2 to E4 and E6 to E8:

• E2: oxalyl-CoA decarboxylase YfdU from Methylobacterium extorquens AM1 (GenBank: ACS38885, GI: 240007659, MexAM1_META1p0990) or YfdU from E. coli (eg GenBank: NP_416874, GI: 16130305);
• E3: 2-hydroxycarbonyl-CoA synthase from strain DSM 45062 (SEQ ID NO 2)
• E4: thioesterase from TesB from E. coli (eg GenBank: YP_488744, GI: 388476558);
• E6: CoA-dependent glyoxylic acid dehydrogenase from Methylobacterium extorquens AM1 (GenBank: ACS39924, GI: 240008698, MexAM1_META1p2129);
• E7: acetoacetyl-CoA-hydrolase / thioesterase from TEII _srf from Bacillus subtilis (eg GenBank: WP_003234568, GI: 489327274) or YbgC from Haemophilus influenza ATCC 51907 (GenBank: P44679, GI: 1175571, HI_0386);
• E8: 3-oxocarboxylic acid decarboxylase from acetoacetate decarboxylase from Clostridium acetobutylicum ATCC 824 (GenBank: AAA63761, GI: 144711).

shows the methylotrophic metabolism and the stoichiometries for the synthesis of 2-hydroxyisobutyric acid from formic acid using the example of the strain Methylobacterium extorquens AM1 after expression of the enzymes E2 to E4 and E6 to E8.

In the case of the strain Methylobacterium extorquens AM1, in the assimilatory metabolism, reduced C1 compounds such as formic acid are introduced into the serine pathway at the stage of formaldehyde ( Chistoserdova et al. 2009, Annu. Rev. Microbiol. 63: 477-499 ).

The formyl-CoA required for the condensation reaction according to the invention for 2-hydroxyisobutyryl-CoA is prepared by means of the enzymes E6 and E2 from the glyoxylate formed in the serine pathway via oxalyl-CoA. Acetone is recovered from the acetoacetyl-CoA ester also formed in the methylotrophic metabolism. E7 catalyzes the cleavage into CoA and acetoacetate. Thereafter, acetoacetate is decarboxylated by E8 to acetone and CO ₂ . Formyl CoA and acetone are then condensed by E3 to the 2-hydroxyisobutyryl CoA ester, which is cleaved by E4 into CoA and the desired product 2-hydroxyisobutyric acid.

This is followed by a sequence protocol according to WIPO St. 25. This can be downloaded from the official publication platform of the DPMA.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• DE 102006017760 A1 [0004]
• WO 2009/156214 A1 [0004]
• DE 102012216904 A1 [0004]
• WO 2014/044674 A2 [0004]

Cited non-patent literature

• https://www.dsmz.de/de/kataloge/katalog-microorganisms.html [0019]
• Widmann et al. 2010, BMC Biochem. 11: 9 [0031]
• Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993 [0035]
• Altschul et al. (1990) J. Mol. Biol. 215: 403-410 [0035]
• http://www.ncbi.nim.nih.gov. [0035]
• Tseng et al. 2009, Appl. Environ. Microbiol. 75: 3137-3145 [0038]
• Bräsen et al. 2005, FEBS Lett. 579: 477-482 [0044]
• Altschul et al. 1990, Journal of Molecular Biology 215: 403-410 [0054]
• Jonsson et al. 2004, Kinetic and mechanistic characterization of the formyl-CoA transferase from Oxalobacter formigenes. J. Biol. Chem. 279: 36003-36012 [0060]
• Sly and Stadtman 1963, Formats metabolism: 1. Formyl coenzyme A, to intermediate in the formate-dependent decomposition of acetyl phosphate in Clostridium kluyveri. J. Biol. Chem. 238: 2632-2638 [0060]
• Padmakumar et al. 1993, A rapid method for the synthesis of methylamlonylcoenzymes A and other CoA esters. Anal. Biochem. 214: 318-320 [0061]
• Yaneva et al. 2012, J. Biol. Chem. 287: 15502-15511 [0067]
• Cracan and Banerjee 2012, Biochemistry 51: 6039-6046 [0068]
• Erb et al. 2008, J. Biol. Chem. 283: 32283-32293 [0068]
• Cracan et al. 2010, J. Biol. Chem. 285: 655-666 [0068]
• Chistoserdova et al. 2009, Annu. Rev. Microbiol. 63: 477-499 [0076]

Claims (13)

Process for the enzymatic preparation of 2-hydroxycarboxylic acids, characterized in that formyl-CoA and a carbonyl compound selected from ketone or aldehyde are incubated in an aqueous reaction solution containing and / or producing an enzyme system containing at least one 2-hydroxycarbonyl-CoA synthase to form a 2-hydroxycarbonyl-CoA ester, wherein the 2-hydroxycarbonyl-CoA ester, the 2-hydroxycarboxylic acid is obtained as the free acid or in the form of their salts. A method according to claim 1, characterized in that the enzyme system comprising 2-hydroxycarbonyl-CoA synthase is an isolated enzyme, an isolated enzyme system and / or a microorganism or a crude extract thereof containing the at least the 2-hydroxycarbonyl-CoA synthase Has or produces enzyme system. A method according to claim 1 or 2, characterized in that is contained as a coenzyme thiamine diphosphate. Method according to one of claims 1 to 3, characterized in that in the reaction solution additionally at least one enzyme which catalyzes the product formation of formyl-CoA and / or a carbonyl compound is included. Method according to one of claims 1 to 4, characterized in that in addition at least one enzyme for converting the 2-hydroxycarbonyl-CoA ester into the acid or its salts is present in the reaction solution. Method according to one of claims 1 to 4, characterized in that the 2-hydroxycarboxylic acid has the general structure of the formula (I): (R ₁ ) (R ₂ ) C (OH) COOH (I), wherein R ₁ and R _{2 may be the} same or different and are independently selected from H and organic radicals having at least the size of a methyl group and represent a straight-chain or branched aliphatic and also an aromatic radical, said 2-hydroxycarboxylic acid may exist as free acid or in lactone form or in salt form with an organic base or an inorganic cation, as well as stereoisomers in D, L and DL form, when R ₁ and R _{2 are} not identical. Method according to one of claims 1 to 6, characterized in that the carbonyl compound has the general formula R ₁ R ₂ C = O, wherein the radicals R ₁ and R _{2 have} the meaning given in formula (I), and in the case of an aldehyde R ₁ or R ₂ always represents a hydrogen atom. Method according to one of claims 1 to 7, characterized in that a ketone and formyl-CoA for the production of 2-hydroxy-2-alkylcarbonylic acids are incubated, wherein the 2-hydroxycarbonyl-CoA synthase to form a 2-hydroxy-2 alkylcarbonyl-CoA ester is capable. A method according to any one of claims 1 to 8, characterized in that a 2-hydroxycarbonyl-CoA synthase is used which has or comprises the amino acid sequence SEQ ID NO: 2 or at least 49% homologues which have the same properties , Method according to one of claims 1 to 9, characterized in that a 2-hydroxycarbonyl-CoA synthase is used which have the amino acid sequences SEQ ID NO: 3 to SEQ ID NO 20. Method according to one of claims 1 to 10, characterized in that microorganisms are cultured which contain one or more nucleic acid sequences which produce at least the 2-hydroxycarbonyl-CoA synthase, or which contain these 2-hydroxycarbonyl-CoA synthase, wherein the Microorganisms in aqueous systems cause the conversion of a ketone or aldehyde and formyl-CoA to a 2-hydroxycarboxylic acid. A method according to claim 11, characterized in that the microorganism comprises one or more nucleic acid sequences which produce enzymes which are further used for the production of Starting compounds ketone or aldehyde and / or formyl-CoA and / or for converting the ester into the corresponding 2-hydroxycarboxylic acid are suitable. 2-hydroxycarbonyl-CoA synthases having the sequences SEQ ID NO 2 to SEQ ID NO 20.

Images