Glycine production by fermentation

Abstract

The present invention relates to a metabolically engineered microorganism for the biological production of glycine or a salt or ester thereof, wherein the genome of the microorganism comprises attenuated expression of a gene encoding an enzyme having glycine cleavage system activity as defined by EC 1.4.1.27, overexpressing a threonine dehydrogenase-dependent pathway as defined by EC 1.1.1.103 and EC 2.3.1.29, and / or overexpressing a threonine aldolase-dependent pathway as defined by either EC 4.1.2.48 or EC 4.1.2.42, or a catalytically active mutant thereof, and the use of the microorganism to produce glycine or one of its salts or esters. The present invention also relates to a fermentation process using the metabolically engineered microorganism to produce glycine or one of its salts or esters.

Description

[Technical Field]

[0001] The present invention relates to the biotechnology industry, and more particularly to metabolically engineered microorganisms and uses of said metabolically engineered microorganisms for the production of glycine or one of its salts or esters. The present invention also relates to fermentation processes using said metabolically engineered microorganisms for the production of glycine or one of its salts or esters. [Background technology]

[0002] Glycine is one of the 22 amino acids that make up proteins. It is the simplest of the alpha amino acids and has the chemical formula C2H5NO2. This amino acid plays many roles in metabolism and is also a precursor for the synthesis of many substances. For example, glycine accounts for one-third of the amino acid content in collagen molecules and can combine with bile acids to form bile salts. Furthermore, glycine is one of the limiting factors in the biosynthesis of glutathione. Glutathione is known for its role as a major antioxidant and is involved in hemoglobin synthesis and creatine metabolism. Glycine also functions as a neurotransmitter, particularly in glutaminergic neurotransmission, and is important for cognition and sleep quality.

[0003] Studies have also shown that this amino acid may mitigate the harmful effects of methionine derivatives in cardiovascular disease and may be involved in extending lifespan. Epidemiological studies have shown an inverse correlation between plasma glycine concentrations and the prevalence of acute myocardial infarction, diabetes, hypertension, and obesity (Ding et al., 2015). Furthermore, glycine is widely used in the food industry for its preservative properties, as a flavor enhancer due to its sweetness, and in animal nutrition. Glycine is also used in the pharmaceutical and plant health industries, such as the pesticide and herbicide industries. For example, China is the world's leading producer of glycine, with an annual production of 600 kT in 2014, more than 80% of which was used for the production of glyphosate (Zeng et al., 2016). According to a Wendisch study, the annual production of glycine in the feed and food industry in 2018 was estimated at 22,000 tonnes per year (Wendisch, 2019).

[0004] Nevertheless, regardless of the industrial sector under consideration (chemicals, food, animal feed, cosmetics, pharmaceuticals), glycine is the last amino acid to be produced exclusively by chemical synthesis from precursors of fossil origin (Tonouchi et al., 2016). Various chemical processes have been developed to synthesize this non-chiral amino acid on an industrial scale. The most frequently used process is the amination of monochloroacetic acid in the presence of ammonia and a chemical catalyst to obtain glycine and ammonium chloride (Orten and Hill, 1931). The Strecker method (Strecker, 1850), which allows the synthesis of this amino acid by reacting an aldehyde with ammonium chloride in the presence of potassium cyanide, is another well-known industrial process. A third process corresponds to the hydrolysis of hydantoin in the presence of an organic solvent (Boyd and Robson, 1935). However, this hydrolysis process has the disadvantage that hydantoin is synthesized from hydrogen cyanide and formaldehyde. These two substances are particularly toxic and are classified as carcinogens, mutagens and reproductive toxins.

[0005] Other synthetic processes for glycine, which is of particular interest in the food and pharmaceutical markets, have been developed but are not widely used. For example, the synthesis of glycine has been described from the rhodium-catalyzed reductive amination of glyoxylic acid, a chemical synthesis from glycinonitrile, ammonia, and carbon dioxide, and a chemical synthesis using potassium phthalimide and monochloroacetic acid to produce glycine and phthalic acid.

[0006] The increasing problem of pollution, combined with the continuing rise in greenhouse gas emissions and the growth of the world's population, necessitates a revision of the economic model based on fossil resources. Thus, one of the great challenges facing society is to move from an economy based on the use of petroleum-derived materials and energy to equivalents derived from renewable biomass.

[0007] In view of the above, there is a clear need to develop new technological solutions to enable glycine production that meets consumer demands for the use of biological products, environmentally responsible behavior, and limited environmental impact. It is therefore clear that there is a need to develop biological production of glycine, particularly on an industrial scale, with sufficient purity and at a reasonable price to meet the needs of the agri-food and pharmaceutical industries. DETAILED DESCRIPTION OF THE INVENTION

[0008] The inventors have unexpectedly and surprisingly developed metabolically engineered microorganisms that are capable of efficiently producing glycine or a salt or ester thereof by fermentation of a suitable medium containing a carbon source, particularly a simple carbon source. These metabolically engineered microorganisms, their uses, and methods for producing glycine or a salt or ester thereof by fermentation according to the present invention are described throughout this specification.

[0009] The objective of the present invention is to produce glycine using biological systems as cell factories, in particular by overexpressing the glycine production pathway and attenuating or suppressing the glycine degradation pathway.

[0010] Therefore, another object of the present invention is to biologically produce glycine from renewable carbon sources using microorganisms, which has advantages over environmentally unfriendly chemical processes and potentially reduces the toxic by-products and energy costs of chemical processes.

[0011] The present invention relates to a metabolically engineered microorganism for the biological production of glycine or a salt or ester thereof, in particular from threonine as a direct precursor, the genome of which comprises: (A) attenuation of the expression of genes encoding enzymes having glycine cleavage system activity as defined by EC 1.4.1.27, in particular enzymes having glycine decarboxylase activity as defined by EC 1.4.4.2 and aminomethyltransferase activity as defined by EC 2.1.2.10, and (B) overexpression of a gene encoding an enzyme having L-threonine 3-dehydrogenase activity as defined by EC 1.1.1.103 and glycine c-acetyltransferase activity as defined by EC 2.3.1.29, and / or (C) Overexpression of a gene encoding an enzyme having L-threonine aldolase activity as defined by EC 4.1.2.48, or a mutant of this enzyme, EC 4.1.2.42, or EC 4.1.2.49.

[0012] According to the present invention, loss of the glycine cleavage system activity encoded by the gcv operon results in excretion of glycine into the medium (Plamann, 1983). Therefore, this modification, combined with the following, can efficiently produce glycine from threonine as a substrate. Overexpression of threonine degradation pathway I (called TDGI), corresponding to genes encoding enzymes with L-threonine 3-dehydrogenase activity, which converts threonine to L-2-aminooxobutanoic acid, and genes encoding enzymes with glycine c-acetyltransferase activity (or 2-amino-3-ketobutyrate coenzyme A ligase activity), which converts L-2-aminooxobutanoic acid to glycine and acetyl-CoA; and / or · Overexpression of threonine degradation pathway II (TDGII), which corresponds to the gene encoding L-threonine aldolase, which cleaves threonine to glycine and acetaldehyde.

[0013] To increase the glycine yield, glyoxylate can be converted to glycine, which is obtained from acetyl-CoA formed as a by-product in threonine degradation pathways I and II, by overexpression of genes encoding enzymes with L- or D-amino acid dehydrogenase activity as defined by EC 1.4.99.1. or EC 1.4.1.9, respectively, or enzymes with glyoxylate alanine transaminase activity.

[0014] In the context of the present invention, the expressions "metabolically engineered microorganism," "recombinant microorganism," and "genetically modified microorganism" are used interchangeably to mean that the microorganism of the present invention is not found in nature and has been modified by the introduction of new genetic elements and / or the deletion or alteration of endogenous genetic elements of the microorganism. Selection pressure may be applied to such microorganisms by site-directed mutagenesis or genomic recombination in combination with growth in selective media.

[0015] In the context of the present invention, an enzymatic activity is also designated by reference to a gene that encodes an enzyme having such activity.

[0016] The use of gene designations is not limited to a particular organism, but covers corresponding genes and proteins in other organisms (e.g., microorganisms, functional analogs, functional variants, and functional fragments thereof (as long as they maintain enzymatic activity)).

[0017] In the context of the present invention, the term "precursor" refers to an initial or intermediate production substrate that is converted into a product by an enzyme to ultimately produce glycine.

[0018] In the context of the present invention, the term "endogenous gene" refers to a gene that was present in a microorganism in a wild-type strain before genetic modification. An endogenous gene can be overexpressed by introducing a heterologous sequence in addition to or replacing an endogenous regulatory element, by replacing its own promoter with the strongest promoter, or by introducing one or more additional copies of the gene into a chromosome or plasmid. Endogenous genes can also be modified to regulate their expression. For example, mutations can be introduced into the promoter sequence to modify expression, or heterologous sequences can be introduced in addition to or replacing the endogenous regulatory element. Regulation of an endogenous gene can result in upregulation and / or enhancement of the activity of the gene product, or downregulation and / or attenuation of the activity of the endogenous gene product. Another way to enhance the expression of an endogenous gene is to introduce one or more additional copies of the gene into a chromosome or plasmid. Conversely, attenuation of the expression of an endogenous gene can be achieved by deleting the gene from the chromosome.

[0019] In the context of the present invention, the terms "heterologous gene" or "exogenous gene" are used interchangeably and refer to a gene that has been introduced into a microorganism by means well known to those skilled in the art, but that does not naturally occur in a wild-type microorganism. Microorganisms can express endogenous genes when introduced into the microorganism along with all the factors that enable expression in the host microorganism. Transforming microorganisms with exogenous DNA is a routine procedure for those skilled in the art. Exogenous genes are either integrated into the host chromosome or expressed extrachromosomally via a plasmid or vector. Various plasmids with different origins of replication and intracellular copy numbers are all known in the art (e.g., Dykxhoorn et al., 1996 or Woodall, 2003). The sequence of an exogenous gene can be adjusted for expression in a host microorganism. In fact, those skilled in the art are aware of the concept of codon usage bias and how to adjust nucleic acid sequences according to a particular codon usage bias without changing the predicted protein.

[0020] In the context of the present invention, the term "overexpression" means an increase in the expression of a gene or protein, such as an enzyme, compared to an unmodified microorganism. The increase in enzyme expression is achieved by increasing the expression of the gene encoding said enzyme. The increase in gene expression can be carried out by any technique known to those skilled in the art. In this regard, mention may be made of the implementation of a strong promoter upstream of the nucleic acid intended to be overexpressed, or the introduction of several copies of said nucleic acid into the genome, between a promoter, in particular a strong promoter, and a terminator.

[0021] In the context of the present invention, the terms "attenuation," "underexpression," and "suppression" are used interchangeably and refer to a reduction or complete inhibition / suppression of the expression of a gene or protein, e.g., an enzyme, compared to the wild type, i.e., a protein, particularly a functionally effective protein (e.g., an enzyme with enzymatic activity), is not produced, or the protein is produced but is nonfunctional / inactive. The reduction, attenuation, or suppression of enzyme expression is achieved by reducing or inhibiting the expression of the gene encoding the enzyme. The reduction, attenuation, or suppression of gene expression can be carried out by any technique known to those skilled in the art. In this regard, particular mention can be made of the implementation of a weak promoter upstream of the coding sequence of the gene intended to be underexpressed. Also included are nucleic acids encoding mutants of the enzyme that are less active than the original enzyme or that are degraded more rapidly in cells than the original enzyme. Mutants of the original enzyme that are degraded more rapidly than the original enzyme include degron-tagged enzymes. These degron-tagged enzymes contain an additional degradation signal amino acid sequence, which acts as a kill signal to degrade the enzyme. The degradation can be either (i) ubiquitin-independent degradation or (ii) ubiquitin-dependent degradation. It can also include reducing the expression of a transcriptional activator of the gene of interest.

[0022] In the context of the present invention, the term "inducible promoter" is used to refer to a promoter whose activity is induced, i.e., whose activity increases when: In the presence of one or more specific metabolites. The higher the concentration of the metabolite in the medium, the higher the promoter activity. Or One or more specific metabolites are at low or absent concentrations. These metabolites are different from the metabolites whose increased presence induces promoter activity. The lower the metabolite concentration in the medium, the higher the promoter activity.

[0023] In the context of the present invention, the term "repressible promoter" is used to refer to a promoter whose activity can be repressed, i.e., whose activity is reduced when: In the presence of one or more specific metabolites. The higher the concentration of the metabolite in the medium, the lower the promoter activity. Or One or more specific metabolites are present at low concentrations or are absent. These metabolites are different from the metabolites whose increased presence represses promoter activity. The lower the metabolite concentration in the medium, the lower the promoter activity.

[0024] In the context of the present invention, the term "activity" of an enzyme is used interchangeably with the term "function" and refers to its ability to catalyze a desired reaction.

[0025] The term "reduced activity" or "attenuated activity" of an enzyme refers to a specific catalytic activity of an enzyme that is reduced or inhibited by a mutation in the amino acid sequence and / or a decrease in the concentration of the enzyme in a cell, achieved by a mutation in the nucleotide sequence, deletion of the corresponding associated gene, or degron tagging of the enzyme.

[0026] The term "increased activity" of an enzyme refers to increased catalytic activity of a particular enzyme and / or increased amount / availability of the enzyme in a cell, achieved, for example, by overexpression of the gene encoding the enzyme.

[0027] In the context of the present invention, the term "variant" or "functional variant" includes enzymes that may exhibit significant sequence changes compared to the sequences specifically described in this application, but that still retain the original enzymatic activity. This also means that the sequence of the enzyme may contain fewer amino acids than the original enzyme, but said truncated enzyme may still retain the original enzymatic activity.

[0028] In the context of the present invention, the articles "a" and "an" are used to refer to one or to more than one (e.g., at least one) unit of the grammatical object of the article. For example, "an element" refers to at least one element, i.e., one or more elements.

[0029] The term "about" or "approximately" when used in reference to measurable values such as amounts, durations and other similar values should be understood to include an uncertainty of ±20% or ±10%, preferably ±5%, more preferably ±1%, and especially preferably ±0.1% of the specified value.

[0030] The term "isolated" in the context of the present invention should be understood as synonymous with being separated or extracted from its environment or natural state. For example, an isolated nucleic acid or peptide is one that has been extracted from the natural environment in which it is normally found, such as a living plant or animal. Thus, a nucleic acid or peptide that occurs naturally in a living animal is not an isolated nucleic acid or peptide within the meaning of the present invention. On the other hand, if the same nucleic acid or peptide is partially or completely separated from other components present in its natural environment, it is itself "isolated" within the meaning of the present invention. An isolated nucleic acid or peptide may exist in a substantially purified form, or it may exist in a non-native environment, such as a host cell.

[0031] In the context of the present invention, a "nucleotide sequence encoding an amino acid" refers to any nucleotide sequence that encodes an amino acid sequence, including degenerate nucleotide sequences that make it possible to obtain said amino acid sequence. A nucleotide sequence that encodes a protein, RNA or cDNA may optionally contain introns.

[0032] In the context of the present invention, the terms "coding," "coding for," "code," or "codes for" refer to the inherent property of particular nucleotide sequences in polynucleotides, such as genes, cDNAs, and mRNAs, which serve as templates for the synthesis of other polymers and macromolecules in biological processes, have a defined nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and result in a biological property. Thus, a gene encodes a protein when transcription and translation of its corresponding mRNA results in the production of the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA and generally described in sequence listings and databases, and the non-coding strand, which is used as a template for a gene or cDNA, may be referred to as coding for a protein or other product in that gene or cDNA.

[0033] In the context of the present invention, the term "polynucleotide" is defined as a chain of nucleotides. Nucleic acids are polymers of nucleotides. Therefore, the terms nucleic acid and polynucleotide used in the context of the present invention are interchangeable. In the fields of molecular biology and genetic engineering, it is well known that nucleic acids are polynucleotides and can be hydrolyzed into monomers. The monomeric form of nucleotides can be hydrolyzed into nucleosides. The term polynucleotide used in the context of the present invention refers, without limitation, to any type of nucleic acid molecule, i.e., a nucleic acid molecule obtained by any means available in the art, including recombinant means, i.e., cloning a nucleic acid sequence from a recombinant library or the genome of a cell using conventional cloning techniques such as PCR, or synthesis.

[0034] Within the meaning of the present invention, the terms "peptide," "polypeptide," "protein," and "enzyme" are used interchangeably and refer to compounds consisting of amino acid residues covalently linked by peptide bonds. Proteins, by definition, contain at least two amino acids, with no limit on the maximum number of amino acids. Enzymes are proteins that catalyze biochemical reactions, particularly within cells. Polypeptides include peptides and / or proteins, which contain two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains, commonly referred to in the art as peptides, oligopeptides, or oligomers, and to longer chains, commonly referred to in the art as proteins, which come in many varieties. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, mutant polypeptides, modified polypeptides, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0035] In the context of the present invention, the terms "homologous" and "identical" refer to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If a position in each of the two compared sequences is occupied by the same base or monomeric amino acid subunit (e.g., if a position in each of the two DNA molecules is occupied by adenine), the molecules are homologous or identical at that position. The percentage identity between two sequences is a function of the number of corresponding positions shared by the two sequences, and corresponds to this number divided by the number of positions compared multiplied by 100. For example, if 6 out of 10 positions in two matched sequences are identical, the two sequences are 60% identical. Comparisons are usually performed by aligning the sequences to obtain maximum identity / homology.

[0036] In the context of the present invention, a "vector" is a molecular structure that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphipathic compounds, plasmids, and viruses. Thus, the term "vector" refers, for example, to an autonomously replicating plasmid or virus. The term is also intended to include non-plasmid or non-viral compounds that allow the transfer of nucleic acid into a cell, such as polylysine compounds, liposomes, etc.

[0037] In the context of the present invention, the term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to the nucleotide sequence to be expressed. Expression vectors include, inter alia, cis-acting expression elements and other elements for expression provided by a host cell or an in vitro expression system. Expression vectors in the sense of the present invention include all those known in the art, such as cosmids, plasmids (e.g., naked or liposomally contained plasmids), and viruses (e.g., lentiviruses, retroviruses, adenoviruses and adeno-associated viruses).

[0038] In the context of the present invention, the term "promoter" is defined as a DNA sequence that is recognized by the synthetic machinery of a cell or introduced synthetic machinery and is necessary to initiate the specific transcription of a polynucleotide sequence.

[0039] In the context of the present invention, the term "promoter / regulatory sequence" refers to a nucleic acid sequence operably linked to the promoter / regulatory sequence and necessary for the expression of a polynucleotide. In some cases, this sequence is the promoter sequence, and in other cases, this sequence also includes enhancer sequences and other regulatory elements useful for the expression of the polynucleotide sequence. The promoter / regulatory sequence may, for example, be a sequence that allows tissue-specific expression of the polynucleotide, i.e., that occurs preferentially in that tissue.

[0040] In the context of the present invention, a "constitutive" promoter is a polynucleotide sequence that, when operably linked to a polynucleotide, causes expression of the polynucleotide under most or all physiological conditions in a cell.

[0041] In the context of the present invention, the term "heterologous expression" refers to the expression of an exogenous gene in a host cell or organism when said exogenous gene is introduced into said cell or organism together with all the elements allowing its expression in said host. The techniques allowing the introduction (or transformation) of DNA into a host are well known to those skilled in the art and include, in particular, membrane permeabilization by application of an electric field (electroporation), thermal means (application of heat shock) or chemical means.

[0042] Preferably, the subject of the present invention is a recombinant microorganism as defined above that produces glycine or one of its salts or esters, having the following technical characteristics, alone or in combination:

[0043] Threonine may be L-threonine, D-threonine, or a mixture thereof.

[0044] The genome of the microorganism of the present invention further comprises attenuated expression of a gene encoding an enzyme having dihydrolipoyl dehydrogenase activity as defined by EC 1.8.1.4, preferably attenuated, but not completely inhibited / suppressed expression, such that dihydrolipoyl dehydrogenase activity can be quantified / monitored.

[0045] Genes encoding enzymes with glycine decarboxylase activity as defined by EC 1.4.4.2 are repressed.

[0046] - Genes encoding enzymes with aminomethyltransferase activity as defined by EC 2.1.2.10 are repressed.

[0047] The genome of the microorganism of the invention further comprises the overexpression of a gene encoding an enzyme with acetylating aldehyde dehydrogenase activity as defined by EC 1.2.1.10, capable of converting acetaldehyde to acetyl-CoA.

[0048] The genes of the microorganism of the present invention further include attenuation or suppression of a gene encoding an enzyme having D-amino acid oxidase activity as defined by EC 1.4.99.

[0049] The genome of the microorganism of the present invention further comprises the overexpression of a gene encoding an enzyme with D-serine / D-alanine / glycine transporter activity as defined by TCDB 2.A.3.1.7.

[0050] The genome of the microorganism of the present invention further comprises attenuation, or even repression, of expression of genes encoding enzymes having extracellular threonine export activity (i.e., RhtC) as defined by TCDB 2.A.76.1.2, in particular threonine / homoserine export activity (RhtA) as defined by TCDB 2.A.7.3.6, and homoserine / homoserine lactone / β-hydroxynorvaline export permease activity (RhtB) as defined by TCDB 2.A.76.1.1.

[0051] The genome of the microorganism of the invention further comprises the overexpression of a gene encoding an enzyme with threonine synthase activity as defined by EC 4.2.3.1.

[0052] The genome of the microorganism of the invention further comprises the overexpression of a gene encoding an enzyme with glycine dehydrogenase activity as defined by EC 1.4.1.10, such as the L-alanine dehydrogenase of M. tuberculosis (Usha et al., 2002).

[0053] The genome of the microorganism of the invention further comprises the overexpression of a gene encoding an enzyme with D-amino acid dehydrogenase activity as defined by EC 1.4.99.1.

[0054] The genome of the microorganism of the invention further comprises the overexpression of a gene encoding an enzyme with glyoxylate alanine transaminase activity as defined by EC 2.6.1.44.

[0055] The mutant with L-threonine aldolase activity as defined by EC:4.1.2.48 is the constitutive enzyme H126F mutant of Escherichia coli, i.e. ItaEH126F as described by Fesko (Fesko, 2016), or an equivalent mutant of another microorganism.

[0056] An enzyme with glycine decarboxylase activity as defined by EC 1.4.4.2 is encoded by the sequence shown in SEQ ID NO: 1 of E. coli or any sequence sharing at least 90% identity with said sequence, or by a sequence corresponding to SEQ ID NO: 2 or any sequence sharing at least 90% identity with said sequence, or a sequence corresponding to an equivalent enzyme with the same enzymatic activity in another microorganism.

[0057] The enzyme with aminomethyltransferase activity as defined by EC 2.1.2.10 is encoded by the sequence shown in SEQ ID NO: 3 of E. coli or any sequence sharing at least 90% identity with said sequence, or by a sequence corresponding to SEQ ID NO: 4 or any sequence sharing at least 90% identity with said sequence, or a sequence corresponding to an equivalent enzyme with the same enzymatic activity in another microorganism.

[0058] The enzyme having L-threonine 3-dehydrogenase activity as defined by EC 1.1.1.103 is encoded by the sequence shown in SEQ ID NO: 5 of E. coli or any sequence sharing at least 90% identity with said sequence, or by a sequence corresponding to SEQ ID NO: 6 or any sequence sharing at least 90% identity with said sequence, or a sequence corresponding to an equivalent enzyme with the same enzymatic activity in another microorganism.

[0059] The enzyme having L-threonine aldolase activity as defined by EC 4.1.2.48 is encoded by the sequence shown in SEQ ID NO: 7 of E. coli or any sequence sharing at least 90% identity with said sequence, or by a sequence corresponding to SEQ ID NO: 8 or any sequence sharing at least 90% identity with said sequence, or a sequence corresponding to an equivalent enzyme with the same enzymatic activity in another microorganism.

[0060] The enzyme having dihydrolipoyl dehydrogenase activity as defined by EC 1.8.1.4 is encoded by the sequence shown in SEQ ID NO: 9 of E. coli or any sequence sharing at least 90% identity with said sequence, or by a sequence corresponding to SEQ ID NO: 10 or any sequence sharing at least 90% identity with said sequence, or a sequence corresponding to an equivalent enzyme with the same enzymatic activity in another microorganism.

[0061] The enzyme having acetylating aldehyde dehydrogenase activity as defined by EC 1.2.1.10 is encoded by the sequence shown in SEQ ID NO: 11 of E. coli or any sequence sharing at least 90% identity with said sequence, or by a sequence corresponding to SEQ ID NO: 12 or any sequence sharing at least 90% identity with said sequence, or a sequence corresponding to an equivalent enzyme with the same enzymatic activity in another microorganism.

[0062] The enzyme having threonine synthase activity as defined by EC 4.2.3.1 is encoded by the sequence shown in SEQ ID NO: 13 of E. coli or any sequence sharing at least 90% identity with said sequence, or by a sequence corresponding to SEQ ID NO: 14 or any sequence sharing at least 90% identity with said sequence, or a sequence corresponding to an equivalent enzyme with the same enzymatic activity in another microorganism.

[0063] The enzyme having glycine dehydrogenase activity as defined by EC 1.4.1.10 is encoded by the sequence shown in SEQ ID NO: 15 of Streptomyces phaechromogenes or any sequence sharing at least 90% identity with said sequence, or by a sequence corresponding to SEQ ID NO: 16 or any sequence sharing at least 90% identity with said sequence, or a sequence corresponding to an equivalent enzyme with the same enzymatic activity in another microorganism.

[0064] An enzyme having D-amino acid dehydrogenase activity as defined by EC 1.4.99.1 is encoded by the sequence shown in SEQ ID NO: 17 of E. coli or any sequence sharing at least 90% identity with said sequence, or by a sequence corresponding to SEQ ID NO: 18 or any sequence sharing at least 90% identity with said sequence, or a sequence corresponding to an equivalent enzyme with the same enzymatic activity in another microorganism.

[0065] The enzyme having glyoxylate alanine transaminase activity as defined by EC 2.6.1.44 is encoded by the sequence shown in SEQ ID NO: 19 of E. coli or any sequence sharing at least 90% identity with said sequence, or by a sequence corresponding to SEQ ID NO: 20 or any sequence sharing at least 90% identity with said sequence, or a sequence corresponding to an equivalent enzyme with the same enzymatic activity in another microorganism.

[0066] The H126F variant of the enzyme having L-threonine aldolase activity as defined by EC 4.1.2.48 corresponds to the amino acid sequence shown in SEQ ID NO: 21 of E. coli or any sequence sharing at least 90% identity with said sequence, or to SEQ ID NO: 22 or any sequence sharing at least 90% identity with said sequence, or to an equivalent enzyme with the same enzymatic activity in another microorganism.

[0067] The enzyme having glycine C-acetyltransferase activity as defined by EC 2.3.1.29 is encoded by the sequence shown in SEQ ID NO: 23 of E. coli or any sequence sharing at least 90% identity with said sequence, or by a sequence corresponding to SEQ ID NO: 24 or any sequence sharing at least 90% identity with said sequence, or a sequence corresponding to an equivalent enzyme with the same enzymatic activity in another microorganism.

[0068] An enzyme having D-serine / D-alanine / glycine transport activity as defined by TCDB 2.A.3.1.7 is encoded by the sequence shown in SEQ ID NO: 25 of E. coli or any sequence sharing at least 90% identity with said sequence, or by a sequence corresponding to SEQ ID NO: 26 or any sequence sharing at least 90% identity with said sequence, or a sequence corresponding to an equivalent enzyme with the same enzymatic activity in another microorganism.

[0069] The enzyme with threonine / homoserine export activity as defined by TCDB 2.A.7.3.6 is encoded by the sequence shown in SEQ ID NO: 27 of E. coli or any sequence sharing at least 90% identity with said sequence, or by a sequence corresponding to SEQ ID NO: 28 or any sequence sharing at least 90% identity with said sequence, or a sequence corresponding to an equivalent enzyme with the same enzymatic activity in another microorganism.

[0070] The enzyme having homoserine / homoserine lactone / β-hydroxynorvaline efflux permease activity is encoded by the sequence shown in SEQ ID NO: 29 of E. coli or any sequence sharing at least 90% identity with said sequence, or by a sequence corresponding to SEQ ID NO: 30 or any sequence sharing at least 90% identity with said sequence, or a sequence corresponding to an equivalent enzyme having the same enzymatic activity in another microorganism.

[0071] An enzyme having threonine efflux permease activity as defined by TCDB 2.A.76.1.2 is encoded by the sequence shown in SEQ ID NO: 31 of E. coli or any sequence sharing at least 90% identity with said sequence, or by a sequence corresponding to SEQ ID NO: 32 or any sequence sharing at least 90% identity with said sequence, or a sequence corresponding to an equivalent enzyme with the same enzymatic activity in another microorganism.

[0072] The enzyme having L-amino acid dehydrogenase activity as defined by EC 1.4.1.9 is encoded by the sequence shown in SEQ ID NO: 33 of Bacillus subtilis or any sequence sharing at least 90% identity with said sequence, or by a sequence corresponding to SEQ ID NO: 34 or any sequence sharing at least 90% identity with said sequence, or a sequence corresponding to an equivalent enzyme with the same enzymatic activity in another microorganism.

[0073] The salt forms of glycine are the ammonium, potassium or sodium salts of glycine.

[0074] The ester derived from glycine is selected from the group consisting of glycine ethyl ester, glycine methyl ester, glycine ethyl ester hydrochloride, and glycine methyl ester hydrochloride.

[0075] A microorganism is any lower unicellular organism that can produce glycine or one of its salts or esters by introducing a gene, a nucleic acid or a chimeric vector of the invention according to the invention.

[0076] The microorganism is a bacterium, advantageously of the family Enterobacteriaceae or Corynebacteriaceae, preferably of the genus Escherichia, Pantoea, Corynebacterium or Brevibacterium, more particularly Escherichia coli, Pantoea ananatis or Corynebacterium glutamicum.

[0077] the microorganism is a fungus, advantageously from the Ascomycota family, preferably from the genus Saccharomyceta or the subphylum Taphrinicotina, more particularly from the subphylum Saccharomycotina, the subphylum Pezizomycotina or the class Archaeorhizomycetes, more advantageously from the class Saccharomycetes, the class Eurotiomycetes, the superclass Leotiomyceta, the class Pezizomycetes or the order Archaeorhizomycetales, preferably Saccharomyces cerevisiae, Talaromyces versatilis, versatilis, and Aspergillus niger.

[0078] In the context of the present invention, the expression "any sequence sharing at least 90% identity with a sequence (nucleic or peptide sequence)" corresponds to any sequence that shares at least 90%, or 91%, 92%, 93%, 94%, 95%, or even 96%, 97%, 98% or 99% identity with said sequence.

[0079] In the context of the present invention, the expressions "equivalent enzyme in another microorganism" or "enzyme with equivalent enzymatic activity in another microorganism" mean an enzyme from a first microorganism capable of catalyzing a reaction on a substrate or a series of reactions on a series of substrates, said enzyme from said first microorganism having the same enzymatic properties (reaction rate, substrate specificity, etc.) or related / similar properties as an enzyme from a second microorganism different from the first microorganism. In other words, this involves the specific recognition of a substrate, in particular the active or catalytic site, and its subsequent conversion into a product by an enzyme from a microorganism different from the microorganism from which said specific substrate is derived. In particular, in the context of the present invention, this expression does not refer to dual enzyme specificity or coenzymes.

[0080] In particular, the synthetic pathway for glycine production according to the present invention can be carried out by microorganisms that have increased threonine production, as described above, such as Corynebacterium, Brevibacterium, and Escherichia coli, particularly Escherichia coli.

[0081] Alternatively, as described above, the engineered pathway for glycine production according to the present invention can be implemented in a microorganism that exhibits increased or reduced threonine production, such as the bacterium Escherichia coli (Debabov, 2003), the yeast Saccharomyces cerevisiae (Farfan et al., 1999), or the filamentous fungi Aspergillus niger, Trichoderma reesei, or Talaromyces versatilis. In this case, the genome of the microorganism of the present invention is modified as described above, but the following method ensures threonine production in the microorganism, particularly increased threonine production.

[0082] (i) Overexpression of aspartate kinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase, and threonine synthase.

[0083] (ii) modifying aspartate kinase enzymes to make them insensitive to production inhibition that may occur due to lysine, methionine, and / or threonine (i.e., modifying the enzymes to make them insensitive to negative feedback induced by lysine, methionine, and / or threonine), and modifying homoserine kinase enzymes to make them insensitive to threonine inhibition (i.e., modifying the enzymes to make them insensitive to negative feedback induced by threonine); and (iii) Inhibition of metabolic pathways that divert the aspartate-homoserine biosynthetic pathway away from threonine synthesis, e.g., inhibition of the methionine pathway.

[0084] In particular, overexpression of aspartate kinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase, and threonine synthase can be achieved by expressing the enzymes from multicopy plasmids under the control of a constitutive promoter or an appropriate inducible promoter. Alternatively, overexpression of the enzymes can be achieved by expressing the relevant genes under a strong, non-repressible promoter. Aspartate kinase can be made resistant to inhibition by aspartate-derived amino acids by introducing appropriate mutations into its amino acid sequence (Omori et al., (1993); Huo et al., (1996); Chen et al., (2011)). Homoserine kinase can be made insensitive to threonine by introducing appropriate mutations into its amino acid sequence. The entry points into metabolic pathways that hijack the homoserine biosynthetic pathway are catalyzed by enzymes with O-succinylhomoserine or O-acetylhomoserine synthase activity (entry points into methionine biosynthesis) or diaminopimelate decarboxylase activity (entry points into lysine biosynthesis). Deleting the genes encoding proteins with these enzyme activities prevents the formation of amino acids derived from aspartate, lysine, and / or methionine, thus promoting the formation of homoserine. Consequently, deleting the metA, thrB, and lysA genes in E. coli (or equivalent enzymes in other microorganisms) increases the flux of homoserine toward threonine synthesis (i.e., attenuates or even inhibits the diversion of homoserine into competing pathways). For example, the enhancement of the threonine pathway in E. coli can be achieved by using the bifunctional aspartate kinase-homoserine dehydrogenase mutant ThrA. S345F (insensitive to threonine inhibition) with asd, thrB, and thrC, or by overexpression of the monofunctional aspartate kinase mutant LysC. E250K This is achieved by overexpression of the genes encoding (lysine-insensitive), asd, thrB, and thrC (all E. coli genes) ( Lee et al., 2007 ; Dong et al., 2012 ).

[0085] The microorganism of the present invention may have an attenuated or absent ability to excrete threonine, thereby increasing the availability of this amino acid within the cell. For example, if the microorganism is Escherichia coli, its genome may further be deficient in the threonine export transporters rhtA, rhtB, and / or rthC (Kruse et al., 2002).

[0086] According to the present invention, acetyl-CoA, a by-product of the reaction catalyzed by an enzyme with glycine C-acetyltransferase activity (or 2-amino-3-ketobutyrate coenzyme A ligase), is "recycled" / used to produce glycine via glyoxylate. Activation of the glyoxylate shunt and expression of an enzyme with glycine dehydrogenase activity as defined by EC 1.4.1.10 or glyoxylate alanine transaminase activity as defined by EC 2.6.1.44 are then required to ensure the conversion of glyoxylate to glycine via D-amino acid dehydrogenase activity as defined by EC 1.4.99, alanine glyoxylate transaminase as defined by EC 2.6.1.44, glutamate-glyoxylate aminotransferase as defined by EC 2.6.1.4, or serine-glyoxylate aminotransferase as defined by EC 2.6.1.45.

[0087] Acetyl-CoA can also be produced from acetaldehyde, a by-product of the cleavage of threonine to glycine, which cleavage is catalyzed by an enzyme with L-threonine aldolase activity as defined by EC 4.1.2.48 or one of its variants. In this case, acetyl-CoA is "recycled" / used via the glyoxylate shunt and expression of an enzyme with glycine dehydrogenase activity as defined by EC 1.4.1.10, or an enzyme with glyoxylate-alanine transaminase activity as defined by EC 2.6.1.44, or a serine-glyoxylate aminotransferase as defined by EC 2.6.1.4, ensuring the production of glycine.

[0088] The present invention also relates to the use of said microorganisms for the production and / or secretion and / or generation of glycine or a salt or ester thereof. The salt form of glycine is the ammonium, potassium or sodium salt of glycine, and The ester derived from glycine is selected from the group consisting of glycine ethyl ester, glycine methyl ester, glycine ethyl ester hydrochloride, and glycine methyl ester hydrochloride.

[0089] Preferably, the present invention relates to the use of a metabolically engineered microorganism as described above, in which glycine or one of its salts or esters is produced from a carbon source, advantageously a simple carbon source, preferably selected from the group consisting of pentoses or hexoses or disaccharides, more particularly glucose, sucrose, xylose, arabinose, ribose, mannose, galactose, fructose and mixtures thereof, advantageously glucose.

[0090] Furthermore, the present invention relates to the use of metabolically engineered microorganisms for the biological production of glycine or a salt or ester thereof as described above for the production and / or secretion and / or purification of acetyl-CoA.

[0091] The present invention also relates to a method for producing glycine or a salt or ester thereof, comprising the steps of: (a) culturing a metabolically engineered microorganism according to the present invention in a suitable culture medium containing a carbon source, thereby producing and accumulating glycine or a salt or ester thereof in the culture medium and / or in the cells of the microorganism; and (b) recovering glycine or one of its salts which accumulates in the culture medium and / or in the cells of the microorganism and which is ultimately present in salt form, in particular in the form of the ammonium, potassium or sodium salt.

[0092] In particular, the salt form of glycine is the ammonium, potassium or sodium salt of glycine, and the ester derived from glycine is selected from the group consisting of glycine ethyl ester, glycine methyl ester, glycine ethyl ester hydrochloride, and glycine methyl ester hydrochloride.

[0093] Preferably, a subject of the present invention is a process as defined above for producing glycine or one of its salts or esters, having, alone or in combination, the following technical features:

[0094] The method further comprises step (c) purifying the glycine or one of its salts or esters.

[0095] In step (a), acetyl-CoA, a co-metabolite of glycine, is produced and used as a substrate for the production of glycine. In particular, the conversion of glyoxylate to glycine is ensured by the expression of an enzyme with glycine dehydrogenase activity as defined by the glyoxylate shunt and EC 1.4.1.10 or an enzyme with glyoxylate-alanine transaminase activity as defined by EC 2.6.1.4 or EC 2.1.6.40.

[0096] The carbon source is selected from the group consisting of simple carbon sources, advantageously pentoses or hexoses or disaccharides, more particularly glucose, sucrose, xylose, arabinose, ribose, mannose, galactose, fructose and mixtures thereof, advantageously glucose.

[0097] The carbon source is glucose.

[0098] The method of the present invention is carried out in a bioreactor.

[0099] The process of the present invention can be carried out in batch or fed-batch mode.

[0100] The recovery of glycine or one of its salts or esters generally involves the steps of cell separation and product purification, concentration and drying, respectively.

[0101] · Ultrafiltration and centrifugation can be used to separate the cells from the fermentation medium.

[0102] Optimizing cell separation by adding additives such as inorganic acids or alkaline salts or by heating the culture broth, as described in US Pat. No. 5,017,480, WO 01 / 72689 or CN 108084041.

[0103] To separate glycine or one of its salts or esters, various ion exchange chromatography methods can be applied before or after biomass removal. This includes, in particular, the use of primary cation exchange resins, which allow the separation of products according to their isoelectric points. In this case, the resin is loaded with the solution, and after increasing the pH (for example, by adding ammonium hydroxide), the retained products are eluted separately in the eluate. It also includes the use of ion exchange chromatography methods using fixed-bed or simulated moving-bed resins.

[0104] Achieving adequate product purity may require a combination of various chromatographic steps.

[0105] The refining process may include a drying step using suitable drying means such as a spray granulator, spray dryer, drum dryer, tunnel dryer, etc.

[0106] A concentrated solution of glycine or one of its salts or esters, or a mixture thereof, can be obtained by heating the fermentation liquid to 130°C under reduced pressure with steam using a conventional concentrator or thin-film evaporator.

[0107] In the context of the present invention, the production level of glycine or one of its salts or esters is expected to be at an industrial level, advantageously such that the concentration of the microorganism of the present invention in the culture medium is 10 mg or more in 48 hours in fed-batch mode from 20 g / L of a carbon source, advantageously 20 g / L of glucose, preferably 50 mg, 100 mg, 150 mg, 200 mg or 1 g or more in 48 hours in fed-batch mode from 20 g / L of a carbon source, advantageously 20 g / L of glucose, and even 250 mg, 300 mg, 350 mg, 400 mg, 500 mg, 1 g, 1.25 g, 2 g or more in 48 hours in fed-batch mode from 20 g / L of a carbon source, advantageously 20 g / L of glucose, and even 5 g, 10 g, 15 g or more in 48 hours in fed-batch mode from 20 g / L of a carbon source, advantageously 20 g / L of glucose. g, 20 g, 25 g, 1 g, 50 g, 75 g, 100 g, 150 g, 200 g or more. At these concentrations, under the conditions of the present invention, glycine or one of its salts or esters remains soluble and does not exhibit any toxicity to the microorganism of the present invention.

[0108] As can be seen from the above, the microorganisms of the present invention or the microorganisms implemented in the methods or uses of the present invention have been genetically modified to ensure efficient production and / or secretion of glycine or salts or esters thereof at industrial levels.

[0109] Furthermore, the production of glycine or a salt thereof by fermentation offers the following technical and economic advantages:

[0110] The process uses renewable and inexpensive carbon sources and is carried out in aqueous solution at room temperature and atmospheric pressure, which consumes less energy than high-pressure processes.

[0111] · No toxic products, organic solvents or petroleum-based resources are used.

[0112] Production of high purity glycine for the food, nutraceutical, chemical and cosmetic industries.

[0113] The present invention is illustrated by the following examples, but is not limited thereto. [Example]

[0114] Example 1: Metabolic engineering of E. coli to produce glycine according to the present invention 1. Materials and Methods a. Plasmid construction All plasmids used in this study were constructed using the commercially available NEBuilder HIFI DNA Assembly Kit (New England Biolabs) by isothermal assembly reactions (Gibson et al., 2009) according to the manufacturer's instructions. For construction of pMW12, the E. coli kbl-tdh operon was amplified using primer pair MW15 and MW16 and assembled into the pACT3 plasmid (Dykxhoorn et al., 1996) at the SacI and HindIII sites. For construction of pMW26 / pMW27, the wild-type or H126F variant of the E. coli L-threonine aldolase gene ltaE was synthesized by IDT (Iowa, USA) and assembled into pACT3 between the SacI and HindIII sites. To construct the plasmids pMW30 / pMW31 containing the synthetic operon kbl-tdh-ltaE or kbl-tdh-ltaE-H126F, IDT (Iowa, US) synthesized kbl-tdh and ltaE or ltaE-H126F as an operon with RBS BBa_BB035 (Englund et al., 2016) between kbl-tdh and ltaE or ltaE-H126F, and assembled them into pACT3 between the SacI and HindIII sites. E. coli TOP10 (Invitrogen) was used as the host strain for plasmid construction. Plasmid pYN7 has been described elsewhere (see patent document EP 0152830 A1 1985 by Nakagawa et al.). It contains the pBR322 ori, the ampicillin resistance gene Ap r Equipped with natural P thrLABC It contains the thrA*, thrB, and thrC genes under the control of the promoter. Mutation of thrA* results in the mutant Thr AS345F occurs (Lee et al., 2007).

[0115] b. Strain construction Unless otherwise noted, the E. coli strain used in this study was BW25113. E. coli mutant strains were constructed by P1 phage transduction. P1 lysate preparation and P1 phage transduction were performed as described elsewhere (Thomason et al., 2007). A strain from the Keio collection (Baba et al., 2006) carrying a single gene deletion and a kanamycin resistance cassette flanked by ERT sites was used as the donor strain. Successful transduction events were confirmed by colony PCR. Removal of the antibiotic resistance marker, catalyzed by FLP-recombinase flippase, was performed by transformation with the pCP20 plasmid (Cherepanov & Wackernagel, 1995), followed by colony PCR confirmation of removal.

[0116] E. coli strain 472T23 pYN7 (K-12 derived) was purchased from ATCC under number 98081. It was constructed by (Kruse et al., 2002; Debabov, 2003D6) and contains mutations in thrC* and ilvA* that lead to the inactivation of homoserine kinase and threonine deaminase, and pYN7. Deletion of rthABC, encoding the threonine exporter (Livshits, 2003 #11032), and deletion of gcvP, encoding the glycine decarboxylase of the glycine cleavage pathway, were performed by PI transduction as described above.

[0117] C. Glycine production in E. coli For glycine production, plasmids pMW12, pMW26, pMW27, pMW30, and pMW31 were transformed into E. coli BW25113 or E. coli BW25113 ΔgcvP. Empty pACT3 was used as a negative control. Precultures of each transformed strain were grown overnight at 37°C and 200 rpm in 50-ml centrifuge tubes using 5 ml of M9 minimal medium supplemented with 2% (w / v) glucose and chloramphenicol (40 μg / ml). The next day, the precultures were inoculated into 25 ml of M9 medium supplemented with 2% (w / v) glucose and chloramphenicol (40 μg / ml) in baffled Erlenmeyer flasks, and the initial OD was reached. 600 The initial OD600 was adjusted to 0.2. This was diluted to an initial OD600 of 0.05-0.2 and grown at 37°C and 200 rpm. When the OD600 reached 0.6-1.0, IPTG was added to a final concentration of 1 mM to induce the cell culture. 1 ml samples were taken at several time points. When necessary, 25 mM threonine was added to the M9 glucose medium simultaneously with induction.

[0118] d. Glycine detection by LC-MS The concentrations of glycine and threonine in the culture medium were measured using a Dionex ThermoFisher UltiMate 3000 system (ThermoFisher Scientific, Waltham, USA), which includes a binary pump, online degassing unit, autosampler, column oven, and RS diode array detector, coupled with a ThermoFisher MSQ Plus mass spectrometer (ThermoFisher Scientific, Waltham, USA). Chromatographic separation was performed on a Poroshell 120 Hilic-Z column (2.1 mm × 150 mm, 2.7 μm P) maintained at 25°C at a flow rate of 0.5 ml / min. The mobile phase consisted of solution A (HO: 200 mM ammonium formate pH 3, 90%:10% (v / v)) and solution B (acetonitrile: 200 mM ammonium formate pH 3, 90%:10% (v / v)). To separate glycine from threonine, a gradient elution program was optimized: 0–7 min, 95%–80% solution B, 7–7.5 min, 80%–60% solution B, 7.5–10 min, 60% solution B, 10–10.5 min, 60%–95% solution B, and 10.5–14 min: solution B. The total run time was 14 min. L-alanine-2,3,3,3-d4 was added to the sample as an internal standard to a final concentration of 3.8 mM, and then diluted 1:20 with solution B. The sample injection volume was 10 μl. Data acquisition and analysis were performed using a Dionex Chromeleon 7 (ThermoFisher Scientific, Waltham, USA).

[0119] 2.Results The results of biotransformation of glucose to glycine by various strains grown for 48 h in M9 medium containing 20 g / l glucose are shown in Table 1.

[0120] [Table 1]

[0121] Data shown are the mean of two biological replicates. *Values are the mean + SD of two independent biological experiments. $ bd is below detection and glycine <0.2 mM. ￡ nt stands for not tested.

[0122] In the metabolically engineered microorganisms Gly2 to Gly5, expression of both threonine degradation pathways I and II did not promote glycine production. However, deletion of the gcvP gene, which inactivates the glycine cleavage system (GCS), which catalyzes the degradation of glycine to methylfolate, CO2, and NH3, resulted in the glycine-producing strain Gly6, which produced small amounts of glycine (28 mg of glycine from 20 g / L glucose in 48 h).

[0123] For the metabolically engineered microorganism, number Gly7, in which GCS was deleted and the tdh-kbl pathway was overexpressed, glycine production was found to be significantly increased, reaching levels in the range of 300-500 mg / l.

[0124] Similarly, in strain Gly9, which also lacks GCS but overexpresses the ltaE-dependent threonine degradation pathway, 48 h of growth in M9 containing 20 g / l glucose increased the glycine titer to 286 mg / l, and this titer was further increased by expression of ltaE* encoding the LtaEH126F mutant. However, overexpression of both tdh-kbl and ltaEH126F did not appear to enhance glycine production compared with overexpression of either tdh-kbl or ltaEH126F alone.

[0125] To assess the critical role of threonine synthesis in glycine production, two complementary experiments were performed. First, 25 mM exogenous threonine was added to cultures of strains GLY1 to GLY10. The results clearly demonstrate an increase in glycine production, as exemplified by strain GLY8. Glycine production increased fourfold in the presence of threonine compared to its absence, supporting the fact that stimulation of the threonine pathway is critical for glycine production.

[0126] Meanwhile, the threonine-producing strain (472T23 pYN7) was used as the recipient for glycine production. In this recipient strain, the threonine operon is expressed from the pYN7 plasmid. pYN7 is a pBR322-based plasmid carrying thrA*, thrB, and thrC under the control of the native promoter. Furthermore, the thrA* gene contains a mutation encoding the ThrAS345F variant, which is insensitive to feedback inhibition by threonine (Lee et al., 2007). In this strain, the threonine export genes rhtA, rhtB, and rhtC were deleted (Gly11) to prevent threonine export, and the gcvP gene was also deleted (Gly12) to inactivate the glycine cleavage system. While Gly11 did not produce glycine, Gly13 produced 278 mg / L of glycine from 20 g / L of glucose within 48 hours, supporting the importance of the GCS deletion for glycine production.

[0127] Overexpression of tdh-kbl in Gly12 yielded Gly13, which further increased glycine production to 454 mg / L. Similarly, overexpression of the ltaE* encoding LtaEH126F mutant in Gly13 yielded Gly14, which produced over 1000 mg / L of glycine from 20 g / L glucose in 48 h. In contrast, glycine production in Gly16, which overexpressed both pathways (tdh-kbl and ltaE), was not as good as that in Gly14 and Gly15, which overexpressed one of the two pathways individually.

[0128] (References) Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, KA, Tomita, M., Wanner, BL, & Mori, H. (2006 ). Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: The Keio collection. In Molecular Systems Biology. (Vol. 2). Boyd, WJ, & Robson, W. (1935). The synthesis of amino-acids: Piperidine and diethylamine as catalysts in the condensation of aromatic aldehydes with hydantoins. Biochem J. 29, 542-545. Chen Z., Rappert S., Sun J., & Zeng AP. (2011). Integrating molecular dynamics and co-evolutionary analysis for reliable target prediction and deregulation of the allosteric inhibition of aspartokinase for amino acid production. J Biotechnol. 154(4):248-54. Cherepanov, PP, & Wackernagel, W. (1995). Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. In Gene. 158(1), 9-14. Debabov, V.G. (2003). The threonine story. Adv Biochem Eng Biotechnol. 79, 113-136. Ding Y., Svingen GF, Pedersen ER, Gregory JF, Ueland PM, Tell GS, & Nygard OK. (2015). Plasma Glycine and Risk of Acute Myocardial Infarction in Patients With Suspected Stable Angina Pectoris. J Am Heart Assoc. 5(1). Dong, X., Quinn, P.J., & Wang, X. (2012). Microbial metabolic engineering for L-threonine production. Subcell Biochem. 64, 283-302. Dykxhoorn, DM, St. Pierre, R., & Linn, T. (1996). A set of compatible tac promoter expression vectors. In Gene. 177(1-2), 133-136. Englund, E., Liang, F., & Lindberg, P. (2016). Evaluation of promoters and ribosome binding sites for biotechnological applications in the unicellular cyanobacterium Synechocystis sp. CCP 6803. Scientific Reports. 6(1), 36640. Farfan, MJ, Aparicio, L., & Calderon, IL. (1999). Threonine overproduction in yeast strains carrying the HOM3-R2 mutant allele under the control of different inducible promoters. Appl Environ Microbiol. 65, 110-116. Fesko, K. (2016). Threonine aldolases: perspectives in engineering and screening the enzymes with enhanced substrate and stereo specificities. Appl Microbiol Biotechnol. 100, 2579-2590. Gibson, DG, Young, L., Chuang, RY, Venter, JC, Hutchison, CA, & Smith, HO (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods. 6(5), 343-345. Huo X, & Viola RE. (1996). Functional group characterization of homoserine kinase from Escherichia coli. Arch Biochem Biophys. 330(2):373-9. Kruse, D., Kramer, R., Eggeling, L., Rieping, M., Pfefferle, W., Tchieu, JH, Chung, YJ, Jr Saier, MH, & Burkovski, A. (2002). Influence of threonine exporters on threonine production in Escherichia coli. Appl Microbiol Biotechnol. 59, 205-210. Lee, KH, Park, JH, Kim, TY, Kim, HU, & Lee, SY. (2007). Systems metabolic engineering of Escherichia coli for L-threonine production. Mol. Syst. Biol. 3, 149. Omori K., Imai Y., Suzuki S., & Komatsubara S. (1993). Nucleotide sequence of the Serratia marcescens threonine operon and analysis of the threonine operon mutations which alter feedback inhibition of both aspartokinase I and homoserine dehydrogenase I. J Bacteriol. 175(3):785-94. Orten, JM, & Hill, RM. (1931). A Simple Method for the Preparation of Glycine. Journal of the American Chemical Society. 53, 2797-2799. Plamann, MD, Rapp, WD, & Stauffer GV. (1983). Escherichia coli K12 mutants defective in the glycine cleavage enzyme system. Mol Gen Genet. 192:15-20. Strecker, A. (1850). The synthesis of lactic acid and a new glycine homologue. Ann. Chem. Pharm. 75, 27. Thomason, LC, Costantino, N., & Court, DL. (2007). E. coli Genome Manipulation by P1 Transduction. In Current Protocols in Molecular Biology. 1.17.1-1.17.8. Tonouchi, N., & Hisao I. (2016) Present global situation of amino acids in industry. Amino Acid Fermentation. 3-14. Usha, V., Jayaraman, R., Toro, JC, Hoffner, SE, and Das, KS. (2002). Glycine and alanine dehydrogenase activities are catalyzed by the same protein in Mycobacterium smegmatis: upregulation of both activities under microaerophilic adaptation. Can J Microbiol. 48, 7-13. Wendisch VF. (2020). Metabolic engineering advances and prospects for amino acid production. Metab Eng. 58:17-34. Woodall, CA. (2003). Plasmid Vectors. Methods in Molecular Biology 235. Zeng, YZ, Li, L., & Demopoulos, GP. (2016) Process for glycine production by antisolvent crystallization using its phase equilibria in the ethylene glycol-NH4Cl-water system. Industrial & Engineering Chemistry Research. 55(8): 2426-2437.

Claims

1. 1. A metabolically engineered microorganism for the biological production of glycine or a salt or ester thereof, wherein the genome of the microorganism comprises: a. Attenuation of the expression of genes encoding enzymes with glycine cleavage system activity as defined by EC 1.4.1.27, in particular enzymes with glycine decarboxylase activity as defined by EC 1.4.4.2 and aminomethyltransferase activity as defined by EC 2.1.2.10, and b. Overexpression of a gene encoding an enzyme having L-threonine 3-dehydrogenase activity as defined by EC 1.1.1.103 and glycine c-acetyltransferase activity as defined by EC 2.3.1.29, and / or c. Overexpression of a gene encoding an enzyme with L-threonine aldolase activity as defined by EC 4.1.2.48 or a mutant of this enzyme, EC 4.1.2.42 or EC 4.1.2.49; metabolically engineered microorganisms, including

2. 10. The metabolically engineered microorganism of claim 1, 10. A metabolically engineered microorganism, wherein the genome further comprises attenuated expression of a gene encoding an enzyme having dihydrolipoyl dehydrogenase activity as defined by EC 1.8.1.

4.

3. The metabolically engineered microorganism of claim 1 or 2, A metabolically engineered microorganism, wherein the genome further comprises overexpression of a gene encoding an enzyme having acetylating aldehyde dehydrogenase activity as defined by EC 1.2.1.

10.

4. The metabolically engineered microorganism of any one of claims 1 to 3, A metabolically engineered microorganism, characterized in that the genome further comprises overexpression of a gene encoding an enzyme having threonine synthase activity as defined by EC 4.2.3.

1.

5. The metabolically engineered microorganism of any one of claims 1 to 4, The genome contains the following enzymes: an enzyme having glycine dehydrogenase activity as defined by EC 1.4.1.10, and / or an enzyme having D-amino acid oxidase activity as defined by EC 1.4.99., and / or an enzyme having glyoxylate alanine transaminase activity as defined by EC 2.6.1.44, and / or an enzyme having glycine C-acetyltransferase activity as defined by EC 2.3.1.29, and / or enzymes with L-amino acid dehydrogenase activity as defined by EC 1.4.1.9, A metabolically engineered microorganism further comprising overexpression of a gene encoding

6. The metabolically engineered microorganism of any one of claims 1 to 5, The genome contains the following enzymes: - have D-serine / D-alanine / glycine transporter activity as defined by TCDB 2.A.3.1.7, and / or - an enzyme having threonine efflux permease activity as defined by TCDB 2.A.76.1.1, A metabolically engineered microorganism, further comprising attenuated expression of a gene encoding

7. The metabolically engineered microorganism of any one of claims 1 to 6, A metabolically engineered microorganism, characterized in that the mutant of the enzyme having L-threonine aldolase activity as defined by EC 4.1.2.48 is the H126F mutant of the constitutive enzyme of Escherichia coli or an equivalent mutant of another microorganism.

8. The metabolically engineered microorganism of any one of claims 1 to 7, A metabolically engineered microorganism characterized by the attenuation, or even suppression, of expression of genes encoding enzymes having extracellular threonine export activity (i.e., RhtC) as defined by TCDB 2.A.76.1.2, in particular threonine / homoserine export activity (RhtA) as defined by TCDB 2.A.7.3.6, and homoserine / homoserine lactone / β-hydroxynorvaline export permease activity (RhtB) as defined by TCDB 2.A.76.1.

1.

9. 9. The metabolically engineered microorganism of claim 1, an enzyme having glycine decarboxylase activity as defined by EC 1.4.4.2 is encoded by the sequence shown in SEQ ID NO: 1 of E. coli or any sequence sharing at least 90% identity with said sequence, or by a sequence corresponding to SEQ ID NO: 2 or any sequence sharing at least 90% identity with said sequence, or a sequence corresponding to an equivalent enzyme with the same enzymatic activity in another microorganism; an enzyme with aminomethyltransferase activity as defined by EC 2.1.2.10 is encoded by the sequence shown in SEQ ID NO: 3 of E. coli or any sequence sharing at least 90% identity with said sequence, or a sequence corresponding to SEQ ID NO: 4 or any sequence sharing at least 90% identity with said sequence, or a sequence corresponding to an equivalent enzyme with the same enzymatic activity in another microorganism; an enzyme having L-threonine 3-dehydrogenase activity as defined by EC 1.1.1.103 is encoded by the sequence shown in SEQ ID NO: 5 of E. coli or any sequence sharing at least 90% identity with said sequence, or by a sequence corresponding to SEQ ID NO: 6 or any sequence sharing at least 90% identity with said sequence, or by a sequence corresponding to an equivalent enzyme with the same enzymatic activity in another microorganism; an enzyme having L-threonine aldolase activity as defined by EC 4.1.2.48 is encoded by the sequence shown in SEQ ID NO: 7 of E. coli or any sequence sharing at least 90% identity with said sequence, or a sequence corresponding to SEQ ID NO: 8 or any sequence sharing at least 90% identity with said sequence, or a sequence corresponding to an equivalent enzyme with the same enzymatic activity in another microorganism; an enzyme having dihydrolipoyl dehydrogenase activity as defined by EC 1.8.1.4 is encoded by the sequence shown in SEQ ID NO: 9 of E. coli or any sequence sharing at least 90% identity with said sequence, or a sequence corresponding to SEQ ID NO: 10 or any sequence sharing at least 90% identity with said sequence, or a sequence corresponding to an equivalent enzyme with the same enzymatic activity in another microorganism; an enzyme having acetylating aldehyde dehydrogenase activity as defined by EC 1.2.1.10 is encoded by the sequence shown in SEQ ID NO: 11 of E. coli or any sequence sharing at least 90% identity with said sequence, or a sequence corresponding to SEQ ID NO: 12 or any sequence sharing at least 90% identity with said sequence, or a sequence corresponding to an equivalent enzyme with the same enzymatic activity in another microorganism; the enzyme having threonine synthase activity as defined by EC 4.2.3.1 is encoded by the sequence shown in SEQ ID NO: 13 of E. coli or any sequence sharing at least 90% identity with said sequence, or by a sequence corresponding to SEQ ID NO: 14 or any sequence sharing at least 90% identity with said sequence, or a sequence corresponding to an equivalent enzyme with the same enzymatic activity in another microorganism; the enzyme having glycine dehydrogenase activity as defined by EC 1.4.1.10 is encoded by the sequence shown in SEQ ID NO: 15 of Streptomyces phaechromogenes or any sequence sharing at least 90% identity with said sequence, or by a sequence corresponding to SEQ ID NO: 16 or any sequence sharing at least 90% identity with said sequence, or a sequence corresponding to an equivalent enzyme with the same enzymatic activity in another microorganism, an enzyme having D-amino acid dehydrogenase activity as defined by EC 1.4.99.1 is encoded by the sequence shown in SEQ ID NO: 17 of E. coli or any sequence sharing at least 90% identity with said sequence, or a sequence corresponding to SEQ ID NO: 18 or any sequence sharing at least 90% identity with said sequence, or a sequence corresponding to an equivalent enzyme with the same enzymatic activity in another microorganism; an enzyme having glyoxylate alanine transaminase activity as defined by EC 2.6.1.44 is encoded by the sequence shown in SEQ ID NO: 19 of E. coli or any sequence sharing at least 90% identity with said sequence, or a sequence corresponding to SEQ ID NO: 20 or any sequence sharing at least 90% identity with said sequence, or a sequence corresponding to an equivalent enzyme with the same enzymatic activity in another microorganism, the H126F variant of an enzyme with L-threonine aldolase activity as defined by EC 4.1.2.48 corresponds to the amino acid sequence shown in SEQ ID NO: 21 of E. coli or any sequence sharing at least 90% identity with said sequence, or to SEQ ID NO: 22 or any sequence sharing at least 90% identity with said sequence, or to an equivalent enzyme with the same enzymatic activity in another microorganism, the enzyme with threonine / homoserine export activity as defined by TCDB 2.A.7.3.6 is encoded by the sequence shown in SEQ ID NO: 27 of E. coli or any sequence sharing at least 90% identity with said sequence, or by a sequence corresponding to SEQ ID NO: 28 or any sequence sharing at least 90% identity with said sequence, or by a sequence corresponding to an equivalent enzyme with the same enzymatic activity in another microorganism; The enzyme having homoserine / homoserine lactone / β-hydroxynorvaline efflux permease activity is encoded by the sequence shown in SEQ ID NO: 29 of E. coli or any sequence sharing at least 90% identity with said sequence, or by a sequence corresponding to SEQ ID NO: 30 or any sequence sharing at least 90% identity with said sequence, or by a sequence corresponding to an equivalent enzyme with the same enzymatic activity in another microorganism. A metabolically engineered microorganism characterized by:

10. 10. The metabolically engineered microorganism of claim 1, In the genome, amplifying the expression of at least one enzyme activity selected from the group consisting of phosphoenolpyruvate carboxylase, isocitrate lyase, pyruvate carboxylase and hexose cotransporter permease; and / or reducing at least one enzyme activity selected from the group consisting of lactate dehydrogenase, alcohol dehydrogenase, acetate kinase, phosphate acetyltransferase, pyruvate oxidase, isocitrate lyase, fumarase, 2-oxoglutarate dehydrogenase, pyruvate kinase, malic enzyme, phosphoglucose isomerase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, pyruvate-formate lyase, succinic semialdehyde dehydrogenase, sugar-transporting phosphotransferase, ketohydroxyglutarate aldolase, homoserine-O-succinyltransferase, homoserine kinase, homoserine efflux transporter, diaminopimelate decarboxylase, and / or methylglyoxal synthase; A metabolically engineered microorganism characterized by:

11. The metabolically engineered microorganism of any one of claims 1 to 10, wherein the microorganism comprises: a bacterium, preferably of the family Enterobacteriaceae or Corynebacteriaceae, preferably of the genus Escherichia coli, Pantoea, Corynebacterium or Brevibacterium, more particularly Escherichia coli, Pantoea ananatis or Corynebacterium glutamicum, or a fungus, preferably of the phylum Ascomycota, preferably of the genus Saccharomycetes or the subphylum Taphrinae, more particularly of the subphylum Saccharomycetes, the subphylum Chauhanophyta or the class Archaeoryzomycetes, more preferably of the class Saccharomycetes, the class Eurotium, the superclass Zucchiniomycetes, the class Pezizomycetes or the order Archaeoryzomycetales, preferably of the species Saccharomyces cerevisiae, Talaromyces versatilis or Aspergillus niger, A metabolically engineered microorganism characterized by:

12. 12. The metabolically engineered microorganism of claim 1, coli, the genome of which is at least one gene selected from the group consisting of ppc, pck, aceA, galP, asd, thrA, metL, lysC, all from Escherichia coli, pycA from Lactococcus lactis, and pycE from Corynebacterium glutamicum is overexpressed, and / or At least one gene selected from the group consisting of dhA, adhE, ackA, pta, poxB, focA, pflB, sad, gabABC, sfcA, maeB, ppc, pykA, pykF, mgsA, sucAB, ptsI, ptsG, pgi, fumABC, aldA, lldD, iclR, metA, lysA, eda, rthA, rthB, and rthC has been deleted. A metabolically engineered microorganism characterized by:

13. 13. A method of using the metabolically engineered microorganism of any one of claims 1 to 12 for the production of glycine or a salt or ester thereof.

14. 1. A method for producing glycine or a salt or ester thereof, comprising the steps of: (a) culturing the metabolically engineered microorganism according to any one of claims 1 to 12 in a culture medium containing a carbon source, and producing and accumulating glycine or one of its salts or esters in the culture medium and / or cells; (b) recovering glycine or one of its salts or esters accumulated in the culture medium and / or the cells of the microorganism; A method comprising:

15. 15. The method of claim 14, further comprising the step (c) of purifying the glycine or salt or ester thereof.

16. 16. The method of claim 14 or 15, 10. The method of claim 9, wherein the carbon source is a pentose or hexose or a disaccharide, preferably selected from the group consisting of glucose, sucrose, xylose, arabinose, ribose, mannose, galactose, fructose, and mixtures thereof.

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