Production of glycine by fermentation

Abstract

The present invention relates to a metabolically engineered microorganism for the biological production of glycine or a salt or ester thereof, the genome of which comprises an attenuated expression of a gene encoding an enzyme having a glycine cleavage system activity as defined by E.C.1.4. 1.27, and the overexpression of a threonine dehydrogenase-dependent pathway as defined by EC E.C.1. 1.1. 1.103 and E.C.2. 3.1. 1.29 and / or a threonine aldolase-dependent pathway as defined by E.C.4.1. 2.48 or EC 4.1. 2.42 or any catalytically active variant thereof, as well as the use of said metabolically engineered microorganism for the production of glycine or any one salt or ester thereof. The invention also relates to a fermentation method for producing glycine or any salt or ester thereof by using the metabolically engineered microorganism.

Description

Technical Field

[0001] The present invention relates to the biotechnology industry, and more particularly to metabolically engineered microorganisms for producing glycine or any salt or ester thereof, and uses of the metabolically engineered microorganisms. The present invention also relates to a fermentation method for producing glycine or any salt or ester thereof using the metabolically engineered microorganisms. Background Art

[0002] Glycine is one of the 22 proteinogenic amino acids. It is the simplest α-amino acid, with the chemical formula C2H5NO2. This amino acid has numerous metabolic roles and serves as a precursor for the synthesis of numerous compounds. For example, glycine comprises one-third of the amino acid content in collagen molecules and can also bind to bile acids to form bile salts. Furthermore, glycine is one of the limiting factors in glutathione biosynthesis and is known to function as a major antioxidant, participating in hemoglobin synthesis and creatine metabolism. Glycine also functions as a neurotransmitter, particularly in glutaminergic neurotransmission, which is important for cognition and sleep quality.

[0003] Research has also shown that this amino acid can mitigate the harmful effects of methionine derivatives on cardiovascular disease and may be associated with a longer lifespan. Epidemiological studies have shown that plasma glycine levels are negatively correlated with the incidence of acute myocardial infarction, as well as 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 for its sweetening properties, and in animal nutrition. Glycine is also used in the pharmaceutical and plant quarantine industries, such as pesticides and herbicides. For example, China is the world's leading producer of glycine, with production reaching 600,000 tons in 2014, over 80% of which was used for glyphosate production (Zeng et al., 2016). According to a review by Wendisch, the annual production of glycine in the feed and food industry was estimated to be 22,000 tons in 2018 (Wendisch, 2019).

[0004] However, regardless of the industrial sector considered (i.e., chemicals, food, animal feed, cosmetics, pharmaceuticals), glycine is the last amino acid produced exclusively by chemical synthesis from fossil-derived precursors (Tonouchi et al., 2016). Different chemical methods have been developed for the industrial-scale synthesis of this achiral amino acid. The most commonly used industrial method involves the amination of monochloroacetic acid in the presence of ammonia and a chemical catalyst to produce glycine and ammonium chloride (Orten and Hill, 1931). Another well-known industrial method is the synthesis of glycine via the Strecker process (Strecker, 1850), which allows the synthesis of this amino acid via the reaction of an aldehyde with ammonium chloride in the presence of potassium cyanide. A third method involves the hydrolysis of hydantoins in the presence of organic solvents (Boyd and Robson, 1935). However, a disadvantage of this hydrolysis method is that hydantoins are synthesized from hydrogen cyanide and formaldehyde, two particularly toxic products classified as carcinogens, mutagens, and toxic to reproduction.

[0005] Given the primary interest in glycine, particularly in the food and pharmaceutical markets, other synthetic methods have been developed but are less commonly used. For example, the synthesis of glycine has been described as follows: reductive amination of glyoxylates in the presence of rhodium as a catalyst, chemical synthesis from aminoacetonitrile, ammonia, and carbon dioxide, or even chemical synthesis using potassium phthalimide and monochloroacetic acid to produce glycine and phthalic acid.

[0006] The growing pollution problem, coupled with increasing greenhouse gas emissions and a growing world population, forces us to revise our economic model based on fossil resources. Therefore, one of the main challenges facing society is to shift from an economy based on the use of materials and energy derived from petroleum to one derived from renewable biomass.

[0007] In conclusion, it is clear that there is a need to develop new technological solutions that would allow the production of glycine to respond to the consumer's desire to use products of biological origin, in order to achieve ecologically responsible behavior and limit their impact on the environment. Therefore, there remains a clear need to develop an efficient production of glycine by biological means, in particular on an industrial scale, with a purity level sufficient to meet the needs of the agri-food and pharmaceutical industries, and at an affordable price. Summary of the Invention

[0008] The inventors unexpectedly and surprisingly developed metabolically engineered microorganisms that can ensure the effective production of glycine or its salt or ester by fermenting a suitable culture medium comprising a carbon source (particularly a simple carbon source). These metabolically engineered microorganisms or recombinant microorganisms of the present invention, their purposes, and the method for producing glycine or its salt or ester by fermentation are described throughout the specification sheets.

[0009] The object of the present invention is to produce glycine using a biological system as a cell factory. This object is achieved in particular by overexpressing a glycine production pathway and attenuating or inhibiting a glycine degradation pathway.

[0010] Therefore, another object of the present invention is to use microorganisms to bioproduce glycine from renewable carbon sources, which may have advantages over environmentally unfriendly chemical methods, reducing toxic byproducts and energy costs of chemical methods.

[0011] The present invention relates to a metabolically engineered microorganism for the biological production of glycine or a salt or ester thereof, in particular for production from threonine as a direct precursor, the genome of the metabolically engineered microorganism comprising:

[0012] (A) attenuated expression of a gene encoding an enzyme having glycine cleavage system activity as defined in EC 1.4.1.27, in particular an enzyme having glycine decarboxylase activity as defined in EC 1.4.4.2 and an enzyme having aminomethyltransferase activity as defined in EC 2.1.2.10; and

[0013] (B) overexpression of a gene encoding an enzyme having L-threonine 3-dehydrogenase activity as defined in EC 1.1.1.103 and an enzyme having glycine c-acetyltransferase activity as defined in EC 2.3.1.29; and / or

[0014] (C) Overexpression of a gene encoding an enzyme having L-threonine aldolase activity as defined in EC 4.1.2.48 or a variant of the enzyme as defined in EC 4.1.2.42 or EC 4.1.2.49.

[0015] According to the present invention, the loss of the glycine cleavage system activity encoded by the gcv operon is accompanied by the secretion of glycine into the culture medium (Plamann, 1983). Thus, by combining this modification with the following, efficient production of glycine from threonine as a substrate is obtained:

[0016] - overexpression of the threonine degradation pathway I (called TDGI), which corresponds to a gene encoding an enzyme having L-threonine 3-dehydrogenase activity, which converts threonine into L-2-amino-oxobutyrate; and a gene encoding an enzyme having glycine c-acetyltransferase activity (or 2-amino-3-ketobutyrate CoA ligase activity), which converts L-2-amino-oxobutyrate into glycine and acetyl-CoA; and / or

[0017] - Overexpression of the threonine degradation pathway II (TDGII), which corresponds to the gene encoding L-threonine aldolase, the enzyme that cleaves threonine into glycine and acetaldehyde.

[0018] To increase glycine production, overexpression of a gene encoding an enzyme having L- or D-amino acid dehydrogenase activity as defined in EC 1.4.99.1 or EC 1.4.1.9, respectively, or an enzyme having glyoxylate-alanine transaminase activity enables conversion of glyoxylate into glycine, which is obtained from acetyl-CoA formed as a by-product in threonine degradation pathways I and II.

[0019] In the context of the present invention, the terms "metabolic engineered microorganism," "recombinant microorganism," and "genetically modified microorganism" are used interchangeably to refer to microorganisms according to the present invention that are not found in nature but are modified by the introduction of new genetic elements and / or by deletion or modification of endogenous genetic elements of the microorganism. Such microorganisms can be subjected to selective pressure by combining site-directed mutagenesis or genomic recombination with cultivation in selective media.

[0020] In the context of the present invention, enzymatic activity is also indicated by reference to a gene encoding an enzyme having such activity.

[0021] The use of gene markers is not limited to a specific organism, but covers all corresponding genes and proteins in other organisms (such as microorganisms, functional analogs, functional variants and functional fragments thereof) as long as they retain enzymatic activity.

[0022] In the context of the present invention, the term "precursor" refers to an initial or intermediate production substrate that is enzymatically converted to a product to ultimately produce glycine.

[0023] In the context of the present invention, the term "endogenous gene" refers to a gene present in a microorganism in a wild-type strain before any genetic modification. Endogenous genes can be overexpressed by increasing or replacing endogenous regulatory elements, or by replacing their own promoters with the strongest promoters, or by introducing one or more extra gene copies into chromosomes or plasmids. Endogenous genes can also be modified to regulate their expression. For example, mutations can be introduced into promoter sequences to modify expression, or heterologous sequences can be introduced to increase or replace endogenous regulatory elements. The regulation of endogenous genes can result in increasing and / or enhancing the activity of gene products, or alternatively, decreasing and / or weakening the activity of endogenous gene products. Another method of enhancing endogenous gene expression is to introduce one or more extra gene copies into chromosomes or plasmids. On the contrary, the weakening of endogenous gene expression can be obtained by deleting this gene from chromosomes.

[0024] In the context of the present invention, the terms "heterologous gene" or "exogenous gene" are used interchangeably and refer to a gene that is introduced into a microorganism by means well known to those skilled in the art, and that does not naturally occur in wild-type microorganisms. If an exogenous gene is introduced into a microorganism that has all the elements that allow it to be expressed in the host microorganism, the microorganism can express the exogenous gene. Transforming a microorganism with exogenous DNA is a routine task for those skilled in the art. The exogenous gene can be integrated into the host chromosome or expressed extrachromosomally via a plasmid or vector. A variety of plasmids with different origins of replication and copy numbers in cells are known in the art (e.g., see Dykxhoorn et al., 1996 or Woodall, 2003). The sequence of the exogenous gene can be adjusted to allow its expression in the host microorganism. In fact, those skilled in the art are aware of the concept of codon usage bias and how to adjust the nucleic acid sequence for a specific codon usage bias without modifying the derived protein.

[0025] In the context of the present invention, the term "overexpression" refers to an increase in the expression of a gene or protein (e.g., enzyme) compared to an unmodified microorganism. The increase in enzyme expression is achieved by increasing the expression of the gene encoding the enzyme. The increase in gene expression can be achieved by all technologies well known to those skilled in the art. In this respect, it can be particularly mentioned that a strong promoter is implemented upstream of the nucleic acid to be overexpressed, or multiple copies of the nucleic acid are introduced into the genome between a promoter (particularly a strong promoter) and a terminator.

[0026] In the context of the present invention, the terms "attenuation", "underexpression" and "repression" are used interchangeably and refer to a reduction in the expression of a gene or protein (e.g., an enzyme) compared to the wild type, or even complete inhibition / repression, i.e., no protein is produced, in particular a functionally effective protein (e.g., an enzyme with enzymatic activity), or the protein is produced but non-functional / inactive. The reduction, attenuation or repression of enzyme expression can be achieved by reducing or inhibiting the expression of the gene encoding the enzyme. The reduction, attenuation or repression of gene expression can be achieved by all techniques known to those skilled in the art. In this regard, it is particularly useful to mention the implementation of a weak promoter upstream of the coding sequence of the gene to be underexpressed. It is also possible to mention the use of nucleic acids encoding variants of the enzyme that are less active than the original enzyme or that are degraded faster in the cell than the original enzyme. Variants of the original enzyme that are degraded faster than the parent enzyme include enzymes with degron tags. These degron-tagged enzymes include an added protein degradation signal amino acid sequence that acts as a kill signal, causing the enzyme to undergo degradation, which can be (i) ubiquitin-independent degradation or (ii) ubiquitin-dependent degradation. Mention may also be made of the reduction of the expression of transcriptional activators of the gene of interest.

[0027] In the context of the present invention, the term "inducible promoter" is used to define a promoter whose activity is induced, that is to say increased, when:

[0028] - the presence of one or more specific metabolites. The higher the concentration of the metabolite in the culture medium, the stronger the promoter activity; or

[0029] - The presence of one or more metabolites at low concentrations or in the absence of one or more metabolites. These metabolites are different from those whose presence increases and induces promoter activity. The lower the concentration of the metabolite in the culture medium, the greater the promoter activity.

[0030] In the context of the present invention, the term "repressible promoter" is used to define a promoter whose activity is repressed, that is to say whose activity is reduced, when:

[0031] - the presence of one or more specific metabolites. The higher the concentration of the metabolite in the culture medium, the weaker the promoter activity; or

[0032] - The presence of one or more metabolites at low concentrations or in the absence of one or more metabolites. These metabolites are different from those whose presence increases and represses promoter activity. The lower the concentration of the metabolite in the culture medium, the weaker the promoter activity.

[0033] In the context of the present invention, the term "activity" of an enzyme is used interchangeably with the term "function" to refer to the ability of an enzyme to catalyze a desired reaction.

[0034] The term "reduced activity" or "attenuated activity" of an enzyme refers to a reduction or even inhibition of the specific catalytic activity of the enzyme obtained by amino acid sequence mutation, and / or a reduction in the concentration of the enzyme in the cell obtained by nucleotide sequence mutation or by deletion of the corresponding related gene or by degradation determinant tagging of the enzyme.

[0035] The term "increased activity" of an enzyme refers to an increase in the specific catalytic activity of the enzyme and / or an increase in the amount / availability of the enzyme in the cell, eg obtained by overexpression of the gene encoding the enzyme.

[0036] In the context of the present invention, the term "variant" or "functional variant" includes enzymes that may exhibit substantial sequence modifications compared to the sequences specifically described in this application, but still retain the original enzymatic activity. This also means that the sequence of the enzyme may contain fewer amino acids than the original sequence, but the truncated enzyme still retains the original enzymatic activity.

[0037] In the context of the present invention, the indefinite articles "a" and "an" are used to refer to one or more (eg, at least one) units of the grammatical object of the article. For example, "an element" means at least one element, that is, one or more elements.

[0038] The term "about" or "approximately", when used to refer to measurable values, such as quantities, durations and other similar values, must be understood to include a measurement uncertainty of ±20% or ±10%, preferably ±5%, even more preferably ±1%, and particularly preferably ±0.1% of the specified value.

[0039] The term "isolated" in the context of the present invention is understood to be synonymous with being removed or extracted from its environment or natural state. For example, an isolated nucleic acid or peptide is a nucleic acid or peptide extracted from a normally existing natural environment (e.g., a living plant or animal). Thus, a nucleic acid or peptide naturally occurring in a living animal is not an isolated nucleic acid or peptide within the meaning of the present invention, but the same nucleic acid or peptide itself, partially or completely separated from other elements present in its natural environment, is "isolated" within the meaning of the present invention. An isolated nucleic acid or peptide may exist in a substantially purified form, or may exist in a non-natural environment, such as a host cell.

[0040] In the context of the present invention, "nucleotide sequence encoding an amino acid sequence" refers to all nucleotide sequences encoding an amino acid sequence, including degenerate nucleotide sequences that make it possible to obtain said amino acid sequence. A nucleotide sequence encoding a protein or RNA or cDNA may optionally include introns.

[0041] In the context of the present invention, the term "encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA or mRNA) to serve as a template for the synthesis of other polymers and macromolecules in biological processes, said other polymers and macromolecules having a defined nucleotide sequence (such as rRNA, tRNA and mRNA) or a defined amino acid sequence, and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to the gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is typically described in sequence listings and databases, and the non-coding strand, which serves as a template for transcription of a gene or cDNA, may be said to encode a protein or other product of the gene or cDNA.

[0042] In the context of the present invention, the term "polynucleotide" is defined as a chain of nucleotides. In addition, nucleic acids are polymers of nucleotides. Therefore, the terms nucleic acid and polynucleotide used in the context of the present invention are interchangeable. It is well known that in the fields of molecular biology and genetic engineering, nucleic acids are polynucleotides that can be hydrolyzed into monomers. Monomeric nucleotides can be hydrolyzed into nucleosides. As used in the context of the present invention, the term polynucleotide refers to, but is not limited to, any type of nucleic acid molecule, that is, a nucleic acid molecule that can be obtained by any means available in the art, including by recombinant means, i.e., cloning nucleic acid sequences from a recombinant library or a cell genome using common cloning techniques such as PCR or by synthesis.

[0043] In the meaning of the present invention, the terms "peptide", "polypeptide", "protein" and "enzyme" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. By definition, a protein contains at least two amino acids, and there is no limit on the maximum number of amino acids. An enzyme is a protein that catalyzes a biochemical reaction (especially in a cell). Polypeptides include, indiscriminately, a variety of peptides and / or proteins, which themselves include two or more amino acids interconnected by peptide bonds. As used herein, the term refers to short chains, which are also commonly referred to in the art as peptides, oligopeptides and oligomers, and long chains, which are commonly referred to in the art as proteins (there are many types of proteins). "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variant polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0044] In the context of the present invention, the terms "homologous" and "identical" refer to sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in each of the two compared sequences is occupied by the same base or monomeric amino acid subunit (for example, when a position in each of the two DNA molecules is occupied by adenine), then the molecules are homologous or identical with respect to that position. The percent identity between the two sequences is a function of the number of corresponding positions shared by the two sequences and corresponds to that number divided by the number of positions compared and multiplied by 100. For example, if 6 out of 10 positions are identical in the two matched sequences, then the two sequences are 60% identical. Typically, comparisons are made by aligning the two sequences to give maximum identity / homology.

[0045] In the context of the present invention, a "vector" is a molecular construct that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" refers to, for example, an autonomously replicating plasmid or virus. The term should also be understood to include non-plasmid or non-viral compounds that facilitate transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like.

[0046] In the context of the present invention, the term "expression vector" refers to a vector comprising a recombinant polynucleotide, which comprises expression control sequences operably linked to the nucleotide sequence to be expressed. Expression vectors particularly comprise cis-acting expression elements; other expression elements may be provided by the host cell or in vitro expression system. Expression vectors within the meaning of the present invention include all vectors known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate a recombinant polynucleotide.

[0047] In the context of the present invention, the term "promoter" is defined as a DNA sequence recognized by the synthetic machinery of the cell or introduced synthetic machinery and is necessary to initiate specific transcription of a polynucleotide sequence.

[0048] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence necessary for the expression of a polynucleotide to which the promoter / regulatory sequence is operably linked. In some cases, the sequence may be a promoter base sequence, while in other cases, the sequence may also include enhancer sequences and other regulatory elements useful for polynucleotide expression. The promoter / regulatory sequence may be, for example, a sequence that allows for tissue-specific expression of the polynucleotide, i.e., that is, is preferentially present in that tissue.

[0049] In the context of the present invention, a "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide, causes the polynucleotide to be expressed under most or all physiological conditions of the cell.

[0050] In the context of the present invention, the term "heterologous expression" refers to the expression of exogenous genes in a host cell or organism if the exogenous genes are introduced into said cell or said organism and all elements allow their expression in the host. Techniques allowing the introduction of DNA into a host (or transformation) are well known to those skilled in the art and include, in particular, membrane permeabilization by applying an electric field (electroporation), by thermal methods (application of heat shock) or by chemical methods.

[0051] Preferably, the subject of the present invention is a recombinant microorganism for producing glycine or any salt or ester thereof as defined above, which has the following technical features, which may be present alone or in combination:

[0052] - threonine is L-threonine, D-threonine or a mixture thereof;

[0053] - the genome of the microorganism of the present invention further comprises attenuated expression of a gene encoding an enzyme having dihydroacyl dehydrogenase activity as defined in EC 1.8.1.4, advantageously attenuating its expression but not completely inhibiting / repressing it, so that the dihydroacyl dehydrogenase activity can be quantified / observed;

[0054] - repression of a gene encoding an enzyme having glycine decarboxylase activity as defined in EC 1.4.4.2;

[0055] - repression of a gene encoding an enzyme having aminomethyltransferase activity as defined in EC 2.1.2.10;

[0056] - the genome of the microorganism according to the invention further comprises the overexpression of a gene encoding an enzyme having acetylating aldehyde dehydrogenase activity as defined in EC 1.2.1.10, which allows the conversion of acetaldehyde into acetyl-CoA;

[0057] The genome of the microorganism according to the invention may also comprise an attenuation or even repression of a gene encoding an enzyme having D-amino acid oxidase activity as defined in EC 1.4.99.-.

[0058] - the genome of the microorganism of the present invention further comprises overexpression of a gene encoding an enzyme having D-serine / D-alanine / glycine transporter activity as defined in TCDB 2.A.3.1.7;

[0059] - the genome of the microorganism of the present invention further comprises attenuating or even repressing the expression of a gene encoding an enzyme having extracellular threonine efflux activity as defined in TCDB 2.A.76.1.2 (i.e., RhtC), in particular threonine / homoserine export activity as defined in TCDB 2.A.7.3.6 (RhtA), homoserine / homoserine lactone / β-hydroxynorvaline efflux permease activity as defined in TCDB 2.A.76.1.1 (RhtB);

[0060] - the genome of the microorganism according to the invention further comprises overexpression of a gene encoding an enzyme having threonine synthase activity as defined in EC 4.2.3.1;

[0061] - the genome of the microorganism according to the invention further comprises the overexpression of a gene encoding an enzyme having glycine dehydrogenase activity as defined in EC 1.4.1.10, such as the L-alanine dehydrogenase of Mycobacterium tuberculosis (Usha et al., 2002);

[0062] - the genome of the microorganism according to the invention further comprises overexpression of a gene encoding an enzyme having D-amino acid dehydrogenase activity as defined in EC 1.4.99.1.;

[0063] - the genome of the microorganism according to the invention further comprises overexpression of a gene encoding an enzyme having glyoxylate-alanine aminotransferase activity as defined in EC 2.6.1.44;

[0064] - the variant of the enzyme having L-threonine aldolase activity as defined in EC:4.1.2.48 is the H126F variant of the Escherichia coli constitutive enzyme, i.e., ItaEH126F as described by Fesko (Fesko, 2016); or an equivalent variant of another microorganism;

[0065] - an enzyme having glycine decarboxylase activity as defined in EC 1.4.4.2, encoded by the sequence shown in SEQ ID NO: 1 of Escherichia coli, or any sequence having at least 90% identity thereto, or corresponding to SEQ ID NO: 2 or any sequence having at least 90% identity thereto, or corresponding to an equivalent enzyme having the same enzymatic activity in another microorganism;

[0066] - the enzyme having aminomethyltransferase activity as defined in EC 2.1.2.10 is encoded by the sequence shown in SEQ ID NO: 3 of Escherichia coli, or any sequence having at least 90% identity thereto, or corresponds to SEQ ID NO: 4 or any sequence having at least 90% identity thereto, or corresponds to an equivalent enzyme having the same enzymatic activity in another microorganism;

[0067] - the enzyme having L-threonine 3-dehydrogenase activity as defined in EC 1.1.1.103 is encoded by the sequence shown in SEQ ID NO: 5 of Escherichia coli or any sequence having at least 90% identity thereto, or corresponds to SEQ ID NO: 6 or any sequence having at least 90% identity thereto, or corresponds to an equivalent enzyme having the same enzymatic activity in another microorganism;

[0068] - an enzyme having L-threonine aldolase activity as defined in EC 4.1.2.48 encoded by the sequence shown in SEQ ID NO: 7 of Escherichia coli or any sequence having at least 90% identity thereto, or corresponding to SEQ ID NO: 8 or any sequence having at least 90% identity thereto, or corresponding to an equivalent enzyme having the same enzymatic activity in another microorganism;

[0069] - the enzyme having dihydroacyl dehydrogenase activity as defined in EC 1.8.1.4 is encoded by the sequence shown in SEQ ID NO: 9 of Escherichia coli or any sequence having at least 90% identity thereto, or corresponds to SEQ ID NO: 10 or any sequence having at least 90% identity thereto, or corresponds to an equivalent enzyme having the same enzymatic activity in another microorganism;

[0070] - the enzyme having acetylating aldehyde dehydrogenase activity as defined in EC 1.2.1.10 is encoded by the sequence shown in SEQ ID NO: 11 of Escherichia coli, or any sequence having at least 90% identity thereto, or corresponds to SEQ ID NO: 12 or any sequence having at least 90% identity thereto, or corresponds to an equivalent enzyme having the same enzymatic activity in another microorganism;

[0071] - the enzyme having threonine synthase activity as defined in EC 4.2.3.1 is encoded by the sequence shown in SEQ ID NO: 13 of Escherichia coli, or any sequence having at least 90% identity thereto, or corresponds to SEQ ID NO: 14 or any sequence having at least 90% identity thereto, or corresponds to an equivalent enzyme having the same enzymatic activity in another microorganism;

[0072] - an enzyme having glycine dehydrogenase activity as defined in EC 1.4.1.10 encoded by the sequence shown in SEQ ID NO: 15 of Streptomyces phaechromogenes, or any sequence having at least 90% identity thereto, or corresponding to SEQ ID NO: 16 or any sequence having at least 90% identity thereto, or to an equivalent enzyme having the same enzymatic activity in another microorganism;

[0073] - the enzyme having D-amino acid dehydrogenase activity as defined in EC 1.4.99.1 is encoded by the sequence shown in SEQ ID NO: 17 of Escherichia coli, or any sequence having at least 90% identity thereto, or corresponds to SEQ ID NO: 18 or any sequence having at least 90% identity thereto, or corresponds to an equivalent enzyme having the same enzymatic activity in another microorganism;

[0074] - an enzyme having glyoxylate-alanine aminotransferase activity as defined in EC 2.6.1.44, encoded by the sequence shown in SEQ ID NO: 19 of Escherichia coli, or any sequence having at least 90% identity thereto, or corresponding to SEQ ID NO: 20 or any sequence having at least 90% identity thereto, or corresponding to an equivalent enzyme having the same enzymatic activity in another microorganism;

[0075] - the H126F variant of the enzyme having L-threonine aldolase activity as defined in EC 4.1.2.48 corresponds to the amino acid sequence shown in SEQ ID NO: 21 of Escherichia coli, or any sequence having at least 90% identity thereto, or to SEQ ID NO: 22 or any sequence having at least 90% identity thereto, or to an equivalent enzyme having the same enzymatic activity in another microorganism;

[0076] - an enzyme having glycine C-acetyltransferase activity as defined in EC 2.3.1.29 encoded by the sequence shown in SEQ ID NO: 23 of Escherichia coli, or any sequence having at least 90% identity thereto, or corresponding to SEQ ID NO: 24 or any sequence having at least 90% identity thereto, or corresponding to an equivalent enzyme having the same enzymatic activity in another microorganism;

[0077] - the enzyme having D-serine / D-alanine / glycine transporter activity as defined in TCDB 2.A.3.1.7 is encoded by the sequence of SEQ ID NO: 25 of Escherichia coli or any sequence having at least 90% identity thereto, or corresponds to SEQ ID NO: 26 or any sequence having at least 90% identity thereto, or corresponds to an equivalent enzyme having the same enzymatic activity in another microorganism;

[0078] - the enzyme having threonine / homoserine export activity as defined in TCDB 2.A.7.3.6 is encoded by the sequence shown in SEQ ID NO: 27 of Escherichia coli or any sequence having at least 90% identity thereto, or corresponds to SEQ ID NO: 28 or any sequence having at least 90% identity thereto, or corresponds to an equivalent enzyme having the same enzymatic activity in another microorganism;

[0079] - an enzyme having homoserine / homoserine lactone / β-hydroxynorvaline efflux permease activity encoded by the sequence represented by SEQ ID NO: 29 of Escherichia coli or any sequence having at least 90% identity thereto, or corresponds to SEQ ID NO: 30 or any sequence having at least 90% identity thereto, or corresponds to an equivalent enzyme having the same enzymatic activity in another microorganism;

[0080] - an enzyme having threonine efflux permease activity as defined in TCDB 2.A.76.1.2, encoded by the sequence shown in SEQ ID NO: 31 of Escherichia coli, or any sequence having at least 90% identity thereto, or corresponding to SEQ ID NO: 32 or any sequence having at least 90% identity thereto, or corresponding to an equivalent enzyme having the same enzymatic activity in another microorganism;

[0081] - an enzyme having L-amino acid dehydrogenase activity as defined in EC 1.4.1.9 encoded by the sequence shown in SEQ ID NO: 33 of Bacillus subtilis or any sequence having at least 90% identity thereto, or corresponding to SEQ ID NO: 34 or any sequence having at least 90% identity thereto, or corresponding to an equivalent enzyme having the same enzymatic activity in another microorganism;

[0082] - the salt form of glycine is glycine ammonium salt, glycine potassium salt or glycine sodium salt;

[0083] - an ester derived from glycine selected from the group consisting of glycine ethyl ester, glycine methyl ester, glycine ethyl ester hydrochloride and glycine methyl ester hydrochloride;

[0084] - the microorganism is any lower unicellular organism into which a gene, nucleic acid or chimeric vector according to the present invention can be introduced for producing glycine or any salt or ester thereof;

[0085] - the microorganism is a bacterium, advantageously belonging to the family Enterobacteriaceae or Corynebacterium, preferably to the genus Escherichia, Pantoea, Corynebacterium or Brevibacterium, more particularly of the species Escherichia coli, Pantoea ananatis or Corynebacterium glutamicum;

[0086] - The microorganism is a fungus, advantageously from the family of Ascomycota, preferably from the group of Saccharomycetes or Exocomycetes, more particularly from the subdivision Saccharomycetes, Pezizomycetes or Archaeometebromycetes, more advantageously from the class Saccharomycetes, Eurotium, Leotiomyceta, Pezizomycetes or Archaeometebromycetes, preferably its species is Saccharomyces cerevisiae, Talaromyces diversicolor, Aspergillus niger.

[0087] In the context of the present invention, the expression "any sequence that is at least 90% identical to a sequence (nucleic acid sequence or peptide sequence)" corresponds to any sequence that is at least 90%, or 91%, 92%, 93%, 94%, 95% or even 96%, 97%, 98%, or 99% identical to said sequence.

[0088] In the context of the present invention, the expression "equivalent enzyme in another microorganism" or "enzyme having the same enzymatic activity in another microorganism" refers to an enzyme of a first microorganism capable of catalyzing a reaction on a substrate or a set of related reactions on a set of substrates, the enzyme of the first microorganism having the same enzymatic properties (reaction rate, substrate specificity, etc.) or related / similar properties as an enzyme of a second microorganism different from the first microorganism. In other words, this involves the specific recognition of the substrate (particularly the active site or catalytic site) by an enzyme derived from a microorganism different from the microorganism from which the specific substrate is derived, followed by its conversion into a product. In particular, in the context of the present invention, this expression does not imply dual enzyme specificity nor coenzyme.

[0089] In particular, as previously described, the synthetic pathway for producing glycine according to the present invention can be implemented in microorganisms having increased threonine production. For example, bacteria that may be mentioned are Corynebacterium or Brevibacterium, Escherichia, in particular Escherichia coli.

[0090] Alternatively, as previously mentioned, the engineered pathway for producing glycine according to the present invention can be implemented in microorganisms that exhibit increased threonine production or have low threonine production. For example, we can mention the bacterium Escherichia coli (Debabov, 2003), the yeast Saccharomyces cerevisiae (Farfan et al., 1999), the filamentous fungi Aspergillus niger, Trichoderma reesei or various Talaromyces. In this case, the genome of the microorganism of the present invention is modified as described above, but the production of threonine in the microorganism is also ensured by the following means, in particular the increase of threonine production:

[0091] (i) overexpression of the enzymes aspartate kinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase, and threonine synthase;

[0092] (ii) modifying the enzyme aspartate kinase so as to make it insensitive to product inhibition that may be caused by lysine, methionine and / or threonine (i.e. modifying the enzyme so as to make it insensitive to negative feedback caused by lysine, methionine and / or threonine), and modifying the enzyme homoserine kinase so as to make it insensitive to inhibition by threonine (i.e. modifying the enzyme so as to make it insensitive to negative feedback caused by threonine), and

[0093] (iii) Suppression of metabolic pathways that separate the aspartate-homoserine biosynthesis pathway from threonine synthesis, such as attenuation of the methionine pathway.

[0094] In particular, the overexpression of aspartate kinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase and threonine synthase can be achieved by expressing these enzymes from a multicopy plasmid under the control of a constitutive promoter or a suitable inducible promoter. Alternatively, the overexpression of the enzyme can be obtained by expressing the related gene under a strong and non-repressible promoter. By introducing appropriate mutations in the amino acid sequence of aspartate kinase, aspartate kinase can be made immune to the inhibition of amino acids derived from aspartate (Omori et al., (1993); Huo et al., (1996); Chen et al., (2011)). By introducing appropriate mutations in the amino acid sequence of homoserine kinase, homoserine kinase can be made insensitive to threonine. The entry point of the metabolic pathway that hijacks the homoserine biosynthetic pathway is catalyzed by an enzyme with O-succinyl homoserine or O-acetyl homoserine synthase activity (entering methionine biosynthesis) or diaminopimelate active decarboxylase (entering lysine biosynthesis). Deletion of genes encoding proteins with such enzymatic activity prevents the formation of amino acids derived from aspartate, lysine, and / or methionine, thereby promoting the formation of homoserine. Thus, deletion of metA, thrB, and lysA genes (or equivalent enzymes in other microorganisms) in E. coli can achieve a higher flux of homoserine to threonine synthesis (i.e., reduce or even inhibit the transfer of homoserine to competitive pathways). For example, increasing the enzymatic activity of the threonine pathway in E. coli can be achieved by overexpressing a mutant ThrA encoding a bifunctional aspartate kinase-homoserine dehydrogenase. S345F (insensitive to threonine inhibition) and the genes for asd, thrB, and thrC, or by overexpression of the monofunctional aspartate kinase mutant LysC E250K (lysine insensitive), asd, thrB, and thrC (all E. coli genes) were obtained (Lee et al., 2007; Dong et al., 2012).

[0095] The microorganism of the present invention may also have a weakened ability to export threonine or no ability to export threonine, which increases the intracellular availability of this amino acid. For example, when the microorganism is Escherichia coli, its genome may also comprise a deletion of the threonine efflux transporters rhtA, rhtB and / or rhtC (Kruse et al., 2002).

[0096] According to the present invention, acetyl-CoA is a by-product of a reaction catalyzed by an enzyme having glycine C-acetyltransferase activity (or 2-amino-3-ketobutyrate-CoA ligase) and is "recycled" / used to produce glycine from glyoxylate, and it is then necessary to activate the glyoxylate shunt and express an enzyme having glycine dehydrogenase activity as defined in EC 1.4.1.10 or glyoxylate-alanine aminotransferase activity as defined in EC 2.6.1.44 to ensure that glyoxylate is converted to glycine by a D-amino acid dehydrogenase activity as defined in EC 1.4.99.- or an alanine-glyoxylate aminotransferase as defined in EC 2.6.1.44 or a glutamate-glyoxylate aminotransferase as defined in EC 2.6.1.4 or a serine-glyoxylate aminotransferase as defined in EC 2.6.1.45.

[0097] Acetyl-CoA can also be produced from acetaldehyde, a byproduct of the cleavage of threonine to glycine catalyzed by an enzyme having L-threonine aldolase activity as defined in EC 4.1.2.48 or a variant of such an enzyme. In this case, acetyl-CoA is "recycled" / used to ensure glycine production via the glyoxylate shunt and by expressing an enzyme having glycine dehydrogenase activity as defined in EC 1.4.1.10, or an enzyme having glyoxylate-alanine aminotransferase activity as defined in EC 2.6.1.44, or a glutamate-glyoxylate aminotransferase as defined in EC 2.6.1.4, or a serine-glyoxylate aminotransferase as defined in EC 2.6.1.45.

[0098] The present invention also relates to the use of the aforementioned microorganisms for the production and / or secretion and / or purification of glycine or its salts or esters. In particular:

[0099] - the salt form of glycine is glycine ammonium salt, glycine potassium salt or glycine sodium salt; and

[0100] - 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.

[0101] Preferably, the present invention relates to the use of a metabolically engineered microorganism as described above, wherein glycine or any salt or ester thereof is produced from a carbon source, advantageously a simple carbon source, preferably a pentose or hexose or disaccharide, more particularly chosen from the group consisting of glucose, sucrose, xylose, arabinose, ribose, mannose, galactose, fructose and mixtures thereof, advantageously glucose.

[0102] Furthermore, the present invention also relates to the use of the metabolically engineered microorganisms as described previously for the bioproduction of glycine or its salts or esters in the production and / or secretion and / or purification of acetyl-CoA.

[0103] The present invention also relates to a method for producing glycine or any salt or ester thereof, comprising the steps of:

[0104] a) culturing the metabolically engineered microorganism according to the present invention in a suitable culture medium comprising a carbon source to produce and accumulate glycine or any salt or ester thereof in the culture medium and / or in the cells of the microorganism; and

[0105] b) recovering glycine or any salt thereof, in particular ammonium, potassium or sodium salt, which has accumulated in the culture medium and / or cells of the microorganism and is ultimately present in the form of a salt.

[0106] in particular:

[0107] - the salt form of glycine is glycine ammonium salt, glycine potassium salt or glycine sodium salt; and

[0108] - 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.

[0109] Preferably, the subject of the present invention is a process for producing glycine or any salt or ester thereof as defined above, having the following technical features, which may be present alone or in combination:

[0110] - the method further comprises the step c) purifying glycine or any salt or ester thereof;

[0111] - in step a), acetyl-CoA is produced as a co-metabolite of glycine and used as a substrate for the production of glycine, in particular via the glyoxylate shunt and expressing an enzyme having glycine dehydrogenase activity as defined in EC 1.4.1.10, or an enzyme having glyoxylate-alanine transaminase activity as defined in EC 2.6.1.4 or EC 2.1.6.40, to ensure the conversion of glyoxylate into glycine;

[0112] - the carbon source is a simple carbon source, advantageously a pentose or hexose or disaccharide, more particularly chosen from the group consisting of glucose, sucrose, xylose, arabinose, ribose, mannose, galactose, fructose and mixtures thereof;

[0113] - the carbon source is glucose;

[0114] - The method of the present invention is carried out in a bioreactor;

[0115] - The method of the present invention is carried out in batch or fed-batch mode;

[0116] - Recovery of glycine or any of its salts or esters generally comprises steps of cell separation and purification, concentration and drying of the product, respectively;

[0117] -Ultrafiltration and centrifugation can be used to separate cells from the fermentation medium;

[0118] - adding additives (such as inorganic acids or alkaline salts) or heating the culture medium to optimize cell separation, as described in US Pat. No. 5,017,480; WO 01 / 72689 or CN 108084041;

[0119] Various chromatographic methods of ion exchange can be used to separate glycine or one of its salts before or after removal of the biomass, including in particular the use of primary cation exchange resins, which facilitate separation of the products according to their isoelectric points, wherein the resin is loaded with a solution and the retained product is eluted separately after raising the pH in the eluent (e.g. by adding ammonium hydroxide); the use of ion exchange chromatography using fixed-bed or simulated moving-bed resins;

[0120] - It may be necessary to combine different chromatographic steps to achieve sufficient product purity;

[0121] - the purification process may further comprise a drying step, which may involve any suitable drying apparatus, such as a spray granulator, a spray dryer, a drum dryer, a rotary dryer and a tunnel dryer;

[0122] - A concentrated solution of glycine or any salt or ester thereof, or a mixture thereof can be obtained by heating the fermentation broth under reduced pressure using steam at 130°C using a general concentrator or a thin film evaporator.

[0123] In the context of the present invention, the production level of glycine or any salt or ester thereof is expected to be at an industrial level, advantageously, the concentration in the culture medium of the microorganism of the present invention is greater than or equal to 10 mg in 48 hours in fed-batch mode, with a carbon source of 20 g / L, advantageously 20 g / L of glucose; preferably, greater than or equal to 50 mg, 100 mg, 150 mg, 200 mg or 1 g in 48 hours in fed-batch mode, with a carbon source (advantageously glucose) of 20 g / L; or even greater than or equal to 10 mg, 100 mg, 150 mg, 200 mg or 1 g in 48 hours in fed-batch mode, with a carbon source (advantageously glucose) of 20 g / L; In some embodiments, the present invention provides a method for producing a microorganism comprising: producing a microorganism in a fed-batch mode at a concentration of at least 250 mg, 300 mg, 350 mg, 400 mg, 500 mg, 1 g, 1.25 g, 1.5 g, 2 g in 48 hours at a carbon source of 20 g / L, advantageously 20 g / L of glucose; or even in a fed-batch mode at a concentration of at least 5 g, 10 g, 15 g, 20 g, 25 g, 1 g, 50 g, 75 g, 100 g or 150 g, 200 g in 48 hours at a carbon source of 20 g / L, advantageously 20 g / L of glucose. At these concentrations and under the conditions according to the method of the invention, glycine or any salt or ester thereof remains soluble and does not exhibit any toxicity to the microorganism of the present invention.

[0124] As can be seen above, the microorganism according to the invention or the microorganism implemented in the use or method according to the invention is genetically modified to ensure efficient production and / or secretion of glycine or any salt or ester thereof at industrial level.

[0125] Furthermore, the production of glycine or any of its salts by fermentation offers the following technical and economic advantages:

[0126] - a process that uses renewable and inexpensive carbon sources in aqueous solution at room temperature and is carried out at ambient pressure, which consumes less energy than high-pressure processes;

[0127] - does not use toxic products, organic solvents or petroleum-based resources; and

[0128] -Production of high-purity glycine for use in the food, dietary supplement, chemical and pharmaceutical, and cosmetics industries. DETAILED DESCRIPTION

[0129] The present invention is illustrated by the following examples, but is not limited thereto.

[0130] Example

[0131] Example 1: Metabolic Engineering of Escherichia coli for Glycine Production According to the Present Invention

[0132] 1. Materials and Methods

[0133] a. Plasmid construction

[0134] All plasmids used in this study were constructed using the commercial NEBuilder HIFI DNA assembly kit (New England Biolabs) by isothermal assembly reactions (Gibson et al., 2009) according to the manufacturer's instructions. For the construction of pMW12, the kbl-tdh operon of E. coli was amplified using primers MW15 and MW16 and assembled into the plasmid pACT3 at the SacI and HindIII sites (Dykxhoorn et al., 1996). For the construction of pMW26 / pMW27, the E. coli l-threonine aldolase gene ltaE wild type or its mutant H126F was synthesized by IDT (Iowa, USA) and assembled into the plasmid pACT3 between the SacI and HindIII sites. For the construction of plasmids pMW30 / pMW31 carrying the synthetic operons kbl-tdh-ltaE or kbl-tdh-ltaE-H126F, kbl-tdh and ltaE or ltaE-H126F were synthesized by IDT (Iowa, USA) into an operon carrying the RBS BBa_BB035 between kbl-tdh and ltaE or ltaE-H126F (Englund et al., 2016) and assembled into the plasmid pACT3 between the SacI and HindIII sites. Escherichia coli TOP10 (Invitrogen) was used as the host strain for plasmid construction. Plasmid pYN7 has been described elsewhere (see patent EP0152 830A1 of Nakagawa et al., 1985). It has the pBR322 ori, the ampicillin resistance gene Ap r , and carries thrA*thrB thrC genes (affected by its natural P thrLABC promoter). Mutations in thrA* result in variant ThrA S345F (Lee et al., 2007).

[0135] b. Strain construction

[0136] Unless otherwise stated, the E. coli strain used in this study was BW25113. The E. coli mutant was constructed by P1 phage transduction. Preparation of P1 lysates and P1 phage transduction were performed as described elsewhere (Thomason et al., 2007). Strains from the Keio collection (Baba et al., 2006) carrying single gene deletions and FRT site-flanked kanamycin resistance cassettes were used as donor strains. Successful transduction events were confirmed by colony PCR. Excision of the antibiotic resistance marker catalyzed by the FLP-recombinase flippase (Cherepanov & Wackernagel, 1995) was performed by transformation with the pCP20 plasmid and subsequent colony PCR verification of excision.

[0137] Escherichia coli strain 472T23 pYN7 (K-12 background) was purchased from ATCCC with the number 98081. TM It was constructed by (Kruse et al., 2002; Debabov, 2003D 6) and contains mutations at thrC* and ilvA* that inactivate homoserine kinase and threonine deaminase as well as pYN7. Deletion of rthABC (Livshits, 2003#11032), encoding a threonine exporter, and gcvP, encoding a glycine decarboxylase of the glycine cleavage pathway, was performed by PI transduction as described above.

[0138] c. Glycine production in E. coli

[0139] In order to produce glycine, plasmids pMW12, pMW26, pMW27, pMW30 and pMW31 were transformed into Escherichia coli BW25113 or Escherichia coli BW25113ΔgcvP. Empty pACT3 was used as a negative control. The pre-culture of each transformed strain was grown overnight (o / n) at 37°C and 200rpm in a 50ml centrifuge tube using 5ml M9 minimal medium supplemented with 2% (w / v) glucose and chloramphenicol (40 μg / ml). The next day, the pre-culture was inoculated in 25ml M9 medium supplemented with 2% (w / v) glucose and chloramphenicol (40 μg / ml) in a 250ml baffled conical flask to an initial OD of 0. 600 Dilute to an initial OD of 0.2 600 The OD value was 0.05–0.2 and incubated at 37°C and 200 rpm. 600 When the cell culture reaches 0.6-1.0, IPTG is added to a final concentration of 1 mM. At several time points, 1 ml samples are taken. When necessary, 25 mM threonine is added to the M9 glucose medium at the same time as induction.

[0140] d. LC-MS detection of glycine

[0141] Glycine and threonine concentrations in the culture medium were determined on a Dionex ThermoFisher UltiMate 3000 system consisting of a binary pump, online degasser, autosampler, column oven, and RS diode array detector (ThermoFisher Scientific, Waltham, USA). The system was interfaced with a ThermoFisher MSQ mass spectrometer (ThermoFisher Scientific, Waltham, USA). Chromatographic separation was performed on a Poroshell 120 Hilic-Z column (2.1 mm x 150 mm, 2.7 μm P), maintained at 25°C, with a flow rate of 0.5 ml / min. The mobile phase consisted of solution A (H₂O: 200 mM ammonium formate, pH 3, 90%:10% (v / v)) and solution B (acetonitrile: 200 mM ammonium formate, pH 3, 90%:10% (v / v)). The gradient elution program was optimized for the separation of glycine and threonine: 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, 10.5-14 min, 95% Solution B, for a total run time of 14 min. Samples were supplemented with L-alanine-2,3,3,3-d4 as an internal standard to a final concentration of 3.8 mM and then diluted 1:20 in Solution B. The sample injection volume was 10 μl. Data acquisition and analysis were performed using a Dionex Chromeleon 7 (ThermoFisher Scientific, Waltham, USA).

[0142] 2. Results

[0143] The results of bioconversion of glucose to glycine by different strains cultured in M9 medium containing 20 g / l glucose for 48 hours are shown in Table 1.

[0144]

[0145]

[0146] Data shown are the means of two biological replicates *Values ​​are the means + SD of two independent biological experiments; $ bd = below detection, i.e., <0.2 mM glycine; ￡ nt: Not tested.

[0147] Regarding metabolically engineered microorganisms Gly2 to Gly5, expression of threonine degradation pathways I and II did not promote glycine production. On the other hand, removal of the gcvP gene inactivated the glycine cleavage system (GCS) that catalyzes the degradation of glycine to methylfolate, CO2, and NH3, resulting in a glycine producer, Gly6, that can produce small amounts of glycine (28 mg glycine from 20 g / l glucose in 48 hours).

[0148] Regarding the metabolically engineered microorganism numbered Gly7 lacking GCS, in which the tdh-kbl pathway was overexpressed, it was found that the production of glycine was significantly increased to a level of 300-500 mg / l glycine.

[0149] Similarly, in the Gly9 strain, which also carries a GCS deletion but in which the ltaE-dependent threonine degradation pathway is overexpressed, glycine titers rose to 286 mg / l after 48 hours of growth on M9 containing 20 g / l glucose, and these titers were even higher by expression of taE* encoding the LtaEH126F variant. On the other hand, overexpression of both tdh-kbl and ltaEH126F did not appear to improve glycine production compared to overexpression of either tdh-kbl or ltaEH126F alone.

[0150] To assess whether the promotion of threonine synthesis is crucial for glycine production, two complementary experiments were conducted. In one experiment, 25 mM exogenous threonine was added to cultures of strains GLY1 to GLY10. The results clearly demonstrated an increase in glycine production. For example, in strain GLY8, glycine production increased fourfold in the presence of threonine compared to the absence of threonine, confirming the crucial role of enhanced threonine pathways in glycine production.

[0151] On the other hand, threonine production strain (472T23 pYN7) is used as the receptor for glycine production. In this receptor strain, the threonine operon is expressed by plasmid pYN7. pYN7 is a plasmid based on pBR322, which carries thrA*, thrB and thrC under the control of its natural promoter. In addition, the thrA* gene includes a mutation encoding the ThrAS345F variant, which is no longer sensitive to feedback inhibition by threonine (Lee et al., 2007). In this strain, threonine export genes rhtA, rhtB and rhtC are deleted (Gly11) to prevent threonine export, and gcvP is deleted to inactivate the glycine cleavage system (Gly12). Although Gly11 does not produce glycine, GLY13 produced 278mg / l from 20g / l glucose in 48 hours, confirming the importance of missing GCS in glycine production.

[0152] Overexpression of tdh-kbl in Gly12 produced Gly13, further increasing glycine production to 454 mg / L. Similarly, overexpression of ltaE*, encoding the LtaEH126F variant, in Gly13 produced Gly14, which produced over 1000 mg / L of glycine from 20 g / L glucose in 48 hours. On the other hand, glycine production in Gly16, which overexpresses both pathways (tdh-kbl and ltaE), appeared to be no better, or even lower, than in Gly14 and Gly15 strains overexpressing either pathway alone.

[0153] References

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Claims

1. A metabolically engineered microorganism for the bioproduction of glycine or a salt or ester thereof, the genome of the microorganism comprising: a. encoding an enzyme having glycine cleavage system activity as defined in EC1.4.1.27, in particular, expression of a gene encoding an enzyme having glycine decarboxylase activity as defined in EC1.4.4.2 and aminomethyltransferase activity as defined in EC2.1.2.10 is reduced; and b. overexpression of a gene encoding an enzyme having L-threonine 3-dehydrogenase activity as defined in EC 1.1.1.103 and an enzyme having glycine c-acetyltransferase activity as defined in EC 2.3.1.29; and / or c. Overexpression of a gene encoding an enzyme having L-threonine aldolase activity as defined in EC 4.1.2.48 or a variant of the enzyme as defined in EC 4.1.2.42 or EC 4.1.2.

49.

2. The metabolically engineered microorganism according to claim 1, characterized in that Its genome further comprises attenuated expression of a gene encoding an enzyme having dihydroacyl dehydrogenase activity as defined in EC 1.8.1.

4.

3. The metabolically engineered microorganism according to claim 1 or 2, characterized in that Its genome also comprises overexpression of a gene encoding an enzyme having acetylating aldehyde dehydrogenase activity as defined in EC 1.2.1.

10.

4. The metabolically engineered microorganism according to any one of the preceding claims, characterized in that Its genome also comprises overexpression of a gene encoding an enzyme having threonine synthase activity as defined in EC 4.2.3.

1.

5. The metabolically engineered microorganism according to any one of the preceding claims, characterized in that Its genome also includes overexpression of genes encoding: - an enzyme having glycine dehydrogenase activity as defined in EC 1.4.1.10; and / or - an enzyme having D-amino acid oxidase activity as defined in EC 1.4.99.-.; and / or - an enzyme having glyoxylate-alanine aminotransferase activity as defined in EC 2.6.1.44; and / or - an enzyme having glycine C-acetyltransferase activity as defined in EC 2.3.1.29; and / or - an enzyme having L-amino acid dehydrogenase activity as defined in EC 1.4.1.

9.

6. The metabolically engineered microorganism according to any one of the preceding claims, characterized in that Its genome also includes decreased expression of genes encoding: - an enzyme having D-serine / D-alanine / glycine transporter activity as defined in TCDB 2.A.3.1.7; and / or - an enzyme having threonine efflux permease activity as defined in TCDB 2.A.76.1.

2.

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

8. The metabolically engineered microorganism according to any one of the preceding claims, characterized in that Its genome also includes attenuating or even repressing the expression of genes encoding enzymes having extracellular threonine efflux activity as defined in TCDB 2.A.76.1.2 (i.e., RhtC), in particular threonine / homoserine export activity (RhtA) as defined in TCDB 2.A.7.3.6, homoserine / homoserine lactone / β-hydroxynorvaline efflux permease activity (RhtB) as defined in TCDB 2.A.76.1.

1.

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

10. The metabolically engineered microorganism according to any one of the preceding claims, characterized in that Its genome also includes: - increasing the expression of at least one enzyme activity selected from the group consisting of: phosphoenolpyruvate carboxylase, isocitrate lyase, pyruvate carboxylase and hexose symporter permease; and / or - reducing the expression of 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, succinate semialdehyde dehydrogenase, sugar-transporting phosphotransferase, ketohydroxyglutarate aldolase, homoserine-O-succinyltransferase, homoserine kinase, homoserine efflux transporter, diaminopimelate decarboxylase and / or methylglyoxal synthase.

11. The metabolically engineered microorganism according to any one of the preceding claims, characterized in that It is: - a bacterium, preferably belonging to the family Enterobacteriaceae or Corynebacterium, preferably to the genus Escherichia, Pantoea, Corynebacterium or Brevibacterium, more particularly the species Escherichia coli, Pantoea ananatis or Corynebacterium glutamicum; - a fungus, preferably belonging to the family Ascomycota, preferably to the group Ascomycota or Exocomycota, more particularly to the subdivision Saccharomyces, Pezizomycetes or Archaeometrium, more preferably to the classes Saccharomyces, Eurotium, Hamsteria, Pezizomycetes or Archaeometrium, preferably the species being Saccharomyces cerevisiae, Talaromyces diversicolor, Aspergillus niger.

12. A metabolically engineered microorganism according to any one of the preceding claims, characterized in that It is Escherichia coli, and in its genome: - overexpression of 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; pycE from Corynebacterium glutamicum; and / or - deletion of at least one gene selected from the group consisting of ldhA, 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, rthC.

13. Use of the metabolically engineered microorganism according to any one of claims 1 to 12 for producing glycine or any salt or ester thereof.

14. A method for producing glycine or any 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 comprising a carbon source to produce and accumulate glycine or any salt or ester thereof in the culture medium and / or cells of the microorganism; and b) recovering glycine or any salt thereof accumulated in the culture medium and / or cells of the microorganism.

15. The method according to claim 14, characterized in that It also comprises the step c) of purifying glycine or any salt or ester thereof.

16. The method according to claim 14 or 15, characterized in that The carbon source is a pentose or hexose or disaccharide, advantageously selected from the group consisting of glucose, sucrose, xylose, arabinose, ribose, mannose, galactose, fructose and mixtures thereof, preferably glucose.

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