Method for improving the production and yield of 2,4-dihydroxybutyrate

Abstract

The present invention relates to an improved method for preparing 2,4-dihydroxybutyrate from a carbon source via homoserine, the method comprising a two-step pathway comprising: a first step of converting the primary amino acid group of homoserine to a carbonyl group and catalyzing it with an enzyme having homoserine transaminase activity as defined by EC 2.6.1.1, EC 2.6.1.42, or EC 2.6.1.57 to obtain 2-oxo-4-hydroxybutyrate; and a second step of reducing the obtained 2-oxo-4-hydroxybutyrate (OHB) to 2,4-dihydroxybutyrate by catalyzing it with an enzyme having OHB reductase activity, wherein the enzyme having OHB reductase activity is lactate dehydrogenase as defined by EC 1.1.1.27 or EC 1.1.1.28, or malate dehydrogenase as defined by EC 1.1.1.37, EC 1.1.1.82, or EC 1.1.1.299, the method comprising a two-step pathway, At least one enzyme having aldolase activity as defined in 4.1.2.28 is inactivated to prevent the degradation of OHB.

Description

Technical Field

[0001] The present invention relates to an improvement over prior art methods for preparing 2,4-dihydroxybutyrate via homoserine from a carbon source. This method comprises a two-step route, where the first step involves converting the primary amino acid group of homoserine to a carbonyl group to obtain 2-oxo-4-hydroxybutyrate (OHB), and the second step involves reducing the obtained 2-oxo-4-hydroxybutyrate to 2,4-dihydroxybutyrate. The improvement of this method lies in reducing and even eliminating the loss of OHB in order to optimize the pathway flux from the carbon source to the target compound, i.e., DHB. The DHB thus produced is particularly used in the production / synthesis of 2-hydroxy-4-(methylthio)butyrate (HMTB) and / or the hydroxy analogs of selenomethionine (HMSeBA or HMSeB) according to the present invention. The present invention relates to a modified microorganism for producing 2,4-DHB via homoserine from a carbon source in a two-step route in which the pathway flux from the carbon source to the target compound, i.e., DHB, is optimized, and also relates to a method for producing 2,4-DHB by culturing such a modified microorganism.

Background Art

[0002] 2,4-Dihydroxybutyric acid (also referred to as 2,4-DHB or DHB) is a very economically important compound. DHB can be easily converted to α-hydroxy-γ-butyrolactone in an aqueous medium by adjusting to an appropriate pH value. α-Hydroxy-γ-butyrolactone is a major precursor for the production of the methionine substitute 2-hydroxy-4-(methylthio)-butyrate (HMTB) (US2009 / 318715) which has a large market in the animal nutrition field, or for the production of the hydroxy analogs of selenomethionine (HMSeBA or HMSeB; EP1778706 B1). Currently, α-hydroxy-γ-butyrolactone can be industrially prepared by two different chemical methods, which are i) A method for deriving γ-butyrolactone from γ-butyrolactone by a multi-step chemical process including halogenation of the α-position of γ-butyrolactone and subsequent substitution of the halogen atom with a hydroxyl group in an alkaline medium (Deck et al., 2008), and ii) A two-step chemical reaction from HMTB, namely alkylation followed by hydrolysis (EP2627645 B1) That is the case.

[0003] Rising oil prices have created a need to produce DHB from renewable resources. Microorganisms have the ability to convert biomass-derived raw materials such as sugars and organic acids into a wide variety of compounds (Werpy & Petersen, 2004). As biochemical and genomic information increases, it is becoming possible to modify microorganisms to overproduce naturally occurring metabolic intermediates in high yield and productivity (Bailey, 1991). Optimizing production microorganisms often requires rational engineering of metabolic networks, which ensures, in particular, the overexpression of enzymes necessary for the biosynthesis of target metabolites and the mitigation of product feedback inhibition. Another possibility is the realization of novel enzyme systems that catalyze the production of industrially important non-natural metabolites.

[0004] Similar to DHB, OHB is a non-natural product in microorganisms. In reference WO 2014 / 009435, a method for converting homoserine to 2,4-DHB using a two-step enzymatic conversion involving 2-oxo-4-hydroxybutyrate (OHB) reduction catalyzed by a modified malate dehydrogenase was first described in a modified microorganism. However, this DHB production pathway is not optimized, and carbon flow, particularly OHB as an enzyme substrate, may be diverted from the DHB production pathway to other competing pathways, resulting in a reduced final DHB yield. However, since OHB is a non-natural metabolite in microorganisms, it is necessary to identify and ultimately eliminate the generation of these potential competitors, which are not apparent to those skilled in the art. In other words, to optimize the DHB production yield by a two-step enzymatic conversion via 2-oxo-4-hydroxybutyrate (OHB), as described in the prior art, • Optimizing the pathway flux from the carbon source to the target compound by overexpressing restriction enzymes of the pathway, and / or • To attenuate and remove unwanted reactions that lose carbon as by-products such as organic acids and CO2, and / or • Eliminate the competitive pathways of common intermediates between the secondary pathway and the production pathway. This will be necessary. [Overview of the project] [Problems that the invention aims to solve]

[0005] Therefore, it is crucial to identify potential pathways that avoid OHB loss and promote pathway changes from DHB production to OHB. However, as mentioned above, since OHB is a non-natural product in microbial metabolism, one might expect that, in response to the presence of this exceptional metabolite, which has not been proposed or described in the prior art, there may be a presumed OHB consumption pathway, or that it may be activated accordingly, in relation to DHB production and other uses. Thus, methods for ensuring optimized DHB production are not obvious to those skilled in the art.

[0006] As mentioned above, the two-step pathway for producing 2,4-DHB from homoserine (i.e., the one described in WO2014 / 009435) is particularly observed to involve the co-production of formaldehyde and formic acid along with DHB. However, formaldehyde is widely known to be highly cytotoxic and mutagenic, while formic acid can cause problems in the downstream processing of DHB. Therefore, suppressing the production of these co-products would lead to an enhancement of the DHB production pathway and further promote the production of HMTB and / or HMSeB, especially the chemical production (i.e., non-microbial) of HMTB and / or HMSeB.

[0007] Therefore, it is necessary to reduce or even eliminate carbon flow loss in order to optimize the production of DHB and its by-products, such as HMTB and / or HMSeB, while simultaneously suppressing the production of secondary products that are particularly harmful / toxic to the producing microorganisms. The present invention aims to satisfy these requirements. [Means for solving the problem]

[0008] Therefore, one of the objectives of the present invention is an improved method for the preparation of 2,4-DHB from a carbon source via homoserine, and particularly from homoserine. • The first step is to convert the primary amino acid group of homoserine to a carbonyl group to obtain OHB, and • The second step involves reducing the obtained OHB to 2,4-DHB. This involves a two-step pathway (see Figure 1). Here, the pathway flux from the carbon source to the target compound is optimized, and OHB in particular, as an enzyme substrate, is not repurposed from the DHB production pathway, but rather chemically produced (i.e., independently of microorganisms) of HMTB and / or HMSeB in particular, by extending the HMTB and / or HMSeB production pathway.

[0009] The inventors have unexpectedly and surprisingly observed that inactivation, deletion, or modification of the enzymatic activity of so-called aldolase enzymes, or preferably pyruvate aldolase class I and class II enzymes, according to EC 4.1.2.28, results in higher DHB production, and even higher HMTB and / or HMSeB production, than that obtained by chemical production methods (i.e., non-microbial) or by carrying out the microorganisms described in prior art, particularly in reference WO2014 / 009435. Furthermore, this inactivation, deletion, or modification of the enzymatic activity of aldolase enzymes according to EC 4.1.2.28 reduces and / or prevents the generation of toxic byproducts such as formaldehyde.

[0010] Therefore, the present invention relates to a method for preparing 2,4-dihydroxybutyrate and / or further HMTB and / or HMSeB, in particular a method for chemically producing HMTB and / or HMSeB from a carbon source via homoserine, and especially from homoserine, wherein the method is The first step involves converting the primary amino acid group of homoserine to a carbonyl group and catalyzing it with an enzyme having homoserine transaminase activity as defined by EC 2.6.1.1, EC 2.6.1.42, or EC 2.6.1.57 to obtain 2-oxo-4-hydroxybutyrate, The second step is to reduce the obtained 2-oxo-4-hydroxybutyrate (OHB) to 2,4-dihydroxybutyrate by catalysis with an enzyme having OHB reductase activity, wherein the enzyme having OHB reductase activity is a lactate dehydrogenase as defined by EC 1.1.1.27 or EC 1.1.1.28, or a malate dehydrogenase as defined by EC 1.1.1.37, EC 1.1.1.82 or EC 1.1.1.299, It has a two-step path, At least one enzyme having aldolase activity as defined by EC 4.1.2.28 is inactivated to prevent the degradation of OHB.

[0011] In fact, Bouzon reported that OHB is produced by the condensation of pyruvate and formaldehyde using pyruvate-dependent aldolase (Bouzon et al., 2017). Several aldolases with slow substrate specificity have been reported to catalyze the condensation of pyruvate and formaldehyde to produce OHB, which is a non-natural intermediate in a synthetic pathway that enables CO2 assimilation by bacteria (Bouzon et al., 2017), but it was unclear whether the loss of these slow-substrate-specific enzymes promotes DHB production.

[0012] The present invention provides a method for producing 2,4-DHB and / or, further HMTB and / or HMSeB, particularly a chemical production method for HMTB and / or HMSeB, which provides an improved / optimized preparation method for 2,4-dihydroxybutyric acid, and further HMTB and / or HMSeB, preventing the decomposition of OHB, particularly to formaldehyde, and then to formic acid (see Figure 2). This leads to a significant improvement in yield, volume productivity, and DHB concentration, and / or, further HMTB and / or HMSeB concentration.

[0013] Therefore, inactivating at least one enzyme having aldolase activity as defined by EC 4.1.2.28 significantly improves the initial substrate yield (e.g., glucose) and also avoids the purification of 2,4-DHB, as well as the production of HMTB and / or HMSeB, and in particular the accumulation of formic acid, an organic acid that can interfere with the chemical production methods of HMTB and / or HMSeB.

[0014] Therefore, the present invention enables a significant improvement in carbon yield and, consequently, a significant reduction in the cost of any biotechnology process carried out using the method and / or modified microorganisms according to the present invention.

[0015] The carboxylic acids cited in this application are similarly named in the form of their salts (e.g., 2,4-dihydroxybutyrate) or acids (e.g., 2,4-dihydroxybutyric acid).

[0016] In this specification, enzyme activity is also specified by referring to the gene encoding the enzyme having such activity. The use of the gene name is not limited to a specific organism and includes all corresponding genes and proteins in other organisms (as long as the enzyme activity is retained, for example, microorganisms, functional analogs, functional mutants, and functional fragments thereof).

[0017] In the present invention, the expression "genetically modified microorganism" means that the microorganism according to the present invention does not exist in nature and has been modified by the introduction of new genetic elements and / or by the deletion and / or modification of endogenous genetic elements of the microorganism. This modification can be carried out by genome editing, in vivo evolution, genome recombination, induced or random mutagenesis, or other strain engineering techniques known to those skilled in the art.

[0018] In the present invention, the terms "inactivation", "modification", and "deletion" are used interchangeably and refer to the non-production of the corresponding protein, or, for example, the implementation of gene silencing techniques corresponding to transcriptional gene silencing or post-transcriptional gene silencing, the introduction of mutations into the promoter sequence controlling the expression of the gene encoding the protein and / or the coding sequence of the protein, the shift of the reading frame causing the introduction of an early termination codon into the coding sequence of the protein, or the generation of a non-functional corresponding protein obtained by the deletion of the gene encoding the protein and / or the gene encoding the promoter controlling the expression of the gene encoding the protein.

[0019] In the context of the present invention, the terms "improved", "optimized", "enhanced", and "increased" are used synonymously and refer to higher activity including enzyme activity and / or a greater amount of compound production compared to the same "non-improved" microorganism.

[0020] In the context of this invention, the terms “identical” and “homologous” are used synonymously and refer to sequence identity between two polypeptides. If, in each of the two sequences being compared, a certain position is occupied by the same monomeric amino acid subunit, then those molecules are homologous or identical at that position. Percentage identity between two sequences is a function of the number of identical molecules at corresponding positions in the two sequences, divided by the number of positions being compared and multiplied by 100. For example, if 6 out of 10 positions in two paired sequences are identical, the two sequences are 60% identical. In principle, this comparison is performed by aligning the two sequences to obtain the greatest possible homology / identity. Various bioinformatics tools known to those skilled in the art, such as BLAST and FASTA, may be used to align nucleic acid sequences.

[0021] The terms "coding" or "coding for," "code" or "code for," are used synonymously and refer to the inherent property of a particular nucleotide sequence in a polynucleotide such as a gene, cDNA, or mRNA to function as a template for the synthesis of other polymers having a specific amino acid sequence, and the resulting biological properties. Thus, a gene codes for a protein if the transcription and translation of the mRNA corresponding to that gene produces that protein in a cell or other biological system. Examples include both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is generally listed in sequence listings or databases, and the non-coding strand, which is used as a template for the transcription of a gene or cDNA, that codes for the protein or other product of that gene or cDNA. Therefore, in the context of this invention, the expression "substantial homology" includes homology with respect to structure and / or amino acid components and / or biological activity. Methods for performing sequence alignment and determining sequence identity are known to those skilled in the art, can be performed without requiring excessive experimentation, and the calculation of identity values ​​is readily apparent. For example, see Ausubel et al. (1995) and the ALIGN program (Dayhoff (1978)). There are many algorithms for aligning sequences and determining sequence identity, such as Needleman et al.'s (1970) homology alignment algorithm, Smith et al.'s (1981) local homology algorithm, Pearson et al.'s (1988) similarity search method, Smith-Waterman algorithm (Shpaer (1997)), BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990)). Computer programs that use these algorithms are also available, such as ALIGN or Megalign (DNASTAR) software, WU-BLAST-2 (Altschul et al. (1996)), GAP, BESTFIT, and BLAST (Altschul et al. (1997)).Examples include FASTA mentioned above, TFASTA available in Genetics Computing Group (GCG) package version 8 (Madison, Wisconsin, USA), and CLUSTAL from Intelligenetics' (Mountain View, California) PC / Gene program. Those skilled in the art can determine appropriate parameters for measuring alignment, including the algorithm necessary to achieve the maximum alignment over the lengths of the sequences being compared. Preferably, sequence identity is determined using default parameters determined by the program. Specifically, sequence identity can be determined using the Smith-Waterman homology search algorithm (Shpaer, 1997) implemented in the MSPRCH program (Oxford Molecular) using an affine gap search with the following search parameters: a gap-open penalty of 12 and a gap-extension penalty of 1. Preferably, paired amino acid comparison can be performed using the GAP program of the GCG sequence analysis software package from Genetics Computer Group, Inc. (Madison, Wisconsin), employing the Blosum 62 amino acid substitution matrix, with a gap weight of 12 and a length weight of 2. For optimal alignment of the two amino acid sequences, the contiguous segment of the mutant amino acid sequence may have additional or deleted amino acid residues compared to the reference amino acid sequence. The contiguous segment used for comparison with the reference amino acid sequence contains at least 20 consecutive amino acid residues, but may contain 30, 40, 50, or more. Correction for the increased sequence identity due to gaps in the derivative amino acid sequence can be performed by assigning a gap penalty.

[0022] In this invention, the term "carbon source" refers to nutrients that supply the carbon necessary for enzyme production according to the present invention. This so-called "carbon source" is a small molecule with a molecular weight of 500 g / mol or less, such as a monosaccharide, diphoroside, or polyol (e.g., glucose).

[0023] In the present invention, the expression "via homoserine" means that homoserine is an intermediate substrate in the DHB production pathway and / or further an intermediate substrate in the chemical production method of HMTB and / or HMSeB.

[0024] In the present invention, the amino acid substitution is a "conservative amino acid substitution" using L-amino acids, in which one amino acid is substituted with another amino acid that is biologically similar. A conservative amino acid substitution is a substitution in which the overall charge, hydrophobicity / hydrophilicity, and / or steric bulk of the substituted amino acid is conserved. Examples of conservative substitutions include substitutions between the following groups: namely, Gly / Ala, Val / Ile / Leu, Lys / Arg, Asn / Gln, Glu / Asp, Ser / Cys / Thr, and Phe / Trp / Tyr. The derivative may differ by only 1 to 10 amino acid residues, such as those that differ by 6 to 10 amino acid residues, 5 amino acid residues, 4 amino acid residues, 3 amino acid residues, 2 amino acid residues, or 1 amino acid residue.

[0025] In the present invention, the expression "functional variant" includes an enzyme that retains the original enzyme activity even if significant sequence modifications are observed compared to the sequence specifically described in the present application. This expression also includes cases where the sequence of the enzyme has fewer amino acids than the original sequence, but the truncated enzyme retains the original enzyme activity.

[0026] In the present invention, the expression "improving activity and / or substrate affinity" means that the enzyme before mutation was unable to use the substrate and / or synthesized the reaction product at a maximum specific rate at least 3 times lower, and / or had an affinity for homoserine or OHB at least 3 times lower, and / or had a maximum specific activity for the natural substrate at least 3 times higher, and / or had an affinity for the natural substrate at least 3 times higher.

[0027] In the present invention, "nucleic acid sequence" refers to a single-stranded or double-stranded DNA or RNA molecule, preferably a DNA molecule. In this specification, "isolated DNA" refers to DNA that does not exist in nature, or that no longer exists in the natural environment in which it originally existed. Examples include DNA coding sequences associated with other regulatory factors within a chimeric gene, DNA transplanted into another host cell, or artificially synthesized DNA sequences having a nucleotide sequence different from that of a natural DNA sequence.

[0028] In the context of the present invention, the term “microorganism” is intended to mean any lower single-celled organism into which a chimeric gene(s), nucleic acid(s), or vector(s) according to the present invention can be introduced to produce 2,4-DHB.

[0029] In the context of this invention, the expression "functionally linked to one another" means that the elements of a chimeric gene are linked to one another in such a way that the function of one of these elements is influenced by the function of the other elements. For example, a promoter is functionally linked to a coding sequence if it can influence the expression of that coding sequence. The construction of a chimeric gene and the assembly of its various elements according to this invention can be carried out using techniques well known to those skilled in the art. The selection of regulatory elements constituting a chimeric gene is essentially dependent on the host organism in which they must function, and those skilled in the art can select regulatory elements that function in a given host organism.

[0030] In the context of this invention, the term "functional" means capable of functioning within a specific host organism.

[0031] Preferably, the present invention relates to a two-step method for preparing 2,4-dihydroxybutyrate from a carbon source via homoserine, and particularly from homoserine, wherein at least one enzyme having aldolase activity as defined by EC 4.1.2.28 is inactivated to prevent the degradation of OHB, and the method is characterized by having the following features individually or in combination:

[0032] The enzyme realized in the first step is catalyzed by an enzyme encoded by an endogenous gene or a heterologous gene.

[0033] The enzyme realized in the second step is catalyzed by an enzyme encoded by an endogenous gene or a heterologous gene.

[0034] The enzyme having aldolase activity is catalyzed by an enzyme encoded by an endogenous gene or a heterologous gene.

[0035] The enzyme having aldolase activity is encoded by the sequence described in SEQ ID NO: 1 or SEQ ID NO: 3, or has the sequence described in SEQ ID NO: 2 or SEQ ID NO: 4, or has any sequence having at least 50% homology, preferably at least 60% homology, at least 70% homology, or at least 80% homology, preferably at least 90% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology to SEQ ID NO: 1, 3, 2, or 4.

[0036] The enzyme that converts the primary amino acid group of homoserine to a carbonyl group to obtain OHB may be homoserine transaminase, homoserine dehydrogenase, or homoserine oxidase.

[0037] Enzymes possessing homoserine transaminase activity can be identified from among enzymes possessing aspartate transaminase (EC 2.6.1.1) activity, branched-chain amino acid transaminase (EC 2.6.1.42) activity, or aromatic amino acid transaminase (EC 2.6.1.57) activity.

[0038] The carbon source is selected from the group consisting of monosaccharides, diphorosides, polyols, and mixtures thereof.

[0039] The carbon source is selected from the group consisting of glucose, sucrose, lactose, glycerol, and mixtures thereof.

[0040] The carbon source is glucose.

[0041] • Enzymes possessing homoserine transaminase activity are encoded by the genes iIvE, tyrB, aspC, araT, bcaT, alaC, and ARO8. Preferably, the enzyme possessing homoserine transaminase activity may be the branched-chain amino acid transaminase Ec-IlvE from E. coli, and Ll-BcaT from Lactococcus lactis, and the homoserine transaminase activity may be the alanine-α-ketoglutarate transaminase Ec-AlaC from E. coli, more preferably the mutant Ec-AlaC. A142P Y275D These may be aromatic amino acid transaminases derived from Escherichia coli, Ec-TyrB, Ll-AraT derived from L. lactis, and Sc-Aro8 derived from Saccharomyces cerevisiae, or aspartate transaminases derived from Escherichia coli, Ec-AspC, or homologs thereof.

[0042] The enzyme having homoserine transaminase activity is encoded by the sequences described in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 126, SEQ ID NO: 128, or corresponds to SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 127, SEQ ID NO: 129, or corresponds to any sequence having at least 50% homology to SEQ ID NOs: 5-16, preferably 60% homology, at least 70% homology, at least 80% homology, preferably at least 90% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology.

[0043] The enzyme having OHB reductase activity is (D)-lactate dehydrogenase, preferably (D)-lactate dehydrogenase derived from Escherichia coli; (L)-lactate dehydrogenase, preferably (L)-lactate dehydrogenase derived from Lactococcus lactis, Orictalagus cuniculus, Diobacillus stearothermophilus, or Bacillus subtilis; (L)-malate dehydrogenase, preferably (L)-malate dehydrogenase derived from Escherichia coli, or homologs thereof.

[0044] The enzyme having OHB reductase activity is a lactate dehydrogenase having at least one mutation at position V17, Q85, E89, I226, or A222, wherein the said position is L-lactis Lactate dehydrogenase as defined with reference to tA (SEQ ID NO: 17 or 18), and / or malate dehydrogenase having at least one mutation at position I12, R81, M85, D86, V93, G179, T211, or M227, wherein the position is a malate dehydrogenase as defined with reference to Escherichia coli Mdh (SEQ ID NO: 19 or 20), where the native amino acid at the position is substituted by any of the other 19 naturally occurring protein-constituting amino acids, namely alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0045] Enzymes possessing OHB reductase activity are lactate dehydrogenases containing at least one mutation at position V17, Q85N, or I226V as defined with reference to L-lactis tA (SEQ ID NO: 17 or 18), and / or malate dehydrogenases containing at least one mutation at position I12V, R81A, M85Q, D86S, or G179D as defined with reference to Escherichia coli Mdh (SEQ ID NO: 19 or 20).

[0046] The enzyme having OHB reductase activity is a lactate dehydrogenase encoded by the sequence described in SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO: 25, or the sequence corresponding to SEQ ID NO: 22, SEQ ID NO: 24, or SEQ ID NO: 26, or any sequence having at least 50%, preferably at least 60%, at least 70%, at least 80%, preferably at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with SEQ ID NOs: 21-26.

[0047] The enzyme having OHB reductase activity is malate dehydrogenase encoded by the sequences described in SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, or sequences corresponding to SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, or any sequence having at least 50%, preferably at least 60%, at least 70%, more preferably at least 80%, more preferably at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more preferably at least 99% homology with the sequences of SEQ ID NOs: 27-44.

[0048] The enzyme having OHB reductase activity is encoded by the sequence described in SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, or SEQ ID NO: 81, or the sequence corresponding to SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, or SEQ ID NO: 82, or any sequence having at least 50%, preferably at least 60%, at least 70%, more preferably at least 80%, preferably at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more preferably at least 99% homology with the sequences of SEQ ID NOs: 45-82.

[0049] The enzymes according to the present invention having the same activity (any of the OHB reductases), or the enzyme that obtains OHB by converting the primary amino acid group of homoserine to a carbonyl group, or the enzyme having aldolase activity, have at least about 50%, 70%, or 85% amino acid sequence identity, preferably at least about 85% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, even more preferably at least about 95% amino acid sequence identity, and even more preferably 98% or 99% amino acid sequence identity.

[0050] • The activity of the enzyme catalyzing the first and / or second step of the method of the present invention is improved. This improvement can be measured by the enzyme assay described in the examples. The improvement of the enzyme can be achieved by at least one mutation, the mutation(s) i) improves the activity and / or substrate affinity of the mutant enzyme to homoserine or OHB, respectively, and / or ii) decreases the activity and / or substrate affinity of the mutant enzyme to the native substrate.

[0051] The present invention also relates to a method for preparing hydroxymethionine (HMTB) and / or hydroxyselenomethionine (HMSeB) from a carbon source via homoserine, and particularly from homoserine. This method comprises a two-step pathway and prevents the degradation of OHB by inactivating at least one enzyme having aldolase activity as defined in EC 4.1.2.28, as defined above. In fact, 2,4-DHB is converted to HMTB or HMSeB by a cyclization reaction by adding sodium mercaptan or lithium methaneselenoate, as described in EP2054382 and EP1778706.

[0052] The present invention also relates to a chimeric gene in which at least one promoter functional in a host organism, a polynucleotide encoding any of the enzymes that catalyze the first and second steps of the method defined according to the present invention, and a terminator element functional in the same host organism are functionally linked to each other. The various elements that a chimeric gene may contain are, firstly, elements that control the transcription, translation, and maturation of a protein, such as a promoter, a sequence encoding a signal peptide or transit peptide, or a terminator element constituting a polyadenylation signal, and secondly, a polynucleotide encoding a protein.

[0053] The promoters that may be included in the chimeric gene according to the present invention are either constitutive promoters or inductive promoters. For example, promoters used for expression in bacteria can be selected from the promoters listed below. For expression in Escherichia coli, examples include lac, trp, lpp, phoA, recA, araBAD, prou, cst-I, tetA, cadA, nar, tac, trc, lpp-lac, Psyn, cspA, PL, PL-9G-50, PR-PL, T7, [lambda]PL-PT7, T3-lac, T5-lac, T4 gene 32, nprM-lac, VHb, and protein A promoter, or otherwise the Ptrp promoter (WO99 / 64607). For expression in Gram-positive bacteria such as Corynebacterium or Streptomyces, examples include the PtipA promoter or the PS1 promoter and PS2 (FR91 / 09870) promoter, or the promoters described in EP0629699A2. Examples of expression targets in yeast and fungi include the K. lactis PLAC4 promoter or the K. lactis Ppgk promoter (FR91 / 05294), the Trichoderma liesey tef1 or cbh1 promoter (WO94 / 04673), the Penicillium funiclosum his, csl or apf promoter (WO00 / 68401), and the Aspergillus niger gla promoter.

[0054] According to the present invention, the chimeric gene may also include other regulatory sequences located between the promoter and the coding sequence, such as transcription activators (enhancers).

[0055] Therefore, in certain embodiments, the chimeric gene of the present invention comprises at least a promoter regulatory sequence functional in the host organism, functionally linked in the transcription direction, a nucleic acid sequence encoding a polynucleotide encoding any enzyme that catalyzes the first and second steps of the method defined according to the present invention, and a terminator regulatory sequence functional in the host organism.

[0056] The present invention also relates to cloning vectors and / or expression vectors comprising a chimeric gene or nucleic acid sequence according to the present invention. The vector according to the present invention is used to transform a host organism and to express in this organism any enzyme that catalyzes the first and / or second step(s) of the method according to the present invention. The vector may be a plasmid, cosmid, bacteriophage, or virus. Preferably, the transformation vector of the present invention is a plasmid. Generally, the main features of the vector are its ability to self-maintain and self-replicate within the host organism's cells, particularly due to the presence of an origin of replication, and its ability to express any enzyme that catalyzes the first and / or second step(s) of the method according to the present invention. To stably transform the host organism, the vector may be incorporated into the genome. The selection of such a vector, and the technique of inserting the chimeric gene according to the present invention into this vector, are in the general knowledge of those skilled in the art. Advantageously, the vector used in the present invention also includes a chimeric gene encoding a selection marker in addition to the chimeric gene according to the present invention. This selection marker allows for the selection of an effectively transformed host organism, i.e., a host organism into which the vector has been incorporated. According to certain embodiments of the present invention, the host organism to be transformed is a bacterium, yeast, or fungus. Available selection markers include, for example, markers containing antibiotic resistance genes such as the hygromycin phosphotransferase gene. Other markers include genes that complement nutritional requirements, such as the pyrA, pyrB, pyrG, pyr4, arg4, argB, and trpC genes, the molybdopterin synthase gene, and the acetamidase gene. Also included are genes encoding easily identifiable enzymes such as the GUS enzyme, or genes encoding pigments or enzymes that control pigment production in transformed cells. In particular, such selection marker genes are described in Japanese patent applications WO91 / 02071, WO95 / 06128, WO96 / 38567, and WO97 / 04103.

[0057] The improved / optimized DHB and / or further HMTB and / or HMSeB production, particularly the chemical production method of HMTB and / or HMSeB according to the present invention, can be carried out in wild-type microorganisms (i.e., non-genetically modified microorganisms) or genetically modified microorganisms that have already been genetically modified, i.e., genetically modified in a manner different from that described in the present invention. Such genetic modification may result in an increase in the production / concentration of intermediate products involved in DHB production, and / or further in the production / concentration of intermediate products involved in the chemical production of HMTB and / or HMSeB. For example, a microorganism genetically modified to produce homoserine in a higher content / yield than wild-type microorganisms may be used.

[0058] More specifically, a modified microorganism for a method of preparing 2,4-dihydroxybutyrate and / or further HMTB and / or HMSeB from a carbon source via homoserine, and particularly from homoserine, wherein the method is The first step involves converting the primary amino acid group of homoserine to a carbonyl group and catalyzing this process with an enzyme having homoserine transaminase activity as defined by EC 2.6.1.1, EC 2.6.1.42, or EC 2.6.1.57 to obtain 2-oxo-4-hydroxybutyrate, The second step involves reducing the obtained 2-oxo-4-hydroxybutyrate (OHB) to 2,4-dihydroxybutyrate by catalysis with an enzyme having OHB reductase activity, The enzyme having OHB reductase activity is a lactate dehydrogenase as defined by EC 1.1.1.27 or EC 1.1.1.28, or a malate dehydrogenase as defined by EC 1.1.1.37, EC 1.1.1.82, or EC 1.1.1.299. Step 2, A method comprising a two-step path, At least one enzyme having aldolase activity as defined by EC 4.1.2.28 is inactivated to prevent the degradation of OHB.

[0059] Preferably, the present invention relates to a modified microorganism for the improved / optimized production of DHB from a carbon source via homoserine, and / or further for the production of HMTB and / or HMSeB (particularly a chemical production method for HMTB and / or HMSeB), wherein the production method comprises a two-step pathway defined as having the following features alone or in combination, wherein the degradation of OHB is prevented by inactivation of at least one enzyme having aldolase activity as defined in EC 4.1.2.28. The enzymes involved in the two steps described above are as stated above. The microorganisms are preferably fungi, such as those of the genera Penicillium, Aspergillus, and especially those of the genera Aspergillus, Chrysosporium, or Trichoderma. The microorganisms are yeasts, particularly yeasts of the Saccharomyces, Pichiaceae, or Schizosaccharomyces families, most preferably Saccharomyces cerevisiae, Schizosaccharomyces pombe, Cluyveromyces lactis, Cluyveromyces marcianas, or Pichia jadini, Pichia stipitis, or Pichia pastris. The microorganisms are bacteria, preferably selected from the Enterobacteriaceae, Clostridaceae, Bacillaceae, Streptomycesaceae, Streptococciaceae, Methylobacteriaceae, and Corynebacteriaceae, most preferably Escherichia coli, Bacillus subtilis, Corynebacteria, Clostridium acetobutyricum, Methylobacterium extroquens, or Lactobacillus lactis.

[0060] In specific embodiments, the present invention also relates to a modified microorganism comprising at least one chimeric gene according to the present invention, which is incorporated into the genome or retained on an extrachromosomal genetic element, for example, on a plasmid. In a more specific aspect of the present invention, the transformed host organism comprises an expression vector comprising a nucleic acid of the present invention encoding a polypeptide that converts a primary amino acid group of homoserine to a carbonyl group to obtain OHB, and / or a polypeptide that reduces OHB in 2,4-DHB, a chimeric gene comprising a nucleic acid encoding a polypeptide that converts a primary amino acid group of homoserine to a carbonyl group to obtain OHB, and / or OHB reductase, or a polypeptide that converts a primary amino acid group of homoserine to a carbonyl group to obtain OHB, or a nucleic acid encoding a polypeptide having OHB reductase activity.

[0061] In a further aspect of the present invention, a synthetic pathway for converting homoserine to DHB and / or further producing HMTB and / or HMSeB, particularly a method for the chemical production of HMTB and / or HMSeB, is expressed in a microorganism with enhanced homoserine production, i.e., having higher homoserine production than the parent organism or wild-type organism. Enhanced homoserine production in microorganisms can be achieved by i) overexpressing aspartate kinase, aspartate semialdehyde dehydrogenase, and homoserine dehydrogenase enzymes, ii) making the aspartate kinase enzyme insensitive to product inhibition that may be caused by lysine, methionine, or threonine, and iii) deleting metabolic pathways branching off from the homoserine biosynthesis pathway. Overexpression of aspartate kinase, aspartate semialdehyde dehydrogenase, and homoserine dehydrogenase can be achieved by expressing the enzymes from a multicopy plasmid under the control of an appropriate constitutive or inducible promoter. Alternatively, overexpression of these enzymes can also be achieved by deleting transcriptional repressors that restrict the transcription of genes encoding aspartate kinase, aspartate semialdehyde dehydrogenase, and homoserine dehydrogenase. Aspartate kinase can be made insensitive to inhibition by aspartate-derived amino acids by introducing appropriate mutations into its amino acid sequence. The entry points to metabolic pathways branching off from the homoserine biosynthesis pathway are catalyzed by enzymes possessing O-succinyl homoserine or O-acetyl homoserine synthase activity (entry point to methionine biosynthesis), homoserine kinase activity (entry point to threonine biosynthesis), or diaminopimelate decarboxylase activity (entry point to lysine biosynthesis). By deleting the genes encoding proteins with these enzymatic activities, the production of aspartate-derived amino acids is suppressed and homoserine production is promoted (Figure 1).

[0062] Deletion of the metA, thrB, and lysA genes in Escherichia coli attenuates pathways branching off from the homoserine biosynthesis pathway. Enzyme activity enhancement of the homoserine pathway in Escherichia coli can be achieved, for example, by overexpression of the bifunctional aspartate kinase-homoserine dehydrogenase mutants thrA S345F (insensitive to threonine inhibition) and asd (both genes derived from Escherichia coli), or by overexpression of the monofunctional aspartate kinase mutant lysC E250K (insensitive to lysine), asd (both genes derived from Escherichia coli), and the homoserine dehydrogenase gene HOM6 derived from S. cerevisiae.

[0063] The microorganisms of the present invention can reduce the ability to excrete homoserine, thereby increasing the intracellular availability of this amino acid. To reduce homoserine excretion from cells, the permease capable of excreting homoserine can be deleted. Such permeases can be identified by overexpressing a genomic library in the microorganism and culturing the microorganism at inhibitory concentrations of homoserine or structurally similar amino acids such as threonine, leucine, and aspartic acid (Zakataeva et al. (1999)). Genes whose overexpression promotes growth when the concentration of any of the above amino acids is elevated are likely to be involved in homoserine excretion.

[0064] In a further aspect, the microorganism Escherichia coli of the present invention has a deletion in the homoserine efflux transporters rhtA, rhtB, and / or rhtC.

[0065] Therefore, the microorganism of the present invention exhibits increased expression of at least one enzyme activity selected from phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, isocitrate lyase, pyruvate carboxylase, and hexose cotransporter permease, and / or decreased expression of at least one enzyme activity selected from lactate dehydrogenase, alcohol dehydrogenase, acetate kinase, phosphate acetyltransferase, pyruvate oxidase, isocitrate lyase, fumarase, 2-oxoglutarate dehydrogenase, pyruvate kinase, malate enzyme, phosphoglucose isomerase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, pyruvate formate lyase, succinate semialdehyde dehydrogenase, sugar transporter phosphotransferase, ketohydroxyglutarate aldolase, homoserine-O-succinate transferase, homoserine kinase, homoserine efflux transporter, diaminopimephosphate decarboxylase, and / or methylglyoxal synthase. In a further aspect, the microorganism Escherichia coli of the present invention overexpresses at least one gene selected from ppc, pck, aceA, galP, asd, thrA, metL, lysC derived from Escherichia coli, and pycA derived from Lactococcus lactis, and / or deletes at least one gene selected from IdhA, adhE, ackA, pta, poxB, focA, pflB, sad, gabABC, sfcA, maeB, ppc, pykA, pykF, mgsA, sucAB, ptsl, ptsG, pgi, fumABC, aldA, lldD, iclR, metA, thrB, lysA, eda, rthA, rthB, rthC.

[0066] The present invention relates to a method for producing 2,4-DHB and / or further HMTB and / or HMSeB, and more particularly to a method for chemically producing HMTB and / or HMSeB from 2,4-DHB produced by the present invention, wherein the method is • Culturing the modified microorganism of the present invention in an appropriate culture medium, • To recover 2,4-DHB from the culture medium, The method includes the step of further purification of 2,4-DHB. The method according to the present invention can also produce HMTB and / or HMSeB by adding a methyl sulfur donor or a methyl selenium donor to 2,4-DHB.

[0067] Product separation and purification are crucial elements that significantly impact the overall process efficiency and product cost. Product recovery methods typically involve the steps of cell separation, product purification, concentration, and drying, respectively.

[0068] Cell separation: Ultrafiltration and centrifugation can be used to separate cells from fermentation media. Cell separation from fermentation media is often complicated when the viscosity of the medium is high. Therefore, to optimize cell separation, additives such as inorganic acids or alkali salts may be added, or the culture medium may be heated.

[0069] Product Recovery: Before or after biomass removal, various ion exchange chromatography methods can be applied for the separation of DHB and / or HMTB and / or HMSeB. These methods include the use of primary cation exchange resins that facilitate product separation according to their isoelectric point. Typically, the resin is filled with a solution, and the retained products are eluted individually after an increase in the pH of the eluate (e.g., by the addition of ammonium hydroxide). Other methods include the use of ion exchange chromatography with fixed-bed or pseudo-mobile-bed resins. In some cases, different chromatography steps may need to be combined to achieve the desired product purity. These purification methods are more economical compared to costly crystallization steps and can offer additional advantages and flexibility regarding the morphology of the final product. [Brief explanation of the drawing]

[0070] [Figure 1]This diagram illustrates a two-step pathway for producing 2,4-DHB, and further HMTB and HMSeB, from homoserine, comprising a first step of converting the primary amino acid group of homoserine to a carbonyl group to obtain OHB, and a second step of reducing the obtained OHB to 2,4-DHB. [Figure 2] This is a diagram of the OHB degradation pathway. [Figure 3] This figure shows the in vivo DHB consumption and formic acid production after incubation with wild-type E. coli cells and "single aldolase-deficient" E. coli cells with DHB. [Figure 4] This figure shows the in vivo DHB consumption and formic acid production after incubation with wild-type E. coli cells and "multiple aldolase-deficient" E. coli cells with DHB. [Figure 5] This diagram shows the in vivo production of DHB and formic acid during glucose fermentation in an aldolase-deficient E. coli DHB-producing strain. [Modes for carrying out the invention]

[0071] Examples 1. Materials and Methods 1.1 Strains and Culture Conditions Details of the bacterial strains used in the examples are shown in Table 1.

[0072] [Table 1]

[0073] Unless otherwise specified (as described below), Escherichia coli K-12 strain MG1655 substrain (ATCC number 47046) was cultured in M9 medium containing 0.2% D-xylose as a carbon source. Where necessary (as described below), 50 mM DHB (a racemic mixture of L-type and D-type DHB ammonium) was also added to this medium.

[0074] 1.2 Gene deletion The genes encoding pyruvate aldolase (i.e., the aldolase according to the present invention) were deleted in Escherichia coli K-12 strain MG1655 substrain using the P1 transduction adaptation protocol (Thomason et al., 2007) for yagE, yjhH, garL, dgoA, yfaU, and eda, and the CrispR-Cas9 method (Jiang et al., 2015) for mhpE.

[0075] 1.3 P1 trait introduction Preparation of lysate 25 μL of sterile glucose solution (20% w / v) and 25 μL of sterile CaCl21M were added to 5 mL of lysogenic culture medium (LB). To this mixture, 100 μL of saturated culture medium from the KEIO collection (Baba et al., 2006) containing the target deletion was inoculated, and the mixture was cultured with shaking at 37°C. After 45 minutes, 100 μL of P1 stock was added, and the mixture was cultured for 3 hours or until lysis was complete. The lysated culture was centrifuged at 4000 g at 4°C for 10 minutes, and the supernatant was filtered and sterilized. The lysate should not lose efficiency even when stored at 4°C for several years.

[0076] Trait introduction One mL of saturated culture medium of the target strain was centrifuged and resuspended with 100 μL of sterile P1 salt solution (10 mM CaCl2, 5 mM MgSO4) and 100 μL of P1 lysate containing the desired mutation. The cells were incubated at 30°C for 30 minutes without stirring, then 100 μL of sterile 1 M sodium citrate solution was added to stop transduction, followed by 1 mL of LB medium. The cells were incubated at 37°C for 1 hour and then seeded in LB kanamycin (50 μg / mL).

[0077] Removal of kanamycin-resistant cassettes Considering that the kanamycin-resistant cassette in the KEIO collection has an FRT site adjacent to each side, this cassette can be easily disrupted using the Flp enzyme (Cox, 1983) derived from Saccharomyces cerevisiae, encoded by the pCP20 plasmid (Cherepanov and WackeRNAgel, 1995). Cells possessing the kanamycin-resistant cassette instead of the target gene can be transformed with pCP20 using the protocol described in other literature (Chung et al., 1989).

[0078] Following transduction, multiple clones were collected on LB Kan plates and inoculated into 1 mL of LB medium. When the OD600 of the culture medium reached approximately 0.3, the cells were centrifuged and resuspended in TSS solution (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 10 g / L PEG 10 kDa, 5 g / L DMSO, and 220 mM MgCl). After leaving the cells on ice for 15 minutes, 100-150 ng of pCP20 plasmid preparation was added. After leaving the cells on ice for 15 minutes, a heat shock of 42°C for 1 minute was administered to the cells, followed by the addition of 1 mL of LB medium. Transexpression of the beta-lactamase encoded by pCP20 was performed with stirring at 30°C for 1-2 hours before seeding on LB ampicillin (100 μg / mL).

[0079] Several clones were taken from LB ampicillin plates, inoculated into 1 mL of LB medium, and incubated at 42°C for 6–8 hours. Several microliters of the culture solution were spread onto LB plates and incubated overnight at 37°C.

[0080] Several clones were individually isolated from LB plates, streaked onto LB Amp, LB kan, and LB plates, and clones that lost resistance to both ampicillin and kanamycin were counter-selected. Deletions in these antibiotic-susceptible clones were confirmed by colony PCR using OneTaq® 2X Master Mix with Standard Buffer (BioLabs) with primers listed in Table 1, following the manufacturer's protocol. Gene deletions were confirmed by PCR amplification using primers listed in Table 3 in all strains with one of the multiple deletions.

[0081] [Table 2]

[0082] [Table 3]

[0083] 1.4 Deletion by CrispR-Cas9 The deletion of mphE was performed using CrispR-Cas9, as described in other literature (Jiang et al., 2015). First, the target strain was transformed using a pCas plasmid containing the Cas9 encoding gene and a lambda red recombination mechanism, as previously described. The plasmid pTarMHPE, which generates gRNA, was constructed based on pTargetF and amplified using primers TM60 and TM34. Accuracy of the amplification was confirmed using primer TM35 with the Sanger sequencing service provided by Eurofins Genomics.

[0084] Construction of donor DNA Repair fragments were obtained from the MG1655 substrain of the genomic E. coli K-12 strain. Briefly, the mhpE region was amplified from a genomic DNA preparation of wild-type E. coli MG1655 using primers TM32 and TM33. The resulting fragment was 1873 bp long and, after gel purification (BioBasic EZ-10 Spin Column DNA Gel Extraction Kit), the 5' end was phosphorylated using T4 polynucleotide kinase (BioLabs). The vector used was pZE13 (Expressys), digested with EcoRV (BioLabs) and dephosphorylated with Antarctic phosphatase (BioLabs). Subsequently, the vector and insert were ligated using HiT4 DNA Ligase (BioLabs). The quality of the insert was confirmed by sequencing using primers TM61 and TM62.

[0085] The obtained circular plasmid (pMHPE) was amplified using primers TM36 and TM37, and the target gene was linearized and deleted. Subsequently, gel purification, phosphorylation, and ligation were performed to obtain pMHPEΔ, in which the coding sequence was deleted from the genomic region of mhpE. Finally, CrispR-Cas9 donor DNA was amplified from pMHPEΔ using primers TM32 and TM33.

[0086] Gene disruption using CrispR-Cas9 Cells containing the pCas plasmid were introduced with 100 ng of pTarget and 400 ng of donor DNA by electroporation. Briefly, 50 μL of a culture of the target strain cultured overnight was inoculated into 5 mL of LB Kan (50 μg / mL) and arabinose (10 mM), and cultured at 30°C for 3-4 hours, or until the OD600 reached 0.5-0.7. Then, 2 mL was taken, allowed to stand on ice to form a pellet, and washed once with 1 mL of ice-cold ddH2O and once with 1 mL of ice-cold 10% glycerol. The cells were resuspended in 50 μL of ice-cold 10% glycerol, and transformation was performed using 100 ng of pTarget and 400 ng of donor DNA under conditions of 2 mm cuvette, 2.5 kV, 200 Ω, and 25 μF. The cells were immediately resuspended, and the state of oxygen (SOC) was allowed to stand overnight at 30°C. All transformation reaction solutions were seeded onto LB kanamycin spectinomycin plates. Before repairing the plasmid and checking for any remaining mutations as needed, the primers mhpE_cas_F and mhpE_cas_R were used to confirm that the genome editing had been performed correctly.

[0087] 1.5 Cloning and characterization of the gene encoding pyruvate aldolase How to create a clone Seven genes encoding pyruvate aldolase were cloned into the pET28 vector (Addgene). Briefly, as shown in Table 4, the genes were amplified from the start codon to the stop codon, without the 3' end. Except for eda and mhpE, this base expansion allowed for the addition of an AvrII restriction site to the 5' end and an MfeI site to the 3' end of the genes. For eda and mhpE, different restriction site pairs, specifically NdeI / XhoI and BamHI / HindIII, were used. The amplicons were gel-purified (BioBasic gel extraction kit) before digestion with the corresponding restriction enzymes (BioLabs). The pET28 vector was amplified with primers that would allow for future corresponding ligation, and the amplicons were digested with 1 μL of DpnI (BioLabs), followed by cleanup (BioBasic gel extraction kit) and digestion with cloning restriction enzymes. The digested product was purified and dephosphorylated using Antarctic Phosphatase (BioLabs). Vector amplification allowed for the addition of the MfeI site and the removal of a portion of the T7 tag and MCS carried by the vector. For mhpE insertion, the pET28 vector was used directly for digestion.

[0088] The insert and vector were ligated using HiT4 DNA ligase (Biolabs), and the resulting cells were transformed into NEB® 5-alpha cells (BioLabs). Positive clones were amplified by PCR using primers TM89 and TM90 to confirm the correct insert length, which was then validated by sequencing.

[0089] [Table 4]

[0090] Production and purification of recombinant proteins E. coli BL21(DE3) strain was transformed with a pET-28a(+) plasmid containing an aldolase gene (yagE, yjhH, yfaU, garL, dgoA, or eda). Isolated colonies were cultured overnight at 37°C and 200 rpm in 14 mL culture tubes containing 5 mL of LB medium with 50 μg / mL kanamycin. When the initial OD600 nm was approximately 0.1, the cells were transferred to 500 mL Erlenmeyer tubes containing 100 mL of fresh LB medium with 50 μg / mL kanamycin. The culture was incubated at 37°C and 200 rpm until the OD600 nm reached approximately 0.6. At this stage, 1 mM IPTG was added to the culture medium and incubated at 37°C and 200 rpm for 3 hours. The cells were centrifuged. The pellet was resuspended in lysesis buffer (Tris-HCl 50 mM, pH 7.5, NaCl 300 mM, imidazole 5 mM), sonicated at 30% for 30 seconds for 4 cycles, and cooled on ice-ethanol mixture (Fisherbrand) for 30 seconds during this time. The cell lysates were centrifuged at 14800 rpm for 15 minutes, and the supernatant was collected and purified. His-tagged proteins were purified using cobalt TALON His Tag purification resin (GE Healthcare). The proteins were eluted with 400 μL of Tris-HCl, 50 mM, pH 7.5, NaCl 300 mM, imidazole 200 mM. The purified enzymes were concentrated in 50 mM Tris-HCl buffer using 10 kDa Amicon Ultra (Millipore). Considering that 1 mg corresponds to 1 OD at 260 nm, protein purification was confirmed by SDS-PAGE, and the protein concentration was measured using a spectrophotometer (Epoch). 2 Measured using Biotek.

[0091] 1.5 Synthesis of 2-oxo-4-hydroxybutyric acid (OHB) OHB was synthesized from D-homoserine, as previously reported (Walther et al., 2018). Briefly, 125 mM D-homoserine was incubated with porcine kidney D-amino acid oxidase (1.25 U / mL, Sigma Aldrich) and bovine liver catalase (4400 U / mL, Sigma Aldrich) in 100 mM Tris buffer (pH 7.8) at 37°C for 90 minutes. The reaction mixture was purified using an Amicon® ultracentrifuge filter (cutoff 10 kDa, Millipore) to remove the enzymes.

[0092] OHB was quantified by mixing 100 μL of purified reaction solution at pH 6.5 with 1 mL of 1 M boric acid and 1 M sodium arsenate. The reaction solution was incubated at room temperature for 30 minutes, and the absorbance was measured at 325 nm. The relationship between absorbance and ketone concentration was calibrated using pyruvate solution of known concentration (Wellner and Lichtenberg, 1971).

[0093] 1.6 Kinetic properties of pyruvate aldolase in OHB Aldolase activity is measured by a binding enzyme assay, where excess LdhA is collected using a microplate reader (Epoch). 2 NADH was detected by oxidation at 37°C and 340 nm using a biotope (Biotek). The assay was performed in 250 μL of Tris-HCl containing 0.2 mM NADH, 50 mM, pH 7.5, 5 mM MgCl2 buffer, with varying concentrations of OHB from 0 to 13 mM. Since E. coli LDH is not commercially available, this enzyme was produced from E. coli BL21(DE3) strain transformed with the pET-28a(+) plasmid containing E. coli ldhA, as described above. The transformed cells were cultured in 500 mL of Erlenmeyer medium containing 100 mL of self-inducible ZYM-5052 medium (Studier, 2005) containing 50 μg / mL kanamycin, and cultured overnight at 37°C and 200 rpm.

[0094] 2. Evidence of the OHB degradation pathway The results are shown in Figure 3. This data indicates that when E. coli is cultured in M9 medium containing 0.2% D-xylose in the presence of 50 mM DHB, a considerable amount of formic acid accumulates. The fact that more than 70% of the consumed DHB was detected as formic acid further highlights the potential importance of this OHB degradation pathway (Figure 3).

[0095] 3. Identification of OHB aldolase Assuming that E. coli possesses OHB aldolase activity, we explored databases (particularly Ecocyc) and literature to identify potential so-called pyruvate aldolase enzymes. This research identified at least seven aldolases of this type that share the versatility to potentially react with OHB. He et al. (2020) constitutively expressed these aldolases and condensed pyruvate and formaldehyde into OHB. While aldolases catalyze reversible reactions, their in vivo functions may differ. In addition, although the genes encoding these pyruvate aldolases are present in the E. coli genome, it is unclear whether they are expressed, or if so, whether they can cleave OHB into pyruvate and formaldehyde in vivo. Therefore, based on previous literature reviews (He, H et al., 2020; Clapes et al., 2010), the inventors listed seven pyruvate aldolases (Table 5) that can cleave OHB into pyruvate and formaldehyde in Escherichia coli or other microorganisms.

[0096] [Table 5]

[0097] Transcriptome analysis during DHB fermentation identified four genes from the aldolase-encoding genes listed in Table 5 that may be involved in OHB degradation (see Table 6). In particular, the expression of YagE and YjhH, which encode aldolases, was most elevated, and this elevation coincided with the maximum formic acid production during fermentation.

[0098] [Table 6]

[0099] Therefore, the inventors introduced systematic deletions one by one and evaluated the effect of these single deletions and combined deletions on the outcome of formic acid production from DHB.

[0100] 4. Evaluation of the kinetic properties of pyruvate aldolase in relation to OHB To target the most efficient OHB aldolase within the cell, five types of aldolases were characterized. OHB affinity (Km value) and catalytic efficiency (Kcat / Km value) were determined for YagE, YjhH, RhmA, DgoA, and GarL aldolases. These results are shown in Table 7. The aldolase encoded by yfaU exhibited the highest catalytic efficiency (kcat / Km), and this enzyme showed 10 times better affinity than YagE and YjhH aldolases. Furthermore, yfaU expression remained unchanged during DHB fermentation. These results may suggest that YagE and YjhH could be candidates for the optimal OHB aldolase under these DHB production conditions.

[0101] [Table 7]

[0102] 5. The ability of the body to degrade OHB due to the deletion of the gene encoding pyruvate aldolase. To evaluate the ability to degrade OHB in vivo, and thus DHB in vivo, experiments were conducted in which DHB was cultured with E. coli cells under aerobic conditions in M9 medium containing 0.2% glucose. Formic acid production was measured in strains in which one of the genes encoding these aldolases was deleted. The results are shown in Figure 3. Simultaneous deletion of yagE and yjhH significantly reduced DHB consumption and formic acid production (see Figure 4), indicating that these enzymes primarily contribute to the reduction of OHB during DHB production by fermentation (see Figure 5). However, deletion of the seven aldolases listed in Table 1 completely eliminated OHB production from DHB (see Figure 4).

[0103] These results showed a significant decrease in formic acid, so the inventors next evaluated whether deleting yagE and yjhH in modified E. coli expressing the DHB production pathway would improve DHB production during glucose fermentation. pZA23-thrA S345F _alaC A142P Y275D _mdh R81A M85E I12V G179D D86S In these modified strains containing plasmids (i.e., strains 1160, 1526, 1527, and 1528, see Table 1), mutations in thrA alleviate threonine feedback inhibition to thrA, and mutations in alaC are obtained through evolutionary modification in vivo, resulting in AlaC with 100-fold higher catalytic activity for homoserine. A142PY275D Mutants were obtained (Bouzon et al., 2017), and mutations in E. coli mdh were generated by protein modification (Frazao et al., 2018), resulting in MDH mutants optimized to reduce OHB.

[0104] Inactivation of yjhH (strain 1526) significantly increased DHB (>10%) production by increasing glucose yield and simultaneously eliminating formic acid production (see Figure 5). This effect was less pronounced after inactivation of yagE (strain 1527). These results are consistent with aldolase biochemical data indicating that the catalytic efficiency of YjhH is higher than that of YagE.

[0105] However, when both yjhH and yagE were deleted (strain 1528), DHB production improved further (>15%). The inventors concluded that this synergistic effect of dual inactivation is due to the fact that the deletion of one aldolase is compensated for by an increase in the other aldolase. Note that under DHB production conditions (i.e., glucose is limited), formic acid can be consumed by the cell as a carbon source, so DHB yield is the only relevant performance indicator.

[0106] 6. Conclusion Among all OHB aldolases found in E. coli, YagE aldolase and YjhH aldolase have been clearly shown to most efficiently degrade OHB in vivo, thereby partially removing OHB from the DHB pathway. Conversely, other aldolases such as RhmA aldolase and GarL aldolase appear to have very high activity in condensing formaldehyde and pyruvate into OHB, but have no function whatsoever towards OHB in vivo.

[0107] Finally, in the application of DHB production from OHB, the inventors believe that inactivation of yjhH and / or yagE (both EC 4.1.2.28) leads to a significant improvement in DHB production, particularly in glucose yield, and more precisely, by eliminating formic acid production.

[0108] [References] Altschul, SF, Gish, W., Miller, W., Myers, EW & Lipman DJ, 1990. Basic local alignment search tool. J Mol Biol 215, 403-10. Altschul, SF & Gish, W., 1996. Local alignment statistics. Methods Enzymol 266, 460-80. Altschul, S. F., Madden, T.L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389-402. Ausubel, M., Brent, R., Kingston, R.E, Moore, D.D., Seidman, J.G., Smith, J.A., & Struhl, K., 1995. Current Protocols in Molecular Biology, Chapter 19. Greene Publishing and Wiley-Interscience, New York. Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K.A., Tomita, M., Wanner, B.L., Mori, H., 2006. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2. Bailey, J. E., 1991. Toward a science of Metabolic Engineering. Science 252, 1668-1975. Bouzon, M., Perret, A., Loreau, O., Delmas, V., Perchat, N., Weissenbach, J., Taran, F. & Marliere, P., 2017. A synthetic alternative to canonical one-carbon metabolism. Acs Synthetic Biology. 61520-1533. Cherepanov, P.P., Wackernagel, W., 1995. Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene 158, 9-14. Chung, C.T., Niemela, S.L., Miller, R.H., 1989. One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc. Natl. Acad. Sci. U.S.A. 86, 2172-2175. Clapes P., Fessner W.D., Sprenger G.A. & Samland A.K. 2010. Recent progress in stereoselective synthesis with aldolases. Curr Opin Chem Biol. 14, 154-67. Cox, M.M., 1983. The FLP protein of the yeast 2-eum plasmid: Expression of a eukaryotic genetic recombination system in Escherichia coli. Proc. Natl. Acad. Sci. USA 5. Dayhoff, M.O., Schwartz, R.M. & Orcutt, B.C., 1978. A model of evolutionary change in proteins. Atlas of Protein Sequence and Structure. Deck, P., Exner, K. & Buschhaus, B. 2008. Method for the production of D,L-hydroxy-4-alkylthio butyric acid. Edited by B. A G. Frazao C., Topham C., Malbert Y., Francois J. M. & Walther T. 2018. Rational engineering of a malate dehydrogenase for microbial production of 2,4-dihydroxybutyric acid via homoserine pathway Biochem J 475, 3887-3901. He H., Hoper R., Dodenhoft M., Marliere P. & Bar-Even A. 2020. An optimized methanol assimilation pathway relying on promiscuous formaldehyde-condensing aldolases in E. coli. Metab Eng. 60, 1-13. Jiang, Y., Chen, B., Duan, C., Sun, B., Yang, J. & Yang, S., 2015. Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System. Appl Environ Microbiol 81, 2506-2514. Needleman, S. B. & Wunsch, C. D., 1970. A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol 48, 443-53. Pearson, W. R & Lipman, D. J., 1988. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A 85, 2444-8. Shpaer, E. G., 1997. GeneAssist. Smith-Waterman and other database similarity searches and identification of motifs. Methods Mol Biol 70, 173-87. Smith, T.F. & Waterman M. S., 1981. Adv. Appl. Math. 2, 482. Studier FW. (2005) Protein production by auto-induction in high-density shaking cultures. Prot. Exp. Pur. 41, 207-234. Thomason, L.C., Costantino, N., Court, D.L., 2007. E. coli Genome Manipulation by P1 Transduction. CP Molecular Biology 79. Walther, T., Calvayrac, F., Malbert, Y., Alkim, C., Dressaire, C., Cordier, H., Francois, J.M., 2018. Construction of a synthetic metabolic pathway for the production of 2,4-dihydroxybutyric acid from homoserine. Metabolic Engineering 45, 237-245. Wellner, D., Lichtenberg, L.A., 1971. [218a] Assay of amino acid oxidase, in: Methods in Enzymology. Elsevier, pp. 593-596. Werpy, T. & Petersen, G., 2004. Top Value Added Chemicals from Biomass: Volume I -- Results of Screening for Potential Candidates from Sugars and Synthesis Gas. Pacific Northwest National Laboratory (PNNL) and National Renewable Energy Laboratory (NREL). Zakataeva N.P., Aleshin V.V., Tokmakova I.L., Troshin P.V. & Livshits V.A., 1999. The novel transmembrane Escherichia coli proteins involved in the amino acid efflux. FEBS Lett. 452, 228-32.

Claims

1. A method for preparing 2,4-dihydroxybutyrate from a carbon source via homoserine, The first step involves converting the primary amino acid group of homoserine to a carbonyl group and catalyzing it with an enzyme having homoserine transaminase activity as defined by EC 2.6.1.1, EC 2.6.1.42, or EC 2.6.1.57 to obtain 2-oxo-4-hydroxybutyrate, The second step involves reducing the obtained 2-oxo-4-hydroxybutyrate (OHB) to 2,4-dihydroxybutyrate by catalysis with an enzyme having OHB reductase activity, The enzyme having OHB reductase activity is a lactate dehydrogenase as defined by EC 1.1.1.27 or EC 1.1.1.28, or a malate dehydrogenase as defined by EC 1.1.1.37, EC 1.1.1.82, or EC 1.1.1.

299. Step 2, A method comprising a two-step path, At least one enzyme having aldolase activity as defined by EC 4.1.2.28 is inactivated to prevent the degradation of OHB. method.

2. The enzyme having aldolase activity is It is coded by the sequence described in SEQ ID NO: 1 or SEQ ID NO: 3, or The sequence described in Sequence ID No. 2 or Sequence ID No. 4, or any sequence that encodes an enzyme having aldolase activity and has at least 50% homology to the said sequence, The method according to claim 1.

3. The enzyme having homoserine transaminase activity is encoded by the genes iIvE, tyrB, aspC, araT, bcaT, alaC, and ARO8. The method according to claim 1 or 2.

4. The enzyme having homoserine transaminase activity is - The sequence described in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 126, SEQ ID NO: 128, or any sequence that encodes an enzyme having homoserine transaminase activity and has at least 50% homology to the aforementioned sequence, or - Corresponding to sequence numbers 6, 8, 10, 12, 14, 16, 127, 129, or any sequence having at least 50% homology to the sequence, The method according to claim 3.

5. The enzyme having OHB reductase activity is (D)-lactate dehydrogenase derived from Escherichia coli, (L)-lactate dehydrogenase derived from Lactococcus lactis, Oryctolagus cuniculus, Diobacillus stearothermophilus, or Bacillus subtilis, (L)-malate dehydrogenase derived from Escherichia coli, or homologs thereof. The method according to any one of claims 1 to 4.

6. Enzymes that possess OHB reductase activity are, Lactate dehydrogenase comprising at least one mutation at positions V17, Q85, E89, I226, or A222, wherein the positions are defined with reference to L-lactis tA (SEQ ID NO: 18), or Malate dehydrogenase comprising at least one mutation at position I12, R81, M85, D86, V93, G179, T211, or M227, wherein the position is defined with reference to Escherichia coli Mdh (SEQ ID NO: 20). That is, The method according to claim 5.

7. The enzyme having the aforementioned OHB reductase activity is - Coded by the sequence described in Sequence ID No. 45, Sequence ID No. 47, Sequence ID No. 49, Sequence ID No. 51, Sequence ID No. 53, Sequence ID No. 55, Sequence ID No. 57, Sequence ID No. 59, Sequence ID No. 61, Sequence ID No. 63, Sequence ID No. 65, Sequence ID No. 67, Sequence ID No. 69, Sequence ID No. 71, Sequence ID No. 73, Sequence ID No. 75, Sequence ID No. 77, Sequence ID No. 79, or Sequence ID No. 81, or any sequence having at least 50% homology to the said sequence, - Corresponds to sequence numbers 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, or 82, or any sequence having at least 50% homology to the aforementioned sequence. The method according to claim 5 or 6.

8. A modified microorganism for a method of preparing 2,4-dihydroxybutyrate from a carbon source via homoserine, wherein the method is The first step involves converting the primary amino acid group of homoserine to a carbonyl group and catalyzing it with an enzyme having homoserine transaminase activity as defined by EC 2.6.1.1, EC 2.6.1.42, or EC 2.6.1.57 to obtain 2-oxo-4-hydroxybutyrate, The second step involves reducing the obtained 2-oxo-4-hydroxybutyrate (OHB) to 2,4-dihydroxybutyrate by catalysis with an enzyme having OHB reductase activity, The enzyme having OHB reductase activity is a lactate dehydrogenase as defined by EC 1.1.1.27 or EC 1.1.1.28, or a malate dehydrogenase as defined by EC 1.1.1.37, EC 1.1.1.82, or EC 1.1.1.

299. Step 2, A method comprising a two-step path, At least one enzyme having aldolase activity as defined by EC 4.1.2.28 is inactivated to prevent the degradation of OHB. Modified microorganisms.

9. The enzyme having homoserine transaminase activity, the enzyme having OHB reductase activity, and the enzyme having aldolase activity are described in any one of claims 2-8. The modified microorganism according to claim 8.

10. Homoserine production is improved compared to the same unmodified microorganism. The modified microorganism according to claim 8 or 9.

11. The activity of aspartate kinase, aspartate semialdehyde dehydrogenase, and / or homoserine dehydrogenase is improved. Modified microorganism according to any one of claims 8 or 10

12. The production of 2,4-dihydroxybutyrate - The same unmodified microorganism, and / or Modified microorganisms obtained by a method comprising: a first step of converting the primary amino acid group of homoserine to a carbonyl group to obtain 2-oxo-4-hydroxybutyrate; and a second step of reducing the obtained 2-oxo-4-hydroxybutyrate to obtain 2,4-dihydroxybutyrate. It has improved compared to A modified microorganism according to any one of claims 8-11.

13. Preferably, the bacteria are selected from the Enterobacteriaceae, Clostridae, Bacillaceae, Streptomycesaceae, Streptococciaceae, Methylobacteriaceae, and Corynebacteriaceae. Most preferably, these are Escherichia coli, Bacillus subtilis, Corynebacterium, Clostridium acetobutyricum, Methylobacterium extroquens, or Lactobacillus lactis. Preferably, the yeast is selected from Saccharomyces, Pichia, and Schizosaccharomyces. Most preferably, budding yeast, fission yeast, Cluyveromyces lactis, Cluyveromyces marcianas, Pichia jadini, Pichia stipitis, or Pichia pastris, or Preferably, the fungus is selected from Penicillium, Aspergillus, Chrysosporium, or Trichoderma. A modified microorganism according to any one of claims 8-12.

14. The expression of at least one enzyme selected from phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, isocitrate lyase, pyruvate carboxylase, and hexose cotransporter permease is increased, and / or The expression of at least one enzyme activity selected from lactate dehydrogenase, alcohol dehydrogenase, acetate kinase, phosphate acetyltransferase, pyruvate oxidase, isocitrate lyase, fumarase, 2-oxoglutarate dehydrogenase, pyruvate kinase, malate enzyme, phosphoglucose isomerase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, pyruvate formate lyase, succinate semialdehyde dehydrogenase, sugar transport phosphotransferase, ketohydroxyglutarate aldolase, homoserine-O-succinate transferase, homoserine kinase, homoserine efflux transporter, diaminopimephosphate decarboxylase, and / or methylglyoxal synthase is reduced. A modified microorganism according to any one of claims 8-13.

15. The modified microorganism according to claim 14, which is an Escherichia coli that overexpresses at least one gene selected from among ppc, pck, aceA, galP, asd, thrA, metL, lysC from Escherichia coli and pycA from Lactococcus lactis, and / or deletes at least one gene selected from among IdhA, adhE, ackA, pta, poxB, focA, pflB, sad, gabABC, sfcA, maeB, ppc, pykA, pykF, mgsA, sucAB, ptsl, ptsG, pgi, fumABC, aldA, lldD, iclR, metA, thrB, lysA, eda, rthA, rthB, rthC.

16. A method for producing 2,4-dihydroxybutyrate, - Culturing the modified microorganism described in any one of claims 8-15 in an appropriate culture medium, - To recover 2,4-dihydroxybutyrate from the culture medium, A method that includes the following steps.

17. The aforementioned 2,4-dihydroxybutyrate is further purified. The method according to claim 16.

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