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  • C12P13/00—Preparation of nitrogen-containing organic compounds
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  • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
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  • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
  • C12Y102/01013—Glyceraldehyde-3-phosphate dehydrogenase (NADP+) (phosphorylating) (1.2.1.13)
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  • C12Y106/99—Oxidoreductases acting on NADH or NADPH (1.6) with other acceptors (1.6.99)
  • C12Y106/99003—NADH dehydrogenase (1.6.99.3)

Definitions

• the present invention relates to the microbiological industry, and specifically to the bioindustrial production of metabolites such as L-serine and derivatives using genetically modified bacteria. More specifically the present disclosure describes recombinant host cells producing the metabolites from glyceraldehyde-3-phosphate through a metabolic pathway engineered to reduce intracellular accumulation of NADH and/or hydroxy glutarate (HGA) negative impacting on the metabolite production. Further disclosed are recombinant polynucleotides encoding recombinant polypeptides of the pathway, and cell cultures of the host cell which when cultured in fermentation methods produce the metabolites. Further disclosed are fermentation compositions the host cells and/or the metabolites thereof resulting from such methods also known as biobased compositions of metabolites.
• HGA hydroxy glutarate
• W02004/108894 discloses methods for producing amino acids using a genetically modified bacteria, including disclosure of a polypeptide similar to SEQ ID NO: 10 of this disclosure.
• W02021/081185 describes microbial organisms having increased availability of co-factors, such as NADPH, for increasing production of various products.
• W02020/0107626 describes methods of producing L-amino acids comprising culturing altered bacterial cells having increased amounts of NADPH as compared to unaltered bacterial cells whereby L-amino acids yields from said altered bacterial cells are greater than yields from unaltered bacterial cells.
• WO2021/195705 describes recombinant microorganisms for producing biological hydrogen and nucleic acid constructs and processes for modifying microorganisms for enabling the production of hydrogen. In this process it is suggested to replace gapA gene in E. coli with the gapC gene from Clostridium acetobutylicum.
• CN103436504A describing a construction method and application of corynebacterium glutamicum strain resistant to the feedback inhibition by L-serine, by mutating a 3-phosophoglycerate dehydrogenase resistant to feedback inhibition by L-serine.
• the 3-phosophoglycerate dehydrogenase has a minor similarity to SEQ. ID NO: 10 of this disclosure.
• HGA hydroxyglutarate
• IDH isocitrate dehydrogenase
• SerA phosphoglycerate dehydrogenase
• HGA oxidases such as LghO (previously YgaF) in E. coli 3 and D- hydroxyglutarate dehydrogenase (D2DHH) in many Pseudomonas species 4 , HGA is oxidized back to KGA. The generated reducing equivalents are donated to cytochrome c which in turn is channelized to Electron transport chain for ATP generation or to oxygen to produce hydrogen peroxide 3 .
• SerA has been classified into three types namely Type 1, Type 2 and Type 3 based on the presence of different protein domains 1 . It is known that some SerA homologues do not have KGA reduction activity. However, such SerA proteins do not necessarily belong to one specific type or class of enzyme. For example, SerA from rat, M. tuberculosis, C. glutamicum and B. subtilis does not seem to have HGA accumulation 4 whereas human SerA belonging to the same class has a tendency of HGA accumulation 5 . It is still not known how SerA without the promiscuous KGA reduction activity overcome the free energy barrier to drive the reaction forward efficiently.
• SerA is the first key step of L-serine biosynthesis, therefore in order to overproduce L-serine, SerA from E. coli is overexpressed in most of the published works 6 . Since the oxidation of 3-PG has been coupled with reduction of KGA (figure 1 B), increased L-serine production leads to higher accumulation of HGA 7 . The obvious strategy to avoid HGA accumulation is to overexpress heterologous SerA which does not have activity towards KGA as it has been shown in C. glutamicum 4,6 or to co-express HGA oxidase to recycle HGA back to KGA as previously demonstrated.
• One objective of the present invention is to provide means allowing a more efficient production of metabolites from glyceraldehyde-3-phosphate, such as L-serine and derivatives thereof, microbial cells. More particularly, it is an object of the present invention to provide means allowing the production of metabolites from glyceraldehyde-3-phosphate, such as L-serine or derivatives thereof at higher nominal yield and improved mass yield.
• the disclosure describes a cell culture, comprising the host cell described herein and a growth medium.
• the disclosure describes a method for producing metabolite from 3- phosphoglycerate comprising: a) culturing the cell culture described herein at conditions allowing the host cell to produce the metabolite from 3-phosphoglycerate; and b) optionally recovering and/or isolating the metabolite from 3-phosphoglycerate.
• the disclosure describes a fermentation composition comprising biobased metabolites of the cell culture described herein and the metabolite from 3-phosphoglycerate, wherein at least 20% by weight of the carbon is biobased.
• the present invention provides in a fifth aspect a genetically engineered bacterium which has been modified to have an increased expression of a polypeptide having D-3-phosphoglycerate dehydrogenase activity (SerA) and a decreased production of hydroxyglutarate (HGA) compared to an otherwise identical bacterium that does not carry said modification.
• SerA D-3-phosphoglycerate dehydrogenase activity
• HGA hydroxyglutarate
• the present invention provides in a sixth aspect a method for producing L-serine comprising: cultivating the bacterium as described in the fifths aspect in a culture medium.
• Figure 1 A. The reaction catalyzed by SerA.
• the native reaction includes oxidation of PGA while promiscuous reaction involves reduction of KGA.
• the potential reason for HGA production by SerA is that the overall Gibbs free energy of the reaction is reduced from 33 to 4.5 kJ/mol 4 .
• FIG. 1 HGA concentrations detected in batch fermentation supernatants after 24h incubation.
• the strain expressing NADH oxidase (Nox) accumulates 8 times less HGA than the strain without NADH oxidase expression.
• Figure 3 A. Schematic of NADH recycling for L-serine production via RocG.
• Figure 6a to 6c Plasmid maps of all the plasmids used in the examples 1 to 5.
• Figure 7 Plasmid maps of plasmids used in Example 6.
• Figure 8 L-serine concentrations detected in batch fermentation supernatants after 24-hour incubation.
• the strain expressing truncated serA from rat presents increased L-serine production.
• Figure 9 L-serine concentrations detected in batch fermentation supernatants after 24-hour incubation of strains expressing different NADPH glyceraldehyde 3 phosphate dehydrogenases.
• Figure 10 Plasmid map of plasmid used in Example 8.
• Figure 11 L-serine concentrations detected in batch fermentation supernatants after 24-hour incubation. Strain expressing Nox from L. parakefiri shows increased L-serine production.
• Figure 12 Plasmids maps of all plasmids used in Example 9.
• Figure 13a to 13d L-serine and HGA concentrations in batch fermentation supernatants after 24-hour incubation of strains over expressing pgl and zwf in combination with serA from C. glutamicum in gapC and gapA backgrounds, and serA from E. coli in gapC and gapA backgrounds.
• Figure 14 shows effects of replacing NADH-dependent glyceraldehyde-3-phosphate dehydrogenase (gapA) with NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (gapC and gdpl) when expressing serA with no KGA reducing activity.
• gapA NADH-dependent glyceraldehyde-3-phosphate dehydrogenase
• gapC and gdpl NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase
• Figure 15 shows the pathway for L-serine, including the steps from 3-phosohoglycerate.
• Figure 16 shows the HMP shunt pathway employing glucose-6-phosphate dehydrogenase (zwf) and 6-phosphogluconolactonase (pgl).
• heterologous or recombinant or “genetically modified” and their grammatical equivalents as used herein interchangeably about nucleotides, polypeptides and cells refers to entities "derived from a different species or cell".
• a heterologous or recombinant polynucleotide gene is a gene in a host cell not naturally containing 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 not naturally containing the polypeptide, i.e. the polypeptide is from a different species or cell type than the host cell.
• host cells refer to host cells comprising and expressing heterologous or recombinant polynucleotides.
• recombinant or “non-naturally occurring” when used with reference to, e.g., a host cell, nucleic acid, or polypeptide, refers to a material, or a material corresponding to the natural or native form of the material, that has been modified in a manner that would not otherwise exist in nature, or is identical thereto but produced or derived from synthetic materials and/or by manipulation using recombinant techniques.
• Non-limiting examples include, among others, recombinant host cells expressing genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise expressed at a different level.
• Heterologous as used herein means that a polypeptide is normally not found in or made (i.e. expressed) by the host organism, but derived from a different species.
• % identity is used herein about the relatedness between two amino acid sequences or between two nucleotide sequences usings standard alignment software known in the art, and applying settings as instructed for the software, including gaps, to achieve the maximum percent identity/similarity/homology and, if necessary, considering any conservative substitutions according to the NCIUB rules (hftp://www.chem. qmul.ac.uk/iubmb/misc/naseq.html; NC-IUB, Eur. J. Biochem. (1985)) as part of the sequence identity.
• Percentage of sequence identity can be used herein to refer to comparisons between an amino acid sequence and a reference amino acid sequence.
• % sequence identify is calculated from the two amino acid sequences as follows: The sequences are aligned using Version 9 of the Genetic Computing Group's GAP (global alignment program), using the default BLOSUIVI62 matrix (see below) with a gap open penalty of -12 (for the first null of a gap) and a gap extension penalty of -4 (for each additional null in the gap). After alignment, percentage identity is calculated by expressing the number of matches as a percentage of the number of amino acids in the reference amino acid sequence. The following BLOSUIVI62 matrix is used:
• Reference sequence or “reference amino acid sequence” refers to a defined sequence to which another sequence is compared.
• a reference amino acid sequence may, for example, be an amino acid sequence set forth in SEQ. ID NO: 5 or 6.
• substitution refers to modification of the polypeptide by replacing one amino acid residue with another, for instance the replacement of a Serine residue with a Glycine or Alanine residue in a polypeptide sequence is an amino acid substitution.
• substitution refers to modification of the polynucleotide by replacing one nucleotide with another, for instance the replacement of a cytosine with a thymine in a polynucleotide sequence is a nucleotide substitution.
• Constant substitution when used with reference to a polypeptide, refers to a substitution of an amino acid residue with a different residue having a similar side chain, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar class of amino acids.
• an amino acid with an aliphatic side chain may be substituted with another aliphatic amino acid, e.g., alanine, valine, leucine, and isoleucine; an amino acid with hydroxyl side chain is substituted with another amino acid with a hydroxyl side chain, e.g., serine and threonine; an amino acid having an aromatic side chain is substituted with another amino acid having an aromatic side chain, e.g., phenylalanine, tyrosine, tryptophan, and histidine; an amino acid with a basic side chain is substituted with another amino acid with a basic side chain, e.g., lysine and arginine; an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain, e.g., aspartic acid or glutamic acid; and a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively.
• another aliphatic amino acid
• Non-conservative substitution when used with reference to a polypeptide, refers to a substitution of an amino acid in a polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affects (a) the structure of the peptide backbone in the area of the substitution (e.g., serine for glycine), (b) the charge or hydrophobicity, or (c) the bulk of the side chain.
• an exemplary non-conservative substitution can 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.
• pathway or “biosynthetic pathway” or “metabolic pathway” as used herein interchangeably refers to one or more enzymes acting in concert in a live cell to convert one or more substrate precursors into a chemical product.
• a pathway may include one enzyme or multiple enzymes acting in sequence or in combination.
• a pathway including only one enzyme may also herein be referred to as "bioconversion” in particular relevant for embodiments where a host cell is fed with a precursor or substrate exogenously to be converted by the enzyme into a desired end product.
• Enzymes are characterized by having catalytic activity, which can change the chemical structure of the substrate(s).
• An enzyme may have more than one substrate and produce more than one product.
• the enzyme may also depend on cofactors, which can be inorganic chemical compounds or organic compounds (co-factor and/or co-enzymes) which may or may not be considered part of the pathway.
• cofactors which can be inorganic chemical compounds or organic compounds (co-factor and/or co-enzymes) which may or may not be considered part of the pathway.
• cofactors can be inorganic chemical compounds or organic compounds (co-factor and/or co-enzymes) which may or may not be considered part of the pathway.
• cofactors can be inorganic chemical compounds or organic compounds (co-factor and/or co-enzymes) which may or may not be considered part of the pathway.
• in vivo refers to within a living cell or organism, including, for example animal, a plant, or a microorganism.
• in vitro refers to outside a living cell or organism, including, without limitation, for example, in a microwell plate, a tube, a flask, a beaker, a tank, a reactor and the like.
• substrate or "precursor” as used herein refers to any compound that can be converted into a different compound.
• substrates and/or precursors include both compounds generated in situ by an enzymatic reaction in a cell or exogenously provided compounds, such as exogenously provided organic molecules which the host cell can metabolize into a desired compound.
• expression includes any step involved in the production of a polypeptide (e.g., encoded enzyme) including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
• a polypeptide e.g., encoded enzyme
• expression vector refers to a DNA molecule, either single- or double stranded, either linear or circular, which comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression.
• Expression vectors include expression cassettes for the integration of genes into a host cell as well as plasmids and/or chromosomes comprising such genes.
• Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as expression vectors.
• Vectors can also refer to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked.
• vector refers to a circular double stranded nucleic acid loop into which additional nucleic acid segments can be ligated.
• Certain other vectors are capable of facilitating the insertion of an exogenous nucleic acid molecule into a genome of a bacterium. Such vectors are referred to herein as "transformation vectors".
• transformation vectors In general, vectors of utility in recombinant nucleic acid techniques are often in the form of plasmids.
• vectors of utility in recombinant nucleic acid techniques are often in the form of plasmids.
• vectors of utility in recombinant nucleic acid techniques are often in the form of plasmids.
• vectors of utility in recombinant nucleic acid techniques are often in the form of plasmids.
• vectors and “vector” can be used interchangeably as the plasmid is one of the most commonly used form of a vector. Large numbers of suitable vectors are known to those of skill in the art and
• host cell refers to any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide to be expressed in the host cell.
• Host cell encompasses any progeny of a parent cell including those that are not identical to the parent cell due to mutations that occur during replication.
• Nucleic acid or “polynucleotide” are used interchangeably herein to denote a polymer of at least two nucleic acid monomer units or bases (e.g., adenine, cytosine, guanine, thymine) covalently linked by a phosphodiester bond, regardless of length or base modification.
• bases e.g., adenine, cytosine, guanine, thymine
• polynucleotide construct refers to a polynucleotide, either single- or double stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, and which comprises a polynucleotide encoding a polypeptide and one or more control sequences.
• operably linked refers to a configuration in which a control sequence is placed at an appropriate position relative to a coding polynucleotide such that the control sequence directs expression of the coding polynucleotide. More generally, “operably linked” refers to a juxtaposition wherein the components described 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 sequence.
• a promoter sequence is "operably-linked” to a gene when it is in sufficient proximity to the transcription start site of a gene to regulate transcription of the gene.
• promoter refers to a sequence of DNA, usually upstream (5') of the coding region of a structural gene, which controls the expression of the coding region by providing recognition and binding sites for RNA polymerase and other factors which may be required for initiation of transcription. The selection of the promoter will depend upon the nucleic acid sequence of interest.
• a suitable “promoter” is generally one which is capable of supporting the initiation of transcription in a bacterium of the invention, causing the production of an mRNA molecule.
• Polypeptide and “protein” are used interchangeably herein to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post- translational modification (e.g., glycosylation, phosphorylation, lipidation, myristylation, ubiquitination, etc.). Included within this definition are D- and L-amino acids, and mixtures of D- and L-amino acids.
• biobased as used herein is used to characterize biobased products wherein: a) the total carbon content of the product is at least 30%, and b) the carbon content of a renewable raw material (biobased) is at least 20%.
• Both fossil and renewable raw materials consist mainly of carbon (C). Carbon occurs in several isotopes. Isotope 14 C is radioactive and occurs naturally in all living organisms (plants, animals, etc.) in a fixed relative concentration which is nearly identical to the relative 14 C concentration in the atmosphere. At this concentration, the radioactivity level of 14 C is 100%. Once an organism is no longer living, this concentration, and thus the radioactivity rate, decays with a half-life of approximately 5700 years. The radioactive 14 C level of an unknown substance can therefore help determine how old the carbon contained in the substance is.
• radioactive 14 C level refers to the total radioactive 14 C level of a given substance, product, or composition, as defined above.
• % biobased carbon When measuring the biobased carbon content, the result may be reported as "% biobased carbon". This indicates the percentage carbon from "natural” (plant or animal by-product) sources versus “synthetic” or “fossil” (petrochemical) sources. For reference, 100 % biobased carbon indicates that a material is entirely sourced from plants or animal by-products and 0 % biobased carbon indicates that a material did not contain any carbon from plants or animal by-products. A value in between represents a mixture of natural and fossil sources. For example: If a product has a radioactive 14 C level of 80%, it means that the product consists of 80% renewable and 20% fossil carbon (C). In other words, the product is 80% biobased.
• L-serine derivative refers to a compound, such as an amino acid, resulting from reaction of L-serine at the amino group or the carboxy group or hydroxyl group, or from the replacement of any hydrogen of L-serine by a heteroatom.
• Non-limiting examples of a “L-serine derivative” include L-cysteine, L-methionine, L-glycine, O-acetylserine, L-tryptophan, thiamine, ethanolamine and ethylene glycol.
• L-serine derivative is described by Chemical Entities of Biological Interest (ChEBI) [https://www.ebi.ac.uk/chebi/init.do], for example, under ChEBI ID CHEBI:84135.
• V overexpression of a native enzyme converting a side product of an enzyme in the metabolite pathway into a substrate of a metabolite pathway enzyme consuming NADH or NADPH;
• VL expression of a second heterologous NADPH producing enzyme which is not comprised in the metabolite pathway;
• the present inventors have found that inhibiting accumulation of NADH in the cell improves the downstream efficiently of pathways producing metabolites from 3-phosphoglycerate, in particular L-serine and/or derivatives thereof, so in a further embodiment the genetic modification of the host cell comprises expression of the first heterologous NADPH generating enzyme converting glyceraldehyde 3-phosphate into 1,3-bisphosphoglycerate or a downstream precursor in the metabolite pathway.
• the present inventors have also found that it is particularly advantageous and synergistic for the host cell production of metabolites from 3-phosphoglycerate, to combine two or more of the modifications I) to VII), supra, and in a further embodiment the genetic modification of the host cell comprises: a) expression of the first heterologous NADPH generating enzyme converting glyceraldehyde 3- phosphate into 1,3-bisphosphoglycerate or a downstream precursor in the metabolite pathway; and b) expression of the heterologous metabolite pathway enzyme having a reduced or eliminated NADH consuming side activity compared to a corresponding metabolite pathway enzyme native to the host cell.
• the present inventors have also found that it is particularly advantageous and synergistic for the host cell production of metabolites from 3-phosphoglycerate, to combine three or more of the modifications I) to VII), supra, and in a further embodiment the genetic modification of the host cell comprises: a) expression of the first heterologous NADPH generating enzyme converting glyceraldehyde 3- phosphate into 1,3-bisphosphoglycerate or a downstream precursor in the metabolite pathway; b) expression of the heterologous metabolite pathway enzyme having a reduced or eliminated NADH consuming side activity compared to a corresponding metabolite pathway enzyme native to the host cell; and c) expression of the heterologous enzyme converting NADH to NAD+.
• the genetic modification of the host cell comprises: a) expression of the first heterologous NADPH generating enzyme converting glyceraldehyde 3- phosphate into 1,3-bisphosphoglycerate or a downstream precursor in the metabolite pathway; b) expression of the heterologous metabolite pathway enzyme having a reduced or eliminated NADH consuming side activity compared to a corresponding metabolite pathway enzyme native to the host cell; c) expression of the heterologous enzyme converting NADH to NAD+; and d) expression of the heterologous enzyme and/or overexpression of the native enzyme converting the side product of the enzyme in the metabolite pathway into the substrate of the metabolite pathway enzyme consuming NADH or NADPH.
• the genetic modification of the host cell comprises: a) expression of the first heterologous NADPH generating enzyme converting glyceraldehyde 3- phosphate into 1,3-bisphosphoglycerate or a downstream precursor in the metabolite pathway; b) expression of the heterologous metabolite pathway enzyme having a reduced or eliminated NADH consuming side activity compared to a corresponding metabolite pathway enzyme native to the host cell; c) expression of the heterologous enzyme converting NADH to NAD+; d) expression of the heterologous enzyme and/or overexpression of the native enzyme converting the side product of the enzyme in the metabolite pathway into a substrate of the metabolite pathway enzyme consuming NADH or NADPH; and e) expression of the second heterologous NADPH
• the first heterologous NADPH generating enzyme, the heterologous enzyme having a reduced or eliminated NADH consuming side activity, the heterologous enzyme converting the side product into a pathway substrate and/or the second heterologous enzyme producing NADPH partially or completely replaces enzymes native to the host cell.
• Such native enzymes can be part of metabolite pathway or not.
• the NADPH pool of the genetically engineered bacterium is increased by the recombinant expression of a NADPH dependent polypeptide having Glyceraldehyde-3- phosphate dehydrogenase activity with or without additional ATP generation.
• the first heterologous NADPH generating enzyme converting glyceraldehyde 3-phosphate into 1,3-bisphosphoglycerate or a downstream precursor in the metabolite pathway can be a bisphoshoglycerate synthase or a glyceraldehyde-3-phosphate dehydrogenase (GAPDH), both converting glyceraldehyde 3-phosphate into 1,3-bisphosphoglycerate; under the co-conversion of NADP+ into NADPH.
• GPDH glyceraldehyde-3-phosphate dehydrogenase
• SEQ ID NOs: 23 to 46 disclose some exemplary glyceraldehyde-3-phosphate dehydrogenases, and in some embodiments the GAPDH enzyme comprises a polypeptide which 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 GAP comprised in any one of SEQ. ID NO: 23 to 46. Particularly the GAPDH of SEQ ID NO: 38 to 46 are useful.
• the GAPDH is GapC or a NADP dependent variant thereof that comprises a polypeptide which 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 GAPDH comprised in SEQ ID NO: 43.
• the NADH pool of the genetically engineered bacterium is reduced by the recombinant expression of a heterologous NADH oxidase, so in a further embodiment the heterologous enzyme converting NADH to NAD+ is a NADH oxidase (Nox).
• SEQ ID NOs: 49 to 56 disclose some exemplary NADH oxidases and in further embodiments the Nox comprises a polypeptide which 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 Nox comprised in any one of SEQ ID NO: 49 to 56.
• the metabolite pathway enzyme having a reduced or eliminated NADH consuming side activity is a 3-phosphoglycerate dehydrogenase (PGDH), also known as a D-3- phosphoglycerate dehydrogenase and the side activity is conversion of a-ketoglutarate (a-KGA) into a-hydroxyglutarate (a-HGA).
• PGDH 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 PGDHs can use a-ketoglutarate (aKG) instead of PHP in the reverse direction to produce a-hydroxyglutarate.
• the PGDH is heterologous and more preferably the heterologous PGDH is overexpressed compared to a native PGDH having a side activity of converting a-ketoglutarate (a- KGA) into a-hydroxyglutarate.
• the overexpression of the heterologous PGDH may be 10% to 10.000% compared to the native PGDH such as 50% to 5000%, such as 100% to 1000%.
• 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 different protein domains 1 . Some D-3-phosphoglycerate dehydrogenases do not have the KGA reduction activity.
• D-3-phosphoglycerate dehydrogenase do not necessarily belong to one type or class of enzyme.
• SerA from Rattus norvegicus, M. tuberculosis, C. glutamicum and B. subtilis does not seem to have HGA accumulation 4
• human SerA has tendency of HGA accumulation although it belongs to the same class 14 .
• Three enzymes have been found to be able to utilize aKG as a substrate: PDGH from E. coli, Pseudomonas stutzeri, and Saccharomyces cerevisiae, which are all type II PGDH's.
• PGDH's are not able to use aKG as a substrate are type I and type III PGDH's.
• the present inventors have found that for each of the three types of PGDH several regions facing the active site contains conserved motifs in both type I and II PHDG's.
• One such motif is the G/ARAGV and GCFCI motif in type I and II PGDHs respectively.
• One distinct, and highly conserved, difference between these two motives is the residue type at the second position. In type I PGDHs this is an arginine residue whereas it is most often a cysteine residue in Type II enzymes.
• col PGDH structures with PHP and aKG bound, respectively it has been found that both the arginine and cysteine residues are facing the active site, indicating that these residues are involved in controlling substrate specificity, and hence the ability to use aKG as a substrate. Further it has been found that using site-directed mutagenesis to substitute the arginyl side chain in M. tuberculosis PGDH with other selected amino acid side chains such as alanine and leucine the removal of the cationic group of Arg 72 in M. tuberculosis PGDH change the specificity from not accepting aKG as a substrate to accepting aKG as a substrate.
• the heterologous PGDH comprises a conserved region facing the active site which comprises the motif G/AXAGV, wherein the underlined residue X is a cationic residue, preferably at host cell intracellular pH, positioned corresponding to position 129 of the PGDH from Mycobacterium tuberculosis (SEQ ID NO: 10).
• Useful PDGH's are those particularly where the underlined X is selected from Arginine, Leucine or Histidine, more particularly Arginine.
• Additionally or alternatively useful heterologous PGDH's are those which comprise a conserved region facing the active site, which does not comprises the motif GCFCI, wherein the underlined cysteine is positioned corresponding to position 129 of the PGDH from Mycobacterium tuberculosis (SEQ. ID NO: 10).
• the heterologous PGDH is mutant PGDH, optionally native to the host cell, modified to reduce or eliminate the NADH consuming side activity compared to the unmodified PGDH, for example by replacing a cysteine positioned corresponding to position 129 of the PGDH from Mycobacterium tuberculosis (SEQ ID NO: 10), optionally in a motif GCFCI, with a cationic residue, such as Arg, Leu or His, particularly Arg.
• the heterologous PGDH can be a type I or type III PGDH, optionally a microbial type I or a type III PGDH and moreover it may be a SerA enzyme.
• SEQ. ID NOs: 1-22 disclose some exemplary D-3-phosphoglycerate dehydrogenases, and particularly useful PGDH enzymes are those which comprise a polypeptide which 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 PGDH comprised in any one of SEQ. ID NO: 1 to 22.
• the PGDH is not producing HGA or is insensitive to L-serine feedback and comprises a polypeptide which 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 PGDH comprised in any one of SEQ ID NO: 6 to 22.
• the PGDH is not producing HGA and comprises a polypeptide which 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 PGDH comprised in any one of SEQ ID NO: 6 to 15.
• the PGDH is a non-HGA producing C.
• the said metabolite pathway enzyme is preferably a glutamate dehydrogenase (GDH)
• GDH glutamate dehydrogenase
• the side product is preferably a- ketoglutarate
• the substrate is glutamate
• the PSPH can be a SerB and in some embodiments comprise a polypeptide which 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 SerB comprised in SEQ. ID NO: 118.
• the host cell further comprises at least 2 copies of one or more polynucleotides encoding one or more metabolite pathway enzymes. In further embodiments, the host cell is further genetically modified to provide an increased amount of a substrate for one or more metabolite pathway enzymes. In further embodiments the host cell is further genetically modified to exhibit increased tolerance towards one or more precursors, substrates, intermediates, or product molecules from the metabolite pathway.
• the cultivation can take place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art.
• suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. from catalogues of the American Type Culture Collection). The selection of the appropriate medium may be based on the choice of host cell and/or based on the regulatory requirements for the host cell. Such media are available in the art.
• the medium may, if desired, contain additional components favouring the host cells over other potentially contaminating microorganisms.
• a suitable nutrient medium comprises a carbon source (e.g.
• a nitrogen source e. g. ammonium sulphate, ammonium nitrate, ammonium chloride, etc.
• an organic nitrogen source e.g. yeast extract, malt extract, peptone, etc.
• inorganic nutrient sources e.g. phosphate, magnesium, potassium, zinc, iron, etc.
• the cultivation process may be a batch process, continuous or fed-batch process, suitably performed at a temperature in the range of 0-100 °C or 10-80 °C, for example, from about 20°C to about 50 °C and/or at a pH, for example, from about 2 to about 10.
• Preferred fermentation conditions for prokaryotic host cells are a temperature in the range of from about 25 °C to about 55 °C and at a pH of from about 3 to about 9. The appropriate conditions are usually selected based on the choice of host cell.
• the method of the disclosure further comprises one or more elements selected from: a) culturing the 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 between 25 to 50 °C; e) culturing the cell culture at a pH of between 3-9; and f) culturing the cell culture for between 10 hours to 30 days.
• the cell culture of the disclosure may be recovered and or isolated using methods known in the art.
• the metabolite may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, spray-drying, or lyophilization.
• the method includes a recovery and/or isolation step comprising separating a liquid phase of the cell or cell culture from a solid phase of the cell or cell culture to obtain a supernatant comprising the metabolite and/or subjecting the supernatant to one or more steps selected from: a) disrupting the cells of the cell culture to release intracellular metabolite into the supernatant; b) separating the supernatant from the solid phase of the cell culture, such as by filtration or gravity separation; c) contacting the supernatant with one or more adsorbent resins to obtain at least a portion of the produced metabolite; d) contacting the supernatant with one or more ion exchange or reversed-phase chromatography columns in order to obtain at least a portion of the metabolite; e) extracting the metabolite; and/or f) precipitating the metabolite by crystallization or evaporating the solvent of the liquid phase; and optionally isolating the
• the method further comprises feeding the cell culture with one or more precursor or substrates in the pathway of the metabolite.
• the method comprises one or more in vitro steps in the process of producing the metabolite.
• the metabolite is not the desired end products further steps may be added to the method described herein either chemically or biologically/enzymatically modifying the metabolite
• the method can further comprise recovering the metabolite and mixing it with one or more carriers, agents, additives, adjuvants and/or excipients to produce a biopesticide composition.
• the present invention also provides methods for production of L- serine or a L-serine derivatives using a genetically engineered bacterium according to the present invention.
• the present invention provides a method for production of L-serine or a L- serine derivatives which comprises cultivating a genetically engineered bacterium as detailed herein in a culture medium.
• the present invention provides a method for producing L-serine.
• the present invention provides a method for producing L-serine, said method comprises cultivating a genetically engineered bacterium as detailed herein in a culture medium.
• the method may further comprise isolating L-serine from the culture medium.
• the present invention provides a method for producing a L-serine derivative.
• the present invention provides a method for producing a L-serine derivative, said method comprises cultivating a genetically engineered bacterium as detailed herein in a culture 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.
• the present invention provides a method for producing L-cysteine.
• the present invention provides a method for producing L-cysteine, said method comprises cultivating a genetically engineered bacterium as detailed 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. Particularly, the present invention provides a method for producing L-methionine; said method comprises cultivating a genetically engineered bacterium as detailed herein in a culture medium. The method may further comprise isolating L-methionine from the culture medium. According to certain embodiments, present invention provides a method for producing L-glycine.
• the present invention provides a method for producing L-glycine; said method comprises cultivating a genetically engineered bacterium as detailed herein in a culture medium. The method may further comprise isolating L-glycine from the culture medium.
• present invention provides a method for producing O-acetylserine.
• the present invention provides a method for producing O- acetylserine, said method comprises cultivating a genetically engineered bacterium as detailed herein in a culture medium.
• the method may further comprise isolating O-acetylserine from the culture medium.
• present invention provides a method for producing L- tryptophan.
• the present invention provides a method for producing L-tryptophan; said method comprises cultivating a genetically engineered bacterium as detailed herein in a culture medium.
• the method may further comprise isolating L-tryptophan from the culture medium.
• present invention provides a method for producing L- thiamine.
• the present invention provides a method for producing L-thiamine, said method comprises cultivating a genetically engineered bacterium as detailed herein in a culture medium.
• the method may further comprise isolating thiamine from the culture medium.
• present invention provides a method for producing ethanolamine.
• the present invention provides a method for producing ethanolamine; said method comprises cultivating a genetically engineered bacterium as detailed herein in a culture medium.
• the method may further comprise isolating ethanolamine from the culture medium.
• present invention provides a method for producing ethylene glycol.
• the present invention provides a method for producing ethylene glycol; said method comprises cultivating a genetically engineered bacterium as detailed herein in a culture medium.
• the method may further comprise isolating ethylene glycol from the culture medium.
• the culture medium employed may be any conventional medium suitable for culturing a bacterium cell in question, and may be composed according to the principles established in the art.
• the medium will usually contain all nutrients necessary for the growth and survival of the respective bacterium, such as carbon and nitrogen sources and other inorganic salts.
• Suitable media e.g. minimal or complex media, are available from commercial suppliers, or may be prepared according to published receipts, e.g. the American Type Culture Collection (ATCC) Catalogue of strains.
• ATCC American Type Culture Collection
• Non-limiting standard medium well known to the skilled person include Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth, MS broth, Yeast Peptone Dextrose, BMMY, GMMY, or Yeast Malt Extract (YM) broth, which are all commercially available.
• suitable media for culturing bacterial cells such as E. coli cells, including minimal media and rich media such as Luria Broth (LB), M9 media, M17 media, SA media, MOPS media, Terrific Broth, YT and others.
• the carbon source may be any suitable carbon substrate known in the art, and in particularly any carbon substrate commonly used in the cultivation of bacteria and/or fermentation.
• Non-limiting examples of suitable fermentable carbon substrates are C5 sugars (such as arabinose or xylose), C6 sugars (such as glucose), acetate, glycerol, plant oils, sucrose, yeast extract, peptone, casamino acids or mixtures thereof.
• a carbon source of particular interest is a C6 sugar such as glucose.
• various ammonium salts such as ammonia and ammonium sulfate, other nitrogen compounds such as amines, a natural nitrogen source such as peptone, soybean-hydrolysate, and digested fermentative microorganism can be used.
• the cultivation can be preferably performed under aerobic conditions, such as by a shaking culture, and by a stirring culture with aeration, at a temperature of about 20 to about 40 °C, such as about 30 to 38 °C, preferably about 37°C.
• the pH of the culture is usually from about 5 and about 9, such as from about 6.5 and 7.5.
• the pH of the culture can be adjusted with ammonia, calcium carbonate, various acids, various bases, and buffers. Usually, 1 to 5- day cultivation leads to accumulation of L-serine in the culture medium.
• L-serine or the L-serine derivative can be collected by conventional method for isolation and purification chemical compounds from a medium.
• Well-known purification procedures include, but are not limited to, centrifugation or filtration, precipitation, ion exchange, chromatographic methods such as e.g. ion exchange chromatography or gel filtration chromatography, and crystallization methods.
• the present invention thus provides L-serine or a L-serine derivative obtainable by a method as detailed herein.
• a fermentation composition which comprises the metabolites of the cell culture described herein and the metabolite from 3-phosphoglycerate, wherein at least 20% by weight of the carbon is biobased.
• the composition comprises at least 50% biobased carbon, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99%, such as at least 100%.
• the composition comprises at least 20% biobased carbon, such as at least 30% biobased carbon, such as at least 40% biobased carbon, such as at least 50% biobased carbon, such as at least 60% biobased carbon, such as at least 70% biobased carbon, such as at least 75% biobased carbon, such as at least 80% biobased carbon, such as at least 85% biobased carbon, such as at least 90% biobased carbon, such as at least 95% biobased carbon, such as 100% biobased carbon.
• biobased carbon such as at least 30% biobased carbon, such as at least 40% biobased carbon, such as at least 50% biobased carbon, such as at least 60% biobased carbon, such as at least 70% biobased carbon, such as at least 75% biobased carbon, such as at least 80% biobased carbon, such as at least 85% biobased carbon, such as at least 90% biobased carbon, such as at least 95% biobased carbon, such as 100% biobased carbon.
• the composition comprises from 20% to 100% biobased carbon, such as from 30% to 100% biobased carbon, such as from 40% to 100% biobased carbon, such as from 50% to 100% biobased carbon, such as from 60% to 100% biobased carbon, such as from 70% to 100% biobased carbon, such as from 75% to 100% biobased carbon, such as from 80% to 100% biobased carbon, such as from 85% to 100% biobased carbon, such as from 90% to 100% biobased carbon, such as from 95% to 100% biobased carbon, such as 100% biobased carbon.
• the composition comprises no more than 50% fossil-based carbon, such as no more than 45%, such as no more than 40%, such as no more than 35%, such as no more than 30%, such as no more than 25%, such as no more than 20%, such as no more than 15%, such as no more than 10%, such as no more than 5%, such as no more than 1% fossil-based carbon.
• 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, for example 94% biobased carbon.
• the fermentation composition may further comprise one or more further compounds or metabolites from the cell culture.
• Such compounds and/or metabolites of the cell culture includes precursors for the metabolite as well as compounds selected from trace metals, vitamins, salts, yeast nitrogen base, carbon source, YNB, and/or amino acids of the fermentation.
• the composition comprises a concentration of the metabolite of at least 1 mg/kg composition, such as at least 5 mg/kg, such as at least 10 mg/kg, such as at least 20 mg/kg, such as at least 50 mg/kg, such as at least 100 mg/kg, such as at least 500 mg/kg, such as at least 1.000 mg/kg, such as at least 5.000 mg/kg, such as at least 10.000 mg/kg, such as at least 50.000 mg/kg.
• the composition is substantially free of a-HGA.
• the composition may also further comprise one or more carriers, agents, additives and/or excipients.
• a DE3 cassette containing T7 polymerase was integrated into the genome of DE3 lysogenization kit (Millipore, Damstadt Germany).
• Glucose M9 minimal media consisted of 2 to 5 g/L glucose, 2mM Glycine, 0.1 mM CaCI 2 , 2.0 mM MgSCU, lx trace element solution, and lx M9 salts.
• the l,000x trace element stock solution consisted of 27 g/L FeCl3*6H2O, 2 g/L ZnCI 2 *4H 2 O, 2 g/L CoCI 2 *6H 2 O, 2 g/L NaMoO 4 *2H 2 O, 1 g/L CaCI 2 *H 2 O, 1.3 g/L CuCI 2 *6H 2 O, 0.5 g/L H 3 BO 3 , and concentrated HCI dissolved in ddH 2 O and sterile filtered.
• the 10x M9 salts stock solution consisted of 68 g/L Na 2 HPO4 anhydrous, 30 g/L KH 2 PO , 5 g/L NaCI, and 10 g/L NH CI dissolved in ddH 2 O and autoclaved.
• the concentration of serine was measured using a Dionex Ultimate 3000 HPLC (High- Performance Liquid Chromatography) equipped with a CHIROBIOTIC® T Chiral (250 x 2.1 mm x 5pm) column (Sigma-Aldrich, St. Louis, MO, USA) and Diode Array Detector (DAD-UV) detector.
• the mobile phase comprised of 60% acetonitrile (v/v) and 0.02% (v/v) formic acid in milliQ. water.
• the mobile phase was delivered at a rate of 1.0 mL/min, and the injection volume was kept at 3 pL for standard and all samples.
• the detection of L-serine was monitored at 205nm.
• Example 1 Effect of replacement of NADH dependent glyceraldehyde-3-phosphate dehydrogenase by NADPH dependent glyceraldehyde-3-phosphate dehydrogenase.
• NADPH dependent glyceraldehyde-3-phosphate dehydrogenases are reported in literature such as from C. acetobutylicum (gapC) 10 or from Bacillus subtilis (gapB) 17 .
• gapC C. acetobutylicum
• gapB Bacillus subtilis
• L-serine is produced in E. coll from three enzymes encoded by serA, serB and serC. All genes were isolated from E. coll MG1655 using primers with respective gene names (Table 2). The 100 pl PCR mixture contained 250 nM of each forward and reverse primer, 250 pM of dNTPs, 2 U of Phusion polymerase, 1 X HF buffer, 1 pl of overnight culture.
• the following two-step PCR protocol was used for the PCR amplification: An initial denaturation step at 98°C for 40, followed by 5 cycles of denaturation at 98°C for 10 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 90 seconds, followed by 20 cycles, where the annealing temperature was increased from 55°C to 65°C.
• the gene products and plasmids were digested using Fast digest enzymes (Thermoscientific, Waltham, MA, USA). About 500 ng of PCR product or 1 pg of plasmids were subjected to digestion by 1 pl each of the restriction enzymes in IX fast digest buffer. The reaction was incubated for 3h and then column purified again.
• the serA PCR product was subjected to double digestion with Ncol and Notl, while the serC PCR product was digested with Ndel and Pad.
• pCDF-Duet was first digested with Ncol and Notl for cut and paste cloning of serA leading to plasmid pCDF-Duet- serA.This plasmid was later used for cut and paste cloning of serC thus generating pCDF-Duet-serA- serC.
• the serB PCR product was cloned in pACYC-Duet vector at Ncol and Pad site leading to pACYC- serB.
• a typical ligation reaction included 1 X T4 ligase buffer 50 ng of plasmid DNA and 100 ng of insert and 0.3 .l/10 pl of T4DNA ligase (Thermoscientific, Waltham, MA, USA).
• gapA was replaced by gopCgene from C. acetobutylicum strain using a cat-sacB based selection system.
• Cat-sacB was inserted using pKD46 harboring exo, beta and gamma genes for recombination. Positive selection for cassette insertion was done by selecting clones for chloramphenicol resistance. The loss of cassette was selected by replica plating of clones on LB-chloramphenicol and LB-sucrose plate containing 15 % sucrose (no NaCI).
• the cat-sacB cassette was amplified using the primers gapC_camsacB_F and R (Table S3).
• reaction mixture and PCR program was the same as described in example 1.
• the extension time was 2 min and 30sec, while the template was 1 pl of overnight culture from E. coll carrying the cat-sacB cassette on its genome. Competent cells were then transformed with 200 ng of gapC-cat-sacB cassette, and were plated on LB-chloramphenicol-ampicillin plates after two hours of regeneration and incubated overnight at 30°C. A single colony was picked and made electrocompetent after 1 h of induction (Example 1) and transformed with gapCgene which was amplified from the C. acetobutylicim genome using the above mentioned PCR program and primers gapC_aF and aR, as mentioned in Table 4.
• strains were transformed with pCDF-Duetl-serAmut-serC and pACYC-serB.
• the resulting glycerol stocks were grown overnight in 2xYT medium containing 0.1 % glucose and supplemented with spectinomycin and chloramphenicol. Overnight cultures were inoculated in fed batch fermentation. The media composition and cultivation conditions were as described previously.
• the strain HM_274 does not carry the gapA::gapC replacement. It accumulated 0,91 g/L HGA in 23h batch fermentation in baffled flask. In a 48h fed-batch fermentation, the same strain accumulated 9,35 g/L HGA. The strain HM_476 does carry the gapA::gapC replacement. It accumulated 0,86 g/L HGA in 23 h batch fermentation in baffled flask. In a 48 h fed-batch fermentation, the same strain accumulated 2,36 g/L HGA. The data for the 23 h timepoint of the batch fermentation and the 48 h fed-batch fermentation is shown in Table 5. The data clearly demonstrate a reduction in HGA production in a strain where gapA is replaced by gapC.
• Table 5 Lower HGA accumulation in strain HM_476 wherein gapA is replaced by gapC compared to HM 274.
• This plasmid was used for cloning nox which was amplified from the Lactobacillus brevis genome. Application of each primer in generating different USER fragments is also mentioned.
• PCR program initial denaturation at 98°C for 40 sec, denaturation at 98°C for 10 sec, annealing 60°C for 30 sec, extension 72°C for 3 min and 30 sec the cycle was repeated 25 times.
• USER fragments 1 to 4 were used for assembling pSEVA27-serACB while fragments 5 and 6 were used for assembling pSEVA27-serACB-Nox.
• the 10 pL USER reaction contained 1 pL of USER enzyme and 1 pL lOx cut smart buffer (New England Biolabs) and 200 ng of each USER fragment. The reaction was incubated at 37°C for 30 min followed by 15 °C for 30 min. The reaction mixture was transformed in chemically competent NEB5 Alpha cells, transformants were grown in SOC media at 37°C before plating on LB-kan plates and incubated at 37°C for overnight.
• Table 7b HGA and serine concentrations detected in supernatants of a 20h fed-batch fermentation.
• ALE-8 accumulated 0,27 g/L HGA 5 h after fermentation start, while ALE-8 expressing RocG accumulated 0,06 g/L HGA.
• ALE-8 accumulated 0,1 g/L HGA and ALE-8 expressing RocG accumulated 0,06 g/L HGA.
• ALE-8 had produced 0,75 g/L L- serine and ALE-8 expressing RocG produced 0,3 g/L L-serine.
• the feedback inhibition was removed by replacing Y463 or N483 to alanine (A) using the site directed mutagenesis protocol explained above and results in vector pCDF-Duetl-serAglut-Y463A-serC and pCDF-Duetl- serAglut-N483A-serC, as well as vector pSEVA27-serAglut-Y463A-serC and pSEVA-serAglut-N483A- serC. Primers are listed in Table 8.
• L-serine production form strains with either gapA or gapC on the genome expressing feedback inhibition insensitive serA from E. coli were compared in fed-batch fermentation over the course of 48 h.
• the L-serine concentrations measured during the fermentation are shown in Figure 4A.
• the strain expressing gapA produced 25 g/L, while the strain expressing gapC produced 35 g/L.
• L-serine production form strains with either gapA or gapC on the genome expressing feedback inhibition insensitive serA from C. glutamicum were compared in fed-batch fermentation over the course of 68 h.
• the L-serine concentrations measured during the fermentation are shown in Figure 4B.
• the strain expressing gapA produced 50 g/L, while the strain expressing gapC produced 100 g/L.
• this data demonstrates that a 40 % increase in L-serine production in fermentation can be achieved from a strain wherein gapA is replaced by gapC and which expressed the L-serine pathway including E. coli serA with promiscuous activity for KGA.
• the gapA::gapC modification boosts L-serine production by 200 % for strains expressing serine pathway including C. glutamicum serA with no promiscuous activity for KGA.
• Example 5 The effect of gapC on the strain expressing serA with no HGA activity.
• 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.
• Example 6 The effect of rat serA in the strain expressing gapC.
• the serA gene from rat is a Type I serA and is found to have negligible activity for production of hydroxyglutarate.
• the gene was ordered as gene fragment from Twist Biosicence (USA). This serA is disclosed herein as SEQ. ID NO: 8.
• SEQ. ID NO: 8 The serA gene was ordered as gene fragment from Twist Biosicence (USA).
• SEQ. ID NO: 8 The serA is disclosed herein as SEQ. ID NO: 8.
• Vector backbone primers oSER_1424 and oSER_1426 bind the sequence upstream and downstream of the serA being replaced.
• the gene fragment with complementary USER overhangs were amplified using primers oSER_1423 and oSER_1425.
• PCR program initial denaturation at 98°C for 40 sec, denaturation at 98°C for 10 sec, annealing 60°C for 30 sec, extension 72°C for 3 min and 30 sec (30 sec/kb) the cycle was repeated 25 times.
• the primer sequences are given in table below.
• the 10 pL USER reaction contained 1 pL of USER enzyme and 1 pL lOx cut smart buffer (New England Biolabs) and 200 ng of each USER fragment. The reaction was incubated at 37°C for 30 min followed by 15 °C for 30 min. The reaction mixture was transformed in chemically competent NEB5 Alpha cells, transformants were grown in SOC media at 37°C before plating on LB-kan plates and incubated at 37°C for overnight.
• MOPS-base medium pH 7.6 was prepared by adding 10 g/L of ammonium sulphate (CAS No. 7783-20-2), 2 g/L of monopotassium phosphate (CAS No. 7778-77), 2 g/L of yeast extract, 40 g/L of MOPS (CAS No. 1132-61-2), and 0.6 g/L of glycine to the desired total medium volume.
• the medium was adjusted to pH 7.6 using a 15% NH3 solution and autoclaved.
• the strains were transformed with pSER_43 plasmid.
• the plasmid contains strong constitutive promoter expressing serine operon with feedback insensitive serA from C. glutamicum (SEQ ID NO: 14).
• the transformants were selected on kanamycin plate.
• the inoculation and shake flask protocol for L-serine production is as mentioned in Example 6.
• Example 9 Effect of increasing NADPH pool by over expression of genes from pentose phosphate pathway
• the pentose phosphate pathway is one of the main sources of providing NADPH pool to bacteria.
• NADPH pool by over-expressing zwf which encodes for Glucose 6 phosphate dehydrogenase or pgl which encodes for 6-phosphogluconolactonase in the pentose phosphate pathway, could enhance the NADPH pool inside the cells.
• zwf which encodes for Glucose 6 phosphate dehydrogenase
• pgl which encodes for 6-phosphogluconolactonase in the pentose phosphate pathway
• Zwf and pgl were amplified from MG1655 genome using the primers mentioned in table below.
• PCR program initial denaturation at 98°C for 40 sec, denaturation at 98°C for 10 sec, annealing 60°C for 30 sec, extension 72°C for 1 min the cycle was repeated 25 times.
• the zw/and pgl genes were cloned below serB in serACB operon. Each gene was cloned in an operon containing either feedback insensitive serA from E. Coll (to check for reduction in production of hydroxyglutarate) or serA from C. glutamicum (to check if the production leads to increase in L-serine production). The constructs are shown in Figure 12.
• Some PGDHs can use a-ketoglutarate (aKG) instead of PHP in the reverse direction to produce a-hydroxyglutarate.
• aKG a-ketoglutarate
• Three enzymes have been found to be able to utilize aKG as a substrate: PDGH from E. coli (REF) , Pseudomonas stutzeri (REF), and Saccharomyces cerevisiae (REF). These three enzymes are all type II PGDH's, and it is hypothesized that type I and type III PGDH's may not be able to use aKG as a substrate and that the ability to use aKG as a substrate is a trait specific to type II PGDH's.
• Example 11 Effect of NADH-dependent glyceraldehyde-3-phosphate dehydrogenase replacement by NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase on intracellular NAD(P)H pool
• gapC SEQ ID NO: 43
• gdpl SEQ ID NO: 36
• serA C. glutamicum/SECt ID NO: 14
• This plasmid contains strong constitutive promoter expressing the serine operon with serA from C. glutamicum (SEQ ID NO: 14).
• the transformants were selected on kanamycin plate.
• the inoculation and shake flask proceeded as described in Example 6.
• 1 mL of biomass was sampled and quenched with 1 mL of 40% ethanol - 0.8% sodium chloride solution, followed by submersion of the samples for 20 seconds in a dry ice - ethanol bath. Then, samples were incubated on ice for 15 minutes, and centrifuged at 11,000 x g for 5 minutes at 4°C. Cell pellets were resuspended in 1 mL of MQ water. Quantification of the intracellular NADP(H) pool was achieved using the [NADP + ]/[NADPH] Quantification Kit (Sigma-Aldrich) according to the manufacturer's instructions.
• SEQ ID NO: 15 Protein sequence of non- HGA producing SerA from Streptomyces sp.
• SEQ ID NO: 42 Protein sequence of NADPH dependent Glyceraldehyde-3-phosphate dehydrogenase from Saccharolobus solfataricus
• Item 1 A genetically engineered bacterium which has been modified to have an increased expression of a polypeptide having D-3-phosphoglycerate dehydrogenase activity (serA) and a decreased production of hydroxyglutarate (HGA) compared to an otherwise identical bacterium that does not carry said modification.
• serA D-3-phosphoglycerate dehydrogenase activity
• HGA hydroxyglutarate
• Item 2 The bacterium according to item 1, wherein the hydroxyglutarate production is reduced by increasing cytosolic NADPH pool and/or reducing cytosolic NADH pool compared to an otherwise identical bacterium that does not carry said modification.
• Item 3 The bacterium according to any of items 1-2, which expresses a polypeptide having D-3- phosphoglycerate dehydrogenase activity and which is selected from the group consisting of SEQ ID NOs: 1 to 22, and polypeptides comprising an amino acid sequence, which has 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%, 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 1-3, wherein the decreased HGA production is achieved by expression of a polypeptide having D-3-phosphoglycerate dehydrogenase activity with reduced or no activity of HGA production.
• Item 5 The bacterium according to any of items 1-4, which expresses a polypeptide having D-3- phosphoglycerate dehydrogenase activity and which is selected from the group consisting of SEQ ID NOs: 6 to 15, and polypeptides comprising an amino acid sequence, which has 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%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 6 to 15.
• Item 6 The bacterium according to any of items 1-5, which expresses a polypeptide having 3-phospho glycerate dehydrogenase activity and which polypeptide is derived from a polypeptide as defined in item 4 and rendered feed back insensitive by truncation of the C terminal domain (e.g. SEQ ID NO:16 and 18) or by site directed mutagenesis of key sites (e.g. SEQ ID NQs:20-22).
• the C terminal domain e.g. SEQ ID NO:16 and 18
• site directed mutagenesis of key sites e.g. SEQ ID NQs:20-22.
• Item 8 The bacterium according to any of items 4 and 7, wherein the resulting reduced growth and redox imbalance upon use of D-3-phosphoglycerate dehydrogenase activity with reduced or no activity of HGA production is solved by increasing cytosolic NADPH pool and/or reducing cytosolic NADH pool.
• Item 9 The bacterium according to any one of items 1-8, in which the NADPH production is increased 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 of items 1-4, which expresses a 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 polypeptides comprising an amino acid sequence, which has at least about 70%, such as at least about Item 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 any of items 1-4, which expresses a 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 polypeptides
• the bacterium according to item 2 which has been modified to enhance the 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).
• glucose-6-phosphate dehydrogenase e.g. SEQ ID NO: 47
• 6-phosphogluconate dehydrogenase e.g. SEQ. ID NO: 48
• Item 12 The bacterium according to item 1-3, which expresses a heterologous polypeptide having NADH oxidase (Nox) activity to reduce the intracellular NADH pool.
• Nox NADH oxidase
• Item 13 The bacterium according to item 12, wherein said heterologous polypeptide having NADH oxidase (Nox) activity is selected from the group consisting of SEQ ID NOs: 49 to 56, and polypeptides comprising an amino acid sequence, which has 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%, at least 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 49 to 56.
• Nox NADH oxidase
• Item 14 The bacterium according to any one of items 1-2, which depletes NADH pool 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 polypeptides comprising an amino acid sequence, which has 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%, 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 1-15, which has been modified to have a decreased 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 carry said modification.
• Item 17 The bacterium according to item 16, wherein the endogenous gene encoding said endogenous NADH dependent polypeptide having glyceraldehyde-3-phosphate dehydrogenase activity is inactivated.
• Item 18 The bacterium according to any one of items 1-17, wherein said polypeptide having D-3- phosphoglycerate dehydrogenase activity is a polypeptide which has reduced activity towards alphaketoglutarate, where reduced activity is measured relative to activity of SEQ ID NO: 16.
• Item 19 The bacterium according to item 18, wherein said polypeptide having D-3-phosphoglycerate dehydrogenase activity is selected from the group consisting of SEQ ID NOs: 16 to 22, and polypeptides comprising an amino acid sequence, which has 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%, 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 1-19, wherein said bacterium belongs to the Enterobacteriaceae family.
• Item 21 The bacterium according to item 20, wherein said bacterium belongs to the genus Escherichia.
• Item 24 The bacterium according to item 23, wherein said bacterium is Corynebacterium glutamicum.
• Item 25 A method for producing L-serine or a L-serine derivative, the method comprises cultivating a bacterium according to any one of items 1-24 in a culture 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.
• 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, wherein the method further comprises isolating L- serine or the L-serine derivative from the culture medium.

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