Method for producing l-methionine by fermentation of a microorganism in a medium comprising a primary alcohol

Abstract

The present invention relates to a method for producing L-methionine comprising the fermentation under aerobic conditions of a genetically modified microorganism which produces more L-methionine than the microorganism before the genetical modification in a medium comprising a primary alcohol upon increasing the activity of the glyoxylate shunt.

Description

[0001] 202400272 Foreign Filings 1

[0002] Method for producing L-methionine by fermentation of a microorganism in a medium comprising a primary alcohol

[0003] The present invention relates to a method for producing L-methionine comprising the fermentation under aerobic conditions of a genetically modified microorganism which produces more L- methionine than the microorganism before the genetical modification in a medium comprising a primary alcohol, such as ethanol.

[0004] 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 feed additive in many feedstuffs for various livestock, where both the racemic and the enantiomerically pure form of methionine may be used. To address the reduction in fossil resources and to decrease the negative environmental impact related to hazardous intermediates and waste, there is growing interest in alternative, more sustainable processes that utilize natural resources. Ethanol from secondary feedstocks is a sustainable alternative carbon source for biotechnological production evaluated in this patent application.

[0005] Organic chemical compounds can be produced by fermentation of strains of microorganisms. Due to their great significance, efforts are constantly being made to improve the preparation process. Improvements to the process may relate to measures concerning fermentation technology, for example stirring and oxygen supply, or to the composition of the nutrient media, such as, for example, selection of the carbon source for fermentation, or to work up to the product form by, for example, ion exchange chromatography, or to the intrinsic performance characteristics of the microorganism itself.

[0006] JAEYOUNG CHA in the publication "Effect of amino acids on the activities of alcohol metabolism enzymes alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH)", JOURNAL OF LIFE SCIENCE, 8 September 2009 (2009-09-08), pages 1-7, investigates the effects of various amino acids on the activity of alcohol-metabolizing enzymes — alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) — using enzyme assays in yeast and rat liver models. It finds that L-methionine and L-arginine enhance the activity of these enzymes. The study concludes that these amino acids could be beneficial as functional ingredients for liver protection and hangover relief in humans. It does not relate to the production of L-methionine using microbial strains.

[0007] CHEN HAILONG ET AL in the publication "The multiple effects of REG1 deletion and SNF1 overexpression improved the production of S-adenosyl-lmethionine in Saccharomyces cerevisiae", MICROBIAL CELL FACTORIES, vol. 21 , no. 1 , 27 August 2022 investigates how deleting the REG1 gene and overexpressing SNF1 in Saccharomyces cerevisiae improves the 202400272 Foreign Filings 2 microbial production of S-adenosyl-L-methionine (SAM). These genetic modifications enhance glucose utilization, reduce ethanol accumulation, boost glycolytic and amino acid metabolism, and ultimately increase SAM yield. Although the fermentation samples were also analyzed for L- methionine production using HPLC, no detection of L-methionine is reported. Thus it does not relate to the production of L-methionine using microbial strains.

[0008] Biosynthetic pathways of carbon utilization as well as amino acid synthesis in wild-type strains are subject to strict metabolic control which ensures that the carbon source is used efficiently, and the amino acids are produced only for the cell's intrinsic needs. An important prerequisite for efficient production processes is therefore the availability of overproducing strains.

[0009] In general, overproducing strains have an increased capacity for performing chemical reactions within the cell contributing and / or causing the increased conversion of the raw material (feed stock) to a desired product like amino acids. Such overproducing microorganisms may be generated by classic mutation / selection processes and / or by modern, more specific, recombinant techniques. Both strategies target to modulate chemical reactions in the cell. The approaches can comprise the activation of enzymes catalyzing the desired chemical reactions, inactivation of enzymes catalyzing unwanted chemical reactions or the modification of regulatory proteins to modulate the chemical reaction network in a cell in favor of the production of the desired product from the desired raw material. As a matter of fact, the generation of efficient overproducing strains require a plurality of different measures.

[0010] The problem underlying the present invention is to provide a method for producing L-methionine comprising the fermentation of a microorganism in media containing a primary alcohol, such as ethanol, as a carbon source under aerobic conditions and to accumulate L-methionine in the medium.

[0011] The problem is solved by a method for producing L-methionine comprising the fermentation under aerobic conditions of a genetically modified microorganism which produces more L-methionine than the microorganism before the genetical modification in a medium comprising a primary alcohol and accumulating L-methionine in the medium to form an L-methionine containing fermentation broth, wherein the microorganism has been further genetically modified so that it comprises a gene encoding a protein having an activity of an alcohol dehydrogenase (EC 1. 1.1.1) which is active under aerobic conditions and a gene encoding a protein having an activity of an aldehyde dehydrogenase (EC 1 .2.1.10) which is active under aerobic conditions or a gene encoding a protein having an activity of both, of an alcohol dehydrogenase (EC 1 .1 .1 .1) and of an aldehyde dehydrogenase (EC 1 .2.1 .10) which is active under aerobic conditions and so that it further comprises an increased activity of an isocitrate lyase (EC 4.1 .3.1) compared to the activity of an isocitrate lyase in the microorganism before the genetical modification and wherein the microorganism is further genetically modified so that comprises an increased activity of a malate 202400272 Foreign Filings 3 synthase reaction (EC 2.3.3.9) compared to the malate synthase reaction in the microorganism before the genetical modification.

[0012] A primary alcohol is a compound having the Formula (1) wherein R is -CH3 or CH3-(CH2)m- and wherein m is 1 , 2 or 3. In the method of the present invention the primary alcohol is preferably ethanol (m = 1).

[0013] In the method according to the present invention the medium may comprise 0.01 - 50 % by weight primary alcohol, preferably ethanol, preferably 0.01 - 25 % by weight primary alcohol, preferably ethanol.

[0014] A microorganism is an organism of microscopic size, which may exist in its single-celled form or as a colony of cells. Microorganisms include most unicellular organisms from all three domains of life. The domains Archaea and Bacteria contain microorganisms only. Among the third domain Eukaryota unicellular organism like yeasts, protists and protozoans belong to the group of microorganisms. Multicellular organisms or parts of them, like cell cultures, are not considered here as microorganisms.

[0015] The genetically modified microorganism subjected to a fermentation under aerobic conditions in the method according to the present invention may belong to the genus Corynebacteriaceae, Enterobacteriaceae, Pseudomonadaceae, Bacillaceae, Yarrowia or Saccharomycetales. In one embodiment, the microorganism belongs to the group of Gamma-Proteobacteria. The microorganism is a member of the group of Enterobacteriaceae, and preferably is member of the species of Escherichia coli.

[0016] The genetically modified microorganism subjected to a fermentation under aerobic conditions in the method according to the present invention may be e.g. a microorganism derived from the following wild type strains:

[0017] Escherichia coli K12 strain MG1655 (ATCC 700926)

[0018] Escherichia coli K12 strain W3110 (ATCC 27325)

[0019] Escherichia coli B strain BL21 (ATCC BAA-1025) Corynebacterium glutamicum strain ATCC 13032 Corynebacterium glutamicum strain ATCC 14067 Corynebacterium glutamicum strain R (JCM 18229) Pseudomonas putida strain KT2440 Bacillus subtilis strain 168 (DSM 23778) 202400272 Foreign Filings 4

[0020] Bacillus subtilis subsp. subtilis strain DSM 10

[0021] Saccharomyces cerevisiae (ATCC 204508 I S288c) (Baker's yeast) Yarrowia lipolytica (CLIB 122 / E 150)

[0022] Komagataella pastoris (DSMZ 70382).

[0023] Preferably, the microorganism is Escherichia coll, Corynebacterium glutamicum, Pseudomonas putida or Bacillus subtilis.

[0024] For clarification, the Enzyme Commission numbers (EC number) are applied here to describe chemical reactions I enzyme activities that are of relevance for the invention. This is a numerical classification scheme for enzymes, based on the chemical reactions they catalyze. As a system of enzyme nomenclature, every EC number is associated with a recommended name for the corresponding enzyme-catalyzed reaction. EC numbers do not specify enzymes but enzyme- catalyzed reactions.

[0025] To increase the contribution of chemical reactions in cells numerous methods and approaches are known. There are methods using wild-type microorganism, auxotrophic strains derived from wildtype strains, mutants lacking regulatory functions or with altered metabolic functions, including reduced or increased activity of enzymes catalyzing desired reactions.

[0026] The activity of an enzyme can be increased for example by measures in the following list: Overexpression of a gene encoding the respective gene, which may be achieved by o Introduction of an additional copy of entire gene(s) or parts of the encoding gene(s) for the enzyme; o Alteration of the ribosome binding site (RBS) or the promotor region of the gene(s); o Introduction or modifications of binding site(s) for regulators on the DNA sequence in the vicinity of regulated gene(s) or within the DNA sequence of the regulated gene(s) encoding the enzyme, e.g. introduction of a strong promoter sequence; o Modification or activation or inactivation of a regulator for the gene encoding the enzyme;

[0027] An exchange or introduction of amino acids forming the polypeptide(s) by changing the gene sequence(s)

[0028] Introducing random mutations into the genome and screening for higher enzyme activities in suitable screening approaches without knowing specific modifications.

[0029] Overexpression of a gene may be generally achieved by increasing the copy number of the gene and / or by functionally linking the gene with a strong promoter and / or by enhancing the ribosomal binding site and / or by codon usage optimization of the start codon or of the whole gene or a combination comprising a selection of all these methods. 202400272 Foreign Filings 5

[0030] Generally, it is possible to achieve an overexpression or an increase in the expression of genes in bacteria by selecting strong promoters, for example by replacing the original promoter with a strong, native, originally assigned to other genes promoter or by modifying certain regions of a given, native promoter (for example its so-called -10 and -35 regions) towards a consensus sequence, e.g. as taught by M. Patek et al. (Microbial Biotechnology 6 (2013), 103-117) for C. glutamicum, or by functional linking of the gene with a strong heterologous promoter. A “functional linkage” is understood to mean the sequential arrangement of a promoter with a gene, which leads to a transcription of the gene.

[0031] In other words, a strong promotor generates an increased gene expression compared to the native promotor.

[0032] The genetically modified microorganism fermented under aerobic conditions in the method according to the present invention comprises among others an increased activity of an isocitrate lyase (EC 4.1.3.1), which may be achieved by overexpression of a gene encoding an isocitrate lyase (e.g. aceA) , e.g. by introduction of an additional copy of a gene encoding an isocitrate lyase and / or by functionally linking a gene encoding an isocitrate lyase (e.g. aceA) with a strong promoter.

[0033] The genetically modified microorganism fermented under aerobic conditions in the method according to the present invention further comprises among others an increased activity of a malate synthase reaction (EC 2.3.3.9) compared to the malate synthase reaction in the microorganism before the genetical modification, which may be achieved by overexpression of a gene encoding a malate synthase (e.g. aceB) , e.g. by introduction of an additional copy of a gene encoding a malate synthase and / or by functionally linking a gene encoding malate synthase (e.g. aceB) with a strong promoter.

[0034] The activity of an enzyme can be decreased for example by measures in the following list: Deletion of entire gene(s) or parts of the encoding gene(s) for the enzyme Alteration of the ribosome binding site (RBS) or the promotor region of the gene(s) An exchange or introduction of amino acids forming the polypeptide(s) by changing the gene sequence(s) Introducing a frame shift mutation Deletion, modification or activation or inactivation of a regulator for the gene encoding the enzyme Introduction, deletions or modifications of binding site(s) for regulators on the DNA sequence in the vicinity of regulated gene(s) or within the DNA sequence of the regulated gene(s) encoding the enzyme

[0035] Introducing an antisense RNA construct binding to the mRNA of the gene encoding the enzyme 202400272 Foreign Filings 6

[0036] Introducing random mutations into the genome and screening for decreased enzyme activities in suitable screening approaches without knowing specific modifications

[0037] Altered regulatory functions can be achieved for example by measures in the following list: Reduced expression of gene(s) encoding proteins with regulatory function by alteration of the ribosome binding site (RBS) or the promotor region of the gene

[0038] An exchange or introduction of amino acids forming the polypeptide(s) by changing the sequence(s) of genes encoding proteins with regulatory function Introducing frame shift mutations in genes encoding proteins with regulatory function Introduction, deletion or modification of the binding site of the regulator on the DNA sequence in the vicinity of regulated genes or within the DNA sequence of the regulated genes

[0039] Introducing an antisense RNA construct binding to the mRNA of the regulator Introducing random mutations into the genome and screening for the altered regulator function in suitable screening approaches without knowing specific modifications

[0040] In the method according to the present invention the increased activity of the isocitrate lyase and the increased activity of the malate synthase in the genetically modified microorganism may also be achieved by decreasing the activity, e.g. inactivation or deletion, of a regulator gene of the glyoxylate shunt. The inactivated or deleted regulator gene of the glyoxylate shunt may be the icIR gene encoding the isocitrate lyase regulator.

[0041] For the utilization of C2 carbon units like ethanol the glyoxylate cycle is applied in many organisms. This cycle is a variation of the tricarboxylic acid cycle (TCA cycle), also named glyoxylate shunt or glyoxylate bypass and is considered as an anabolic pathway. The glyoxylate shunt provides chemical reactions for the formation of one malate molecule from two molecules of acetyl-CoA. It bypasses the oxidative decarboxylation steps of the TCA cycle and conserves carbon skeletons for biomass and product generation.

[0042] The glyoxylate shunt comprises two central, dedicated catalytic activities: isocitrate lyase activity according EC4.1.3.1 and malate synthase activity according to EC 2.3.3.9.

[0043] The isocitrate lyase activity comprises the aldol cleavage of isocitrate to succinate and glyoxylate and can be catalyzed by enzymes named isocitrate lyase, isocitritase, isocitratase, isocitric lyase and by enzymes of other names. In Eschericha coli for example the isocitrate lyase enzyme is encoded by the gene aceA which is part of the aceBAK operon.

[0044] The malate synthase activity comprises the condensation of acetyl-CoA with glyoxylate to form (S)- malate and can be catalyzed by enzymes named malate synthase, malate synthase g, malate synthetase, and by enzymes of other names. In Eschericha coli for example the malate synthase 202400272 Foreign Filings 7 enzyme is encoded by the gene aceB which is part of the aceBAK operon (Byrne C, Stokes HW, Ward KA. Nucleic Acids Res. 1988 Oct 11 ;16(19):9342. doi: 10.1093 / nar / 16.19.9342. PMID: 3050899; PMCID: PMC338715.).

[0045] The regulation of the glyoxylate shunt activities is obtained by very different measures and is specific for particular organisms, summarized by Dolan and Welch 2018 (Annu Rev Microbiol. 2018 Sep 8;72:309-330. doi: 10.1146 / annurev-micro-090817-062257).

[0046] In principle, the expression of genes encoding enzymes performing the required activities is regulated by different regulators. In addition, biochemical regulation of enzyme activities occurs at the entry of the glyoxylate shunt to balance the flux of carbon into the shunt via the isocitrate lyase activity in favor of the isocitrate dehydrogenase activity which is the competing activity within the TCA cycle. For example, in Escherichia coli one of the regulators for the expression of the aceA and aceB genes is named IcIR and encoded by the IcIR gene (Cortay JC, Negre D, Galinier A, Duclos B, Perriere G, Cozzone AJ.EMBO J. 1991 Mar;10(3):675-9. doi: 10.1002 / j.1460- 2075.1991. tb07996.x.

[0047] The activity of the glyoxylate shunt may be increased by a variety of measures. For example, Yang et al. (2019, Applied microbiology and biotechnology, 103(11), 4549-4564) describe the increase of the glyoxylate shunt activity by insertion of a strong promoter upstream of the aceBAK operon and by the deletion of the regulator-encoding gene icIR in Escherichia coli. Also Zhu et al. (2019, Biotechnology and applied biochemistry, 66(6), 962-976) described the deletion of the regulatorencoding gene IcIR in Escherichia coli.

[0048] As an alternative approach the inactivation of the IcIR binding sites in the aceBAK regulatory region upstream of the start codon of aceBAK was described by Sunnarborg et al., (1990, Journal of bacteriology, 172(5), 2642-2649).

[0049] The activity of the glyoxylate shunt reactions in Corynebacterium glutamicum was enhanced by insertion of additional gene copies of aceA and aceB into the chromosome. Moreover, e.g. the expression of the genes aceA and aceB from a plasmid was applied for increasing the glyoxylate shunt activities (Zahoor et al., 2014, Journal of biotechnology, 192 Pt B, 366-375).

[0050] As a further example, the isocitrate dehydrogenase activity can be lowered by limiting the expression of the led gene encoding for the enzyme with isocitrate dehydrogenase activity. Again, measures can be applied described above in the definitions. Such an approach was successfully applied by Li and coworkers (Li Y, Huang B, Wu H, Li Z, Ye Q, Zhang YP., ACS Synth Biol. 2016 Nov 18;5(11):1299-1307. doi: 10.1021 / acssynbio.6b00052. Epub 2016 May 5) in Escherichia coli and by Zahoor and coworkers for Corynebacterium glutamicum (Zahoor et al., 2014, Journal of biotechnology, 192 Pt B, 366-375). 202400272 Foreign Filings 8

[0051] The method according to the present invention uses a genetically modified microorganism which produces more L-methionine than the microorganism before the genetical modification. In other words, used in the method according to the present invention is overproducing L-methionine. The term overproducing is applied for microorganism which, in contrast to wild-type microorganisms, have a drastically increased production output, more than the intrinsic needs (overproduction), for the preparation of the desired product from the starting material, i.e. a carbon source, e.g. a sugar and / or an alcohol.

[0052] A wild type of the microorganism is a microorganism that has not been subjected to undirected or directed modifications as described below. Such wild type microorganisms can be obtained from strain collections frequently or isolated from natural habitats. Examples of commonly used wild type strains for microorganisms comprise members of the following list: Escherichia coli K12 strain MG1655 (ATCC 700926) Escherichia coli K12 strain W3110 (ATCC 27325) Escherichia coli B strain BL21 (ATCC BAA-1025) Corynebacterium glutamicum strain ATCC 13032 Corynebacterium glutamicum strain ATCC 14067 Corynebacterium glutamicum strain R (JCM 18229) Pseudomonas putida strain KT2440 Bacillus subtilis strain 168 (DSM 23778) Bacillus subtilis subsp. subtilis strain DSM 10

[0053] Saccharomyces cerevisiae (ATCC 204508 I S288c) (Baker's yeast) Yarrowia lipolytica (CLIB 122 / E 150) Komagataella pastoris (DSMZ 70382)

[0054] L-methionine overproduction by a microorganism may be accomplished by a multitude of different approaches comprising the increase or decrease of enzyme activities as well as of regulatory functions and other measures. All measures target the increase of the capacity of the microorganism to produce L-methionine accompanied by the excretion of L-methionine into the medium and the accumulation of L-methionine into the fermentation broth.

[0055] In different microorganisms the pathways for L-methionine synthesis differ in terms of catalytic reactions that are active, in terms of regulatory mechanisms that were identified and in terms of proteins that are contributing as enzymes, regulators or transport proteins. This caused the successful application of manyfold approaches describing the increase of the capacity of L- methionine production.

[0056] One of the approaches is the application of mutagenesis and selection. In principle, mutants with manifold changes in their genetic material are produced and subsequently exposed to toxic 202400272 Foreign Filings 9 analogues of methionine. Mutants that can survive are frequently carrying mutations (i.e. genetic modifications) causing an increase in the capacity of L-methionine production that is maintained also in the absence of the analogues of methionine. Analogues such as ethionine, selenomethionine, norleucine, and methionine hydroxamate have been used to develop methionine- overproducing strains.

[0057] The application of this strategy for different organisms were described by Lawrence et al. (Lawrence, D. A., Smith, D. A., & Rowbury, R. J. (1968). Genetics, 58(4), 473-492. https: / / doi.Org / 10.1093 / genetics / 58.4.473) for Salmonella typhimurium, by Nakamori et al. (Nakamori, S., Kobayashi, S., Nishimura, T., & Takagi, H. (1999). Applied microbiology and biotechnology, 52(2), 179-185. https: / / doi.org / 10.1007 / s002530051506) for Escherichia coll or by Park et al. (Park, S. D., Lee, J. Y., Sim, S. Y., Kim, Y., & Lee, H. S. (2007). Metabolic engineering, 9(4), 327-336. https: / / doi.Org / 10.1016 / j.ymben.2007.05.001) for Corynebacterium glutamicum.

[0058] In general, a mechanistic understanding of the reasons causing the increased capacity for L- methionine production or of a genetically modified microorganism which produces more L- methionine than the microorganism before the genetical modification is not required for such approaches. Accordingly, many L-methionine overproducing strains are known and available wherein the genetic modifications are unknown and therefore cannot be traced to specific modifications of chemical reactions, enzymes, regulators or other measures.

[0059] In contrast, to the undirected search for L-methionine overproducing microorganisms many directed approaches were described to obtain an increase in L-methionine production capacity. For such approaches the understanding of the L-methionine pathway, transport and regulation as well as the specific aspects connected to the organisms used for L-methionine production is required.

[0060] A detailed descriptions of methionine synthesis pathway and regulations involved in Escherichia coli and Corynebacterium glutamicum were reported by Kumar and Gomes (Kumar D, Gomes J.Biotechnol Adv. 2005 Jan;23(1):41-61 . doi: 10.1016 / j.biotechadv.2004.08.005.). In general, L- methionine, along with L-lysine and L-threonine, is derived from aspartate / oxaloacetate. Sulphur is introduced e.g. in the form of L-cysteine (via cystathionine as intermediate) into L-methionine by trans-sulfuration. The CH3 group of L-methionine originates e.g. from C1 metabolism and is transferred to L-homocysteine by the methionine synthases activity that can by catalyzed by different enzymes.

[0061] Such knowledge can be applied to generate microorganism with an increased L-methionine synthesis compared to the respective unmodified microorganism. Cai et al. (Cai M, Liu Z, Zhao Z, Wu H, Xu M, Rao Z.Biotechnol Adv. 2023 Dec;69:108260. doi: 10.1016 / j.biotechadv.2023.108260. Epub 2023 Sep 20.) provides a comprehensive overview on recent advances in the microbial 202400272 Foreign Filings 10 production of L-methionine. Moreover, strains and processes for fermentative production of L- methionine have been described for E. coli in W02006001616 A1 or W02009043803 A2, or in W02005108561 A2, for example.

[0062] The generation of an L-methionine producer in Corynebacterium glutamicum was described by Qin et al. (Qin, T., Hu, X., Hu, J., & Wang, X. (2015) Biotechnology and applied biochemistry, 62(4), 563-573. https: / / doi.org / 10.1002 / bab.1290) for example.

[0063] Finally, a combination of both strategies, undirected and directed approaches was applied to generate L-methionine producers as described for Corynebacterium glutamicum by Li et al. (Li, Y., Cong, H., Liu, B., Song, J., Sun, X., Zhang, J., & Yang, Q. (2016) Antonie van Leeuwenhoek, 109(9), 1185-1197. https: / / doi.org / 10.1007 / s10482-016-0719-0) and for Pantoea ananatis in US11390896 B2.

[0064] The microorganism used in the method according to the present invention is a genetically modified microorganism which produces more L-methionine than the microorganism before the genetical modification.

[0065] The microorganism that produces methionine may have an increased homoserine O- acetyltransferase activity (EC 2.3.1.31) or an increased homoserine O-succinyltransferase activity (EC 2.3.1 .46) or an increased homoserine kinase activity (EC 2.7.1 .39) or a combination of increased activity of a homoserine O-acetyltransferase, homoserine O-succinyltransferase and / or homoserine kinase. In one specific embodiment of the present invention, the microorganism that produces methionine has an increased homoserine O-succinyltransferase activity (EC 2.3.1.46).

[0066] The microorganism that produces methionine may have a modified metA gene that encodes for a variant of the MetA enzyme having an increased homoserine O-succinyltransferase activity in presence and / or absence of methionine.

[0067] The microorganism may have a deleted or inactivated metJ gene encoding for a MetJ protein having the function of a transcriptional regulator or may have modifications of the DNA binding site(s) for the regulator MetJ in the vicinity of regulated gene(s) or within the DNA sequence of the regulated gene(s).

[0068] The microorganism may have an increased expression of the genes cysP, cysU, cysl / 1 / ; cysA and / or cysM . Alternatively, it may have an increased expression of a cysPUWAM operon coding for the sulfate / thiosulfate importer and thiosulfate specific cysteine synthase.

[0069] In a particular embodiment of the present invention this overproduction of L-methionine may e.g. be achieved in the genetically modified microorganism among others by inactivation or deletion of a 202400272 Foreign Filings 11 metJ gene coding for a DNA-binding transcriptional repressor MetJ, overexpression of a cysJIH operon coding for a sulfite reductase (E.C. 1 .8.1 .2), a phosphoadenosine phosphosulfate reductase (E.C. 1 .8.4.8) and by overexpression of a gene metA* coding for a feedback-resistant homoserine O-succinyltransferase. Examples of metA* genes coding for a feedback-resistant homoserine O-succinyltransferase are provided in W02005108561 A2, in particular in Example 4 of W02005108561 A2. The genetically modified microorganism may further comprise an overexpressed cysPUWAM operon coding for the sulfate / thiosulfate importer and thiosulfate specific cysteine synthase (W02009043372 A1).

[0070] The microorganism used in the method according to the present invention comprises among others a gene encoding a protein having an activity of an alcohol dehydrogenase (EC 1. 1.1.1) which is active under aerobic conditions and a gene encoding a protein having an activity of an aldehyde dehydrogenase (EC 1 .2.1.10) which is active under aerobic conditions or a gene encoding a protein having an activity of both, of an alcohol dehydrogenase (EC 1 .1 .1 .1) and of an aldehyde dehydrogenase (EC 1 .2.1.10) which is active under aerobic conditions.

[0071] Next to the application of sugar (derived) carbon sources obtained by starch hydrolysis alcohols such as ethanol can be applied as carbon source. A potential advantage of using ethanol as carbon source as that it can be derived from feed stocks that do not compete with human nutrition. In addition, the channelling of ethanol into the cellular metabolism can provide advantages in terms of efficiency due to the more direct connection of the entry of the carbon source and the metabolic pathways involved in product formation.

[0072] In principle, the assimilation of ethanol in microorganisms requires two initial enzymatic activities, alcohol dehydrogenase activity and aldehyde dehydrogenase activity. As a result of both activities Acetyl-CoA is generated and can be channeled into the metabolism.

[0073] A wide variety of alcohol dehydrogenase activities were described in literature that can be discriminated in general by using primary alcohols or secondary alcohols as substrate and by transferring electrons to different acceptor molecules. The different activities were classified applying the enzyme nomenclature and EC system.

[0074] Suwannarangsee et al., (Suwannarangsee S, Kim S, Kim OC, Oh DB, Seo JW, Kim CH, Rhee SK, Kang HA, Chulalaksananukul W, Kwon O.AppI Microbiol Biotechnol. 2012 Nov;96(3):697-709. doi: 10.1007 / S00253-011-3866-2. Epub 2012 Jan 17.PMID: 22249723) provide an example from Hansenula polymorpha, describing an alcohol dehydrogenase activity (EC 1 .1.1.1) with the following reaction: alcohol + NAD+ «-> aldehyde + NADH + H+

[0075] Liu et al., (Liu X, Dong Y, Zhang J, Zhang A, Wang L, Feng L., Microbiology (Reading). 2009 Jun;155(Pt 6):2078-2085. doi: 10.1099 / mic.0.027201-0. Epub 2009 Apr 21) provide an example of 202400272 Foreign Filings 12 an alcohol dehydrogenases activity from Geobacillus thermodenitrificans NG80-2, (EC 1.1.1 .2) performing the following reaction: alcohol + NADP+ «-> aldehyde + NADPH + H+

[0076] Rupp and Gorisch, (Rupp M, Gorisch H., Biol Chem Hoppe Seyler. 1988 Jun;369(6):431-9. doi: 10.1515 / bchm3.1988.369.1 .431 .) provide an example of an alcohol dehydrogenase activity from Pseudomonas aeruginosa, an alcohol dehydrogenase (cytochrome c) activity (EC 1 .1 .2.8) with the following reaction: a primary alcohol + 2 an oxidized cytochrome c550 —> an aldehyde + 2 reduced cytochrome c550 + 2 H+

[0077] Moreover, alcohol dehydrogenase activities were described and assigned to the classes EC 1 .1 .5.5 EC1.1.9.1 and 1.1.99.36 using ethanol and other electron accepting substrates.

[0078] The described activities can be performed by a multitude of different enzymes that might be named as alcohol dehydrogenase. Examples of enzymes performing an alcohol dehydrogenase activity can be found in the references describing the enzyme activities.

[0079] A wide variety of aldehyde dehydrogenase activities were described in literature that can be discriminated in general by using different substrates containing an aldehyde function and different molecules that act as electron acceptor. For the assimilation of ethanol e.g. a specific acetaldehyde dehydrogenase activity is widely distributed in nature (EC 1 .2.1 .10).

[0080] The acetaldehyde dehydrogenase activity can be performed by a multitude of different enzymes that might be named as aldehyde dehydrogenase or acetaldehyde dehydrogenase.

[0081] Examples for acetaldehyde dehydrogenase activity carrying enzymes are the EutE protein from Escherichia coli (Kozak BU, van Rossum HM, Niemeijer MS, van Dijk M, Benjamin K, Wu L, Daran JM, Pronk JT, van Maris AJ., FEMS Yeast Res. 2016 Mar;16(2):fow006. doi: 10.1093 / femsyr / fow006. Epub 2016 Jan 26.) or the ALDH protein from Corynebacterium glutamicum (Auchter M, Arndt A, Eikmanns BJ.J Biotechnol. 2009 Mar 10; 140(1 -2):84-91 . doi: 10.1016 / j.jbiotec.2008.10.012. Epub 2008 Nov 12).

[0082] The alcohol dehydrogenase activity and the acetaldehyde dehydrogenase activity can be performed by different enzymes as described above or by a single polypeptide as described for the AdhE protein of Escherichia coli (W. Lorowitz and D. Clark, J Bacteriol 1982 Vol. 152 Issue 2 Pages 935-8; Accession Number: 6752127 PMCID: PMC221556 DOI: 10.1128 / jb.152.2.935- 938.1982).

[0083] The alcohol dehydrogenases activity and the acetaldehyde dehydrogenase activity can be observed in organisms under anaerobic conditions as well as under aerobic conditions. Whereas the mentioned ALDH connected alcohol dehydrogenase activity in Corynebacterium glutamicum 202400272 Foreign Filings 13 can be detected under aerobic conditions, the alcohol dehydrogenase activity connected to the AdhE protein of Escherichia coli can be detected under anaerobic conditions in cells of the wild type.

[0084] The alcohol dehydrogenase activity and the acetaldehyde dehydrogenase activity can be increased to activate the assimilation of ethanol and / or to improve the utilization of ethanol. For such improvements of enzyme activity, the before mentioned measures can be applied.

[0085] E.g. the alcohol dehydrogenase activity in Escherichia coli cells was improved under aerobic conditions by increasing the expression of the adhE gene and by introducing point mutations into the adhE gene causing the change of the amino acid sequence of the AdhE protein. The resulting variant of the AdhE enzyme was present and active under aerobic conditions (C. A. Holland-Staley, K. Lee, D. P. Clark and P. R. Cunningham, J Bacteriol 2000 Vol. 182 Issue 21 Pages 6049-54;

[0086] Accession Number: 11029424 PMCID: PMC94738 DOI: 10.1128 / jb.182.21.6049-6054.2000).

[0087] Preferably, the microorganism used in the method according to the present invention may comprise a gene (SEQ ID NO: 4, SEQ ID NO: 6) encoding a variant of the AdhE enzyme (SEQ ID NO: 3) that is active under aerobic conditions.

[0088] In principle, the use of ethanol for the production of poly hydroxy butyrate was already described (S. Sun, Y. Ding, M. Liu, M. Xian and G. Zhao, Front Bioeng Biotechnol 2020 Vol. 8 Pages 833;

[0089] Accession Number: 32850713 PMCID: PMC7396591 DOI: 10.3389 / fbioe.2020.00833). In addition, the use of ethanol as a carbon source was mentioned for the production of the amino acids L- lysine, L-threonine, L-tryptophane, L-phenylalanine, L-valine, L-leucine, L-isoleucine and L-serine (EP2 192 170 A1).

[0090] In the method according to the present invention the activity of the alcohol dehydrogenase is preferably targeting primary alcohols as substrates according to Formula (1)

[0091] R — OH CO. wherein R is -CH3 or CH3-(CH2)m- and wherein m is 1 , 2 or 3.

[0092] In the method according to the present invention the activity of the aldehyde dehydrogenase is preferably targeting substrates according to Formula (2) 202400272 Foreign Filings 14 wherein R is -CH3 or -CH3-(CH2)m- and wherein m is 1 , 2 or 3.

[0093] In the method according to the present invention the alcohol dehydrogenase activity is preferably targeting substrates according to Formula (1) wherein R is -CH3 and wherein the aldehyde dehydrogenase activity is targeting substrates according to Formula (2) wherein R is -CH3.

[0094] The method according to the present invention may further comprise isolating L-methionine from the L-methionine containing fermentation broth.

[0095] Brief description of the sequences

[0096] SEQ ID NO: 1 : DNA Sequence of plasmid pTZ_E177_a containing a kanamycin resistance gene (kanR) and a p15A origin of replication

[0097] SEQ ID NO: 2: Amino acid sequence of the AdhE protein from E. coli MG1655

[0098] SEQ ID NO: 3: Amino acid sequence of protein variant AdhE-A267T-E568K

[0099] SEQ ID NO: 4: Synthetic DNA sequence coding for AdhE-A267T-E568K

[0100] SEQ ID NO: 5: DNA sequence upstream of the adhE gene

[0101] SEQ ID NO: 6: Full expression unit coding for AdhE-A267T-E568K as ordered for DNA synthesis

[0102] SEQ ID NO: 7: DNA sequence for inactivation of icIR as ordered for DNA synthesis

[0103] SEQ ID NO: 8: PCR primer aceBAK_fw for amplification of the aceBAK operon

[0104] SEQ ID NO: 9: PCR primer aceBAK_rv for amplification of the aceBAK operon

[0105] SEQ ID NO: 10: PCR product covering the genes aceB, ace A and aceK of E. coli MG1655

[0106] A) MATERIALS and METHODS

[0107] Chemicals

[0108] Kanamycin solution from Streptomyces kanamyceticus was purchased from Sigma Aldrich (St. Louis, USA, Cat. no. K0254). If not stated otherwise, all other chemicals were purchased analytically pure from Merck (Darmstadt, Germany), Sigma Aldrich (St. Louis, USA) or Carl-Roth (Karlsruhe, Germany).

[0109] Cultivation for cell proliferation

[0110] If not stated otherwise, cultivation I incubation procedures were performed as follows herewith: a. LB broth (MILLER) from Merck (Darmstadt, Germany; Cat. no. 110285) was used to propagate E. coli strains in liquid medium. The liquid cultures (10 ml liquid medium per 100 ml Erlenmeyer flask with 3 baffles) were incubated in the Infors HT Multitron standard incubator shaker from Infors GmbH (Bottmingen, Switzerland) at 30°C and 200 rpm. 202400272 Foreign Filings 15 b. LB agar (MILLER) from Merck (Darmstadt, Germany, Cat. no. 110283) was used for cultivation of E. coli strains on agar plates. The agar plates were incubated at 30°C in an INCU- Line® mini incubator from VWR (Radnor, USA).

[0111] Determining optical density of bacterial suspensions

[0112] The optical density of bacterial suspensions was determined at 600 nm (OD600) using the BioPhotometer from Eppendorf AG (Hamburg, Germany).

[0113] Centrifugation a. Bacterial suspensions with a maximum volume of 2 ml were centrifuged in 1 .5 ml or 2 ml reaction tubes (e.g. Eppendorf Tubes® 381 OX) using an Eppendorf 5417 R benchtop centrifuge (5 min. at 13.000 rpm). b. Bacterial suspensions with a maximum volume of 50 ml were centrifuged in 15 ml or 50 ml centrifuge tubes (e.g., FalconTM 50 ml Conical Centrifuge Tubes) using an Eppendorf 5810 R benchtop centrifuge for 15 min. at 4.000 rpm.

[0114] DNA isolation

[0115] Plasmid DNA was isolated from E. coli cells using the QIAprep Spin Miniprep Kit from Qiagen (Hilden, Germany, Cat. No. 27106) according to the instructions of the manufacturer.

[0116] Polymerase chain reaction (PCR)

[0117] PCR with a proof reading (high fidelity) polymerase was used to amplify a desired segment of DNA for sequencing or DNA assembly. Non-proof-reading polymerase Kits were used for determining the presence or absence of a desired DNA fragment directly from E. coli colonies. a. The Phusion® High-Fidelity DNA Polymerase Kit (Phusion Kit) from New England BioLabs Inc. (Ipswich, USA, Cat. No. M0530) was used for template-correct amplification of selected DNA regions according to the instructions of the manufacturer. 202400272 Foreign Filings 16

[0118] Table 1 : Thermocycling conditions for PCR with Phusion® High-Fidelity DNA Polymerase Kit from New England BioLabs Inc. b. Taq PCR Core Kit (Taq Kit) from Qiagen (Hilden, Germany, Cat. No.201203) was used to amplify a desired segment of DNA to confirm its presence. The kit was used according to the instructions of the manufacturer.

[0119] Table 2: Thermocycling conditions for PCR with Taq PCR Core Kit (Taq Kit) from Qiagen. c. All oligonucleotide primers were synthesized by Eurofins Genomics GmbH (Ebersberg, Germany). d. As PCR template either a suitably diluted solution of isolated plasmid DNA or of total DNA isolated from a liquid culture or the total DNA contained in a bacterial colony (colony PCR) was used. For said colony PCR the template was prepared by taking cell material with a sterile toothpick from a colony on an agar plate and placing the cell material directly into the PCR reaction tube. The cell material was heated for 10 sec. with 800 W in a microwave oven type Mikrowave & Grill from SEVERIN Elektrogerate GmbH (Sundern, Germany) and then the PCR reagents were added to the template in the PCR reaction tube. e. All PCR reactions were carried out in PCR cyclers type Mastercycler or Mastercycler nexus gradient from Eppendorf AG (Hamburg, Germany). 202400272 Foreign Filings 17

[0120] Restriction enzyme digestion of DNA

[0121] For restriction enzyme digestions either „FastDigest restriction endonucleases (FD)“ (ThermoFisher Scientific, Waltham, USA) or restriction endonucleases from New England BioLabs Inc. (Ipswich, USA) were used. The reactions were carried out according to the instructions of the manufacturer’s manual.

[0122] Determining the sizes of DNA fragments a. The sizes of small DNA fragments (<1000 bps) were usually determined by automatic capillary electrophoresis using the QIAxcel from Qiagen (Hilden, Germany). b. If DNA fragments needed to be isolated or if the DNA fragments were >1000 bps DNA was separated by TAE agarose gel electrophoresis and stained with GelRed® Nucleic Acid Gel Stain (Biotium, Inc., Fremont, Canada). Stained DNA was visualized at 302 nm.

[0123] Purification of PCR amplificates and restriction fragments

[0124] PCR amplificates and restriction fragments were cleaned up using the QIAquick PCR Purification Kit from Qiagen (Hilden, Germany; Cat. No. 28106), according to the manufacturer’s instructions. DNA was eluted with 30 pl 10 mM Tris*HCI (pH 8.5).

[0125] Determining DNA concentration

[0126] DNA concentration was measured using the NanoDrop Spectrophotometer ND-1000 from PEQLAB Biotechnologie GmbH, since 2015 VWR brand (Erlangen, Germany).

[0127] Assembly cloning

[0128] Plasmid vectors were assembled using the “NEBuilder HiFi DNA Assembly Cloning Kit” purchased from New England BioLabs Inc. (Ipswich, USA, Cat. No. E5520). The reaction mix, containing the linear vector and at least one DNA insert, was incubated at 50°C for 60 min. 0.5 pl of Assembly mixture was used for each transformation experiment.

[0129] Transformation of E. coli

[0130] For plasmid cloning, chemically competent “NEB® Stable Competent E. coli (High Efficiency)" (New England BioLabs Inc., Ipswich, USA, Cat. No. C3040) were transformed according to the manufacturer's protocol. Successfully transformed cells were selected on LB agar supplemented with 50 mg / l kanamycin, 20 mg / l chloramphenicol and / or 100 mg / l ampicillin when appropriate. Commercial E. coli chemical or electrocompetent cells were used for cloning and amplification of plasmid DNA (NEB, Catalog # C3019, C3020, C3040) following the manufacturer’s instructions. After the recovery period, cells were spread on LB agar plates with the appropriate antibiotic for selection. After transformation, plates were incubated at 37°C overnight to obtain single colonies. 202400272 Foreign Filings 18

[0131] For electroporation of E. coli competent cells were prepared as follows. A single colony from plate was used to inoculate 10 mL of SOB medium and grown overnight at 37°C, 200 rpm as a preculture. A 1 :100 dilution in 50 mL of fresh SOB medium was inoculated as the main culture and incubated until it reached an ODeoo of 0.7. The culture transferred to a falcon tube and cooled on ice for 30 min. The culture was collected by centrifugation (15 min, 4°C, 4000 rpm). The supernatant was discarded, and the cells were washed twice with 5 mL of ice-cold ddFLO. The pellet was resuspended in 1 mL of ice-cold 10% (v / v) glycerol. The cell suspension was centrifuged (2 min, 4°C, 6000rpm), the supernatant was discarded, and the pelled was resuspended in 100 pL of ice-cold 10% (v / v) glycerol. Aliquots of 40 pL each were prepared and stored at -80°C until use. For electroporation, an aliquot of cells was thawed on ice and DNA was added to it. The cells-DNA mixture was transferred to a 2 mm electroporation cuvette (Sigma-Aldrich, Catalog # Z706088- 50EA) and a 2.5 kV, 200 Q, 25 pF pulse with a 5 ms time constant was triggered using a Gene Pulser Xcell™ (Bio-Rad). The cuvette was placed on ice briefly and 1 mL of SOC medium (SOB medium + 20 mM glucose) was added to the cuvette. The contents were transferred to a 1 .5 mL microcentrifuge tube which was incubated at 37°C for 1 h with shaking for the cells to recover. After recovery, cells were spread on LB agar plates with the appropriate antibiotic for selection. The plates were incubated at 37°C overnight to obtain single colonies.

[0132] Determining nucleotide sequences

[0133] Nucleotide sequences of DNA molecules were determined by Eurofins Genomics GmbH (Ebersberg, Germany) by cycle sequencing, using the dideoxy chain termination method of Sanger et al. (Proceedings of the National Academy of Sciences USA 74, 5463 - 5467, 1977).

[0134] Clonemanager Professional 9 software from Scientific & Educational Software (Denver, USA) was used to visualize and evaluate the sequences as well as for in silico assembly of sequences.

[0135] Glycerol stocks of E. coli strains

[0136] For long time storage of E. coli strains glycerol stocks were prepared. Selected clones were cultivated in 10 ml LB medium supplemented with 2 g / l glucose. Media for growing plasmid containing E. coli strains were supplemented with 50 mg / l kanamycin, 20 mg / l chloramphenicol and / or 100 mg / l ampicillin when appropriate. The medium was contained in 100 ml Erlenmeyer flasks with 3 baffles. It was inoculated with a loop of cells taken from a colony. The culture was then incubated for 18 h at 30°C and 200 rpm. After said incubation period 1 .2 ml 85 % (v / v) sterile glycerol were added to the culture. The obtained glycerol containing cell suspension was then aliquoted in 2 ml portions and stored at -80°C.

[0137] Methionine production in small-scale cultivations

[0138] Precultures of the strains were done in 5.5 ml seed medium (SM). The medium was contained in a 100 ml Erlenmeyer flask with 3 baffles. It was inoculated with 55 pl of a glycerol stock culture and the culture was incubated for 48 h at 37°C and 200 rpm. The composition of the seed medium (SM) is shown in Table 3. 202400272 Foreign Filings 19

[0139] Table 3: Seed medium (SM) After said incubation period the optical densities OD600 of the precultures were determined. The volume, needed to inoculate 10 ml of production medium (PM) to an OD600 of 0.5, was used for inoculation. The main cultures were started by inoculating Erlenmeyer flask (with 3 baffles) containing 9.9 ml production medium (PM) with each 100 pl of the resuspended cells from the precultures. The composition of the production medium (PM) is shown in Table 4. 202400272 Foreign Filings 20

[0140] Table 4: Production medium (PM)

[0141] The main cultures were cultivated for 48 h at 37 °C and 200 rpm in an Infers HT Multitron standard incubator shaker from Infers GmbH (Bottmingen, Switzerland).

[0142] After cultivation the culture suspensions were transferred to a deep well microplate. A part of the culture suspension was suitably diluted to measure the OD600. Another part of the culture was centrifuged and the concentrations of amino acids in the supernatant were analyzed.

[0143] Quantification of amino acids

[0144] The concentration of amino acids in the culture supernatants was determined by ion exchange chromatography using a SYKAM 5433 amino acid analyzer from SYKAM Vertriebs GmbH (Furstenfeldbruck, Germany). As solid phase column with spherical, polystyrene-based cation exchanger (Peek LCA N04 / Na, dimension 150x4.6 mm) from SYKAM was used. Depending on the amino acid the separation takes place in an isocratic run using a mixture of buffers A and B for elution or by gradient elution using said buffers. As buffer A an aqueous solution containing in 20 I 263 g trisodium citrate, 120 g citric acid, 1100 ml methanol, 100 ml 37% HCI and 2 ml octanoic acid (final pH 3.5) was used. As buffer B an aqueous solution containing in 20 I 392 g trisodium citrate, 100 g boric acid and 2 ml octanoic acid (final pH 10.2) was used. The free amino acids were colored with ninhydrin through post-column derivatization and detected photometrically at 570 nm.

[0145] B) EXPERIMENTAL RESULTS

[0146] Example 1 : Construction of the L-methionine producing strain MET3

[0147] The L-methionine producing strain Escherichia coli MET3 is a derivative of the wild type strain Escherichia coli K12 MG1655 (= ATCC 700926). The strain Escherichia coli (Migula) Castellani and Chalmers (ATCC 700926) is commercially available.

[0148] W02005108561 A2 discloses that MG1655 (= ATCC 700926) can be rendered into a L-methionine producing strain by deleting the metJ gene and introducing a mutation into the metA gene, resulting in the allele metA*11 , coding for a feedback resistant homoserine transsuccinylase enzyme.

[0149] Both changes were introduced into MG1655 as described in W02005108561 resulting in strain

[0150] MG1655_DmefJ_meM*11 . 202400272 Foreign Filings 21

[0151] W02009043372 A1 discloses that insertion of the strong promoter Ptrc upstream of the operon cysPUWAM enhances the production of L-methionine. Ptrc was inserted upstream of the operon cysPUWAM of strain MG1655_DmetJ_metA*11 as described in W02009043372 resulting in strain MG1655_DmetJ_metA*1 Ptrc-cysPUWAM. This strain was renamed as MET3.

[0152] Aerobic cultivation of MET3 in glucose containing production medium confirmed its capability of producing L-methionine.

[0153] Example 2: Construction of the plasmid pTZ_adhE-A267T-E568K

[0154] The plasmid pTZ_E177_a was purchased from Trenzyme GmbH, Byk-Gulden-Str. 2, 78467 Konstanz, Germany (SEQ ID NO: 1). It contains a kanamycin resistance gene (kanR) and a p15A origin of replication. It was cut using the restriction endonucleases Zra\ and Asci and the DNA was purified with the „QIAquick PCR Purification Kit“ (Qiagen GmbH, Hilden, Germany).

[0155] The amino acid sequence of the AdhE protein of E. coli MG1655 was obtained from the EcoCyc database (https: / / ecocyc.org / ; gene: adhE = b1241 ; SEQ ID NO: 2).

[0156] The AdhE protein is sensitive to oxidative damage and therefor poorly suited for aerobic ethanol utilization. As shown by Holland-Staley et al. (2000), changing alanine 267 into threonine and glutamate 568 into lysine results in a protein that has both acetaldehyde dehydrogenase (acetylating, EC 1 .2.1 .10) activity and alcohol dehydrogenase (EC 1 .1 .1.1) activity under aerobic conditions. It is thus suitable for aerobic ethanol utilization. Accordingly, both exchanges were introduced in silico into the amino acid sequence of AdhE, resulting in the variant AdhE-A267T- E568K (SEQ ID NO: 3). Using the tools of the ThermoFisher DNA synthesis services (Geneart AG, Am Biopark 11 , 93053 Regensburg, Germany) this amino acid sequence was retranslated into a DNA sequence with an optimized codon usage for gene expression in E. coli and avoidance of recognition sequences for the restriction enzymes BsmBI, Zra\ and Asci (SEQ ID NO: 4). To facilitate transcription and translation of the gene a DNA sequence covering 501 bp from immediately upstream of the adhE gene was obtained from the EcoCyc database (https: / / ecocyc.org / ). Two base pairs at the 3’-end (“tt”) were changed to “cc” (SEQ ID NO: 5). This sequence was combined with SEQ ID NO: 4 and restriction sites for Zra\ and Asci were added at the ends. Additionally, two 30 bp homology regions for Gibson-ZHifi-assembly were appended and flanked by two inwards oriented BsmBI recognition sites (SEQ ID NO: 6).

[0157] The resulting DNA sequence was ordered for gene synthesis from ThermoFisher DNA synthesis services (Geneart AG, Am Biopark 11 , 93053 Regensburg, Germany) as part of a cloning plasmid with an ampicillin resistance gene.

[0158] After delivery, the plasmid containing SEQ ID NO: 6 was cut using BsmBI and the DNA was purified with the „QIAquick PCR Purification Kit“ (Qiagen GmbH, Hilden, Germany). 202400272 Foreign Filings 22

[0159] The purified DNA was joined with the purified DNA of pTZ_E177_a (previously cut with Zra\ and Asci) and the matching sequence ends were assembled using the “NEBuilder HiFi DNA Assembly Cloning Kit” (New England BioLabs Inc., Ipswich, USA, Cat. No. E5520).

[0160] The product was transformed into „NEB Stable Competent E. co / i (High Efficiency)" (New England Biolabs, Ipswich, USA) and cells were grown on LB agar containing 50 mg / l kanamycin. Proper plasmid clones were identified by restriction digestion and DNA sequencing. The resulting plasmid was named pTZ_adhE-A267T-E568K. In production assays it was used to provide enzymatic activity of alcohol dehydrogenase (EC 1.1.1.1) and acetaldehyde dehydrogenase (acetylating, EC 1 .2.1.10) under aerobic cultivation conditions. The empty vector pTZ_E177_a served as a negative control.

[0161] Example 3: Activation of the glyoxylate shunt (by inactivation of the ic / R gene) in E. coli MET3

[0162] Inactivation of the gene IcIR (b4018), coding for the DNA-binding transcriptional repressor IcIR, is a known method for activating the glyoxylate shunt and increasing the corresponding metabolic flux (Yang et al., 2022, Frontiers in bioengineering and biotechnology, 10, 1066651).

[0163] For inactivating the genomic icIR gene in MET3 by homologous recombination a knockout plasmid based on pKO3 was constructed (Link et al., 1997; Journal of bacteriology, 179(20), 6228-6237, DNA sequence available at: https: / / arep.med.harvard.edu / labgc / pKO3v.html).

[0164] Plasmid DNA of pKO3 was digested using the restriction endonucleases Smal and Age I and the DNA was purified using the „QIAquick PCR Purification Kit" (Qiagen GmbH, Hilden, Germany).

[0165] A sequence was designed consisting of 750 bp upstream of the IcIR gene, a replacement sequence for the IcIR CDS and 750 bp downstream of IcIR. Additionally, two 30 bp homology regions for Gibson-ZHifi-assembly were appended and flanked by two inwards oriented BsmBI recognition sites (SEQ ID NO: 7).

[0166] The resulting DNA sequence was ordered for gene synthesis from ThermoFisher DNA synthesis services (Geneart AG, Am Biopark 11 , 93053 Regensburg, Germany) as part of a cloning plasmid with an ampicillin resistance gene.

[0167] After delivery, the plasmid containing SEQ ID NO: 7 was cut using BsmBI and the DNA was purified with the „QIAquick PCR Purification Kit" (Qiagen GmbH, Hilden, Germany). 202400272 Foreign Filings 23

[0168] This DNA was joined with the purified DNA of pKO3 (previously cut with Smai and Agel) and the matching sequence ends were assembled using the “NEBuilder HiFi DNA Assembly Cloning Kit” (New England BioLabs Inc., Ipswich, USA, Cat. No. E5520).

[0169] The product was transformed into „NEB Stable Competent E. coli (High Efficiency)" (New England Biolabs, Ipswich, USA) and cells were grown at 30°C on LB agar containing 20 mg / l chloramphenicol. Proper plasmid clones were identified by restriction enzyme digestion and DNA sequencing. The resulting plasmid was named pKO3_D / c / R.

[0170] For inactivating the IcIR gene, strain MET3 was transformed by pKO3_D / c / R using electroporation. The cells were plated on LB agar plates containing 20 mg / l chloramphenicol and incubated for 24h at 30°C. Single colonies were picked, each resuspended in 1 ml LB medium and diluted 1 :1000. Each 100 pl of diluted cells were spread on LB agar plates containing 20 mg / l chloramphenicol and incubated for 18 h at 43°C. Colonies were picked, spread on LB agar containing 20 mg / l chloramphenicol and incubated again for 18 h at 43°C. A couple of colonies were picked, each resuspended in 1 ml LB medium and diluted 1 :1000. The dilutions were spread on LB agar plates containing 5% sucrose and incubated for 44 h at 30°C. Several colonies were picked and streaked in parallel on LB agar plates containing 20 mg / l chloramphenicol and LB agar plates containing 5% sucrose. Colonies that were chloramphenicol sensitive and sucrose resistant were tested by PCR for having a truncated and thereby inactivated IcIR gene. In proper clones the DNA target region was then amplified by PCR and sequenced. A clone having the inactivated icIR gene was chosen and it was named MET3_D / c / R. It has an activated glyoxylate shunt due to the derepression of aceA, aceB and aceK. In production assays strain MET3 served as a control strain.

[0171] Example 4: Construction of the plasmid pTrc aceBAK

[0172] For increasing the expression of the genes aceB, aceA and aceK in E. coli, the plasmid pTrc_aceBAK was constructed.

[0173] The Plasmid pTrc99a (Amann et al., 1988; Gene. 1988 Sep 30;69(2):301-15, GenBank accession: U13872.1) was cut using the restriction endonucleases Ncoi and Smal and the DNA was purified with the „QIAquick PCR Purification Kit" (Qiagen GmbH, Hilden, Germany).

[0174] A PCR was performed with genomic DNA of E. coli MG1655 as a template, using the DNA primers aceBAK_fw (SEQ ID NO: 8) and aceBAK_rv (SEQ ID NO: 9). The resulting PCR product of 4965 bp covered the genes aceB, aceA and aceK (SEQ ID NO: 10) and the DNA was purified with the „QIAquick PCR Purification Kit" (Qiagen GmbH, Hilden, Germany).

[0175] The purified PCR product was joined with the purified DNA of pTrc99a (previously cut with Ncoi and Smal) and the matching sequence ends were assembled using the “NEBuilder HiFi DNA Assembly Cloning Kit” (New England BioLabs Inc., Ipswich, USA, Cat. No. E5520).

[0176] The product was transformed into „NEB Stable Competent E. coli (High Efficiency)" (New England Biolabs, Ipswich, USA) and cells were grown on LB agar containing 100 mg / l ampicillin. Proper plasmid clones were identified by restriction digestion and DNA sequencing. The resulting plasmid 202400272 Foreign Filings 24 was named pTrc_aceBAK. In production assays it was used to increase the activity of the glyoxylate shunt. The empty vector pTrc99a served as a control.

[0177] Example 5: Transformations with plasmids pTZ_E177_a and pTZ_adhE-A267T-E568K Using electroporation, strains MET3 and MET3_D / c / R were transformed by each of the plasmids pTZ_E177_a and pTZ_ac / hE-A267T-E568K and plated on LB agar containing 50 mg / l of kanamycin. The resulting strains are shown in Table 5.

[0178] Table 5: Strains resulting from electroporation.

[0179] Example 6: Transformations with plasmids pTrc99a and pTrc_aceBAK

[0180] Using electroporation, strains MET3 / pTZ_E177_a and MET3 / pTZ_ac / hE-A267T-E568K were transformed with each of the plasmids pTrc99a and pTrc_aceBAK and plated on LB agar containing 100 mg / l of ampicillin and 50 mg / l of kanamycin. The resulting strains are shown in Table 6. 202400272 Foreign Filings 25

[0181] Table 6: Strains resulting from electroporation.

[0182] Example 7: Impact of providing aerobically active alcohol dehydrogenase (EC 1.1.1.1) and acetaldehyde dehydrogenase (acetylating, EC 1.2.1.10) and / or activating the glyoxylate shunt (by inactivating icIR) on the production of L-methionine in ethanol containing medium under aerobic conditions

[0183] To assess the impact of providing an aerobically active alcohol dehydrogenase (EC 1 .1.1.1) and acetaldehyde dehydrogenase (acetylating, EC 1.2.1.10) and / or activating the glyoxylate shunt (by inactivating icIR) on amino acid production, the strains generated in example 5 were cultivated aerobically in production medium containing ethanol. After cultivating for 48 h at 37°C and 200 rpm the concentration of L-methionine was determined.

[0184] Table 7: Impact of activating the glyoxylate shunt (by inactivating icIR) and / or providing an aerobically active alcohol dehydrogenase and an aerobically active acetaldehyde dehydrogenase (acetylating) on the production of L-methionine from ethanol under aerobic conditions, (n.d. below detection limit)

[0185] In the medium of strain MET3 / pTZ_E177_a no L-methionine was detected. We conclude that MET3 / pTZ_E177_a does not efficiently produce L-methionine from ethanol under aerobic conditions.

[0186] In the medium of strain MET3_D / c / R / pTZ_E177_a also no L-methionine was detected. We conclude that activating the glyoxylate shunt (by inactivating IcIR) does not improve the production of L-methionine from ethanol under aerobic conditions. 202400272 Foreign Filings 26

[0187] Strain MET3 / pTZ_ac / / 7E-A267T-E568K produced a significant amount of L-methionine. We conclude that providing an aerobically active alcohol dehydrogenase (EC 1 .1.1.1) and an aerobically active acetaldehyde dehydrogenase (acetylating, EC 1.2.1.10) significantly improves the production of L-methionine from ethanol under aerobic conditions.

[0188] Strain MET3_D / c / / ? / pTZ_ac / hE-A267T-E568K produced significantly more L-methionine when compared to strain MET3 / pTZ_ac / hE-A267T-E568K. We conclude that combining an aerobically active alcohol dehydrogenase (EC 1.1.1.1) and an aerobically active acetaldehyde dehydrogenase (acetylating, EC 1.2.1.10) with increased glyoxylate shunt reactions (by inactivating icIR) further improves the production of L-methionine from ethanol under aerobic conditions.

[0189] Example 8: Impact of providing aerobically active alcohol dehydrogenase (EC 1.1.1.1) and acetaldehyde dehydrogenase (acetylating, EC 1.2.1.10) and / or activating the glyoxylate shunt (by overexpressing the aceBAK operon) on the production of L-methionine from ethanol under aerobic conditions

[0190] To assess the impact of providing aerobically active alcohol dehydrogenase (EC 1 .1.1.1) and acetaldehyde dehydrogenase (acetylating, EC 1.2.1.10) and / or activating the glyoxylate shunt (by overexpressing the aceBAK operon) on amino acid production, the strains generated in example 6 were cultivated aerobically with ethanol present in the production medium. After cultivating for 48 h at 37°C and 200 rpm the concentration of L-methionine was determined.

[0191] Table 8: Impact of activating the glyoxylate shunt (by overexpressing the aceBAK operon) and / or providing an aerobically active alcohol dehydrogenase and an aerobically active acetaldehyde dehydrogenase (acetylating) on the production of L-methionine from ethanol under aerobic conditions, (n.d. below detection limit)

[0192] In the medium of strain MET3 / pTZ_E177_a / pTrc99a no L-methionine was detected. We conclude that MET3 / pTZ_E177_a / pTrc99a does not efficiently produce L-methionine from ethanol under aerobic conditions. 202400272 Foreign Filings 27

[0193] In the medium of strain MET3 / pTZ_E177_a / pTrc_aceBAK also no L-methionine was detected. We conclude that activating the glyoxylate shunt (by overexpressing the aceBAK operon) does not improve the production of L-methionine from ethanol under aerobic conditions. Strain MET3 / pTZ_ac / hE-A267T-E568K / pTrc99a produced a significant amount of L-methionine. We conclude that providing an aerobically active alcohol dehydrogenase (EC 1 .1 .1 .1) and an aerobically active acetaldehyde dehydrogenase (acetylating, EC 1.2.1.10) significantly improves the production of L-methionine from ethanol under aerobic conditions. Strain MET3 / pTZ_ac / hE-A267T-E568K / pTrc_aceBAK produced significantly more L-methionine when compared to strain MET3 / pTZ_ac / hE-A267T-E568K / pTrc99a. We conclude that combining an aerobically active alcohol dehydrogenase (EC 1 .1 .1 .1) and an aerobically active acetaldehyde dehydrogenase (acetylating, EC 1.2.1.10) with increased glyoxylate shunt reactions (by overexpressing the aceBAK operon) further improves the production of L-methionine from ethanol under aerobic conditions.

Claims

202400272 Foreign Filings 28Claims1 . A method for producing L-methionine comprising the fermentation under aerobic conditions of a genetically modified microorganism which produces more L-methionine than the microorganism before the genetical modification in a medium comprising a primary alcohol and accumulating L-methionine in the medium to form an L-methionine containing fermentation broth, wherein the microorganism has been further genetically modified so that it comprises a gene encoding a protein having an activity of an alcohol dehydrogenase which is active under aerobic conditions and a gene encoding a protein having an activity of an aldehyde dehydrogenase which is active under aerobic conditions or a gene encoding a protein having an activity of both, of an alcohol dehydrogenase and of an aldehyde dehydrogenase which is active under aerobic conditions and so that it further comprises an increased activity of an isocitrate lyase compared to the activity of an isocitrate lyase in the microorganism before the genetical modification and wherein the microorganism is further genetically modified so that comprises an increased activity of a malate synthase reaction compared to the malate synthase reaction in the microorganism before the genetical modification.

2. The method of claim 1 , wherein the microorganism that produces methionine has an increased homoserine O-acetyltransferase activity (EC 2.3.1 .31) OR an increased homoserine O- succinyltransferase activity (EC 2.3.1.46) OR an increased homoserine kinase activity (EC2.7.1 .39) OR a combination of increased activity of a homoserine O-acetyltransferase, homoserine O-succinyltransferase and / or homoserine kinase.

3. The method according to any one of the preceding claims, wherein the microorganism that produces methionine has an increased homoserine O-succinyltransferase activity (EC 2.3.1.46).

4. The method according to any one of the preceding claims, wherein the microorganism that produces methionine is characterized by having a modified metA gene that encodes for variant of the MetA enzyme having an increased homoserine O-succinyltransferase activity in presence and / or absence of methionine.

5. The method according to any one of the preceding claims, wherein the microorganism is characterized by having a deleted or inactivated metJ gene encoding for a MetJ protein having the function of a transcriptional regulator OR having modifications of the DNA binding site(s) for the regulator MetJ in the vicinity of regulated gene(s) or within the DNA sequence of the regulated gene(s).

6. The method according to any one of the preceding claims, wherein the microorganism is characterized by having an increased expression of the genes cysP, cysU, cys l / l / ; cysA and or202400272 Foreign Filings 29 cysM or of a cysPUWAM operon coding for the sulfate / thiosulfate importer and thiosulfate specific cysteine synthase.

7. The method according to any one of the preceding claims, wherein the genetically modified microorganism is a member of the group of Gamma-Proteobacteria.

8. The method according to any one of the preceding claims, wherein the genetically modified microorganism is a member of the group of Enterobacteriaceae.

9. The method according to any one of the preceding claims, wherein the genetically modified microorganism is a member of the species of Escherichia coli.

10. The method according to any one of the preceding claims, wherein the activity of the alcohol dehydrogenase is targeting primary alcohols as substrates according to Formula (1)wherein R is -CH3 or CH3-(CH2)m- and wherein m is 1 , 2 or 3.11 . The method according to any one of the preceding claims, wherein the activity of the aldehyde dehydrogenase is targeting substrates according to Formula (2)OR ^H (2), wherein R is -CH3 or CH3-(CH2)m- and wherein m is 1 , 2 or 3.

12. The method according to any one of the preceding claims, wherein the alcohol dehydrogenase activity is targeting substrates according to Formula (1) wherein R is -CH3 and wherein the aldehyde dehydrogenase activity is targeting substrates according to Formula (2) wherein R is - CH3.

13. The method according to any one of the preceding claims, further comprising isolating L- methionine from the L-methionine containing fermentation broth.

14. The method according to any one of the preceding claims, wherein the primary alcohol is ethanol.

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