WO2017009047A1

WO2017009047A1 - Method for producing l-methionine

Info

Publication number: WO2017009047A1
Application number: PCT/EP2016/065146
Authority: WO
Inventors: Ines Ochrombel; Brigitte Bathe; Marleen Hasselmeyer; Kay Marin; Joanne Louise PEDALL
Original assignee: Evonik Degussa Gmbh
Priority date: 2015-07-13
Filing date: 2016-06-29
Publication date: 2017-01-19

Abstract

The present invention relates to a method for producing L-methionine, in which O-acetyl-L- homoserine is reacted with methyl mercaptan or with a salt of methyl mercaptan, in the presence of a protein having O-acetylhomoserine sulphydrylase activity produced by Corynebacterium humireducens and mutants thereof or in the presence of a microorganism producing such a protein or a cell digest of said microorganism, to give L-methionine.

Description

Method for producing L-methionine The present invention relates to a method for producing L-methionine, in which O-acetyl-L- homoserine is reacted with methyl mercaptan or with a salt of methyl mercaptan, in the presence of a protein having O-acetylhomoserine sulphydrylase activity or in the presence of a microorganism producing a protein having O-acetylhomoserine sulphydrylase activity or a cell digest of said microorganism, to give L-methionine. The present invention further relates to proteins having O- acetylhomoserine sulphydrylase activity, polynucleotides which code for these proteins, recombinant vectors comprising these polynucleotides and also recombinant microorganisms which are transformed by these vectors.

The amino acid methionine is currently industrially produced worldwide in large amounts and is of considerable commercial importance. Methionine is employed in many fields, such as

pharmaceutical, health and fitness products, but particularly as feedstuff additive in many feedstuffs for various livestock, where both the racemic and the enantiomerically pure form of methionine may be used. On an industrial scale, methionine is produced chemically via the Bucherer-Bergs reaction, which is a variant of the Strecker synthesis. In this case, the starting substances

methylmercaptopropionaldehyde (prepared from acrolein and methyl mercaptan), hydrogen cyanide, ammonia and carbon dioxide are reacted to give 5-(2-methylmercaptoethyl) hydantoin (methionine hydantoin), this subsequently being hydrolysed by alkali to give the alkali metal methioninate and the methionine then being liberated by neutralisation with acid. Various other methods can also be used to prepare methionine, for example, the amidocarbonylation reaction, the hydrolysis of proteins or the fermentation of microorganisms producing methionine. In chemical synthesis, methionine is produced as a racemic mixture of D- and L-methionine, whereas L- methionine, or L-configured precursors of the same (L-homoserine for example), can be produced by the fermentation of suitable microorganisms. Use of racemic methionine as feedstuff additive, however, does not require any enantiomer separation. The DL-methionine racemate can be used directly as feedstuff additive since, in natural organisms, the D-methionine is converted into the naturally occurring L-methionine. Hateley et al. disclose a method in which L-methionine is obtained by a chemical route (WO 2007/085514 A2), starting from L-homoserine which can be prepared biotechnologically.

A two-stage biotechnological method for preparing L-methionine is proposed by Kim et al. (WO 2008/013432 A1 ). In a first step here, an L-methionine precursor, O-succinyl-L-homoserine or O- acetyl-L-homoserine, is initially obtained by means of recombinant microorganisms, which are ideally enriched in the culture broth. In the subsequent second step, the L-methionine precursor is reacted with methyl mercaptan in the presence of a protein having O-acylhomoserine

sulphydrylase activity or in the presence of a microorganism producing a protein having O- acylhomoserine sulphydrylase activity or a cell digest of this microorganism, to give L-methionine and the corresponding carboxylic acid, acetate or succinate. In the second step, the reaction of O- acetyl-L-homoserine with methyl mercaptan to give L-methionine and acetate, an O-acetyl sulphydrylase from Corynebacterium glutamicum (MetY_Cg), inter alia, can be used. An improved conversion rate and shortened reaction time in the second step, the reaction of O-acetyl-L- homoserine with methyl mercaptan to give L-methionine and acetate, is achieved by using an enzyme having O-acetylhomoserine sulphydrylase activity, which originates from Rhodobacter sphaeroides or is derived from this enzyme (EP 2 657 249 A2). The best result was achieved with the enzyme derived from the O-acetylhomoserine sulphydrylase from Rhodobacter sphaeroides having the three mutations, I3N, F65Y and V104A (EP 2 657 249 A2, SEQ ID No. 18), referred to below as MetZ_Rsp_l3N_F65Y_V104A (SEQ ID No. 15 or 16 in this document). However, in this enzymatic reaction, equimolar amounts of acetate are formed in addition to L-methionine. This leads to high concentrations of acetate in the course of the fermentation, particularly on an industrial scale. As a kosmotropic/antichaotropic salt, however, acetate has an enhancing effect on hydrophobic interactions in protein structures and thereby reduces their solubility, which can both positively and negatively influence enzyme activities. It is further known that kosmotropic (but also chaotropic) anions can have an inhibitory effect on enzymes at high concentrations (H. Zhao,

Journal of Molecular Catalysis B: Enzymatic 37 (2005) 16-25). Therefore, the O-acetylhomoserine sulphydrylase used in the reaction of O-acetyl-L-homoserine with methyl mercaptan should also have high activities in the presence of relatively high concentrations of acetate. In the enzymatic reaction of O-acetyl-L-homoserine and methyl mercaptan to give L-methionine and acetate, methyl mercaptan is used in the form of its sodium salt, sodium methyl mercaptide (CH3-SNa), inter alia, due to its greater ease of handling. The addition of sodium methyl mercaptide can lead to fluctuations of the pH in the alkaline range. An elevated pH likewise has effects on the structure of the enzyme and thus its activity. A high pH can lead to an irreversible inactivation of the enzyme. This is because changes in pH influence the folding and activity of the enzyme via changes in the charge structure of the protein. At high pH values especially, the cysteine residues of the proteins are chemically modified. No cysteine residues are present in the MetY enzyme of C. glutamicum, whereas the MetY enzyme of Rhodobacter sphaeroides has three cysteine residues. It is of advantage, however, particularly on an industrial scale, if the enzyme used remains stable in its activity even under fluctuating pH conditions.

The object of the present invention is to provide a method for producing L-methionine comprising the enzymatic reaction of O-acetyl-L-homoserine and methyl mercaptan to give L-methionine and acetate, in which the activity of the enzyme used, an O-acetylhomoserine sulphydrylase, at least remains stable at high acetate concentrations and high pH values, particularly at the acetate concentrations and pH values which occur in a method on an industrial scale, or even increase compared to the activity at low acetate concentrations and pH values of about 7.5. Furthermore, the nucleotide sequence of the enzyme to be provided should be characterized by a high expression level in a host cell.

The object is achieved by a method for producing L-methionine which comprises the step of reacting O-acetyl-L-homoserine with methyl mercaptan or with a methyl mercaptan salt in the presence of a protein having O-acetylhomoserine sulphydrylase activity or in the presence of a microorganism producing a protein having O-acetylhomoserine sulphydrylase activity or a cell digest of this microorganism. In this case, the protein having O-acetylhomoserine sulphydrylase activity is selected from the group of O-acetylhomoserine sulphydrylase produced by

Corynebacterium humireducens and mutants thereof having O-acetylhomoserine sulphydrylase activity, in which 1 to 5 amino acids have been mutated or which has been shortened by 1 to 5 amino acids at the C-terminus end.

Suitable as protein having O-acetylhomoserine sulphydrylase activity from Corynebacterium humireducens is a protein which originates from Corynebacterium humireducens DSM 45392 (publicly available at the DSMZ, Leibniz Institut DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH [German Collection of Microorganisms and Cell Cultures], InhoffenstraBe 7 B, 38124 Braunschweig, Germany) and comprises the amino acid sequence according to SEQ ID No. 8 or is encoded by a polynucleotide comprising a nucleotide sequence according to SEQ ID No. 7.

Also suitable are the proteins derived from the abovementioned protein having O-acetylhomoserine sulphydrylase activity from Corynebacterium humireducens, for example a protein comprising the amino acid sequence according to SEQ ID No. 10 (MetY_Ch-421 Stop) or which is encoded by a polynucleotide comprising a nucleotide sequence according to SEQ ID No. 9 (metY_Ch-421 Stop). The amino acid sequence of this protein is shortened by 2 amino acids compared to the protein with the amino acid sequence according to SEQ ID No. 8. A stop codon is in the corresponding nucleotide sequence in the position which follows the codon which codes for the amino acid in position 421 .

A protein that is also suitable comprises the amino acid sequence according to SEQ ID No. 12 (MetY_Ch-L418l) or is encoded by a polynucleotide comprising a nucleotide sequence according to SEQ ID No. 1 1 (metY_Ch-L418l). This protein differs from the protein with the amino acid sequence according to SEQ ID No. 8 in that the amino acid in position 418 of the sequence according to SEQ ID No. 8 has been mutated, namely by the exchange of the amino acid L-leucine by the amino acid L-isoleucine. The object is further achieved by providing a protein having O-acetylhomoserine sulphydrylase activity which comprises the amino acid sequence according to SEQ ID No. 10 (MetY_Ch-421 Stop) or by providing a polynucleotide comprising a nucleotide sequence according to SEQ ID No. 9 (metY_Ch-421 Stop) or by providing a protein having O-acetylhomoserine sulphydrylase activity which comprises the amino acid sequence according to SEQ ID No. 12 (MetY_Ch-L418l) or by providing a polynucleotide comprising a nucleotide sequence according to SEQ ID No. 1 1

(metY_Ch-L418l). Vectors comprising such polynucleotides can be prepared by recombination with the polynucleotides from Corynebacterium humireducens DSM 45392 having the nucleotide sequence according to SEQ ID No. 7 or having the nucleotide sequence according to SEQ ID No. 9 (metY_Ch-421 Stop) or according to SEQ ID No. 1 1 (metY_Ch-L418l). Using these vectors, microorganisms can then be transformed, for example, bacteria from the species Escherichia or Corynebacterium, which produce the abovementioned proteins having O-acetylhomoserine sulphydrylase activity, comprising the amino acid sequences according to SEQ ID No. 8, SEQ ID No. 10 or SEQ ID No. 12.

The method according to the invention can thus also be carried out in a manner such that the reaction of O-acetyl-L-homoserine with methyl mercaptan or with a methyl mercaptan salt takes place in the presence of a microorganism transformed with such vectors or a cell digest of this microorganism.

Example 1 : Production of O-acetylhomoserine sulphydrylases

1.1 O-Acetylhomoserine sulphydrylases and variants thereof from Corynebacterium species

On the basis of the genome sequence of Corynebacterium humireducens (DSM 45392)

NZ_CP005286 and of Corynebacterium glutamicum (ATCC13032) NC_003450, primers were synthesized for the gene sequence of the O-acetylhomoserine sulphydrylase which catalyses the reaction of L-O-acetylhomoserine and methyl mercaptan to give L-methionine and acetate. The chromosome of Corynebacterium humireducens has been isolated via culturing the strain which was obtained from the DSMZ (Leibniz Institute DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, InhoffenstraBe 7 B, 38124 Braunschweig, Germany). The PCR comprised 30 cycles. The denaturing step was carried out at 98°C for 30 seconds, the annealing step at 67°C for 30 seconds and the extension at 72°C for 2 minutes. In this case, the chromosome was used as template and the respective primer pairs of the SEQ ID No. 1 and SEQ ID No.2; SEQ ID No.1 and SEQ ID No. 3; SEQ ID No. 1 and SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6 and also the PCR Premix "Phusion High-Fidelity PCR Master Mix" (Life Technologies GmbH). To analyse the PCR products, DNA sequencing was in each case carried out by Eurofins MWG Operon. The DNA sequences of Corynebacterium humireducens obtained were checked with respect to correctness using the Clone Manager software. The nucleotide sequences according to SEQ ID No. 7, 9 and 11 were thereby confirmed. The DNA sequence of Corynebacterium glutamicum obtained was likewise checked with respect to correctness using the Clone Manager software. The nucleotide sequence according to SEQ ID No. 13 was thereby confirmed. The DNA fragments, which were each obtained by the PCR reactions, are cleaved by the restriction enzymes Notl and Ndel and have been cloned into the pET26b(+) vecktor (Novagen, Germany) (Figure 1 ), which was also cleaved using Notl and Ndel.

The following vectors were thereby prepared:

pET26b(+)_metY_Ch (Fig. 2),

pET26b(+)_metY_Ch_421 Stop (Fig. 3),

pET26b(+)_metY_Ch_L418l (Fig. 4) and

pET26b(+)_metY_Cg (Fig. 5). These vectors have each been transformed in Escherichia coli BL21 (DE3) (New England Biolabs), which were subsequently cultured on LB medium agar plates with 50 mg/l kanamycin. In each case a colony has been selected which was inoculated into 10 ml of LB medium with 50 mg/l kanamycin and cultured at 37°C, 250 rpm for 6 hours. 5 ml of Riesenberg medium (Riesenberg D, et al., J. Biotechnol. 1991 , 20(1 ):17-27) were subsequently treated with 50 mg/l kanamycin and inoculated with 50 μΙ of the growth cell culture and incubated at 28°C, 250 rpm for 16 h. This cell culture was diluted with 200 ml of fresh Riesenberg medium containing 50 μ9/Ι kanamycin in a 2I flask to an OD of 0.15 and was further cultured under identical conditions until an OD of 0.5 was attained (circa 4 h). The start point of the induction of gene expression was then effected by adding 200 μΙ of a 300 mM IPTG stock solution (final concentration 300 μΜ isopropyl^-D-thiogalactopyranoside (IPTG), Sigma-Aldrich, Germany). The induction was carried out at 28°C, 250 rpm for 4 h. The enzyme culture was then harvested (8 ml normalised to an OD=1 ), the supernatant removed by centrifugation (20 min, 4000 rpm, 4°C) and the pelleted cells were washed twice with 800 μΙ of 0.1 M potassium phosphate buffer (pH 7.5) and taken up in 1 ml of buffer. The mechanical cell digestion was carried out in a FastPrep FP120 instrument (QBiogene, Heidelberg), wherein the cells were shaken four times for 30 s at 6.5 m/s in digestion vessels with 300 mg of glass beads (0 0.2-0.3 mm). The crude extract was then centrifuged at 12 000 rpm, 4°C, 20 min, in order to remove undigested cells and cell debris.

The total amount of protein was determined using the Bio-Rad protein quantitation assay (Bio-Rad, USA). The protein expression was moreover confirmed by means of a Coomassie-stained SDS gel. The enzyme homogenate was then used for the enzymatic reaction of O-acetyl-L-homoserine with methyl mercaptan to give L-methionine and acetate.

1.2 O-Acetylhomoserine sulphydrylase from Rhodobacter species The nucleotide sequence disclosed by Kim et al. (EP 2 657 249 A2: SEQ ID No. 22; in this document SEQ ID No. 15), which codes for an enzyme, MetZ_ I3N_F65Y_V104A (EP 2 657 249 A2, derived from an O-acetylhomoserine sulphydrylase from Rhodobacter sphaeroides: SEQ ID No.18; in this document SEQ ID No. 16), was synthesized in Life Technologies Invitrogen GeneArt (Germany) and cloned into the vector pET26b(+) via the Notl and Xbal restriction sites. The resulting vector, pET26(+)_Rsp_metZJ3N_F65Y_V104A, is shown schemiatically in Figure 6.

The cell homogenates were obtained in the same manner as described in Example 1 .1 . using the cloned vector, and were then used in the enzymatic reaction of O-acetyl-L-homoserine with methyl mercaptan to give L-methionine and acetate.

Example 2: Comparison of the expression levels of the O-acetylhomoserine sulphydrylases

The expression levels of the various O-acetylhomoserine sulphydrylases were compared to one another. The enzyme homogenates have been obtained by the methods of Examples 1 .1 and 1 .2. The amount of protein is generally quantified by means of a BioRad protein assay as mentioned in Example 1 .1 . However, it is difficult to determine quantitatively the amount of enzyme protein in the mixture of E.coli proteins, as it is in the form of an enzyme homogenate. Accordingly, in this experiment 10 μg of each enzyme homogenate were applied in each case to an SDS-PAGE (BIO RAD 10% Mini-PROTEAN® TGX™ Precast Gel), in order to compare the expression levels with one another. The result showed that the expression levels of MetY_Ch, MetY_Ch_421 Stop, MetY_Ch_L418l, and MetY_Cg are virtually identical, whereas the MetZ_Rsp_l3N_F65Y_V104A is only expressed about half as much (Figure 7). Example 3: Comparison of the effects of the acetate concentrations on the enzyme activities.

The activities of the enzymes MetY_Ch, MetY_Ch 421 Stop, MetYCh_L418l, MetY_Cg and MetZ_Rsp_l3N_F65Y_V104A were determined at various acetate concentrations from 0 to 1 M. The specific methionine production rate based on the cell homogenate was in each case derived from the enzymatic conversion of 80 g/l O-acetylhomoserine by measuring the methionine concentration.

The enzymatic reaction was carried out at 33°C for 180 min in a 1 ml batch in 0.1 M potassium phosphate buffer (pH 7.5) by the addition of 0.06 ml of sodium methyl mercaptide (CH3-SNa) solution (2 M), 0.086 ml of O-acetyl-L-homoserine solution (0.407 M), 0.01 ml of pyridoxal-5 - phosphate monohydrate (PLP) solution (0.001 M) and 0.1 ml of enzyme homogenate (protein concentration: 0.3 mg/ml) without acetate addition and also by addition of sodium acetate and adjusting to various acetate concentrations. The concentration of O-acetyl-L-homoserine was analysed by HPLC. The rate of methionine formation as quantitative amount produced was based on the cell homogenate protein concentration used. Even in the presence of 0.3 M acetate, the enzyme activities of MetY_Ch, MetY_Ch_421 Stop and MetY_Ch_L418l are significantly increased, whereas the enzyme activity of MetZ_Rsp_l3N_F65Y_V104A is distinctly reduced under the same conditions (Table 1 ). Table 1 : Enzyme activities at pH 7.5 and various acetate concentrations

Example 4: Comparison of the effect of alkaline pH on the enzyme activities in the presence of acetate as reaction product

Under production relevant conditions, the presence of acetate reaction product already accumulated may correspond to a concurrent increase in pH. Both factors together influence the protein structure and therefore the sulphydrylase activity. In particular, a brief increase of the pH can already lead to an irreversible inactivation of the enzyme. In order to investigate the effects of acetate concentration and high pH on the enzyme activities, the activities of MetY_Ch,

MetY_Ch_421 Stop, MetY_Ch_L418l, MetY_Cg and MetZ_Rsp_l3N_F65Y_V104A were measured in the presence of 0 M and 0.3 M acetate at an alkaline pH of 9. The mixture was carried out as described in Example 3. As is evident from Table 2, the sulphydrylase MetY_Ch under relevant production conditions (realistic concentration of 0.3 M acetate and high pH) has the highest specific activities, whereas the enzyme MetY_Ch_421 Stop, MetY_Ch_L418l and MetY_Cg still have significant activities and the activity of MetZ_Rsp_l3N_F65Y_V104A declines extremely sharply.

Table 2: Enzyme activities at pH 9.0 and various acetate concentrations pH 9

Sodium acetate

0 0.3

Enzyme concentration [mol/l]

Ψ

L-Met [μιτιοΙ/minl/mg

Control without enzyme enzyme homogenate 0 0

L-Met [μιτιοΙ/minl/mg

MetY Ch enzyme homogenate 7.3 6.3

L-Met [μιτιοΙ/minl/mg

MetY_Ch_421 Stop enzyme homogenate 6.2 4.7

L-Met [μιτιοΙ/minl/mg

MetY Ch L418I enzyme homogenate 5.9 5.0

L-Met [μιτιοΙ/minl/mg

MetY_Cg enzyme homogenate 8.9 5.5

L-Met [μιτιοΙ/minl/mg

MetZ Rsp_l3N_F65Y_V104A enzyme homogenate 12.2 3.6

Claims

Method for producing L-methionine, comprising the step of reacting O-acetyl-L-homoserine with methyl mercaptan or with a methyl mercaptan salt in the presence of a protein having O-acetylhomoserine sulphydrylase activity or in the presence of a microorganism producing a protein having O-acetylhomoserine sulphydrylase activity or a cell digest of this microorganism, wherein the protein having O-acetylhomoserine sulphydrylase activity is selected from the group of O-acetylhomoserine sulphydrylase produced by Corynebacterium humireducens and mutants thereof having O-acetylhomoserine sulphydrylase activity, in which 1 to 5 amino acids have been mutated or which has been shortened by 1 to 5 amino acids at the C-terminus end.

Method according to Claim 1 , wherein the protein having O-acetylhomoserine sulphydrylase activity comprises the amino acid sequence according to SEQ ID No. 8.

Method according to Claim 2, wherein the protein having O-acetylhomoserine sulphydrylase activity is encoded by a polynucleotide comprising a nucleotide sequence according to SEQ ID No. 7.

Method according to Claim 1 , wherein the protein having O-acetylhomoserine sulphydrylase activity comprises the amino acid sequence according to SEQ ID No. 10.

Method according to Claim 4, wherein the protein having O-acetylhomoserine sulphydrylase activity is encoded by a polynucleotide comprising a nucleotide sequence according to SEQ ID No. 9.

Method according to Claim 1 , wherein the protein having O-acetylhomoserine sulphydrylase activity comprises the amino acid sequence according to SEQ ID No. 12.

Method according to Claim 6, wherein the protein having O-acetylhomoserine sulphydrylase activity is encoded by a polynucleotide comprising a nucleotide sequence according to SEQ ID No. 1 1 .

Method according to Claim 1 , wherein the reaction of O-acetyl-L-homoserine with methyl mercaptan or with a methyl mercaptan salt takes place in the presence of a microorganism transformed with a vector comprising a nucleotide sequence according to SEQ ID No. 7, according to SEQ ID No. 9 or according to SEQ ID No. 1 1 or a cell digest of this microorganism.