Genetically engineered host cells producing L-serine

Abstract

The present invention relates to genetically modified host cells that produce metabolites from 3-phosphoglycerate through a metabolic pathway, the host cells comprising one or more genetic modifications that prevent, reduce, or mitigate adverse effects on metabolite production resulting from intracellular accumulation of NADH and / or hydroxyglutarate (HGA) produced by one or more pathway enzymes.

Description

[Technical Field]

[0001] The present invention relates to the microbial industry, specifically to the bioindustrial production of metabolites, such as L-serine and derivatives, using genetically engineered bacteria. More specifically, the present disclosure describes recombinant host cells that produce metabolites from glyceraldehyde-3-phosphate through a metabolic pathway designed to reduce the intracellular accumulation of NADH and / or hydroxyglutarate (HGA), which have a detrimental effect on metabolite production. Recombinant polynucleotides encoding recombinant polypeptides of the pathway and cell cultures of the host cells that produce the metabolites when cultured in a fermentation process are also disclosed. Fermentation compositions of the host cells and / or their metabolites resulting from such processes, also known as biobased compositions of metabolites, are also disclosed. [Background technology]

[0002] Metabolites such as L-serine produced by genetically engineered host cells are known, for example, from International Publication No. WO 2016120326, which describes the production of L-serine using a genetically engineered microorganism defective in the serine degradation pathway. International Publication No. WO 2004 / 108894 discloses methods for the production of amino acids using genetically engineered bacteria, including disclosure of a polypeptide similar to SEQ ID NO: 10 of the present disclosure. International Publication No. WO 2021 / 081185 describes microorganisms with increased availability of cofactors, such as NADPH, to enhance the production of various products. International Publication No. WO 2020 / 0107626 describes a method for producing L-amino acids, comprising culturing modified bacterial cells with increased amounts of NADPH compared to intact bacterial cells, whereby the yield of L-amino acids from the modified bacterial cells is higher than the yield from intact bacterial cells. International Publication No. WO 2021 / 195705 describes a recombinant microorganism for biologically producing hydrogen and a nucleic acid construct, as well as a process for modifying the microorganism to enable hydrogen production. In this process, the gapA gene in E. coli is proposed to be replaced with the gapC gene from Clostridium acetobutylicum. Chinese Patent No. 103436504A describes a method for constructing and applying a Corynebacterium glutamicum strain resistant to feedback inhibition by L-serine by mutating 3-phosphoglycerate dehydrogenase resistant to feedback inhibition by L-serine. The 3-phosphoglycerate dehydrogenase shares slight similarity with SEQ ID NO: 10 of the present disclosure.

[0003] Furthermore, hydroxyglutarate (HGA) is known to be produced as a by-product in many different organisms, including humans. In humans, it is produced by isocitrate dehydrogenase (IDH) or phosphoglycerate dehydrogenase (SerA). These enzymes oxidize NADH to NAD+ and reduce α-ketoglutarate (KGA) to HGA, as shown in Figure 1a. 1 Accumulation of HGA has been observed in many types of cancer and is classified as an abnormal metabolite. 2 In prokaryotes, the main source of HGA production is SerA. The reason for such promiscuous reaction of SerA is unknown and it was thought to be a random reaction. 3 It has been shown very recently that the production of HGA is coupled to the native SerA reaction to lower the Gibbs free energy. SerA catalyzes the first step in L-serine biosynthesis, in which 3-phosphoglycerate (3-PG) is oxidized to 3-hydroxyphosphopyruvate (3-PY), a highly unfavorable reaction with a Gibbs free energy of over +30 kJ / mol. 4 On the other hand, the other two reactions for L-serine biosynthesis, catalyzed by SerC and SerB, have negative Gibbs free energies (Fig. 1b), making the SerA reaction the rate-determining step. To reduce the energy barrier, the oxidation of 3-PG was shown to be coupled with the reduction of KGA to HGA. HGA oxidases, such as LghO (formerly YgaF) in Escherichia coli (E. coli), are also involved. 3 and D-hydroxyglutarate dehydrogenase (D2DHH) in many Pseudomonas species 4 When activated, HGA is oxidized back to KGA. The resulting reducing equivalents are supplied to cytochrome c, which in turn transports oxygen to the electron transport chain for ATP production or hydrogen peroxide production. 3 .

[0004] SerAs have been classified into three types, namely type 1, type 2, and type 3, based on the presence of various protein domains. 1Some SerA homologs are known to lack KGA-reducing activity. However, such SerA proteins do not necessarily belong to one particular type or class of enzyme. For example, SerA from rat, M. tuberculosis, C. glutamicum, and B. subtilis does not appear to accumulate KGA. 4 However, human SerA, which belongs to the same class, tends to accumulate HGA. 5 It remains unknown how SerA, which lacks promiscuous KGA reduction activity, overcomes the free energy barrier to drive the reaction forward efficiently.

[0005] As mentioned above, SerA is the first major step in L-serine biosynthesis, so SerA from Escherichia coli (E. coli) has been overexpressed in most published studies to overproduce L-serine. 6 Because the oxidation of 3-PG is coupled to the reduction of KGA (Figure 1B), increased L-serine production leads to greater accumulation of KGA. 7 A clear strategy to avoid HGA accumulation has been demonstrated in C. glutamicum. 4、6 The two main strategies are to overexpress a heterologous SerA that has no activity towards KGA, as shown here, or to coexpress an HGA oxidase, as previously shown, to recycle HGA back to KGA.

[0006] Both approaches were tested, and expression of SerA lacking KGA reduction activity resulted in reduced growth and production of L-serine (Examples 5 and 6), whereas coexpression of LghO did not result in reduced levels of HGA accumulation (data not shown). A unique approach was taken, hypothesizing that HGA accumulation may be proportional to the NADH pool available to the cell, and that a reduction in this NADH pool would similarly reduce HGA accumulation. Furthermore, this approach would result in higher L-serine production when combined with overexpression of SerA with absent or reduced KGA activity.

[0007] In the literature, two strategies are applied to reduce the NADH pool: oxidizing NADH to NAD+ using NADH oxidase (Nox) 8 or replacing the NADH-producing reaction with an enzyme that produces NADPH, e.g., by making the NADPH-dependent glutamate dehydrogenase NADH-dependent. 9 or replacing 3-phosphoglycerate dehydrogenase with a heterologous NADPH variant. 10,11 All of the above approaches have been tried in the literature, but their effects on HGA accumulation have never been studied. Furthermore, we show that a reduction in the NADH pool is important for L-serine production from SerA, which lacks KGA reductive activity. Without these approaches, cell growth and L-serine production are hindered. Furthermore, L-serine is a precursor for many amino acids, such as cysteine, methionine, and tryptophan, and overexpression of SerA is a standard strategy to enhance the production of these compounds. 12,13 , the above strategies will also enhance their production. Summary of the Invention

[0008] One object of the present invention is to provide a means for enabling microbial cells to more efficiently produce metabolites from glyceraldehyde-3-phosphate, such as L-serine and its derivatives. More specifically, the object of the present invention is to provide a means for enabling the production of metabolites, such as L-serine or its derivatives, from glyceraldehyde-3-phosphate with higher nominal yields and improved mass yields. This is achieved by the discovery that the production of metabolites, such as L-serine, from glyceraldehyde-3-phosphate can be enhanced by designing a microorganism to prevent, reduce, or mitigate the adverse effects on metabolite production caused by the intracellular accumulation of NADH and / or hydroxyglutarate (HGA) produced by one or more enzymes in the pathway that produces the metabolite, including designing a bacterium to increase the expression of SerA and reduce the production of hydroxyglutarate.

[0009] In a first aspect, the present disclosure describes a genetically engineered host cell that produces a metabolite from 3-phosphoglycerate through a metabolic pathway, the host cell comprising one or more genetic modifications that prevent, reduce, or mitigate adverse effects on metabolite production due to intracellular accumulation of NADH and / or hydroxyglutarate (HGA) produced by one or more pathway enzymes.

[0010] In a second aspect, the present disclosure describes a cell culture comprising the host cells and growth medium described herein.

[0011] In a third aspect, the present disclosure provides a method for producing a metabolite from 3-phosphoglycerate, comprising: a) culturing a cell culture described herein under conditions that allow the host cells to produce 3-phosphoglycerate-derived metabolites; and b) optionally recovering and / or isolating the metabolites from 3-phosphoglycerate A method including:

[0012] In a fourth aspect, the present disclosure describes a fermentation composition comprising bio-based metabolites of a cell culture described herein and 3-phosphoglycerate-derived metabolites, wherein at least 20% by weight of the carbon is bio-based.

[0013] In a fifth aspect, the present invention provides a genetically modified bacterium that has been modified to increase expression of a polypeptide having D-3-phosphoglycerate dehydrogenase activity (SerA) and decrease production of hydroxyglutaric acid (HGA) compared to an identical bacterium that does not possess the modification.

[0014] In a sixth aspect, the present invention provides a method for producing L-serine, comprising culturing the bacterium according to the fifth aspect in a medium. [Brief explanation of the drawings]

[0015] [Figure 1] A. Reaction catalyzed by SerA. The native reaction involves the oxidation of PGA, while the promiscuous reaction involves the reduction of KGA. B. A potential reason for the production of HGA by SerA is that the overall Gibbs free energy of the reaction is reduced from 33 kJ / mol to 4.5 kJ / mol.4 [Figure 2] HGA concentrations detected in batch fermentation supernatants after 24 hours of incubation. The strain expressing NADH oxidase (Nox) accumulated 8-fold less HGA than the strain lacking NADH oxidase expression. [Figure 3] A. Schematic of NADH recycling for RocG-mediated L-serine production. [Figure 4A-4B] L-serine concentrations detected in 48- and 70-h fed-batch fermentation supernatants of E. coli strains expressing native gapA or heterologous gapC, both of which express either the E. coli-derived serACB (A) or the E. coli-derived serCB and C. glutamicum-derived serA (B) L-serine pathways. [Figure 5] Growth curves of genetically engineered bacteria with (serHM_608) and without (serHM_708) gapC expression obtained in a 24-hour batch fermentation in baffled shake flasks. Error bars represent the standard deviation of 2 / 3 biological replicates. [Figures 6a-6c] Plasmid maps of all plasmids used in Examples 1 to 5. [Figure 7] Plasmid maps of the plasmids used in Example 6. [Figure 8] L-serine concentrations detected in batch fermentation supernatants after 24 hours of incubation. Strains expressing truncated serA from rat exhibit increased L-serine production. [Figure 9] L-serine concentrations detected in batch fermentation supernatants after 24 h of incubation of strains expressing different NADPH glyceraldehyde 3-phosphate dehydrogenases. [Figure 10]Plasmid maps of the plasmids used in Example 8. [Figure 11] L-serine concentrations detected in batch fermentation supernatants after 24 hours of incubation. Strains expressing Nox from L. parakefiri show increased L-serine production. [Figure 12] Plasmid maps of all plasmids used in Example 9. [Figures 13a-13d] L-serine and HGA concentrations in batch fermentation supernatants after 24 h of incubation of strains overexpressing pgl and zwf in combination with serA from C. glutamicum in a gapC and gapA background, and SerA from E. coli in a gapC and gapA background. [Figure 14] The effect of replacing NADH-dependent glyceraldehyde-3-phosphate dehydrogenase (gapA) with NADPH-dependent glyceraldehyde-3-phosphate dehydrogenases (gapC and gdp1) is shown when serA, which lacks KGA reduction activity, is expressed. [Figure 15] This shows the pathway to L-serine, including steps from 3-phosphoglycerate. [Figure 16] The HMP shunt pathway using glucose-6-phosphate dehydrogenase (zwf) and 6-phosphogluconolactonase (pgl) is shown. DETAILED DESCRIPTION OF THE INVENTION

[0016] The terms "heterologous" or "recombinant" or "transgenic," and their grammatical equivalents, used interchangeably herein with respect to nucleotides, polypeptides, and cells, refer to entities "from a different species or cell." For example, a heterologous or recombinant polynucleotide gene is a gene in a host cell that does not naturally contain that gene, i.e., the gene is from a different species or cell type than the host cell. A heterologous or recombinant polypeptide is a polypeptide produced in a host cell that does not naturally contain that polypeptide, i.e., the polypeptide is from a different species or cell type than the host cell. The term "host cell," as used herein with respect to a host cell, refers to a host cell that contains and expresses a heterologous or recombinant polynucleotide. In some embodiments, "recombinant" or "non-naturally occurring," e.g., when used in reference to a host cell, nucleic acid, or polypeptide, refers to a material that has been modified in a way not otherwise found in nature, or is identical to it, but corresponds to the natural or native form of that material, produced or derived from synthetic material and / or by manipulation using recombinant technology. Non-limiting examples include recombinant host cells that express genes not found in the native (non-recombinant) form of the cell, or that express native genes that are otherwise expressed at different levels, among others. As used herein, "heterologous" means that the polypeptide is not normally found in or made (i.e., not expressed) by the host organism, but is derived from a different species.

[0017] The term "% identity" is used herein to describe the relationship between two amino acid sequences or two nucleotide sequences using standard alignment software known in the art, applying the settings as directed for the software, including gaps, to achieve the maximum percent identity / similarity / homology, and taking into account any conservative substitutions as part of the sequence identity, as appropriate, in accordance with NCIUB rules (http: / / www.chem.qmul.ac.uk / iubmb / misc / naseq.html; NC-IUB, Eur. J. Biochem. (1985)). Using such standard software, 5' or 3' extensions or insertions (in nucleic acids) or N' or C' extensions or insertions (in polypeptides) do not usually result in a loss of identity, similarity, or homology. Thus, "percent sequence identity," "% sequence identity," and "percent identity" may be used herein to refer to a comparison of an amino acid sequence to a reference amino acid sequence. For example, "% sequence identity," as used herein, is calculated from two amino acid sequences as follows: the sequences are aligned using Genetic Computing Group's GAP (Global Alignment Program) version 9, using the default BLOSUM62 matrix (see below), with a gap opening penalty of -12 (for the first null in a gap) and a gap extension penalty of -4 (for each additional null within that gap). After alignment, the percent identity is calculated by expressing the number of matches as a percentage relative to the number of amino acids in the reference amino acid sequence. The following BLOSUM62 matrix is ​​used: [ka]

[0018] A "reference sequence" or "reference amino acid sequence" refers to a defined sequence to which another sequence is compared. In the context of the present invention, a reference amino acid sequence can be, for example, the amino acid sequence set forth in SEQ ID NO: 5 or 6.

[0019] "Substitution" or "substituted" refers to the modification of a polypeptide by replacing one amino acid residue with another, e.g., replacing a serine residue with a glycine or alanine residue in a polypeptide sequence is an amino acid substitution. When used in reference to a polynucleotide, "substitution" or "substituted" refers to the modification of a polynucleotide by replacing one nucleotide with another, e.g., replacing a cytosine with a thymine in a polynucleotide sequence is a nucleotide substitution.

[0020] " Conservative substitution " when used in relation to polypeptide refers to the substitution of amino acid residue with a different residue with a similar side chain, and therefore typically includes the substitution of amino acid in polypeptide with the amino acid of the same or similar class of amino acid.By way of example and not limitation, the amino acid with aliphatic side chain can be substituted with another aliphatic amino acid, such as alanine, valine, leucine and isoleucine; the amino acid with hydroxyl side chain can be substituted with another amino acid with hydroxyl side chain, such as serine and threonine; the amino acid with aromatic side chain can be substituted with another amino acid with aromatic side chain, such as phenylalanine, tyrosine, tryptophan and histidine; the amino acid with basic side chain can be substituted with another amino acid with basic side chain, such as lysine and arginine; the amino acid with acidic side chain can be substituted with another amino acid with acidic side chain, such as aspartic acid or glutamic acid; and hydrophobic or hydrophilic amino acid can be substituted with another hydrophobic or hydrophilic amino acid, respectively.

[0021] "Non-conservative substitution," when used in reference to a polypeptide, refers to the substitution of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitutions may use amino acids between, rather than within, defined groups and affect (a) the structure of the peptide backbone in the area of ​​substitution (e.g., glycine for serine), (b) the charge or hydrophobicity, or (c) the bulk of the side chain. By way of example and not limitation, representative non-conservative substitutions may be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.

[0022] The terms "pathway" or "biosynthetic pathway" or "metabolic pathway," as used interchangeably herein, refer to one or more enzymes that work together in a living cell to convert one or more substrate precursors into a chemical product. A pathway may include one enzyme or multiple enzymes that function in sequence or in combination. Pathways that include only one enzyme may also be referred to as "bioconversions," and are particularly suited to embodiments in which precursors or substrates are externally supplied to the host cell and enzymatically converted into a desired end product. Enzymes are characterized by having catalytic activity that can change the chemical structure of a substrate. Enzymes may have multiple substrates and produce multiple products. Enzymes may also depend on cofactors, which may be inorganic or organic compounds (cofactors and / or coenzymes) and may or may not be considered part of a pathway.

[0023] As used herein, the term "in vivo" refers to within a living cell or organism, including, for example, an animal, plant, or microorganism.

[0024] As used herein, the term "in vitro" refers to outside a living cell or organism, including but not limited to, for example, in a microwell plate, in a test tube, in a flask, in a beaker, in a tank, in a reactor, etc.

[0025] As used herein, the term "substrate" or "precursor" refers to any compound that can be converted into a different compound. For clarity, substrate and / or precursor include both compounds that are produced in situ by an enzymatic reaction within a cell or exogenously provided compounds, e.g., exogenously provided organic molecules that the host cell can metabolize into a desired compound.

[0026] The term "expression" includes any step that involves the production of a polypeptide (e.g., an encoded enzyme), including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0027] The term "expression vector" refers to a DNA molecule, either single-stranded or double-stranded, linear or circular, containing a polynucleotide encoding a polypeptide, operably linked to a control sequence that confers its expression. Expression vectors include expression cassettes for the integration of a gene into a host cell, as well as plasmids and / or chromosomes containing such a gene. A vector capable of directing the expression of an operably linked gene is referred to herein as an expression vector. A "vector" can also refer to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded nucleic acid loop into which additional nucleic acid segments can be ligated. Certain other vectors can facilitate the insertion of foreign nucleic acid molecules into bacterial genomes. Such vectors are referred to herein as "transformation vectors." Generally, vectors useful in recombinant nucleic acid technology are often in the form of plasmids. Because plasmids are one of the most commonly used forms of vectors, the terms "plasmid" and "vector" can be used interchangeably herein. Many suitable vectors are known to those of skill in the art and are commercially available.

[0028] The term "host cell" refers to any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector containing a polynucleotide to be expressed in the host cell. A host cell includes any progeny of a parent cell, including those that are not identical to the parent cell due to mutations that occur during replication.

[0029] The terms "nucleic acid" or "polynucleotide" are used interchangeably herein and refer to a polymer of at least two nucleic acid monomer units or bases (e.g., adenine, cytosine, guanine, thymine) covalently linked by phosphodiester bonds, regardless of length or base modification.

[0030] The term "polynucleotide construct" refers to a polynucleotide, either single-stranded or double-stranded, isolated from a naturally occurring gene or modified to contain a segment of nucleic acid in a manner not occurring in nature, or synthesized, that includes a polynucleotide encoding a polypeptide and one or more regulatory sequences.

[0031] The term "operably linked" refers to a configuration in which a control sequence is positioned in an appropriate position relative to a coding polynucleotide so that the control sequence directs expression of the coding polynucleotide. More generally, "operably linked" refers to a juxtaposition in which the described components are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. A promoter sequence is "operably linked" to a gene if it is positioned sufficiently close to the transcription start site of the gene that it controls the transcription of the gene.

[0032] As used herein, "promoter" refers to a sequence of DNA, generally upstream (5') of the coding region of a structural gene, that controls expression of the coding region by providing a recognition and binding site for RNA polymerase and other factors that may be required for initiation of transcription. The choice of promoter is determined by the nucleic acid sequence of interest. A suitable "promoter" is generally one that is capable of supporting the initiation of transcription in the bacteria of the invention, resulting in the production of an mRNA molecule.

[0033] "Polypeptide" and "protein" are used interchangeably herein and refer to polymers of at least two amino acids covalently joined by amide bonds, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristylation, ubiquitination, etc.). Included in this definition are D- and L-amino acids, and mixtures of D- and L-amino acids.

[0034] As used herein, the term "bio-based" is used to characterize bio-based products: a) the total carbon content of the product is at least 30%; b) The carbon content from renewable sources (bio-based) is at least 20%.

[0035] As recognized by the Circular Biobased Europe Consortium (CBE Consortium), established in 2021, the development of biobased materials is essential if the EU is to reach its climate goals, such as those set out in the European Green Deal. In the present disclosure, methods are provided for efficiently providing fatty alcohols and fatty aldehydes with a high percentage of biobased carbon.

[0036] Both fossil and renewable sources consist primarily of carbon (C). Carbon exists in several isotopes. Isotopes 14 C is radioactive and in the atmosphere 14 It occurs naturally in all living organisms (plants, animals, etc.) at a fixed relative concentration that is approximately the same as the relative concentration of C. At this concentration,14 The radioactivity level of C is 100%. Once it becomes unviable for living organisms, this concentration, and therefore the radioactivity rate, decays with a half-life of approximately 5700 years. 14 The C level can help determine how old the carbon contained in the material is.

[0037] "Young" carbon (0-10 years old) derived from renewable sources, e.g., plants or animals, is the carbon that is in the atmosphere. 14 Relative concentrations of C and nearly identical isotopes 14 C relative concentration and therefore the radioactivity of such young carbon 14 The C level is approximately 100%.

[0038] Such isotopes from synthetic and fossil sources have an age of approximately 5700 years. 14 "Old" carbon (millions of years old) from synthetic or fossil (petrochemical) sources isotopes because it is far beyond the half-life of C. 14 Carbon from synthetic or fossil sources is therefore isotopically 14 The relative concentration of C is about 0%, so the radioactivity of such old carbon 14 C levels are around 0%.

[0039] In one embodiment, the term "radioactive 14 "C Level" means the total radioactivity of a particular substance, product or composition as defined above. 14 Refers to C level.

[0040] Isotopes 14 The C method can be used to determine the concentration of carbon in young (renewable) materials compared to the concentration in older (fossil) resources. The carbon content of renewable feedstocks is called the "biobased carbon content." The carbon content or "biobased carbon content" of renewable feedstocks can be determined as described below.

[0041] When measuring biobased carbon content, the results may be reported as "% biobased carbon." This indicates the percentage of carbon derived from "natural" (plant or animal by-product) sources versus "synthetic" or "fossil" (petrochemical) sources. For reference, 100% biobased carbon indicates that the material is sourced entirely from plant or animal by-products, while 0% biobased carbon indicates that the material contained no carbon derived from plant or animal by-products. Values ​​in between represent a mixture of natural and fossil sources. For example: Radioactivity of the product 14 A C level of 80% means that the product is made up of 80% renewable feedstock and 20% fossil carbon (C). In other words, the product is 80% biobased. Analytical measurements are sometimes expressed as "percent modern carbon (pMC)." This is the percentage of modern carbon measured in a sample relative to a modern standard (NIST 4990C). 14 The % biobased carbon content is the ratio of carbon dioxide in the air today to the carbon dioxide in the atmosphere. 14 It is calculated from pMC by applying a small adjustment factor to C. 14 It is important to note that all internationally recognized standards that use C assume that the plant or biomass feedstock was obtained from the natural environment. pMC can be analyzed by standard test methods, such as ASTM D6866.

[0042] The terms "nucleotide sequence" and "polynucleotide" are used interchangeably herein.

[0043] As used throughout this specification, the terms "comprise" and "include" and accompanying terms and variations thereof, such as "comprises," "comprising," "includes," and "including," are to be interpreted inclusively. These words are intended to convey that other elements or integers not specifically stated may be included, where the context so permits.

[0044] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to one or at least one) of the grammatical object of the article. By way of example, "an element" may mean one element or more than one element.

[0045] Terms such as "preferably," "generally," "particularly," and "typically" are not used herein to limit the scope of the specified invention or to imply that a particular feature is critical, essential, or even essential to the structure or function of the specified invention. Rather, these terms are merely intended to highlight alternative or additional features that may or may not be available in a particular embodiment of the invention.

[0046] As used herein, the term "cell culture" refers to a medium containing a large number of host cells as described herein. A cell culture may contain a single strain of host cells or may contain two or more separate host cell strains. The medium may be any medium that may contain a recombinant host, such as a liquid medium (i.e., culture broth) or a semi-solid medium, and may contain additional components, such as a carbon source, a nitrogen source, a phosphate source, vitamins, trace elements, salts, amino acids, nucleic acid bases, etc.

[0047] As used herein, the term "endogenous" or "native" refers to a gene or polypeptide within a host cell that originates from the same host cell.

[0048] As used herein, the terms "substantially" or "approximately" or "about" refer to a reasonable deviation around a value or parameter such that the value or parameter does not change significantly. These terms referring to deviations from a value should be interpreted as including deviations from that value if the deviation does not negate the meaning of the deviating value. For example, in connection with a reference numerical value, a term of degree can include a range of values ​​plus or minus 10% from that value. For example, deviations from a value can include the specified value plus or minus a certain percentage from that value, such as 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% plus or minus from the specified value.

[0049] Where numerical limits or ranges are stated herein, the endpoints are included, and all values ​​and subranges within the numerical limits or ranges are specifically included as if expressly written out.

[0050] As used herein, the term "and / or" is intended to represent an inclusive "or." The phrase X and / or Y is intended to mean X or Y, as well as both X and Y. Furthermore, the phrase X, Y and / or Z is intended to mean X, Y and Z alone or any combination of X, Y and Z.

[0051] The term "isolated," as used herein with respect to a compound, refers to any compound that has been placed, through human intervention, in a form or environment different from that in which it is found in nature. Isolated compounds include, but are not limited to, compounds of the present disclosure in which the ratio of the compound to other components with which it is associated in nature is increased or decreased. In important embodiments, the amount of the compound is increased relative to other components with which it is associated in nature. In one embodiment, a compound of the present disclosure can be isolated in pure or substantially pure form. In this context, a substantially pure compound means that the compound has been separated from other extraneous or unwanted substances that are present from the beginning of the compound's production or that are generated during the production process. Such a preparation of a substantially pure compound contains less than 10%, e.g., less than 8%, e.g., less than 6%, e.g., less than 5%, e.g., less than 4%, e.g., less than 3%, e.g., less than 2%, e.g., less than 1%, e.g., less than 0.5%, by weight, of other extraneous or unwanted substances normally associated with the naturally expressed or recombinantly expressed compound. In one embodiment, the isolated compound is at least 90% pure, such as at least 91% pure, for example at least 92% pure, such as at least 93% pure, for example at least 94% pure, such as at least 95% pure, for example at least 96% pure, such as at least 97% pure, for example at least 98% pure, such as at least 99% pure, for example at least 99.5% pure, such as 100% pure, by weight.

[0052] The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from a mature, spliced ​​mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as a mature, spliced ​​mRNA.

[0053] As used herein, the term "GAPDH" refers to NAD + or NADP+ GapA refers to the enzyme glyceraldehyde-3-phosphate dehydrogenase, which converts glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate while simultaneously converting NAD to NADH or NADPH, respectively. + GapC is an example of a GAPDH that produces 1,3-bisphosphoglycerate while simultaneously converting NADP to NADH. + This is an example of a GAPDH that produces 1,3-bisphosphoglycerate while simultaneously converting 1,3-bisphosphoglycerate to NADPH.

[0054] As used herein, the term "Nox" refers to the conversion of NADH to NAD + This refers to the NADH oxidase enzyme that converts

[0055] As used herein, the term "PGDH" or "3-PGDH" or "PHGDH" refers to the enzyme 3-phosphoglycerate dehydrogenase, which catalyzes the conversion of 3-phosphoglycerate to 3-phosphohydroxypyruvate with the simultaneous reduction of NAD to NADH. An example of a PGDH is SerA in the serine pathway.

[0056] As used herein, the term "GDH" refers to the glutamate dehydrogenase enzyme, which catalyzes the conversion of α-ketoglutarate to glutamate.

[0057] As used herein, the term "deletion," in the context of polynucleotides and genes, refers to the manipulation of a gene so that it is no longer expressed in a host cell. "Deletion" or "deleted," when used in reference to a polypeptide, refers to the modification of the polypeptide by removing one or more amino acids within the reference polypeptide. Deletions can include the removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, up to 10% of the total number of amino acids comprising the polypeptide, or up to 20% of the total number of amino acids, while retaining enzymatic activity and / or improving the properties of the recombinant enzyme. Deletions can be directed to internal and / or terminal portions of the polypeptide, and in various embodiments, deletions can include contiguous or non-contiguous segments.

[0058] As used herein, the term "disruption" refers to manipulating either a gene or the machinery involved in the expression of that gene so that the gene is not expressed in a host cell.

[0059] As used herein, the term "attenuated" refers to the manipulation of a gene or any mechanism involved in the expression of that gene, thereby reducing the expression of the gene compared to the expression in the absence of that manipulation.

[0060] "Insertion" or "inserted," when used in reference to a polypeptide, refers to the modification of a polypeptide by the addition of one or more amino acids to a reference polypeptide. An insertion can include the addition of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids. Insertions can be made in an internal portion of the polypeptide, or at the carboxy or amino terminus. Insertions can be a contiguous segment of amino acids or can be separated by one or more amino acids in the reference polypeptide.

[0061] As used herein, the phrase "a bacterium that has been modified to increase expression of a polypeptide having D-3-phosphoglycerate dehydrogenase activity (serA) and decrease production of HGA" means that the bacterium has been modified such that a) the recombinant bacterium has a higher level of expression of D-3-phosphoglycerate dehydrogenase (serA) compared to other identical bacteria that do not possess the modification, and b) the recombinant bacterium has a lower production of HGA compared to other identical bacteria that do not possess the modification. It should be understood that "modification" can encompass one or more separate genetic modifications to achieve the described effect.

[0062] The presence or absence of a gene on a bacterial chromosome can be detected by well-known methods, including PCR, Southern blotting, etc. Furthermore, the level of gene expression can be estimated by measuring the amount of mRNA transcribed from the gene using various well-known methods, including Northern blotting, quantitative RT-PCR, etc. The amount of protein encoded by the gene can be measured by well-known methods, including SDS-PAGE followed by immunoblotting assay (Western blotting analysis), etc.

[0063] As used herein, "genetically modified bacteria" refers to bacteria into which genetic modifications have been introduced, such as the introduction of a new gene, an additional copy of a gene, a modified gene, or a change in the sequence controlling gene expression. As used herein, "L-serine derivatives" refer to amino acids resulting from the reaction of L-serine with a compound, such as an amino group, a carboxyl group, or a hydroxyl group, or the replacement of any hydrogen in L-serine with a heteroatom. Non-limiting examples of "L-serine derivatives" include L-cysteine, L-methionine, L-glycine, O-acetylserine, L-tryptophan, thiamine, ethanolamine, and ethylene glycol. Further examples of "L-serine derivatives" are described by the Chemical Entities of Biological Interest (ChEBI) [https: / / www.ebi.ac.uk / chebi / init.do], for example, in ChEBI ID CHEBI:84135.

[0064] All methods described herein can be performed in any suitable order of steps unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or representative language (e.g., "such as") provided herein is intended merely to better elucidate the invention and does not impose limitations on the scope of the otherwise claimed invention. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0065] All percentages, ratios, and proportions herein are by weight unless otherwise specified. Weight percentages (also wt.%) of components are based on the total weight of the composition in which the component is included (e.g., of the total reaction mixture), unless specifically stated to the contrary.

[0066] Unless specifically defined herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art of biochemistry, genetics, and microbiology.

[0067] All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, and suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting unless otherwise specified.

[0068] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, and recombinant DNA, which are available to those skilled in the art and are fully explained in the literature. For example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (MJGait Nucleic Acid Hybridization (BD Harries & S. J. Higgins eds. 1984); Transcription And Translation (BD Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (RIFreshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York), specifically, volumes 154 and 155 (Wu et al., eds.) and volume 185, "Gene Expression Technology" (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (JH Miller and MPCalos, eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D.M. Weir and C.C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986).

[0069] Genetically engineered host cells As described in the first aspect above, the genetically engineered host cells described herein produce metabolites from 3-phosphoglycerate through a metabolic pathway that includes one or more genetic modifications that prevent, reduce, or mitigate the adverse effects on metabolite production of intracellular accumulation of NADH and / or hydroxyglutarate (HGA) produced by one or more pathway enzymes. In a preferred embodiment, the metabolite is L-serine or a derivative thereof. The L-serine pathway is shown in Figure 15.

[0070] In some embodiments, the host cell comprises: I. Expression of a first heterologous NADPH-generating enzyme that converts glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate or a downstream precursor in the metabolic pathway; II. Expression of heterologous enzymes that convert NADH to NAD+; III. Expression of heterologous enzymes in metabolic pathways that reduce or eliminate NADH-consuming side activities compared to the corresponding pathway enzymes native to the host cell; IV. Expression of a heterologous enzyme that converts a by-product of a metabolic pathway enzyme into a substrate for an NADH- or NADPH-consuming metabolic pathway enzyme; V. Overexpression of native enzymes that convert by-products of enzymes in metabolic pathways into substrates for NADH- or NADPH-consuming metabolic pathway enzymes; VI. Expression of a second heterologous NADPH-producing enzyme not involved in the metabolic pathway; and / or VII. Overexpression of natural NADPH-producing enzymes not involved in metabolic pathways In some cases, the vector is genetically engineered to contain one or more, optionally two or more, optionally three or more, optionally four or more, optionally five or more, or optionally seven genetic modifications selected from:

[0071] The inventors have found that inhibiting intracellular accumulation of NADH improves the efficiency of downstream pathways that produce metabolites, particularly L-serine and / or its derivatives, from 3-phosphoglycerate, and therefore, in a further embodiment, the genetic modification of the host cell comprises expression of a first heterologous NADPH-generating enzyme that converts glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate or a precursor for the downstream metabolic pathway.

[0072] The present inventors have also found that combining two or more of the above modifications I) to VII) is particularly advantageous and synergistic for a host cell to produce a metabolite from 3-phosphoglycerate, and in a further embodiment, the host cell is genetically modified to: a) expression of a first heterologous NADPH-generating enzyme that converts glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate or a downstream precursor in a metabolic pathway; and b) Expression of heterologous metabolic pathway enzymes that have reduced or eliminated NADH-consuming side activities compared to the corresponding metabolic pathway enzymes native to the host cell. Includes.

[0073] The present inventors have also found that combining three or more of the above modifications I) to VII) is particularly advantageous and synergistic for a host cell to produce a metabolite from 3-phosphoglycerate, and in a further embodiment, the host cell is genetically modified to: a) Expression of a first heterologous NADPH-generating enzyme that converts glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate or a downstream precursor in a metabolic pathway; b) expression of heterologous metabolic pathway enzymes that have reduced or eliminated NADH-consuming side activities compared to the corresponding metabolic pathway enzymes native to the host cell; and c) Expression of a heterologous enzyme that converts NADH to NAD+ Includes.

[0074] The present inventors have also found that combining four or more of the above modifications I) to VII) is particularly advantageous and synergistic for a host cell to produce a metabolite from 3-phosphoglycerate, and in a further embodiment, the host cell is genetically modified to: a) Expression of a first heterologous NADPH-generating enzyme that converts glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate or a downstream precursor in a metabolic pathway; b) expression of heterologous metabolic pathway enzymes that have reduced or eliminated NADH-consuming side activities compared to the corresponding metabolic pathway enzymes native to the host cell; c) expression of a heterologous enzyme that converts NADH to NAD+; and d) Expression of heterologous enzymes and / or overexpression of native enzymes that convert by-products of metabolic pathway enzymes into substrates for NADH- or NADPH-consuming metabolic pathway enzymes. Includes.

[0075] The present inventors have also found that combining five or more of the above modifications I) to VII) is particularly advantageous and synergistic for a host cell to produce a metabolite from 3-phosphoglycerate, and in a further embodiment, the host cell is genetically modified to: a) Expression of a first heterologous NADPH-generating enzyme that converts glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate or a downstream precursor in a metabolic pathway; b) expression of heterologous metabolic pathway enzymes that have reduced or eliminated NADH-consuming side activities compared to the corresponding metabolic pathway enzymes native to the host cell; c) expression of a heterologous enzyme that converts NADH to NAD+; d) Expression of heterologous enzymes and / or overexpression of native enzymes that convert by-products of enzymes in metabolic pathways into substrates for metabolic pathway enzymes that consume NADH or NADPH; and e) Expression of a second heterologous NADPH-producing enzyme and / or overexpression of a native NADPH-producing enzyme, neither of which is involved in the metabolic pathway. Includes.

[0076] In further embodiments, the first heterologous NADPH-generating enzyme, the heterologous enzyme with reduced or eliminated NADH-consuming side activities, the heterologous enzyme that converts side products to pathway substrates, and / or the second heterologous enzyme that produces NADPH partially or completely replaces an enzyme native to the host cell, which may or may not be part of a metabolic pathway.

[0077] In some embodiments, the NADPH pool of a genetically modified bacterium is increased by recombinant expression of an NADPH-dependent polypeptide with glyceraldehyde-3-phosphate dehydrogenase activity, with or without additional ATP generation.

[0078] In further embodiments, the first heterologous NADPH-generating enzyme that converts glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate or a downstream precursor in a metabolic pathway can be bisphosphoglycerate synthase or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), both of which convert glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate while simultaneously converting NADP+ to NADPH. SEQ ID NOs: 23-46 disclose some representative glyceraldehyde-3-phosphate dehydrogenases, and in some embodiments, the GAPDH enzyme comprises a polypeptide that is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to a GAP included in any one of SEQ ID NOs: 23-46. GAPDHs of SEQ ID NOs: 38-46 are particularly useful. In particular, the GAPDH is GapC or an NADP-dependent variant thereof comprising a polypeptide which is at least 20%, such as at least 40%, for example at least 50%, such as at least 60%, for example at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99%, such as 100% identical to the GAPDH contained in SEQ ID NO: 43.

[0079] In some embodiments, the NADH pool of the genetically modified bacterium is reduced by recombinant expression of a heterologous NADH oxidase, and thus in further embodiments, the heterologous enzyme that converts NADH to NAD+ is an NADH oxidase (Nox). SEQ ID NOs: 49-56 disclose some representative NADH oxidases, and in further embodiments, Nox comprises a polypeptide that is at least 20%, such as at least 40%, for example at least 50%, such as at least 60%, for example at least 70%, for example at least 80%, such as at least 90%, for example at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99%, such as 100% identical to a Nox comprised in any one of SEQ ID NOs: 49-56.

[0080] In a further embodiment, the metabolic pathway enzyme with a reduced or eliminated NADH-consuming secondary activity is 3-phosphoglycerate dehydrogenase (PGDH), also known as D-3-phosphoglycerate dehydrogenase, whose secondary activity is the conversion of α-ketoglutarate (α-KGA) to α-hydroxyglutarate (α-HGA). D-3-phosphoglycerate dehydrogenase (PGDH) converts D-3-phosphoglycerate (PGA) to phosphohydroxypyruvate (PHP) in the first step of L-serine biosynthesis. This reaction is reversible, and some PGDH can use α-ketoglutarate (αKG) instead of PHP in the reverse direction to produce α-hydroxyglutarate.

[0081] Preferably, the PGDH is heterologous, and more preferably, the heterologous PGDH is overexpressed compared to native PGDH, which has the secondary activity of converting α-ketoglutarate (α-KGA) to α-hydroxyglutarate. The overexpression of heterologous PGDH can be 10% to 10,000%, for example, 50% to 5000%, for example, 100% to 1000%, compared to native PGDH. PGDH or D-3-phosphoglycerate dehydrogenase (serA) has been classified into three types, namely, type 1, type 2, and type 3, based on the presence of various protein domains. 1 Some D-3-phosphoglycerate dehydrogenases lack KGA-reducing activity. However, such D-3-phosphoglycerate dehydrogenases do not necessarily belong to one type or class of enzyme. For example, SerA from Rattus norvegicus, M. tuberculosis, C. glutamicum, and B. subtilis appear to lack KGA accumulation. 4 However, human SerA has a tendency to accumulate HGA, but it belongs to the same class. 14 Three enzymes have been found to be able to utilize αKG as a substrate: PDGH from E. coli, Pseudomonas stutzeri, and Saccharomyces cerevisiae, all of which are type II PGDHs. We hypothesize that the PGDHs that cannot use αKG as a substrate are type I and type III PGDHs. We found that for each of the three types of PGDHs, several regions facing the active site contain motifs that are conserved in both type I and type II PHDGs. One such motif is the αKG motif in type I and type II PGDHs, respectively. [ka] One clear and highly conserved difference between these two motifs is the type of residue at the second position. In type I PGDH, this is an arginine residue, whereas in type II enzymes, it is most often a cysteine ​​residue. In the structures of M. tuberculosis and E. coli PGDH with PHP and αKG bound, respectively, both arginine and cysteine ​​residues were found to face the active site, suggesting that these residues are involved in controlling substrate specificity, allowing αKG to be used as a substrate. Furthermore, by using site-directed mutagenesis to replace the arginyl side chain in M. tuberculosis PGDH with other selected amino acid side chains, such as alanine and leucine, the cationic group of Arg72 in M. tuberculosis PGDH was found to change its specificity from not accepting αKG as a substrate to accepting αKG as a substrate. However, replacement of the arginyl side chain with another cationic moiety (lysyl side chain) does not result in a change in enzyme specificity. These results clearly demonstrate that the presence of a cationic side chain at the second position of the conserved motif prevents the enzyme from using αKG. Therefore, in a preferred embodiment, heterologous PGDH is constructed using the motif [ka] wherein the underlined residues are [ka] is a cationic residue at a position corresponding to position 129 of PGDH from Mycobacterium tuberculosis (SEQ ID NO: 10), preferably at the intracellular pH of the host cell. Useful PDGHs are particularly [ka] is selected from arginine, leucine or histidine, more particularly arginine. Additionally or alternatively, useful heterologous PGDHs may have the motif [ka] wherein the underlined cysteine ​​is at a position corresponding to position 129 of PGDH from Mycobacterium tuberculosis (SEQ ID NO: 10).

[0082] In a further embodiment, the heterologous PGDH is a mutant PGDH, optionally native to the host cell, that reduces or eliminates NADH-consuming side activities compared to unmodified PGDH, e.g., at a position corresponding to position 129 of PGDH from Mycobacterium tuberculosis (SEQ ID NO: 10), optionally containing the motif [ka] is modified by replacing the cysteine ​​with a cationic residue such as Arg, Leu or His, particularly Arg.

[0083] In some embodiments, the heterologous PGDH may be type I or type III PGDH, optionally a microbial type I or type III PGDH, or even a SerA enzyme.

[0084] SEQ ID NOs: 1-22 disclose some representative D-3-phosphoglycerate dehydrogenases, particularly useful PGDH enzymes are those comprising a polypeptide which is at least 20%, such as at least 40%, for example at least 50%, such as at least 60%, for example at least 70%, for example at least 80%, such as at least 90%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, such as 100% identical to a PGDH comprised in any one of SEQ ID NOs: 1-22. More specifically, the PGDH does not produce HGA or is insensitive to L-serine feedback, and comprises a polypeptide that is at least 20%, such as at least 40%, for example at least 50%, for example at least 60%, for example at least 70%, for example at least 80%, for example at least 90%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, or for example 100% identical to a PGDH comprised in any one of SEQ ID NOs: 6 to 22. In a further embodiment, the PGDH does not produce HGA and comprises a polypeptide that is at least 20%, for example at least 40%, for example at least 50%, for example at least 60%, for example at least 70%, for example at least 80%, for example at least 90%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, or for example 100% identical to a PGDH comprised in any one of SEQ ID NOs: 6 to 15. In a still further embodiment, the PGDH is a non-HGA producing C. glutamicum PGDH or a derivative thereof, comprising a polypeptide which is at least 20%, such as at least 40%, for example at least 50%, for example at least 60%, such as at least 70%, for example at least 80%, for example at least 90%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, for example 100% identical to the PGDH contained in SEQ ID NO: 14, or 19-21, in particular SEQ ID NO: 14.

[0085] In a further embodiment, both the heterologous PGDH, particularly serA from C. glutamicum (SEQ ID NO: 14, or 19-21), and the heterologous GAPDH, particularly GapC (SEQ ID NO: 43), are included. As indicated above, the present invention is based, inter alia, on the finding that L-serine production can be enhanced by, for example, increasing SerA expression and reducing hydroxyglutarate (HGA) production.

[0086] The reduction in HGA production can be achieved by a modification that increases the cytosolic NADPH pool and / or decreases the cytosolic NADH pool compared to an otherwise identical bacterium that does not carry said modification.

[0087] Reduced HGA production can also be achieved by modifications that result in the expression of a polypeptide having D-3-phosphoglycerate dehydrogenase activity, resulting in reduced or no HGA production.

[0088] Thus, the present invention provides genetically modified bacteria, particularly bacteria capable of producing L-serine, wherein the bacteria have been modified to enhance expression of a polypeptide having D-3-phosphoglycerate dehydrogenase activity (serA) and to reduce production of HGA compared to an otherwise identical bacterium that does not carry the modification.

[0089] In embodiments in which the enzyme converts a by-product of an enzyme in a metabolic pathway into a substrate for the metabolic pathway enzyme, the metabolic pathway enzyme is preferably glutamate dehydrogenase (GDH), the by-product is preferably α-ketoglutarate, and the substrate is glutamate. In more specific embodiments, the GDH comprises a polypeptide that is at least 20%, such as at least 40%, for example at least 50%, such as at least 60%, for example at least 70%, for example at least 80%, such as at least 90%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, or such as 100% identical to a GDH comprised in any one of SEQ ID NOs: 57-66.

[0090] In embodiments in which the host cell expresses a second heterologous NADPH-producing enzyme and / or overexpresses a native NADPH-producing enzyme, neither of which is included in the metabolic pathway, the second heterologous or native NADPH-producing enzyme is preferably glucose-6-phosphate dehydrogenase and / or 6-phosphogluconolactonase, respectively. In more specific embodiments, the glucose-6-phosphate dehydrogenase comprises a polypeptide that is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the glucose-6-phosphate dehydrogenase contained in SEQ ID NO: 47. This glucose-6-phosphate dehydrogenase is also known as "zwf." In a more specific embodiment, the 6-phosphogluconolactonase comprises a polypeptide which is at least 20%, such as at least 40%, for example at least 50%, such as at least 60%, for example at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99%, such as 100% identical to the 6-phosphogluconolactonase contained in SEQ ID NO: 48. This 6-phosphogluconolactonase is also known as "pgl".

[0091] In a further embodiment, the host cell: a) phosphoserine aminotransferase (PSAT), which converts 3-phosphohydroxypyruvate to phosphoserine; and b) Phosphoserine phosphatase (PSPH), which converts phosphoserine to L-serine The host expresses one or more heterologous or native metabolic pathway enzymes selected from:

[0092] The PSAT may be a SerC enzyme, and in some embodiments comprises a polypeptide that is at least 20%, such as at least 40%, for example at least 50%, for example at least 60%, such as at least 70%, for example at least 80%, such as at least 90%, for example at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99%, such as 100% identical to the serC contained in SEQ ID NO: 117.

[0093] The PSPH may be SerB, and in some embodiments comprises a polypeptide that is at least 20%, such as at least 40%, for example at least 50%, such as at least 60%, for example at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99%, such as 100% identical to SerB contained in SEQ ID NO: 118.

[0094] The heterologous enzyme expressed by the host cell described herein can be (i) an enzyme from a species different from the host cell, (ii) a mutant enzyme from a species different from the host cell, and / or (iii) a mutant enzyme native to the host cell. In further embodiments, the host cell further comprises at least one transporter molecule facilitating transport of a metabolite or one of its precursors. In further embodiments, one or more native or endogenous genes of the host cell are attenuated, disrupted, and / or deleted, such as native genes encoding NADH-dependent GAPDH or α-HGA-producing PHDH. In further embodiments, the host cell further comprises at least two copies of one or more polynucleotides encoding one or more metabolic pathway enzymes. In further embodiments, the host cell is further genetically engineered to increase the amount of substrate for one or more metabolic pathway enzymes. In further embodiments, the host cell is further genetically engineered to exhibit increased tolerance for one or more precursor, substrate, intermediate, or product molecules from a metabolic pathway.

[0095] In further embodiments, the host cell of any one of the preceding claims is a prokaryotic cell, optionally a bacterium. The prokaryotic cell can be of the phylum Pseudomonadota, optionally the class Gammaproteobacteria, optionally the family Enterobacteriaceae, optionally the genus Escherichia, or optionally a species of Escherichia coli. In some embodiments, the genetically modified bacterium belongs to the family Enterobacteriaceae. In some embodiments, the genetically modified bacterium belongs to the genus Escherichia. In some embodiments, the genetically modified bacterium is Escherichia coli. Additionally or alternatively, the prokaryotic cell is of the phylum Actinomycetota, optionally of the class Actinobacteria, optionally of the family Corynebacteriaceae, optionally of the genus Corynebacterium, or optionally of Corynebacterium glutamicum. In some embodiments, the genetically modified bacterium is of the species Corynebacterium glutamicum.

[0096] In one embodiment, the genetically modified bacterium is engineered to reduce hydroxyglutarate production by increasing the cytosolic NADPH pool and / or decreasing the cytosolic NADH pool relative to an otherwise identical bacterium that does not possess said modification. In another embodiment, the genetically modified bacterium is engineered to reduce hydroxyglutarate production by expression of a polypeptide having D-3-phosphoglycerate dehydrogenase activity with reduced or no hydroxyglutarate production activity.

[0097] In a separate aspect, the present disclosure describes a cell culture comprising the host cells and growth medium described herein, and a method for producing a metabolite derived from 3-phosphoglycerate by culturing the cell culture under conditions that allow the host cells to produce the metabolite; and optionally recovering and / or isolating the metabolite. Suitable growth media for prokaryotic cells are well known in the art. The cell culture can be cultured in a nutrient medium under conditions suitable for producing the metabolite and / or its precursor and / or for increasing cell number using methods known in the art. For example, the culture can be cultured in a suitable medium under conditions that allow the host cells to grow and / or proliferate, by shake flask culture, or by small-scale or large-scale fermentation (including continuous fermentation, batch fermentation, fed-batch fermentation, or solid-state fermentation) in a laboratory or industrial fermentor, and optionally recovered and / or isolated.

[0098] Culturing can be carried out using procedures known in the art in an appropriate nutrient medium containing carbon and nitrogen sources and inorganic salts. Suitable media are available from commercial suppliers or can be prepared according to published recipes (e.g., from catalogs of the American Type Culture Collection). The selection of an appropriate medium may be based on the choice of host cell and / or on regulatory requirements for the host cell. Such media are available in the art. The medium may contain additional components, if necessary, that favor the host cells over other potentially contaminating microorganisms. Thus, in one embodiment, an appropriate nutrient medium includes a carbon source (e.g., glucose, maltose, molasses, starch, cellulose, xylan, pectin, lignocellulolytic biomass hydrolysate, etc.), a nitrogen source (e.g., ammonium sulfate, ammonium nitrate, ammonium chloride, etc.), an organic nitrogen source (e.g., yeast extract, malt extract, peptone, etc.), and an inorganic nutrient source (e.g., phosphate, magnesium, potassium, zinc, iron, etc.). Culturing of the host cells can be carried out for a period of about 0.5 to about 30 days. The culturing process may be a batch process, a continuous process, or a fed-batch process, and may suitably be carried out at a temperature in the range of 0 to 100°C or 10 to 80°C, e.g., about 20°C to about 50°C, and / or at a pH of, e.g., about 2 to about 10. Preferred fermentation conditions for prokaryotic host cells are a temperature in the range of about 25°C to about 55°C and a pH of about 3 to about 9. Suitable conditions are typically selected based on the choice of host cell. Thus, in one embodiment, the method of the present disclosure further comprises: a) cultivating a cell culture in a nutrient medium; b) culturing the cell culture under aerobic or anaerobic conditions c) culturing the cell culture under agitation; d) culturing the cell culture at a temperature of 25-50°C; e) culturing the cell culture at a pH of 3 to 9; and f) culturing the cells for 10 hours to 30 days It contains one or more elements selected from:

[0099] The cell culture of the present disclosure can be harvested and isolated using methods known in the art. For example, metabolites can be recovered from the nutrient medium by conventional procedures, including, but not limited to, centrifugation, filtration, spray drying, or freeze-drying. In certain embodiments, the method comprises separating the liquid phase of the cells or cell culture from the solid phase of the cells or cell culture to obtain a supernatant containing metabolites and / or separating the supernatant: a) disrupting cells in a cell culture to release intracellular metabolites into the supernatant; b) separating the supernatant from the solid phase of the cell culture, for example by filtration or sedimentation; c) contacting the supernatant with one or more adsorption resins to obtain at least a portion of the metabolites produced; d) contacting the supernatant with one or more ion exchange or reverse phase chromatography columns to obtain at least a portion of the metabolites; e) extracting metabolites; and / or f) precipitating the metabolites by crystallization or evaporating the liquid solvent; and optionally isolating the metabolites by filtration or sedimentation; and subjecting the resulting mixture to one or more steps selected from the group consisting of: thereby recovering and / or isolating the metabolites. The method comprises a recovery and / or isolation step comprising:

[0100] In one embodiment, the method further comprises the step of supplying the cell culture with one or more precursors or substrates in the metabolic pathway.

[0101] In another embodiment, the method includes one or more in vitro steps in the process of producing a metabolite. Further steps of chemically or biologically / enzymatically modifying the metabolite may be added to the methods described herein, particularly if the metabolite is not the desired end product.

[0102] The method may further include recovering the metabolites and mixing the metabolites with one or more carriers, agents, additives, adjuvants and / or excipients to produce a biopesticide composition.

[0103] Methods of the Invention In particular embodiments, the present invention also provides methods for producing L-serine or an L-serine derivative using the genetically modified bacteria according to the present invention. In particular, the present invention provides methods for producing L-serine or an L-serine derivative, comprising culturing a genetically modified bacterium as described herein in a medium. According to certain embodiments, the present invention provides methods for producing L-serine. In particular, the present invention provides methods for producing L-serine, comprising culturing a genetically modified bacterium as described herein in a medium. The method may further comprise isolating the L-serine from the medium. According to certain embodiments, the present invention provides methods for producing an L-serine derivative. In particular, the present invention provides methods for producing an L-serine derivative, comprising culturing a genetically modified bacterium as described herein in a medium. The L-serine derivative may be selected from the group consisting of L-cysteine, L-methionine, L-glycine, O-acetylserine, L-tryptophan, thiamine, ethanolamine, and ethylene glycol. The method may further comprise isolating the L-serine derivative from the culture medium. According to certain embodiments, the present invention provides a method for producing L-cysteine. In particular, the present invention provides a method for producing L-cysteine, the method comprising culturing a genetically modified bacterium as described herein in a culture medium. The method may further comprise isolating L-cysteine ​​from the culture medium. According to certain embodiments, the present invention provides a method for producing L-methionine. In particular, the present invention provides a method for producing L-methionine, the method comprising culturing a genetically modified bacterium as described herein in a culture medium. The method may further comprise isolating L-methionine from the culture medium. According to certain embodiments, the present invention provides a method for producing L-glycine. In particular, the present invention provides a method for producing L-glycine, the method comprising culturing a genetically modified bacterium as described herein in a culture medium. The method may further comprise isolating L-glycine from the culture medium.According to certain embodiments, the present invention provides a method for producing O-acetylserine. In particular, the present invention provides a method for producing O-acetylserine, said method comprising culturing a genetically modified bacterium as described herein in a culture medium. The method may further comprise isolating the O-acetylserine from the culture medium. According to certain embodiments, the present invention provides a method for producing L-tryptophan. In particular, the present invention provides a method for producing L-tryptophan; said method comprises culturing a genetically modified bacterium as described herein in a culture medium. The method may further comprise isolating the L-tryptophan from the culture medium.

[0104] According to certain embodiments, the present invention provides a method for producing L-thiamine. In particular, the present invention provides a method for producing L-thiamine, the method comprising culturing a genetically modified bacterium as described herein in a medium. The method may further comprise isolating the thiamine from the medium. According to certain embodiments, the present invention provides a method for producing ethanolamine. In particular, the present invention provides a method for producing ethanolamine; the method comprises culturing a genetically modified bacterium as described herein in a medium. The method may further comprise isolating the ethanolamine from the medium. According to certain embodiments, the present invention provides a method for producing ethylene glycol. In particular, the present invention provides a method for producing ethylene glycol; the method comprises culturing a genetically modified bacterium as described herein in a medium. The method may further comprise isolating the ethylene glycol from the medium. The medium used may be any conventional medium suitable for culturing the bacterial cells of interest and may be constructed according to principles established in the art. Culture media typically contain all nutrients necessary for the growth and survival of each bacterium, such as carbon and nitrogen sources and other inorganic salts. Suitable media, such as minimal or complex media, are available from commercial suppliers or can be prepared according to published recipes, such as the American Type Culture Collection (ATCC) catalog of strains. Non-limiting standard media well known to those skilled in the art include Luria-Bertani (LB) broth, Sabouraud dextrose (SD) broth, MS broth, yeast peptone dextrose, BMMY, GMMY, or yeast malt extract (YM) broth, all of which are commercially available. Non-limiting examples of media suitable for culturing bacterial cells, such as Escherichia coli (E. coli) cells, include minimal and rich media, such as Luria broth (LB), M9 medium, M17 medium, SA medium, MOPS medium, Terrific broth, and YT. The carbon source can be any suitable carbon substrate known in the art, and in particular any carbon substrate commonly used in bacterial cultivation and / or fermentation.Non-limiting examples of suitable fermentable carbon substrates include C5 sugars (e.g., arabinose or xylose), C6 sugars (e.g., glucose), acetate, glycerol, vegetable oil, sucrose, yeast extract, peptone, casamino acids, or mixtures thereof. A carbon source of particular interest is C6 sugar, such as glucose. Nitrogen sources include various ammonium salts, such as ammonia and ammonium sulfate, other nitrogen compounds, such as amines, natural nitrogen sources, such as peptone, soybean hydrolysate, and digested fermentation microorganisms. Minerals that can be used include potassium monophosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride, and the like. Cultivation is preferably carried out under aerobic conditions, such as by shaking or agitation with aeration, at a temperature of about 20 to about 40°C, e.g., about 30 to 38°C, preferably about 37°C. The pH of the culture is usually about 5 to about 9, e.g., about 6.5 to 7.5. The pH of the culture can be adjusted with ammonia, calcium carbonate, various acids, various bases, and buffers. Typically, L-serine accumulates in the medium after 1 to 5 days of culture. After cultivation, solids, such as cells, can be removed from the medium by centrifugation or membrane filtration. L-serine or an L-serine derivative can be recovered by conventional methods for isolating and purifying compounds from the medium. Well-known purification procedures include, but are not limited to, centrifugation or filtration, precipitation, ion exchange, chromatographic methods, such as ion exchange chromatography or gel filtration chromatography, and crystallization. Thus, the present invention provides L-serine or an L-serine derivative obtainable by the methods described herein.

[0105] In a further aspect, there is provided a fermentation composition comprising metabolites of the cell culture described herein and 3-phosphoglycerate-derived metabolites, wherein at least 20% by weight of the carbon is biobased. In some embodiments, the composition comprises at least 50%, such as at least 55%, for example at least 60%, such as at least 65%, for example at least 70%, such as at least 75%, for example at least 80%, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 99%, such as at least 100% biobased carbon. In another embodiment, the composition comprises at least 20% biobased carbon, such as at least 30% biobased carbon, for example at least 40% biobased carbon, such as at least 50% biobased carbon, for example at least 60% biobased carbon, such as at least 70% biobased carbon, for example at least 75% biobased carbon, such as at least 80% biobased carbon, for example at least 85% biobased carbon, such as at least 90% biobased carbon, for example at least 95% biobased carbon, such as 100% biobased carbon. In yet another embodiment, the composition comprises between 20% and 100% biobased carbon, such as between 30% and 100% biobased carbon, for example between 40% and 100% biobased carbon, such as between 50% and 100% biobased carbon, for example between 60% and 100% biobased carbon, such as between 70% and 100% biobased carbon, for example between 75% and 100% biobased carbon, such as between 80% and 100% biobased carbon, for example between 85% and 100% biobased carbon, for example between 90% and 100% biobased carbon, such as between 95% and 100% biobased carbon, for example 100% biobased carbon. In yet other embodiments, the composition comprises 50% or less fossil carbon, such as 45% or less, such as 40% or less, for example 35% or less, such as 30% or less, for example 25% or less, such as 20% or less, for example 15% or less, such as 10% or less, for example 5% or less, such as 1% or less fossil carbon.In still further embodiments, the composition comprises 90% biobased carbon, 91% biobased carbon, 92% biobased carbon, 93% biobased carbon, 94% biobased carbon, 95% biobased carbon, 96% biobased carbon, 97% biobased carbon, 98% biobased carbon, 99% biobased carbon, or 100% biobased carbon, e.g., 94% biobased carbon. The fermentation composition can further comprise one or more additional compounds or metabolites from the cell culture. Such compounds and / or cell culture metabolites include precursors for metabolites and compounds selected from trace metals, vitamins, salts, yeast nitrogen base, carbon source, YNB, and / or amino acids of the fermentation. In particular, the composition comprises a concentration of metabolites of at least 1 mg / kg of composition, such as at least 5 mg / kg, for example at least 10 mg / kg, for example at least 20 mg / kg, for example at least 50 mg / kg, for example at least 100 mg / kg, for example at least 500 mg / kg, for example at least 1,000 mg / kg, for example at least 5,000 mg / kg, for example at least 10,000 mg / kg, for example at least 50,000 mg / kg. In other embodiments, the composition is substantially free of alpha-HGA. The composition may also further comprise one or more carriers, agents, additives and / or excipients.

[0106] Having generally described the invention, a further understanding can be obtained by reference to certain specific examples, which are provided herein for purposes of illustration only and, unless expressly stated, are not intended to be limiting.

[0107] Example Materials and Methods DE3 Integration To use the pET vector as an expression system, a DE3 cassette containing T7 polymerase was integrated into the genome using a DE3 lysogenization kit (Millipore, Darmstadt, Germany).

[0108] Shake flask media and culture Serine production was examined in M9 minimal medium. Glucose M9 minimal medium consisted of 2–5 g / L glucose, 2 mM glycine, 0.1 mM CaCl2, 2.0 mM MgSO4, 1× trace element solution, and 1× M9 salts. A 1,000× trace element stock solution was prepared by dissolving 27 g / L FeCl3·6H2O, 2 g / L ZnCl2·4H2O, 2 g / L CoCl2·6H2O, 2 g / L NaMoO4·2H2O, 1 g / L CaCl2·H2O, 1.3 g / L CuCl2·6H2O, 0.5 g / L H3BO3, and concentrated HCl in ddH2O and sterile filtering. A 10x M9 salt stock solution was composed of 68 g / L Na2HPO4 anhydrous, 30 g / L KH2PO4, 5 g / L NaCl, and 10 g / L NH4Cl dissolved in ddH2O and autoclaved.

[0109] The medium was filter sterilized and 50 ml was added to a 250 ml sterile baffled shake flask. An overnight culture was used as inoculum, started at an OD of 0.1, and incubated at 37°C and 250 rpm unless otherwise noted.

[0110] Batch and fed-batch fermentation As previously published 15 Batch and fed-batch fermentations were performed in M9-glycine and TPM2 media, respectively. Except for the antibiotic, the medium composition and growth and induction conditions were identical. In fed-batch fermentations, a constant or linear feed rate was maintained but increased stepwise based on the glucose level in the fermentor, as monitored by glucose strips.

[0111] Analysis method Previously published HPLC method 16 was used to quantify glucose, HGA and other organic acids.

[0112] The concentration of serine was measured using a Dionex Ultimate 3000 HPLC (high-performance liquid chromatography) equipped with a CHIROBIOTIC® T chiral (250 × 2.1 mm × 5 μm) column (Sigma-Aldrich, St. Louis, MO, USA) and a diode array detector (DAD-UV). The mobile phase consisted of 60% acetonitrile (v / v) and 0.02% (v / v) formic acid in Milli-Q water. The mobile phase was delivered at a rate of 1.0 mL / min, and the injection volume was maintained at 3 μL for standards and all samples. L-serine detection was monitored at 205 nm.

[0113] Example 1 - Effect of replacing NADH-dependent glyceraldehyde-3-phosphate dehydrogenase with NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase. Many NADPH-dependent glyceraldehyde-3-phosphate dehydrogenases are derived from C. acetobutylicum (gapC) 10 or from Bacillus subtilis (gapB) 17 The native gapA was replaced with gapC from C. acetobutylicum, and hydroxyglutarate production was monitored in shake flasks and fed-batch fermentations.

[0114] Construction of plasmid vectors pCDF-serAmut-serC and pACYC-serB L-serine was produced in E. coli from three enzymes encoded by serA, serB, and serC. The entire gene was isolated from E. coli MG1655 using primers bearing the names of each gene (Table 2). A 100 μl PCR mixture contained 250 nM of each forward and reverse primer, 250 μM dNTPs, 2 U of Phusion polymerase, 1x HF buffer, and 1 μl of overnight culture. The following two-step PCR protocol was used for PCR amplification: an initial denaturation step at 98°C for 40 seconds, followed by five cycles of denaturation at 98°C for 10 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 90 seconds, followed by 20 cycles of increasing the annealing temperature from 55°C to 65°C. After column purification, the gene product and plasmid were digested using Fast Digest enzyme (Thermo Scientific, Waltham, MA, USA). Approximately 500 ng of PCR product or 1 μg of plasmid was digested with 1 μl of each restriction enzyme in 1× fast digest buffer. The reaction was incubated for 3 hours and then column-purified again. The serA PCR product was double-digested with NcoI and NotI, while the serC PCR product was digested with NdeI and PacI. For cut-and-paste cloning of serA, pCDF-Duet was first digested with NcoI and NotI to generate the plasmid pCDF-Duet-serA. This plasmid was later used for cut-and-paste cloning of serC to generate pCDF-Duet-serA-serC. The serB PCR product was cloned into the pACYC-Duet vector at the NcoI and PacI sites to generate pACYC-serB. A typical ligation reaction contained 1× T4 ligase buffer, 50 ng of plasmid DNA and 100 ng of insert, and 0.3 μl / 10 μl of T4 DNA ligase (Thermoff Scientific, Waltham, MA, USA).

[0115] by mutating three residues H344, N346 and N364 to alanine by site-directed mutagenesis 18This eliminated feedback inhibition of serA (Table 3). The master mix was used as described above, with the only change being that it was divided into two equal aliquots, and then forward and reverse primers were added to each aliquot. A total of 100 ng of pCDF-Duet-serA-serC plasmid was used as template. The two-step PCR program consisted of an initial denaturation at 98°C for 40 seconds, denaturation at 98°C for 10 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 4 minutes and 30 seconds. The cycle was repeated five times, after which the two aliquots were mixed and resuspended for an additional 15 cycles. To allow for vector backbone exchange, the NcoI site within serC was removed using the same approach with the primers listed in Table 3.

[0116] [Table 1]

[0117] [Table 2]

[0118] Replacement of gapA with gapC Using a cat-sacB-based selection system, gapA was replaced with the gapC gene from a C. acetobutylicum strain. cat-sacB was inserted using pKD46, which harbors the exo, beta, and gamma genes for recombination. Positive selection for cassette insertion was achieved by selecting clones for chloramphenicol resistance. Loss of the cassette was selected by replica plating of clones on LB-chloramphenicol and LB-sucrose plates containing 15% sucrose (without NaCl). Primers gapC_camsacB_F and R were used to amplify the cat-sacB cassette (Table S3). The reaction mixture and PCR program were the same as those described in Example 1, except for the template and extension time. The extension time was 2 minutes and 30 seconds, and the template was 1 μl of an overnight culture from E. coli carrying the cat-sacB cassette in its genome. Competent cells were then transformed with 200 ng of the gapC-cat-sacB cassette. After 2 hours of regeneration, the cells were plated onto LB-chloramphenicol-ampicillin plates and incubated overnight at 30°C. Single colonies were selected, electrocompetent after 1 hour of induction (Example 1), and transformed with the gapC gene amplified from the C. acetobutylicum genome using the PCR program described above and primers gapC_aF and aR, as described in Table 4. After 2 hours of recovery, the cells were plated onto LB-sucrose plates and incubated at 42°C to allow recovery of the pKD46 plasmid. 24 clones from each experiment were replica-plated onto LB-chloramphenicol and LB plates. Clones that did not grow on LB-chloramphenicol plates were checked for cassette loss by colony PCR and subsequently sequenced by Sanger sequencing.

[0119] [Table 3]

[0120] The strain was then transformed with pCDF-Duet1-serAmut-serC and pACYC-serB. The resulting glycerol stock was grown overnight in 2xYT medium containing 0.1% glucose and supplemented with spectinomycin and chloramphenicol. The overnight culture was incubated in fed-batch fermentation. Medium composition and culture conditions were as previously described.

[0121] result Strain HM_274 does not carry the gapA::gapC substitution. This strain accumulated 0.91 g / L of HGA in a 23-hour batch fermentation in a baffled flask. In a 48-hour fed-batch fermentation, the same strain accumulated 9.35 g / L of HGA. Strain HM_476 does not carry the gapA::gapC substitution. This strain accumulated 0.86 g / L of HGA in a 23-hour batch fermentation in a baffled flask. In a 48-hour fed-batch fermentation, the same strain accumulated 2.36 g / L of HGA. Data for the 23-hour batch fermentation and the 48-hour fed-batch fermentation are shown in Table 5. This data clearly demonstrates a decrease in HGA production in the strain in which gapA was replaced by gapC.

[0122] [Table 4]

[0123] Example 2 - Effect of NADH pool reduction by oxidation of NADH using NADH oxidase (Nox) Materials and Methods (Nox) Construction of plasmids pSEVA27-serAmut-CB and pSEVA27-serAmut-CB-Nox All plasmid manipulations were performed using the Uracil-Specific Removal Reagent (USER) cloning method. PCR was performed using Phusion U Hot Start Polymerase Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). Oligonucleotides were ordered from Integrated DNA Technologies (IDT, Coralville, IA, USA) and are listed in Table 6. Genes serB and serC were amplified from the E. coli MG1655 genome and cloned into the pSEVA27-sl backbone. serAmut was amplified from pCDFDuet-1-serAmut-serC and cloned alongside serB and serC into the pSEVA27-sl backbone. This plasmid was used to clone nox, which was amplified from the Lactobacillus brevis genome. The application of each primer to generate different USER fragments is also noted. The PCR program was: initial denaturation at 98°C for 40 seconds, denaturation at 98°C for 10 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 3 minutes and 30 seconds; this cycle was repeated 25 times. USER fragments 1–4 were used to assemble pSEVA27-serACB, while fragments 5 and 6 were used to assemble pSEVA27-serACB-Nox.

[0124] The 10 μL USER reaction contained 1 μL USER enzyme, 1 μL 10x CutSmart Buffer (New England BioLabs), and 200 ng of each USER fragment. The reaction was incubated at 37°C for 30 minutes, followed by 15°C for 30 minutes. The reaction mixture was transformed into chemically competent NEB5 alpha cells, and the transformants were grown in SOC medium at 37°C before being plated on LB agar plates and incubated overnight at 37°C.

[0125] The next day, single colonies were selected and plated on 2xYT agar medium and incubated at 37°C for 16 hours for plasmid prep and Sanger sequencing. Plasmid maps are provided in Figures 1 and 6. The confirmed plasmids were transformed into E. coli strains for L-serine production. The shake flask protocol described above was used for serine production.

[0126] [Table 5] TIFF2025535809000013.tif85159

[0127] result In batch fermentations performed in baffled flasks, strains co-expressing Nox and SerACB from pSEVA27-serACB-Nox accumulated 0.06–0.07 g / L of HGA after 24 h of incubation, as shown in Figure 2. The strain expressing SerACB alone accumulated 0.4 g / L of HGA. This data indicates that NADH depletion resulting from expression of NADH oxidase results in reduced HGA production.

[0128] Example 3 – Effect of overexpression of NADH-dependent glutamate dehydrogenase To recycle NADPH to NADP+, E. coli and C. glutamicum use the reduction of KGA to glutamate by an NADPH-dependent glutamate dehydrogenase. To switch from NADPH to NADH consumption and thus recycle NADH to NAD+, an NADH-dependent glutamate dehydrogenase can be expressed. There are many NADH-dependent glutamate dehydrogenases known in the literature, and the NADH-dependent glutamate dehydrogenase RocG from Bacillus subtilis was tested.

[0129] Materials and Methods rocG was cloned into the pACYC-serB plasmid using USER cloning, as described above. Primers are given in Table 7a. The PCR program, USER reaction, and transformation were performed as described above, with the only difference being that after the regeneration step, cells were plated on LB-chloramphenicol plates and 2xYT-chloramphenicol medium was used for cell growth. The medium for production performed in shake flasks was as described above.

[0130] [Table 6]

[0131] result In 20-hour shake flask fermentations, ALE-8 and ALE-8 expressing RocG were monitored for HGA and L-serine concentrations.

[0132] [Table 7]

[0133] The strain expressing RocG showed a six-fold reduction in HGA concentration compared to the strain lacking RocG expression. Although serine concentration was also lower, the serine / HGA ratio was almost two-fold better in the +rocG strain.

[0134] Similarly, in a separate experiment, ALE-8 accumulated 0.27 g / L of HGA 5 hours after the start of fermentation, while ALE-8 expressing RocG accumulated 0.06 g / L of HGA. After 48 hours, ALE-8 accumulated 0.1 g / L of HGA, while ALE-8 expressing RocG accumulated 0.06 g / L of HGA. After 48 hours, ALE-8 produced 0.75 g / L of L-serine, while ALE-8 expressing RocG produced 0.3 g / L of L-serine.

[0135] This experiment shows that the additional expression of rocG helps reduce HGA accumulation and its expression, and also reduces L-serine production. However, when comparing the L-serine to HGA ratio in both strains, rocG expression is more favorable.

[0136] Example 4 - Effect of gapC on L-serine production. In this example, we demonstrate that introduction of gapC increases L-serine production in strains expressing serA with KGA reduction activity (E. coli) or serA lacking KGA reduction activity. To demonstrate the first case, an L-serine operon containing feedback-insensitive SerA from E. coli was transformed into an E. coli strain expressing either gapA or gapC from its genome. To demonstrate the latter case, E. coli-derived SerA was replaced with feedback-insensitive serA from C. glutamicum, which lacks KGA reduction activity, in both the pCDF and pSEVA27 vectors.

[0137] Plasmid construction: The construction of pCDF-Duet1-serAmut-serC, pACYC-serB, and pSEVA27-serAmut-serC is described in the previous examples. To replace serAmut (E. coli) in these vectors with serA from Corynebacterium glutamicum, the USER primers listed in Table 8 were used. The PCR program, USER reaction, and transformation were performed as described in the previous examples. Using the site-directed mutagenesis protocol described above, feedback inhibition was eliminated by replacing Y463 or N483 with alanine (A), resulting in vectors pCDF-Duet1-serAglut-Y463A-serC and pCDF-Duet1-serAglut-N483A-serC, and vectors pSEVA27-serAglut-Y463A-serC and pSEVA-serAglut-N483A-serC. The primers are listed in Table 8.

[0138] [Table 8]

[0139] result L-serine-producing strains expressing feedback-insensitive SerA from Escherichia coli (E. coli) and either gapA or gapC on their genomes were compared in a 48-hour fed-batch fermentation. The L-serine concentrations measured during the fermentation are shown in Figure 4A. The gapA-expressing strain produced 25 g / L, while the gapC-expressing strain produced 35 g / L. Furthermore, L-serine-producing strains expressing feedback-insensitive serA from C. glutamicum and either gapA or gapC on their genomes were compared in a 68-hour fed-batch fermentation. The L-serine concentrations measured during the fermentation are shown in Figure 4B. The gapA-expressing strain produced 50 g / L, while the gapC-expressing strain produced 100 g / L.

[0140] First, the data show that a 40% increase in L-serine production can be achieved in fermentation from a strain expressing the KGA-promiscuous E. coli serA-containing L-serine pathway in which gapA is replaced by gapC. Second, the gapA::gapC modification boosts L-serine production by 200% in a strain expressing the KGA-promiscuous C. glutamicum serA-containing serine pathway.

[0141] Example 5 - Effect of gapC on a strain expressing serA that lacks HGA activity. This example demonstrates that enhancing the NADPH pool, for example by expressing gapC, not only enhances L-serine production (Example 4), but also reduces the redox imbalance caused by expression of serA from C. glutamicum, which cannot oxidize NADH but reduces KGA to HGA. Providing additional NADPH (from gapC in this example) likely also provides the NADPH needed to recycle KGA to glutamate. To demonstrate this, the L-serine pathway was expressed in both the gapA (serHM_708) and gapC (serHM_608) strains from vectors pCDF and pACYC, which contain serA from C. glutamicum lacking HGA activity. The two strains, serHM_708 and serHM_608, were compared in batch fermentation experiments.

[0142] As shown in Figure 5, the gapC strain (serHM_608) grows to a final OD of 7, while the gapA strain (serHM_708) grows to a final OD of 3.

[0143] Example 6 - Effect of rat serA on gapC expressing strains. The rat serA gene is a type I serA and exhibits negligible activity in the production of hydroxyglutarate. This gene was ordered as a gene fragment from Twist Biosciences (USA). This serA is disclosed herein as SEQ ID NO: 8. To clone rat serA, the pSEVA serACB vector, which has constitutive promoter strength, was selected. These vectors are well known in the art. The vector backbone primers oSER_1424 and oSER_1426 bind the upstream and downstream sequences of the replaced serA. Primers oSER_1423 and oSER_1425 were used to amplify the gene fragment with complementary USER overhangs.

[0144] The PCR program was: initial denaturation at 98°C for 40 seconds, denaturation at 98°C for 10 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 3 minutes 30 seconds (30 seconds / kb), this cycle was repeated 25 times. The primer sequences are given in the table below.

[0145] [Table 9]

[0146] A 10 μL USER reaction contained 1 μL USER enzyme, 1 μL 10x CutSmart Buffer (New England BioLabs), and 200 ng of each USER fragment. The reaction was incubated at 37°C for 30 minutes, followed by 15°C for 30 minutes. Chemically competent NEB5 alpha cells were transformed with the reaction mixture, and the transformants were grown in SOC medium at 37°C before being plated on LB agar plates and incubated overnight at 37°C.

[0147] The next day, single colonies were selected and plated on 2xYT agar medium, then incubated at 37°C for 16 hours for plasmid prep and Sanger sequencing. The plasmid map is shown in Figure 7. The confirmed plasmid was transformed into an Escherichia coli (E. coli) strain for L-serine production. A truncated form of rat serA was obtained using primers oSER_1424 and oSER_1533. The plasmid obtained above was used as a template. PCR, USER reaction, and transformation protocols were the same as above. Shake flask culture medium and protocols are described below.

[0148] Growth and L-serine titer in production strains transformed with different serA variants were tested in 24-h shake flask experiments. For this purpose, MOPS-based medium (pH 7.6) was prepared by adding 10 g / L ammonium sulfate (CAS No. 7783-20-2), 2 g / L potassium dihydrogen phosphate (CAS No. 7778-77), 2 g / L yeast extract, 40 g / L MOPS (CAS No. 1132-61-2), and 0.6 g / L glycine to the desired total medium volume. The medium was adjusted to pH 7.6 using 15% NH3 solution and autoclaved. Subsequently, 20 mL of sterilized MOPS-based medium was added to a 250 mL sterile shake flask and supplemented with 600 μL of glucose monohydrate (CAS No. 14431-43-7), 80 μL of trace elements, and 200 μL of magnesium sulfate heptahydrate (CAS No. 56-40-6). The OD of the overnight culture was then measured. 600 The optical density of the strain with the lowest optical density was measured and used as inoculum. One mL of the strain with the lowest optical density was inoculated into a shake flask, while the remaining strains were inoculated in similarly small volumes to ensure the same initial biomass was obtained in all shake flasks. These were incubated at 37°C and 250 rpm for 24 hours. Optical density was monitored after 1.5, 3, 4.5, 6, 7.5, and 24 hours of growth. At the same time points, 300 μL of culture was harvested into a 96-deep-well plate and spun down at 3,500 × g for 5 minutes at 4°C. The resulting supernatant was filtered through a 22 μm membrane onto an HPLC plate for subsequent quantification of L-serine.

[0149] [Table 10]

[0150] result As shown in Figure 8, strains expressing rat-derived serA produced surprisingly low titers, indicating that they were not inhibited by L-serine. However, when the regulatory domain was removed using primers oSER_1423 and oSER_1535, a truncated version of the serA rat gene (SEQ ID NO: 144 / SER_1827) produced more L-serine, reaching 2.6 g / L after 24 hours. The amount of L-serine produced by the truncated serA rat variant (SER_1827) was surprisingly higher than that of the non-truncated (SER_1739) and control (SER_351) strains, which produced 0.4 g / L and 1.4 g / L of L-serine, respectively, after 24 hours. This indicates that strains using truncated serA from the Type I taxon can produce significant amounts of L-serine.

[0151] Example 7 - Effect of replacing NADH-dependent glyceraldehyde-3-phosphate dehydrogenase with a variant of NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase Many different variants of NADPH-glyceraldehyde-3-phosphate dehydrogenase are known. Some of them are shown in SEQ ID NOS: 23-46. To demonstrate that NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase can also be used for L-serine production, synthetic gene fragments of SEQ ID NOS: 25 and 36 with additional 500-bp overhangs complementary to the upstream and downstream regions of gapA were ordered from Twist Biosciences (USA). gapA was replaced using the cat-sacB selection system mentioned in Example 1. The primers used to amplify the gene fragment replacing the cat-sacB cassette with the gapA site are given in the table below.

[0152] [Table 11]

[0153] This strain was transformed with the pSER_43 plasmid. The plasmid expresses the serine operon and contains a strong constitutive promoter with the feedback-insensitive serA gene from C. glutamicum (SEQ ID NO: 14). Transformants were selected on kanamycin plates. The inoculation and shake flask protocols for L-serine production were as described in Example 6.

[0154] result As shown in Figure 9, NADPH-glyceraldehyde-3-phosphate dehydrogenase from C. glutamicum and K. lactis, with average titers of 3.62 g / L and 3.65 g / L, was comparable to L-serine production from gapC from C. acetobutylicum, which produced an average of 3.3 g / L of L-serine after 24 hours. Therefore, these variants can also be used to enrich the NADPH pool.

[0155] Example 8 - Effect of NADH pool reduction on the production of L-serine by oxidation of NADH using different variants of NADH oxidase In Example 2, we demonstrated the effect of NADH oxidase (seq ID 49) from Lactobacillus brevis on the reduction of hydroxyglutarate. In this example, we cloned various Nox variants (SEQ ID NOS: 50, 52, and 55) downstream of the serACB operon of serA (non-hydroxyglutarate-producing) from C. glutamicum. In this example, we aimed to analyze whether expression of other variants of NADH oxidase enhanced L-serine production.

[0156] Gene fragments (SEQ ID NOs: 50, 52, 55) were ordered from Twist Biosciences. The cloning strategy was similar to that mentioned in Example 2. The primers used for amplifying the different Nox variants are listed in the table below. The constructs are shown in Figure 10. Inoculation and shake flask experiments were performed as in Examples 6 and 7. An OD of 0.55-0.65 was obtained. 600 The only difference was that the cultures were induced by adding IPTG to a final concentration of 40 μM in the cultures.

[0157] [Table 12]

[0158] [Table 13]

[0159] result: As shown in Figure 11, expression of Nox (SER_1888) from L. parakefiri resulted in an L-serine titer of 1.9 g / L after 24 hours, which was therefore higher than the amount produced by the control strain (SER_1911), which reached 1.6 g / L after 24 hours, suggesting that expression of NADH oxidase from L. parakefiri helps enhance L-serine production.

[0160] Example 9 - Effect of increasing the NADPH pool by overexpressing genes from the pentose phosphate pathway The pentose phosphate pathway is one of the major sources of NADPH for bacteria. In this example, we tested whether increasing the NADPH pool by overexpressing zwf, which encodes glucose-6-phosphate dehydrogenase, or pgl, which encodes 6-phosphogluconolactonase in the pentose phosphate pathway, could enhance the NADPH pool in cells. In this example, we examined the effects of these enzymes in the presence and absence of NADPH-based glyceraldehyde phosphate dehydrogenase (gapC). zwf and pgl were amplified from the MG1655 genome using the primers listed in the table below.

[0161] The PCR program was: initial denaturation at 98°C for 40 seconds, denaturation at 98°C for 10 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 1 minute; this cycle was repeated 25 times.

[0162] Like NADH oxidase, the zwf and pgl genes were cloned under serB in the serACB operon. Each gene was cloned into an operon containing either a feedback-insensitive SerA from E. coli (to examine reduced production of hydroxyglutarate) or a serA from C. glutamicum (to examine whether its production leads to increased L-serine production). The constructs are shown in Figure 12.

[0163] [Table 14]

[0164] The cloning strategy and shake flask testing protocol are the same as in Example 8.

[0165] [Table 15]

[0166] result Overexpression of the gp1 and zwf genes affected L-serine production when combined with serA from C. glutamicum. As can be seen in Figure 13a, overexpression of zwf (SER_1882) in the gapC background resulted in L-serine production of 1.95 g / L, which was higher than the 1.6 g / L produced by the control strain (SER_1911). Furthermore, overexpression of pgl (SER_1890) and zwf (SER_1892) in the gapA background with serA from C. glutamicum enhanced L-serine production with pgl, resulting in a titer of 3.1 g / L. As seen in Figure 13b, this amount of L-serine was higher than that produced by the control strain (SER_1901), which achieved 2.9 g / L of L-serine production after 24 h.

[0167] Similarly, overexpression of pgl and zwf had an effect on L-serine and hydroxyglutarate production when combined with SerA from E. coli. As shown in Figure 13c, strains in the gapC background expressing pgl (SER_1881) and zwf (SER_1883) enhanced L-serine production at titers of 3.4 g / L and 3.5 g / L, respectively, while the control strain (SER_1912) produced 3.2 g / L of L-serine after 24 h. Furthermore, HGA production decreased from 0.96 g / L in the control strain to 0.83 g / L and 0.79 g / L when pgl and zwf were overexpressed, respectively. Similarly, overexpression of pgl (SER_1891) and zwf (SER_1893) in combination with SerA from E. coli in the gapA strain resulted in reduced HGA production. As shown in Figure 13d, HGA decreased from 0.98 g / L in the control strain (SER_1902) to 0.41 g / L and 0.82 g / L when pgl and zwf were overexpressed, respectively. Furthermore, overexpression of pgl in such a gapA background resulted in improved L-serine production, reaching 3.45 g / L after 24 h. This strain produced a higher amount of L-serine than the control strain, which produced 3.1 g / L at the same time point.

[0168] Thus, overexpression of pgl and zwf enhanced L-serine production and reduced HGA production, with the effect being more pronounced in the gapA strain.

[0169] Example 10 - PGDH motif with reduced HGA activity. Some PGDHs can use α-ketoglutarate (αKG) instead of PHP in the reverse direction to produce α-hydroxyglutarate. Three enzymes have been shown to be able to utilize αKG as a substrate: PGDHs from Escherichia coli (REF), Pseudomonas stutzeri (REF), and Saccharomyces cerevisiae (REF). All three enzymes are type II PGDHs; type I and type III PGDHs cannot use αKG as a substrate, and the ability to use αKG as a substrate is hypothesized to be a trait specific to type II PGDHs.

[0170] To examine the conserved residues in type I and type II PGDH, representatives from each class were selected. For type I, PGDH from Mycobacterium tuberculosis (M. tuberculosis) was selected, while PGDH from Escherichia coli (E. coli) was selected for type II. These enzymes were chosen because they have been extensively studied and crystal structures of complexes with related substrates exist for both. Residue conservation was examined using the ConSurf-DB server. Relevant crystal structures: type I (M. tuberculosis, PDB ID: 3DDN) and type II (E. coli, PDB ID: 1YBA) were used as input. The ConSurf workflow was as previously described. Briefly, similar amino acid sequences to each sequence in the provided PDB IDs were collected and multiple alignments were performed using HMMER and MAFFT, respectively. The evolutionary conservation of each amino acid position in the alignment was calculated using the Rate4Site algorithm implemented in the ConSurf web server. This algorithm explicitly took into account the phylogenetic relationships between aligned proteins and the stochastic nature of evolutionary processes. Rate4Site assigned a conservation level for each residue using empirical Bayesian estimation. For visualization purposes, the persistent conservation scores were divided into nine discrete scales, ranging from the most variable positions (grade 1) colored turquoise, through moderately conserved positions (grade 5) colored white, to the most conserved positions (grade 9) colored maroon (not shown). Conservation scores were projected onto the protein / nucleotide sequence and the crystal structure corresponding to the PDB ID used as input.

[0171] result Examination of the conserved amino acid sequences of type I and type II PGDHs showed that several regions facing the active site are conserved in both type I and type II PHDGs. One such motif is [ka] One clear and highly conserved difference between these two motifs was the type of residue at the second position. In type I PGDH, this is an arginine residue, whereas in type II enzymes, it is most often a cysteine ​​residue. Examination of representative type I (M. tuberculosis) and type II (E. coli) PGDH structures with PHP and αKG, respectively, showed that both arginine and cysteine ​​residues face the active site, suggesting that these residues could be involved in controlling substrate specificity and, therefore, the ability to use αKG as a substrate. This was further investigated using site-directed mutagenesis to replace the arginyl side chain in M. tuberculosis PGDH with other selected amino acid side chains, such as alanine and leucine. Removal of the cationic group at Arg72 in M. tuberculosis PGDH converted this enzyme from one that does not accept αKG as a substrate to one that does. However, replacing the arginyl side chain with a lysyl side chain failed to achieve this. These results clearly demonstrate that the presence of a cationic side chain at the second position of the conserved motif prevents the enzyme from using αKG.

[0172] Example 11 - Effect of replacing NADH-dependent glyceraldehyde-3-phosphate dehydrogenase with NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase on the intracellular NAD(P)H pool This example demonstrates that introduction of gapC (SEQ ID NO: 43) or gdp1 (SEQ ID NO: 36) increases the overall NADP(H) pool in strains expressing serA (C. glutamicum / SEQ ID NO: 14), which lacks KGA reduction activity. To demonstrate this, E. coli strains expressing either gapA, gapC, or gdp1 from their genomes were transformed with the pSER_43 plasmid. This plasmid contains a strong constitutive promoter expressing the serine operon containing serA (SEQ ID NO: 14) from C. glutamicum. Transformants were selected on kanamycin plates. Inoculation and shake flasks were performed as described in Example 6. After 24 hours of incubation, 1 mL of biomass was harvested from each strain and quenched with 1 mL of 40% ethanol-0.8% sodium chloride solution. Subsequently, the samples were immersed in a dry ice-ethanol bath for 20 seconds. The samples were then incubated on ice for 15 min and centrifuged at 11,000 × g for 5 min at 4°C. The cell pellet was resuspended in 1 mL of MQ water. The intracellular NADP(H) pool was quantified using an [NADP+] / [NADPH] quantification kit (Sigma-Aldrich) according to the manufacturer's instructions.

[0173] result As shown in Figure 14, replacing NADH-dependent glyceraldehyde-3-phosphate dehydrogenase (gapA) with NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (gapC and gdp1) increases the overall intracellular cofactor pool when overexpressing gdp1, which lacks KGA reduction activity. The effects of gdp1 and gapC on the NADP(H) pool are comparable. Results are compared with those of 4 × 10 cells. 6 NADP in individuals + and the intracellular concentration of NADPH (picomolar).

[0174] Overall, this example also shows that the combined (over)expression of NADPH producing GAPDH, particularly GapC, and non-GAP producing PGDH, particularly serA from C. glutamicum, balances the intracellular NADPH and NADPH pools, allowing for superior L-serine production.

[0175] A list of references cited in the description 1. Grant, GA D-3-phosphoglycerate dehydrogenase. Front. Mol. Biosci. 5, 1-18 (2018). 2. Wei Xu1, 2, 12, Hui Yang1, 2, 12, Ying Liu3, 12, Ying Yang1, Ping Wang1, Se-Hee Kim8, S., Ito8, 10, Chen Yang6, Pu Wang1, 2, Meng-Tao Xiao1, 2, Li-xia Liu5, Wen-qing Jiang1, 2, J., Liu6, Jin-ye Zhang2, Bin Wang4, Stephen Frye9, Yi Zhang8, 10, 11, Yan-hui Xu1, Q. & Lei2, 5, Kun-Liang Guan1, 2, 5, 7,*, Shi-min Zhao1, 2,*, and Yue Xiong1, 2, 8, 11. Control of Embryonic Stem Cell State Richard. Cancer Cell 29, 997-1003 (2012). 3. Kalliri, E., Mulrooney, SB & Hausinger, RP Identification of Escherichia coli YgaF as an L-2-hydroxyglutarate oxidase. J. Bacteriol. 190, 3793-3798 (2008). 4. Zhang, W. et al. Coupling between D-3-phosphoglycerate dehydrogenase and D-2-hydroxyglutarate dehydrogenase drives bacterial L-serine synthesis. Proc. Natl. Acad. Sci. U. S. A. 114, E7574-E7582 (2017). 5. Zhao, G. & Winkler, M. E. A novel alpha-ketoglutarate reductase activity of the serA-encoded 3-phosphoglycerate dehydrogenase of Escherichia coli K-12 and its possible implications for human 2-hydroxyglutaric aciduria. J. Bacteriol. 178, 232-9 (1996). 6. Zhang, X., Xu, G., Shi, J., Koffas, M. A. G. & Xu, Z. Microbial Production of L-Serine from Renewable Feedstocks. Trends Biotechnol. 36, 700-712 (2018). 7. Rennig, M. et al. Industrializing a Bacterial Strain for l -Serine Production through Translation Initiation Optimization. ACS Synth. Biol. 8, 2347-2358 (2019). 8. Geueke, B., Riebel, B. & Hummel, W. NADH oxidase from Lactobacillus brevis: A new catalyst for the regeneration of NAD. Enzyme Microb. Technol. 32, 205-211 (2003). 9. Marx, A., Eikmanns, B. J., Sahm, H., De Graaf, A. A. & Eggeling, L. Response of the Central Metabolism inCorynebacterium glutamicumto the use of an NADH-Dependent Glutamate Dehydrogenase. Metab. Eng. 1, 35-48 (1999). 10. Martinez, I., Zhu, J., Lin, H., Bennett, G. N. & San, K. Y. Replacing Escherichia coli NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with a NADP-dependent enzyme from Clostridium acetobutylicum facilitates NADPH dependent pathways. Metab. Eng. 10, 352-359 (2008). 11. King, Z. A. & Feist, A. M. Optimal cofactor swapping can increase the theoretical yield for chemical production in Escherichia coli and Saccharomyces cerevisiae. Metab. Eng. 24, 117-128 (2014). 12. Niu, H. et al. Metabolic engineering for improving l-tryptophan production in Escherichia coli. J. Ind. Microbiol. Biotechnol. 46, 55-65 (2019). 13. Hashim, Y., Ismail, N., Jamal, P., Othman, R. & Salleh, H. Production of Cysteine: Approaches, Challenges and Potential Solution. Int. J. Biotechnol. Wellness Ind. 3, 95-101 (2014). 14. Zhao, G. & Winkler, M. E. A novel α-ketoglutarate reductase activity of the sera-encoded 3-phosphoglycerate dehydrogenase of Escherichia coli K-12 and its possible implications for human 2-hydroxyglutaric aciduria. J. Bacteriol. 178, 232-239 (1996). 15. Mundhada, H. et al. Increased production of L-serine in Escherichia coli through Adaptive Laboratory Evolution. Metab. Eng. 39, 141-150 (2017). 16. Mundhada, H., Schneider, K., Christensen, H. B. & Nielsen, A. T. Engineering of high yield production of L-serine in Escherichia coli. Biotechnol. Bioeng. 113, 807-816 (2016). 17. Wang, Y., San, K. Y. & Bennett, G. N. Improvement of NADPH bioavailability in Escherichia coli by replacing NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase GapA with NADP+-dependent GapB from Bacillus subtilis and addition of NAD kinase. J. Ind. Microbiol. Biotechnol. 40, 1449-1460 (2013). 18. Al-rabiee, R., Zhang, Y. & Grant, G. A. The Mechanism of Velocity Modulated Allosteric Regulation in D -3-Phosphoglycerate Dehydrogenase. 271, 23235-23238 (1996). 19. Ben Chorin A., Masrati G., Kessel A., Narunsky A., Sprinzak J., Lahav S., Ashkenazy H. and Ben-Tal N. (2020). ConSurf-DB: An accessible repository for the evolutionary conservation patterns of the majority of PDB proteins. Protein Science 29:258-267. 20. Goldenberg O., Erez E., Nimrod G. and Ben-Tal N. (2009). The ConSurf-DB: Pre-calculated evolutionary conservation profiles of protein structures. Nucleic Acids Research (Database issue), 37:D323-D327; PMID: 18971256.

[0176] Sequence Listing The instant application contains a list of sequences contained in Tables A and B below, which have been submitted electronically in ST26 format and are incorporated herein by reference in their entireties.

[0177] [Table 16] TIFF2025535809000026.tif235159TIFF2025535809000027.tif234159TIFF2025535809000028.tif231159TIFF2025535809000029.tif108159

[0178] [Table 17] TIFF2025535809000031.tif234159TIFF2025535809000032.tif237159TIFF2025535809000033.tif236159TIFF20255358090 00034.tif232159TIFF2025535809000035.tif232159TIFF2025535809000036.tif233159TIFF2025535809000037.tif235159 TIFF2025535809000038.tif235159TIFF2025535809000039.tif237159TIFF2025535809000040.tif233159TIFF20255358090 00041.tif238159TIFF2025535809000042.tif235159TIFF2025535809000043.tif236159TIFF2025535809000044.tif235159 TIFF2025535809000045.tif236159TIFF2025535809000046.tif236159TIFF2025535809000047.tif237159TIFF20255358090 00048.tif238159TIFF2025535809000049.tif235159TIFF2025535809000050.tif236159TIFF2025535809000051.tif232159 TIFF2025535809000052.tif232159TIFF2025535809000053.tif236159TIFF2025535809000054.tif234159TIFF20255358090 00055.tif237159TIFF2025535809000056.tif232159TIFF2025535809000057.tif235159TIFF2025535809000058.tif234159

[0179] Items of this disclosure The present disclosure further provides the following embodiments and items: Item 1. A genetically modified bacterium that has been modified to increase expression of a polypeptide having D-3-phosphoglycerate dehydrogenase activity (SerA) and decrease production of hydroxyglutaric acid (HGA) compared to other identical bacteria that do not possess the modification. Item 2. The bacterium according to Item 1, wherein hydroxyglutarate production is reduced by increasing the cytosolic NADPH pool and / or decreasing the cytosolic NADH pool compared to an otherwise identical bacterium that does not possess the modification. Item 3. The bacterium according to any one of Items 1 and 2, which expresses a polypeptide having D-3-phosphoglycerate dehydrogenase activity and comprising a polypeptide selected from the group consisting of SEQ ID NOs: 1 to 22, and an amino acid sequence having at least about 70%, for example, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1 to 22. Item 4. The bacterium according to any one of Items 1 to 3, wherein the reduction in HGA production is achieved by expression of a polypeptide having D-3-phosphoglycerate dehydrogenase activity that has low or no HGA production activity. Item 5. The bacterium according to any one of Items 1 to 4, which expresses a polypeptide having D-3-phosphoglycerate dehydrogenase activity and comprising a polypeptide selected from the group consisting of SEQ ID NOs: 6 to 15, and an amino acid sequence having at least about 70%, for example, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 6 to 15. Item 6.3 - The bacterium according to any of items 1 to 5, which expresses a polypeptide having phosphoglycerate dehydrogenase activity, wherein the polypeptide is derived from the polypeptide defined in item 4 and is made feedback insensitive by truncation of the C-terminal domain (e.g., SEQ ID NOs: 16 and 18) or by site-directed mutagenesis of key sites (e.g., SEQ ID NOs: 20 to 22). Item 7. The bacterium according to any one of Items 1 to 6, which has improved production of L-serine or an L-serine-derived compound. Item 8. The bacterium according to any one of Items 4 and 7, wherein when D-3-phosphoglycerate dehydrogenase activity is used in the presence of low or no activity of HGA production, the resulting decreased growth and redox imbalance are resolved by increasing the cytosolic NADPH pool and / or decreasing the cytosolic NADH pool. Item 9. The bacterium according to any one of items 1 to 8, wherein NADPH production is improved by heterologous expression of an NADPH-dependent polypeptide having glyceraldehyde-3-phosphate dehydrogenase activity, with or without additional ATP generation. Item 10. The bacterium according to any one of Items 1 to 4, which expresses an NADPH-dependent polypeptide having glyceraldehyde-3-phosphate dehydrogenase activity, with or without additional ATP generation, and which is selected from the group consisting of SEQ ID NOs: 23 to 46, and wherein the polypeptide comprises an amino acid sequence having at least about 70%, for example, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 23 to 46. Item 11. The bacterium according to Item 2, which has been modified to enhance expression of the enzyme glucose-6-phosphate dehydrogenase (e.g., SEQ ID NO: 47) and / or 6-phosphogluconate dehydrogenase (e.g., SEQ ID NO: 48). Item 12. The bacterium according to any one of Items 1 to 3, which expresses a heterologous polypeptide having NADH oxidase (Nox) activity so as to reduce the intracellular NADH pool. Item 13. The bacterium according to Item 12, wherein the heterologous polypeptide having NADH oxidase (Nox) activity is selected from the group consisting of SEQ ID NOs: 49 to 56, and the polypeptide comprises an amino acid sequence having at least about 70%, for example at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 49 to 56. Item 14. The bacterium according to any one of Items 1 to 2, wherein the NADH pool is depleted by expressing a heterologous NADH-dependent polypeptide having glutamate dehydrogenase activity. Item 15. The bacterium according to Item 14, wherein the heterologous NADH-dependent polypeptide having glutamate dehydrogenase activity is selected from the group consisting of SEQ ID NOs: 57 to 66, and the polypeptide comprises an amino acid sequence having at least about 70%, for example, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 57 to 66. Item 16. The bacterium according to any one of Items 1 to 15, which has been modified to reduce the expression and / or activity of an endogenous NADH-dependent polypeptide having glyceraldehyde-3-phosphate dehydrogenase activity, compared to an identical bacterium that does not possess the modification. Item 17. The bacterium according to Item 16, wherein the endogenous gene encoding the endogenous NADH-dependent polypeptide having glyceraldehyde-3-phosphate dehydrogenase activity is inactivated. Item 18. The bacterium according to any one of Items 1 to 17, wherein the polypeptide having D-3-phosphoglycerate dehydrogenase activity is a polypeptide with reduced activity on alpha-ketoglutarate, and the activity measured is reduced compared to the activity of SEQ ID NO: 16. Item 19. The bacterium according to Item 18, wherein the polypeptide having D-3-phosphoglycerate dehydrogenase activity is selected from the group consisting of SEQ ID NOs: 16 to 22, and the polypeptide comprises an amino acid sequence having at least about 70%, for example at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 16 to 22. Item 20. The bacterium according to any one of Items 1 to 19, wherein the bacterium belongs to the family Enterobacteriaceae. Item 21. The bacterium according to Item 20, wherein the bacterium belongs to the genus Escherichia. Item 22. The bacterium according to Item 21, wherein the bacterium is Escherichia coli. Item 23. The bacterium according to any one of Items 1 to 19, wherein the bacterium belongs to the genus Corynebacterium. Item 24. The bacterium according to Item 23, wherein the bacterium is Corynebacterium glutamicum. Item 25. A method for producing L-serine or an L-serine derivative, comprising culturing the bacterium according to any one of items 1 to 24 in a medium. Item 26. The method according to Item 25, wherein the L-serine derivative is selected from the group consisting of L-cysteine, L-methionine, L-glycine, O-acetylserine, L-tryptophan, thiamine, ethanolamine, and ethylene glycol. Item 27. The method according to Item 25 or 26, further comprising isolating L-serine or an L-serine derivative from the culture medium.

Claims

1. A genetically engineered host cell that produces a metabolite from 3-phosphoglycerate through a metabolic pathway, the host cell comprising one or more genetic modifications that prevent, reduce, or alleviate adverse effects resulting from intracellular accumulation of NADH and / or hydroxyglutarate (HGA) produced by one or more pathway enzymes on the production of the metabolite.

2. The host cell of claim 1 , wherein the metabolite is L-serine or a derivative thereof.

3. a) expression of a first heterologous NADPH-generating enzyme that converts glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate or a downstream precursor in the metabolic pathway; b) NADH to NAD ＋ Expression of a heterologous enzyme that converts c) expression of a heterologous enzyme in the metabolic pathway that reduces or eliminates NADH-consuming side activities compared to the corresponding pathway enzyme native to the host cell; d) expression of a heterologous enzyme that converts a by-product of an enzyme in the metabolic pathway into a substrate for an NADH- or NADPH-consuming metabolic pathway enzyme; e) overexpression of a native enzyme that converts a by-product of an enzyme in the metabolic pathway into a substrate for an NADH- or NADPH-consuming metabolic pathway enzyme; f) expression of a second heterologous NADPH-producing enzyme not involved in said metabolic pathway; and / or g) Overexpression of a natural NADPH-producing enzyme not included in the metabolic pathway 3. The host cell of claim 1 or 2, comprising one or more, optionally two or more, optionally three or more, optionally four or more, optionally five or more, optionally seven genetic modifications selected from the group consisting of:

4. 4. The host cell of claim 3, wherein the genetic modification comprises expression of the first heterologous NADPH-generating enzyme that produces a precursor for the metabolic pathway.

5. The genetic modification comprises: a) expression of the first heterologous NADPH-generating enzyme that converts glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate or a downstream precursor in the metabolic pathway; and b) expression of said heterologous metabolic pathway enzyme with reduced or eliminated NADH-consuming side activities compared to the corresponding metabolic pathway enzyme native to said host cell; The host cell of claim 4, comprising:

6. The genetic modification comprises: a) expression of said first heterologous NADPH-generating enzyme that converts glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate or a downstream precursor in said metabolic pathway; b) expression of the heterologous metabolic pathway enzyme with reduced or eliminated NADH-consuming side activities compared to the corresponding metabolic pathway enzyme native to the host cell; and c) NADH to NAD ＋ Expression of said heterologous enzyme converting The host cell of claim 5 , comprising:

7. The genetic modification comprises: a) expression of said first heterologous NADPH-generating enzyme that converts glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate or a downstream precursor in said metabolic pathway; b) expression of said heterologous metabolic pathway enzyme with reduced or eliminated NADH-consuming side activities compared to the corresponding metabolic pathway enzyme native to said host cell; c) NADH to NAD ＋ expression of said heterologous enzyme which converts d) Expression of the heterologous enzyme and / or overexpression of the native enzyme that converts the by-product of the enzyme in the metabolic pathway into the substrate of the metabolic pathway enzyme that consumes NADH or NADPH. The host cell of claim 6, comprising:

8. The genetic modification comprises: a) expression of said first heterologous NADPH-generating enzyme that converts glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate or a downstream precursor in said metabolic pathway; b) expression of said heterologous metabolic pathway enzyme with reduced or eliminated NADH-consuming side activities compared to the corresponding metabolic pathway enzyme native to said host cell; c) NADH to NAD ＋ expression of said heterologous enzyme which converts d) expression of the heterologous enzyme and / or overexpression of the native enzyme that converts the by-product of the enzyme in the metabolic pathway into a substrate for the metabolic pathway enzyme that consumes NADH or NADPH; and e) Expression of the second heterologous NADPH-producing enzyme and / or overexpression of the native NADPH-producing enzyme, neither of which is involved in the metabolic pathway. The host cell of claim 7, comprising:

9. a) the first heterologous NADPH-generating enzyme that converts glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate or a downstream precursor in the metabolic pathway partially or completely replaces an NADH-producing enzyme native to the host cell; b) the heterologous metabolic pathway enzyme, with reduced or eliminated NADH-consuming side activities, partially or completely replaces an enzyme native to the host cell; c) the heterologous enzyme that converts the by-product of the enzyme in the metabolic pathway to the substrate for the NADH- or NADPH-consuming metabolic pathway enzyme partially or completely replaces a native enzyme that converts the by-product of the enzyme in the metabolic pathway to the substrate for the NADH- or NADPH-consuming metabolic pathway enzyme; and / or d) the second heterologous enzyme that produces NADPH partially or completely replaces a native NADPH-producing enzyme that is not included in the metabolic pathway; A host cell according to any one of claims 3 to 8.

10. a) the first heterologous NADPH-generating enzyme that converts glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate or a downstream precursor in the metabolic pathway is bisphosphoglycerate synthase or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and NADP ＋ both convert glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate with the simultaneous conversion of b) NADH to NAD ＋ wherein the heterologous enzyme that converts c) the metabolic pathway enzyme having a reduced or eliminated NADH-consuming secondary activity is 3-phosphoglycerate dehydrogenase (PGDH), and the secondary activity is the conversion of α-ketoglutarate (α-KGA) to α-hydroxyglutarate (α-HGA); d) the enzyme in the metabolic pathway that converts the by-product of the enzyme to the substrate of the metabolic pathway enzyme is glutamate dehydrogenase (GDH), the by-product is α-ketoglutarate, and the substrate is glutamate; and / or e) the second heterologous or native NADPH-producing enzyme not involved in the metabolic pathway is glucose-6-phosphate dehydrogenase and / or 6-phosphogluconolactonase; The host cell of claim 9.

11. 11. The host cell of claim 10, wherein the heterologous enzyme is (i) an enzyme from a species different from the host cell, (ii) a mutant enzyme from a species different from the host cell, and / or (iii) a mutant enzyme native to the host cell.

12. 12. A host cell according to claim 10 or 11, comprising a heterologous PGDH.

13. The host cell according to claim 12, wherein the heterologous PGDH is overexpressed compared to a native PGDH that has the side activity of converting α-ketoglutarate (α-KGA) to α-hydroxyglutarate.

14. The host cell according to claim 13, wherein the heterologous PGDH is overexpressed by 10 to 10,000% compared to native PGDH.

15. The heterologous PGDH has the motif 【Chemistry 1】 and a conserved region facing the active site comprising the underlined 【Chemistry 2】 15. The host cell according to any one of claims 12 to 14, wherein is a cationic residue (SEQ ID NO: 10) at a position corresponding to position 129 of the PGDH derived from Mycobacterium tuberculosis.

16. The underlined 【Transformation 3】 The host cell of claim 12 , wherein is selected from arginine, leucine, or histidine.

17. The underlined 【Chemistry 4】 The host cell of claim 12 , wherein is arginine.

18. The heterologous PGDH has the motif 【Transformation 5】 16. The host cell of claim 12, further comprising a conserved region facing the active site that does not contain the underlined cysteine, wherein the underlined cysteine ​​is at a position corresponding to position 129 of the PGDH from Mycobacterium tuberculosis (SEQ ID NO: 10).

19. A host cell described in any one of claims 12 to 18, wherein the heterologous PGDH is a mutant PGDH, optionally native to the host cell, and modified to reduce or eliminate secondary activities that consume NADH compared to the unmodified PGDH.

20. 20. The host cell according to any one of claims 12 to 19, wherein the heterologous PGDH is a type I or type III PGDH, optionally a microbial type I or type III PGDH.

21. 21. The host cell of claim 12, wherein the heterologous PGDH enzyme is a SerA enzyme.

22. a) said GAPDH enzyme comprises a polypeptide which is at least 20%, such as at least 40%, for example at least 50%, such as at least 60%, for example at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99%, such as 100% identical to said GAP comprised in any one of SEQ ID NOs: 23 to 46, optionally SEQ ID NOs: 38 to 46; b) said Nox comprises a polypeptide which is at least 20%, such as at least 40%, for example at least 50%, such as at least 60%, for example at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99%, such as 100% identical to said Nox comprised in any one of SEQ ID NOs: 49-56; c) the PGDH comprises a polypeptide that is at least 20%, such as at least 40%, for example at least 50%, for example at least 60%, for example at least 70%, for example at least 80%, for example at least 90%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, for example 100% identical to the PGDH comprised in any one of SEQ ID NOs: 1 to 22; d) the GDH comprises a polypeptide that is at least 20%, such as at least 40%, for example at least 50%, for example at least 60%, such as at least 70%, for example at least 80%, such as at least 90%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, such as 100% identical to the GDH comprised in any one of SEQ ID NOs: 57 to 66; e) said glucose-6-phosphate dehydrogenase comprises a polypeptide which is at least 20%, such as at least 40%, for example at least 50%, such as at least 60%, for example at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 96%, for example at least 97%, such as at least 98%, for example at least 99%, such as 100% identical to said glucose-6-phosphate dehydrogenase comprised in SEQ ID NO: 47; and / or f) the 6-phosphogluconolactonase comprises a polypeptide which is at least 20%, such as at least 40%, for example at least 50%, such as at least 60%, for example at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99%, such as 100% identical to the 6-phosphogluconolactonase comprised in SEQ ID NO: 48; 22. A host cell according to any one of claims 10 to 21.

23. The host cell of claim 22, comprising a PGDH comprising a polypeptide that is at least 20%, such as at least 40%, for example at least 50%, for example at least 60%, such as at least 70%, for example at least 80%, for example at least 90%, such as at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, for example 100% identical to the PGDH comprised in any one of SEQ ID NOs: 6 to 22.

24. The host cell of claim 23, wherein the PGDH comprises a polypeptide that is at least 20%, such as at least 40%, for example at least 50%, for example at least 60%, such as at least 70%, for example at least 80%, for example at least 90%, for example at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99%, for example 100% identical to the PGDH comprised in any one of SEQ ID NOs: 6 to 15.

25. The host cell of claim 23, wherein the PGDH comprises a polypeptide that is at least 20%, such as at least 40%, for example at least 50%, for example at least 60%, such as at least 70%, for example at least 80%, for example at least 90%, for example at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99%, for example 100% identical to the PGDH comprised in SEQ ID NO: 14, or 19-21.

26. 26. The host cell of any one of claims 22 to 25, further comprising a GAPDH comprising a polypeptide which is at least 20%, such as at least 40%, for example at least 50%, such as at least 60%, for example at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99%, such as 100% identical to said GAPDH comprised in any one of SEQ ID NOs: 38 to 46.

27. 27. The host cell of claim 26, wherein the GAPDH comprises a polypeptide which is at least 20%, such as at least 40%, for example at least 50%, such as at least 60%, for example at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, for example 100% identical to the GAPDH comprised in SEQ ID NO:

43.

28. The host cell according to any one of claims 22 to 27, comprising a PGDH comprising a polypeptide which is at least 20%, such as at least 40%, for example at least 50%, such as at least 60%, for example at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99%, such as 100% identical to the PGDH comprised in SEQ ID NO: 14, or any one of 19 to 21; or a GAPDH comprising a polypeptide which is at least 20%, such as at least 40%, for example at least 50%, such as at least 60%, for example at least 70%, such as at least 80%, for example at least 90%, for example at least 95%, such as at least 96%, for example at least 97%, such as at least 98%, for example at least 99%, such as 100% identical to the GAPDH comprised in SEQ ID NO:

43.

28. a) phosphoserine aminotransferase (PSAT), which converts 3-phosphohydroxypyruvate to phosphoserine; and b) Phosphoserine phosphatase (PSPH), which converts phosphoserine to L-serine 28. The host cell of any one of claims 1 to 27, further expressing one or more metabolic pathway enzymes selected from:

29. a) said PSAT is serC and comprises a polypeptide which is at least 20%, such as at least 40%, for example at least 50%, such as at least 60%, for example at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 96%, for example at least 97%, such as at least 98%, for example at least 99%, such as 100% identical to said serC comprised in SEQ ID NO: 117; and b) said PSPH is serB and comprises a polypeptide which is at least 20%, such as at least 40%, for example at least 50%, such as at least 60%, for example at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, for example 100% identical to said serB comprised in SEQ ID NO: 118; 29. The host cell of claim 28.

30. 30. The host cell of any one of claims 1 to 29, further comprising at least one transporter molecule that facilitates the transport of a metabolite or one of its precursors.

31. 31. The host cell of any one of claims 1 to 30, wherein one or more native or endogenous genes of the cell are attenuated, disrupted, and / or deleted.

32. The host cell of claim 31 , wherein the native gene encodes an NADH-dependent GAPDH or an α-HGA-producing PHDH.

33. 33. The host cell of any one of claims 1 to 32, further comprising at least two copies of one or more polynucleotides encoding one or more metabolic pathway enzymes.

34. 34. The host cell of any one of claims 1 to 33, further genetically modified to provide increased amounts of substrates for one or more metabolic pathway enzymes.

35. 35. The host cell of any one of claims 1 to 34, further genetically modified to exhibit increased tolerance to one or more substrate, intermediate, or product molecules from the metabolic pathway.

36. 36. A host cell according to any one of claims 1 to 35, which is a prokaryotic cell, optionally a bacterium.

37. 37. The host cell of claim 36, wherein the prokaryotic cell is of the phylum Pseudomonadota, optionally the class Gammaproteobacteria, optionally the family Enterobacteriaceae, optionally the genus Escherichia, or optionally Escherichia coli.

38. 37. The host cell of claim 36, wherein the prokaryotic cell is of the phylum Actinomycetota, optionally the class Actinobacteria, optionally the family Corynebacteriaceae, optionally the genus Corynebacterium, or optionally Corynebacterium glutamicum.

39. 37. The host cell of claim 36, further modified to increase expression of a polypeptide having D-3-phosphoglycerate dehydrogenase activity and decrease production of alpha-hydroxyglutarate (alpha-HGA) compared to an otherwise identical bacterium not carrying said modification.

40. 40. The host cell of claim 39, wherein the production of alpha-hydroxyglutarate is reduced by increasing the cytosolic NADPH pool and / or decreasing the cytosolic NADH pool compared to an otherwise identical bacterium that does not possess the modification.

41. 41. The host cell of any one of claims 39 to 40, wherein the host cell expresses a polypeptide having D-3-phosphoglycerate dehydrogenase activity and comprising a polypeptide selected from the group consisting of SEQ ID NOs: 1-22, and an amino acid sequence having at least about 70%, e.g., at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-22.

42. 42. The host cell of claim 41, wherein the host cell expresses a polypeptide having D-3-phosphoglycerate dehydrogenase activity and comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-15, and having at least about 70%, e.g., at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 6-15.

43. 43. The bacterium of any one of claims 39 to 42, wherein the resulting reduced growth and redox imbalance are resolved by increasing the cytosolic NADPH pool and / or decreasing the cytosolic NADH pool when D-3-phosphoglycerate dehydrogenase activity is used in the presence of low or no activity of HGA production.

44. 44. The host cell of any one of claims 39 to 43, which has glyceraldehyde-3-phosphate dehydrogenase activity with or without additional ATP generation and expresses an NADPH-dependent polypeptide selected from the group consisting of SEQ ID NOs: 38-46, and a polypeptide comprising an amino acid sequence having at least about 70%, e.g., at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 38-46.

45. 45. A host cell according to any one of claims 39 to 44, which expresses a heterologous polypeptide having NADH oxidase (Nox) activity so as to reduce the intracellular NADH pool.

46. 46. ​​The host cell of claim 45, wherein the heterologous polypeptide having NADH oxidase (Nox) activity is selected from the group consisting of SEQ ID NOs: 49-56 and polypeptides comprising an amino acid sequence having at least about 70%, e.g., at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 49-56.

47. 47. The host cell of any one of claims 39 to 46, which has been modified to reduce the expression and / or activity of an endogenous NADH-dependent polypeptide having glyceraldehyde-3-phosphate dehydrogenase activity compared to an otherwise identical bacterium that does not possess said modification.

48. 48. The bacterium of claim 47, wherein the endogenous gene encoding the endogenous NADH-dependent polypeptide having glyceraldehyde-3-phosphate dehydrogenase activity is inactivated.

49. 49. The bacterium of claim 48, wherein the polypeptide having D-3-phosphoglycerate dehydrogenase activity is selected from the group consisting of SEQ ID NOs: 16-22 and polypeptides comprising an amino acid sequence having at least about 70%, such as at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 16-22.

50. 50. The bacterium of any one of claims 39 to 49, which belongs to the genus Escherichia or Corynebacterium.

51. 51. A cell culture comprising a host cell according to any one of claims 1 to 50 and a growth medium.

52. 1. A method for producing a metabolite from 3-phosphoglycerate, comprising: a) culturing the cell culture of claim 51 under conditions that allow the host cells to produce the metabolite; and b) optionally recovering and / or isolating said metabolites A method comprising:

53. 53. The method of claim 52, wherein the metabolite is L-serine or a derivative thereof.

54. The recovering and / or isolating step comprises separating a liquid phase of the cells or cell culture from a solid phase of the cells or cell culture to obtain a supernatant containing the metabolites, and separating the supernatant by: a) disrupting the cells of the cell culture to release intracellular metabolites into the supernatant; b) separating the supernatant from the solid phase of the cell culture, for example by filtration or sedimentation; c) contacting the supernatant with one or more adsorption resins to obtain at least a portion of the produced metabolites; d) contacting the supernatant with one or more ion exchange or reverse phase chromatography columns to obtain at least a portion of the metabolites; and e) crystallizing or extracting the metabolites from the supernatant; and f) evaporating the solvent from the supernatant to concentrate or precipitate the metabolites 53. The method of claim 52, comprising subjecting the metabolites to one or more steps selected from the group consisting of:

55. a) culturing said cell culture in a nutrient medium; b) culturing the cell culture under aerobic or anaerobic conditions c) culturing the cell culture under agitation; d) culturing the cell culture at a temperature of 25-50°C; e) culturing the cell culture at a pH of 3 to 9; and f) culturing the cell culture for 10 hours to 30 days 55. The method of any one of claims 52 to 54, further comprising one or more elements selected from:

56. 56. The method of any one of claims 52 to 55, wherein one or more steps of producing the metabolite are carried out in vitro.

57. 57. The method of any one of claims 52 to 56, comprising feeding the cell culture one or more metabolite precursors.

58. 52. A fermentation composition comprising metabolites of the cell culture of claim 51 and 3-phosphoglycerate-derived metabolites, wherein at least 20% by weight of the carbon is biobased.

59. 59. The fermentation composition of claim 58, further comprising one or more compounds selected from trace metals, vitamins, salts, yeast nitrogen base, carbon source, YNB, and / or fermentation amino acids; and wherein the concentration of the metabolites is at least 1 mg per kg of composition.

60. 60. The fermented composition of claim 58 or 59, which is substantially free of α-HGA.

61. 61. The fermentation composition of any one of claims 58 to 60, further comprising one or more carriers, agents, additives and / or excipients.

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