Expression of biomolecules with improved promoter and tir

Abstract

The present disclosure describes genetically modified host cells expressing or overexpressing one or more genes of an L-serine pathway, or an operon comprising such one or more genes, wherein the genes or operon are operably linked to a constitutive promoter having a nucleotide sequence which is 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 constitutive promoter comprised in anyone of SEQ ID NO: 1 to 16.

Description

Expression of biomolecules with improved promoter and TIR.Technical Field

[0001] The present invention relates to the microbiological industry, and specifically to the bioindustrial production of L-serine or derivatives thereof by expressing a 3-phosphoglycerate dehydrogenase (PGDH or SerA) enzyme, optionally in an operaon also including phosphoserine aminotransferase (serC) and a phosphoserine phosphatase (serB) having TIRs and being operably linked to an improved constitutive promoter. More specifically the present disclosure describes genetically modified host cells producing the L-serine or derivatives thereof through a metabolic pathway engineered to improve production of the said L-serine or derivatives thereof. Further disclosed are enzymes and polynucleotides encoding such enzymes of the pathway, polynucleotide constructs for expression of these enzymes and cell cultures of the host cell which when cultured in fermentation methods produce the L-serine or derivatives thereof. Further disclosed are fermentation compositions the host cells and / or the L-serine or derivatives thereof resulting from such methods also known as biobased compositions.Background

[0002] Genetically modified host cells producing metabolites such as L-serine are known e.g. from WO2016120326 describing production of L-serine using genetically engineered microorganisms deficient in serine degradation pathways. 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. CN103436504A describes 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. WQ20247084049, incorporated herein by reference, describes host cells producing L-serine genetically modified to reduce intracellular accumulation of NADH and / or hydroxyglutarate (HGA) thereby reducing the negative impact of these compounds on the production of L- serine and other metabolites. Further art includes US2021095245A1 which relates to modified E. col i, expressing serAmut (Fbr), serB, serC in a method for enhanced production of serine and includes a general passage on promoters, including constitutive promoters, and measures for regulating transcription and translation. Yan Zhang et al; "Engineering of Serine-Deamination pathway, Entner-Doudoroff pathway and pyruvate dehydrogenase complex to improve poly(3- hydroxybutyrate) production in Escherichia coli; Microbial Cell Factories"; vol. 13, 172, 16 December 2014, pages 1-11, relates to modified E. coli expressing Cg / SerA, Ec / SerC, Ec / SerC, SdaA, PDH, PhaABC pathway for production of PHB via serine. Further described is an edd-eda operon regulated by the constitutive promoter J23119. US20190233857A1 relates to modified Cupriavidis necator expressing Cg / Ser A (Fbr), Ec / SerB, Ec / SerC, resp. SerA, SerB, SerC from C. necator, for production of L-serine with no explicit indication of promoter. Chen Lin et al; "Rational design and metabolic analysis of Escherichia coli for effective production of L-tryptophan at high concentration"; Vol. 101, No. 2, 6 September 2016, pages 559-568, relates to production of amino acids in E. coli as host cell using J23119 as constitutive promoter. WO2023093883A1 relates to production of IL-22 in E. coli using J23119. Anderson J.C.: "Promoters / Catalog / Anderson - parts.igem.org" describes the Anderson library. Liow Lu Ting et al; "Characterisation of Constitutive Promoters from the Anderson library in Chromobacterium violaceum ATCC 12472"; Engineering Biology, vol. 3, no. 3, 12 July 2019, pages 57- 66, characterizes promoters of the Anderson library in Chromobacterium violaceum. W02020071538A1 relates to serine producing bacteria, including a general reference to Mutalik et al. 2013. Mutalik V.K. et al; "Precise and reliable gene expression via standard transcription and translation initiation elements"; Nature Methods, vol. 10, no. 4, 1 April 2013, pages 354-360 relates to genetic elements controlling transcription and translation initiation in Escherichia coli.

[0003] However there is a continuous need for optimizing pathways for improving host cell production of L-serine.Summary

[0004] An objective of the invention described herein is to provide means allowing a more efficient production and stable of L-serine and derivatives thereof. More particularly, it is an objective of the of the invention described herein to provide means allowing the production of L-serine at higher nominal yield and improved mass yield.

[0005] PGDH is the first key step of L-serine biosynthesis converting 3-phosphoglycerate into 3- phosphohydroxypyruvate, and therefore efficient and stable expression of this enzymes is crucial to successful product 3-phosphohydroxypyruvate and / or any metabolites thereof. Previous work uses aT7 polymerase-based system to regulate expression of the L-serine pathway (Landberg et al., 2020; Rennig et al., 2019b). This however requires either the use of an inducer (IPTG) or low concentrations of yeast extract in the medium for production of L-serine. However, inducement by IPTG increases the costs and technical complexity of the process and using low concentrations of yeast extract in the medium puts additional burden on the cells to produce all the required metabolites and vitamins leading to lower yield and titres.

[0006] Therefore, different constitutive expression promoters and different Translation Initiation Regions (TIRs) for SerA and SerC were constructed and studied looking to avoid addition of IPTG while maintaining complex media addition level that best suits the productivity, in particular to identify constitutive promoters which provides expression similar to or even surpassing that of the T7 based induction system. Surprisingly, constitutive promoters were identified which were not only similar or even surpassing the expression performance of the T7 based induction system, but they were also superior in being very stable as observed by repetitive fermentation from the biomass of previous fermentation run.

[0007] Accordingly, in a first aspect a genetically modified host cell is provided which expresses or overexpresses one or more genes or an operon comprising one or more genes of an L-serine pathway wherein the gene or operon is operably linked to a constitutive promoter having a nucleotide sequence which is 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 constitutive promoter comprised in SEQ ID NO: 1 to 16.

[0008] In a further aspect a polynucleotide construct is provided which comprises one or more genes or an operon comprising one or more genes of an L-serine pathway operably linked to a constitutive promoter which has a nucleotide sequence which is at least 70 % identity, 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 constitutive promoter comprised in SEQ ID NO: 1 to 16.

[0009] In a further aspect a cell culture is provided which comprises the host cell of the disclosure and a growth medium.

[0010] In a further aspect a method is provided for producing a L-serine or a derivative thereof comprising: a) culturing the cell culture described herein at conditions allowing the cell to produce the L-serine or a derivative thereof; and b) optionally recovering and / or isolating the L-serine or a derivative thereof.

[0011] In a further aspect a fermentation composition is provided which comprises the cell culture described herein and / or the L-serine or the derivative thereof comprised therein.

[0012] In a further aspect a genetically modified TIR is provided comprising a sequence as set forth in anyone of SEQ. ID NO 145 to 170.Brief description of the figures

[0013] Figure 1A to IP shows plasmid maps of vectors with different promoters.

[0014] Figure 2 shows plasmid map of TARSYN construct subjected for TIR optimization.

[0015] Figure 3 shows the TIR region sequence of serA subjected for selection on ampicillin tolerance.

[0016] Figure 4 shows the TIR region sequence of serC subjected for selection on ampicillin tolerance.

[0017] Figure 5 shows Serine production in g / L from pSEVA plasmid containing mentioned constitutive promoters and the comparison with IPTG inducible promoter.

[0018] Figure 6 shows a schematic drawing of randomization and screening of TIR library.

[0019] Figure 7 shows fold de- / increase in protein and serine concentrations of strains, containing the plasmids without bla cassette compared to the control Serl51. Strains were grown in M9 media with 0.5% (w / v) glucose, 2mM glycine, and kanamycin at 37 °C and 250 rpm. Proteomic and HPLC samples were taken after 24 hours.

[0020] Figure 8 shows the serine concentrations of the top 5 strains with the optimized plasmids and Serl51 (positive control). Strains were grown in 250mL bioreactors with minimal media at 37 °C.

[0021] Figure 9 shows the pathway for L-serine, including potential exit shunts.

[0022] Figure 10 shows the HMP shunt pathway employing glucose-6-phosphate dehydrogenase (zwf) and 6-phosphogluconolactonase (pgl).Detailed descriptionDefinitions

[0023] The term "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". For example, 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. Where the terms as used herein about host cells, they refer to host cells comprising and expressing heterologous or recombinant polynucleotides.

[0024] The term "% identity" is used herein about the relatedness between two amino acidsequences 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. 5' or 3' extensions nor insertions (for nucleic acids) or N' or C' extensions nor insertions (for polypeptides) usually result in a reduction of identity, similarity or homology using such standard software.

[0025] The term "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.

[0026] The term "in vivo", as used herein refers to within a living cell or organism, including, for example animal, a plant, or a microorganism.

[0027] The term "in vitro" as used herein 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.

[0028] The term "substrate" or "precursor" as used herein refers to any compound that can be converted into a different compound. For clarity, 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.

[0029] The term "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.

[0030] The term "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 thosethat are not identical to the parent cell due to mutations that occur during replication.

[0031] The term "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.

[0032] The term "operably linked" refers to a configuration in which a control sequence, such as a promoter, is placed at an appropriate position relative to a coding polynucleotide such that the control sequence directs expression of the coding polynucleotide. More generally, 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.

[0033] As used herein, "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.

[0034] "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.

[0035] The term "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%.

[0036] As recognized by the Circular Bio-based Europe Joint Undertaking (CBE Joint Undertaking) established in 2021, developing biobased materials is essential if the EU is to reach its climate targets as set out in the European Green Deal. The present disclosure provides a methodology for efficiently providing fatty alcohols and fatty aldehydes having a high content of biobased carbon (%). Both fossil and renewable raw materials consist mainly of carbon (C). Carbon occurs in several isotopes. Isotope14C is radioactive and occurs naturally in all living organisms (plants, animals, etc.) in a fixed relative concentration which is nearly identical to the relative14C concentration in the atmosphere. At this concentration, the radioactivity level of14C 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. Theradioactive14C level of an unknown substance can therefore help determine how old the carbon contained in the substance is. "Young" carbon (0 to 10 years) derived from renewable raw materials, such as plants or animals, has a relative isotope14C concentration which is nearly identical to the relative14C concentration in the atmosphere and the radioactive14C level of such young carbon is thus about 100%. "Old" carbon (millions of years) derived from synthetic, or fossil (petrochemical) sources is greatly depleted from isotope14C as the age of such synthetic and fossil sources far exceeds the halflife of isotope14C which is approximately 5700 years. Hence, carbon derived from synthetic, or fossil sources has a relative isotope14C concentration around 0% and the radioactive14C level of such old carbon is thus about 0%. In one embodiment the term "radioactive14C level" refer to the total radioactive14C level of a given substance, product, or composition, as defined above. The isotope14C method may be used to determine the concentration of young (renewable) materials in comparison with the concentration of old (fossil) resources. The carbon content of a renewable raw material is referred to as the "biobased carbon content". The carbon content of a renewable raw material or the "biobased carbon content" may be determined as described below. 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 radioactive14C 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. The analytical measurement may be cited as "percent modern carbon (pMC)". This is the percentage of14C measured in the sample relative to a modern reference standard (NIST 4990C). The % Biobased Carbon content is calculated from pMC by applying a small adjustment factor for14C in carbon dioxide in air today. It is important to note that all internationally recognized standards using14C assume that the plant or biomass feedstocks were obtained from natural environments. pMC may be analyzed by a standard test method, such as "ASTM D6866".

[0037] The terms "nucleotide sequence" and "polynucleotide" are used herein interchangeably.

[0038] The term "comprise" and "include" as used throughout the specification and the accompanying items as well as variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. These words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

[0039] The articles "a" and "an" are used herein 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 oneelement or more than one element.

[0040] Terms like "preferably", "commonly", "particularly", and "typically" are not utilized herein to limit the scope of the itemed invention or to imply that certain features are critical, essential, or even important to the structure or function of the itemed invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present invention.

[0041] The term "cell culture" as used herein refers to a culture medium comprising a plurality of the host cells described herein. A cell culture may comprise a single strain of host cells or may comprise two or more distinct host cell strains. The culture medium may be any medium that may comprise a recombinant host, e.g., a liquid medium (i.e., a culture broth) or a semi-solid medium, and may comprise additional components, e.g., a carbon source; a nitrogen source; a phosphate source; vitamins; trace elements; salts; amino acids; nucleobases; and the like.

[0042] Term "endogenous" or "native" as used herein refers to a gene or a polypeptide in a host cell which originates from the same host cell.

[0043] The term "operon" as used herein refers to a functioning unit of DNA containing a cluster of genes under the control of a single regulatory signal or promoter. Operons are commonly found in prokaryotes, such as bacteria, and they allow for the efficient regulation of gene expression. The genes within an operon are typically involved in the same biological pathway or process, and their expression is coordinated so that they can be transcribed together as a single mRNA (messenger RNA) molecule.

[0044] The terms "substantially" or "approximately" or "about", as used herein refers to a reasonable deviation around a value or parameter such that the value or parameter is not significantly changed. These terms of deviation from a value should be construed as including a deviation of the value where the deviation would not negate the meaning of the value deviated from. For example, in relation to a reference numerical value the terms of degree can include a range of values plus or minus 10% from that value. For example, deviation from a value can include a specified value plus or minus a certain percentage from that value, such as plus or minus 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from the specified value.

[0045] Where a numerical limit or range is stated herein, the endpoints are included. Also, all values and sub ranges within a numerical limit or range are specifically included as if explicitly written out.

[0046] The term "and / or" as used herein is intended to represent an inclusive "or". The wording X and / or Y is meant to mean both X or Y and X and Y. Further the wording X, Y and / or Z is intended to mean X, Y and Z alone or any combination of X, Y, and Z.

[0047] The term "isolated" as used herein about a compound, refers to any compound, which by means of human intervention, has been put in a form or environment that differs from the form orenvironment in which it is found in nature. Isolated compounds include but are not limited to compounds of the disclosure for which the ratio of the compounds relative to other constituents with which they are associated in nature is increased or decreased. In an important embodiment the amount of compound is increased relative to other constituents with which the compound is associated in nature. In an embodiment the compound of the disclosure may be isolated into a pure or substantially pure form. In this context a substantially pure compound means that the compound is separated from other extraneous or unwanted material present from the onset of producing the compound or generated in the manufacturing process. Such a substantially pure compound preparation contains less than 10%, such as less than 8%, such as less than 6%, such as less than 5%, such as less than 4%, such as less than 3%, such as less than 2%, such as less than 1 %, such as less than 0.5% by weight of other extraneous or unwanted material usually associated with the compound when expressed natively or recombinantly. In an embodiment the isolated compound is at least 90% pure, such as at least 91% pure, such as at least 92% pure, such as at least 93% pure, such as at least 94% pure, such as at least 95% pure, such as at least 96% pure, such as at least 97% pure, such as at least 98% pure, such as at least 99% pure, such as at least 99.5% pure, such as 100 % pure by weight.

[0048] The term "GAPDH" as used herein refers to a glyceraldehyde-3-phosphate dehydrogenase enzyme converting glyceraldehyde 3-phosphate into 1,3-bisphosphoglycerate; under the coconversion of NAD+or NADP+into NADH or NADPH respectively. GapA is an example of a GAPDH producing 1,3-bisphosphoglycerate under co-conversion of NAD+into NADH, while GapC is an example of a GAPDH producing 1,3-bisphosphoglycerate under co-conversion of NADP+into NADPH.

[0049] The term "Nox" as used herein refers to an NADH oxidase enzyme converting NADH to NAD+.

[0050] The term "PGDH" or "3-PGDH" or "PHGDH" as used herein refers to a 3-phosphoglycerate dehydrogenase enzyme that catalyzes the conversion of 3-phosphoglycerate into 3- phosphohydroxypyruvate, under the simultaneous reduction of NAD+ to NADH. An example of PGDH is SerA in the serine pathway. 3-phosphoglycerate dehydrogenase may also in some art be referred to as 3-phosphopyruvate dehydrogenase.

[0051] The term "PSAT" as used herein refers to a phosphoserine aminotransferase converting 3- phosphohydroxypyruvate into phosphoserine. An example of a PSAT is SerC in the serine pathway.

[0052] The term "PSPH" as used herein refers to a phosphoserine phosphatase (PSPH) converting phosphoserine into L-serine. An example of a PSPH is SerB in the serine pathway.

[0053] The term "GDH" as used herein refers to a glutamate dehydrogenase enzyme that catalyzes the conversion of a-ketoglutarate into glutamate.

[0054] The term "deletion" as used herein 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.

[0055] The term "disruption" as used herein refers to manipulation of a gene or any of the machinery participating in the expression the gene, so that it is no longer expressed in a host cell.

[0056] The term "attenuation" as used herein refers to manipulation of a gene or any of the machinery participating in the expression the gene, so that it the expression of the gene is reduced as compared to expression without the manipulation.

[0057] The term "paralogs" as used herein about genes refers to genes that has arisen through duplication within the same genome and has evolved to perform a similar function compared to its original gene counterpart. These genes, known as paralogous genes, are typically part of a gene family within the same organism and often retain some sequence similarity due to their shared ancestry.

[0058] The term "orthologs" as used herein about genes refers to genes in different species that originated from a common ancestral gene through a speciation event. These genes generally retain similar sequences and functions across different organisms, as they continue to perform equivalent roles in their respective species.AII methods described herein can be performed in any suitable order of steps unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0059] All percentages, ratios and proportions herein are by weight, unless otherwise specified. A weight percent (weight %, also as wt. %) of a component, unless specifically stated to the contrary, is based on the total weight of the composition in which the component is included (e.g., on the total amount of the reaction mixture).

[0060] Unless specifically defined herein, all technical and scientific terms used have the same meaning as commonly understood by a skilled person in the fields of biochemistry, genetics, and microbiology.

[0061] All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being 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, shall prevail. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified.

[0062] Practice of the invention described herein employs, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, and recombinant DNA methodologies, which are available to the person skilled in the art. Such techniquesare explained fully in the literature. See, 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 (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Harries & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, 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, Vols.154 and 155 (Wu et al. eds.) and Vol. 185, "Gene Expression Technology" (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos 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 l-IV (D. M. Weir and C. C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).Genetically engineered host cell

[0063] As described supra, in a first aspect a genetically modified host cell is provided which expresses or overexpresses one or more genes of an L-serine pathway or an operon comprising such one or more genes wherein the gene or operon is operably linked to a constitutive promoter having a nucleotide which is 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 constitutive promoter comprised in SEQ ID NO: 1 to 16. In a preferred embodiment the constitutive promoter has a nucleotide sequence which is at least 95 %, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to the constitutive promoter comprised in SEQ ID NO: 8. In some embodiments the gene encoding the 3-phosphoglycerate dehydrogenase (PGDH or SerA) enzyme producing 3-phosphoglycerate operably linked to the constitutive promoter is comprised in a plasmids of anyone of SEQ ID NO: 17 to 40 or 140 to 144, in particular in any one of SEQ ID NO: 18, 33 to 40 or 140 to 144, in particular in any one of SEQ ID NO: 18, 38 or 39..

[0064] In particular embodiments the one or more genes or operon comprising said genes encode a 3-phosphoglycerate dehydrogenase (PGDH or serA) enzyme producing 3-phosphoglycerate; a phosphoserine aminotransferase (PSAT or serC) converting 3-phosphohydroxypyruvate into phosphoserine; and / or a phosphoserine phosphatase (PSPH or serB) converting phosphoserine into L-serine. In a more specific embodiment the one or more genes or operon comprising said genes encode the PGDH or serA; the PSAT or serC; and the PSPH.

[0065] The one or more of the genes or operon of genes can be heterologous to the host cell.

[0066] In further embodiments the 3-phosphoglycerate dehydrogenase (PGDH or SerA), also known as a D-3-phosphoglycerate dehydrogenase is heterologous to the host cell and has a reduced or eliminated NADH consuming side activity, preferably being conversion of a-ketoglutarate (a-KGA) into a-hydroxyglutarate (a-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 PGDHs can use a-ketoglutarate (aKG) instead of PHP in the reverse direction to produce a-hydroxyglutarate.

[0067] 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 domains1. Some D-3-phosphoglycerate dehydrogenases do not have the KGA reduction activity. However, such D-3-phosphoglycerate dehydrogenase do not necessarily belong to one type or class of enzyme. For example, SerA from Rattus norvegicus, M. tuberculosis, C. glutamicum and B. subtilis does not seem to have HGA accumulation4, whereas human SerA has tendency of HGA accumulation although it belongs to the same class14. 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. It is hypothesized that 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 (SEQ ID NO: 171) and GCFCI (SEQ ID NO: 172) 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. For M. tuberculosis and E. 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. However, replacement of the arginyl side chain with another cationic moiety (lysyl side chain) do not result in a changed enzyme specificity. These results clearly show that it is the presence of a cationic side chain at the second position in theconserved motif which renders the enzyme unable to use aKG. Accordingly, in a preferred embodiment, 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: 50). 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 comprise the motif GCFCI, wherein the underlined cysteine is positioned corresponding to position 129 of the PGDH from Mycobacterium tuberculosis (SEQ ID NO: 50).

[0068] In a further embodiment 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: 50), optionally in a motif GCFCI, with a cationic residue, such as Arg, Leu or His, particularly Arg.

[0069] In some embodiments, 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.

[0070] SEQ ID NOs: 41-63 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: 41 to 63. More particularly 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: 46 to 63. In further embodiments 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: 46 to 55. In still further embodiments 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%, 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% identicalto the PGDH comprised in SEQ ID NO: 54, or 59 to 61 or 63 in particular SEQ ID NO: 54.

[0071] Accordingly, disclosed herein is a genetically engineered bacterium, especially a bacterium having an ability to produce L-serine, wherein said bacterium has been modified to have increased expression of a polypeptide having D-3-phosphoglycerate dehydrogenase activity (SerA) and a decreased production of HGA compared to an otherwise identical bacterium that does not carry said modification. The decreased production of HGA may be realized by a modification increasing the cytosolic NADPH pool and / or reducing the cytosolic NADH pool compared to an otherwise identical bacterium that does not carry said modification. The decreased production of HGA may also be realized by a modification causing expression of a polypeptide having D-3-phosphoglycerate dehydrogenase activity with reduced or no HGA production.

[0072] The genetically modified host cell described herein preferably produces L-serine or a derivative thereof through a metabolic pathway, in some embodiments comprising one or more genetic modifications preventing or reducing or alleviating a negative impact on production of the L-serine from intracellular accumulation of NADH and / or hydroxy glutarate (HGA), produced by one or more pathway enzymes. The pathway for L-serine is shown in figure 9.

[0073] In some embodiments the PSAT or serC comprises a polypeptide sequence 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 serC sequence comprised in SEQ ID NO: 108; and / or the PSPH or serB and comprises a polypeptide sequence 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 sequence comprised in SEQ ID NO: 109.

[0074] In some embodiments the host cell is genetically engineered to express or overexpress one or more further genes of the L-serine pathway and to produce L-serine or a derivative thereof. These one or more further genes may also be heterologous to the host cell. These further genes preferably encodes one or more enzymes selected from selected from: a) a first heterologous NADPH generating enzyme converting glyceraldehyde 3-phosphate into 1,3-bisphosphoglycerate or a downstream precursor in the L-serine pathway; b) a heterologous enzyme converting NADH to NAD+; c) a heterologous enzyme converting a side product of an enzyme in the L-serine pathway into a substrate of a L-serine pathway enzyme consuming NADH or NADPH; d) a native enzyme converting a side product of an enzyme in the L-serine pathway into a substrate of a L-serine pathway enzyme consuming NADH or NADPH;e) a second heterologous NADPH producing enzyme, which is not comprised in the L-serine pathway; and / or f) a native NADPH producing enzyme, which is not comprised in the L-serine pathway.

[0075] The present inventors have further found that inhibiting accumulation of NADH in the cell improves the downstream efficiently of pathways producing L-serine 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 both precursors for 3-phosphoglycerate.

[0076] The present inventors have also found that it is particularly advantageous and synergistic for the host cell production of L-serine or derivatives thereof, to combine two or more of the modifications a) to f), 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 enzyme converting NADH to NAD+.

[0077] The present inventors have also found that it is particularly advantageous and synergistic for the host cell production of L-serine or derivatives thereof, to combine three or more of the modifications a) to f), 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 enzyme converting NADH to NAD+; and c) 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.

[0078] The present inventors have also found that it is particularly advantageous and synergistic for the host cell production of L-serine or derivatives thereof, to combine four more of the modifications a) to f), 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 enzyme converting NADH to NAD+;c) 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 d) expression of the second heterologous NADPH producing enzyme and / or overexpression of the native NADPH producing enzyme, any of which are not comprised in the metabolite pathway.

[0079] In a further embodiment 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 L-serine pathway or not.

[0080] In some embodiments, 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.

[0081] In a further embodiment the first heterologous NADPH generating enzyme converting glyceraldehyde 3-phosphate into 1,3-bisphosphoglycerate or a downstream precursor in the L-serine pathway can be a bisphoshoglycerate synthase or a glyceraldehyde-3-phosphate dehydrogenase (GAPDH), both converting glyceraldehyde 3-phosphate into 1,3-bisphosphoglycerate; under the coconversion of NADP+ into NADPH. SEQ ID NO: 64 to 87 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 GAPDH comprised in any one of SEQ ID NO: 64 to 87. Particularly the GAPDH of SEQ ID NO: 79 to 87 are useful. In particular 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: 84.

[0082] In some embodiments, 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: 90 to 97 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%, suchas 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: 90 to 97.

[0083] In the embodiment where the enzyme converting a side product of an enzyme in the L-serine pathway into the substrate of a L-serine pathway enzyme, the said L-serine pathway enzyme is preferably a glutamate dehydrogenase (GDH), the side product is preferably a-ketoglutarate, and the substrate is glutamate. In a more specific embodiment the GDH 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 GDH comprised in any one of SEQ ID NO: 98 to 107.

[0084] In the embodiment where the host cell expresses a second heterologous NADPH producing enzyme and / or overexpresses a native NADPH producing enzyme, any of which are not comprised in the L-serine pathway, the second heterologous or the native NADPH producing enzyme is preferably a Glucose-6-phosphate dehydrogenase and / or a 6-phosphogluconolactonase, respectively. In a more specific embodiment the glucose-6-phosphate dehydrogenase 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 Glucose-6-phosphate dehydrogenase comprised in SEQ ID NO: 88. 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%, 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 6- phosphogluconolactonase comprised in SEQ ID NO: 89. This 6-phosphogluconolactonase is also known as "pgl".

[0085] In a further embodiment the host cell comprises both a heterologous PGDH as described, supra, in particular a serA comprising a polypeptide sequence 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 serA comprised in any one of SEQ ID NO: 54, or 59 to 61 or 63 and a heterologous GAPDH as described supra, comprising 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 atleast 98%, such as at least 99%, such as 100% identical to the GAPDH sequence comprised in SEQ ID NO: 84 (GapC). As indicated above, the present invention is inter alia based on the finding that the production of L-serine can be enhanced by e.g. increasing expression of SerA and decreasing the production of hydroxyglutarate (HGA).

[0086] In some embodiments the host cell expresses an L-serine biosynthesis operon comprising genes encoding a 3-phosphoglycerate dehydrogenase (PGDH or serA) enzyme, a phosphoserine aminotransferase (serC) and phosphoserine phosphatase (serB), optionally using one or more of the selected promoters (SEQ ID NO: 1-16) and / or Translation Initiation Regions (SEQ ID NO: 145-170) described herein.

[0087] In a further separate aspect Translation Initiation Regions or TIR's are provided which further enhance the expression of genes of the L-serine pathway or the operon comprising one or more of such genes. Certain TIR's have been found to further promote the expression of L-serine pathway genes or operons, in particular for SerA and SerC. The sequences for these TIR's are set forth in SEQ ID NO: 145-170. Accordingly, in a separate aspect a genetically modified host cell is provided which expresses one or more L-serine pathway genes or operon of genes operably linked to a TIR sequence comprising anyone of SEQ ID NO: 145-170. The TIR is preferably positioned upstream of the first, second and / or third codons of the gene to be transcribed for optimal effect. While the TIR is preferably positioned downstream of the promoter operably linked to the gene to be expressed. Accordingly, in some embodiments the host cell described herein further comprises a Translation Initiation Region (TIR) operably linked to a gene of the L-serine operon, such as particularly the gene encoding the PGDH or serA or PSAT or SerC and positioned upstream of the first, second and / or third codons of the PGDH or serA or PSAT or SerC, said TIR comprising the sequences set forth in any of SEQ ID NO: 145 to 170. Further in separate embodiments, the host cell comprises Translation Initiation Region (TIR) operably linked to the gene encoding the PGDH or serA comprising the sequences set forth in any of SEQ ID NO: 145 to 157. In other embodiments the host cell comprises a Translation Initiation Region (TIR) operably linked to the gene encoding the PSAT or serC comprising the sequences set forth in any of SEQ ID NO: 158 to 170.

[0088] The heterologous enzymes expressed by the host cell as described herein can be (i) enzymes from a species different from the host cell, (ii) a mutated enzyme from a species different from the host cell, and / or (iii) a mutated enzyme native to the host cell. In further embodiments the host cell further comprises at least one transporter molecule facilitating transport of the L-serine or derivative thereof or any of its precursors. In further embodiments, one or more native or endogenous genes of the hos cell is attenuated, disrupted and / or deleted, such as a native gene encoding a NADH dependent GAPDH or a a-HGA producing PHDH. In further embodiments, the host cell furthercomprises at least 2 copies of one or more polynucleotides / genes encoding one or more L-serine pathway enzymes. In further embodiments, the host cell is further genetically modified to provide an increased amount of a substrate for one or more L-serine 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 L-serine pathway.

[0089] In further embodiments the L-serine production of the host cells is further enhanced by attenuation, disruption and / or deletion of genes encoding enzymes involved in the degradation of L- serine, such as the genes encoding serine deamidase (e.g. sdaA, sdaB, or tdcG) and glyA in E. coli. or paralogs or orthologs thereof. The genes sdaA, sdaB and tdcG encode L-serine deaminase I (SdaA), L- serine deaminase II (SdaB) and L-serine deaminase III (TdcG), respectively, which are three enzymes carrying out L-serine degradation in the pathway, by converting serine into pyruvate. Further information regarding sdaA, sdaB and tdcG of, e.g., Escherichia coli is available at EcoCyc (www.biocyc.org) under Accession numbers EG10930, EG11623 and G7624, respectively. Representative nucleotide sequences of sdaA, sdaB and tdcG are set forth in SEQ ID NOs: 177, 178, and 179, respectively. The gene glyA encodes serine hydroxymethyltransferase (GlyA) which converts serine to glycine, transferring a methyl group to tetrahydrofolate, thus forming 5,10-methylene- tetrahydrofolate (5,10-mTHF). 5,10-mTHF is the major source of Cl units in the cell, making GlyA a key enzyme in the biosynthesis of purines, thymidine, methionine, choline and lipids. Further information regarding glyA of, e.g., Escherichia coli is available at EcoCyc (www.biocyc.org) under Accession number EG10408. A representative nucleotide sequence of glyA is set forth in SEQ ID NO: 180.

[0090] Accordingly in some embodiments the host cell comprises native genes which involved in the degradation of L-serine, of which at least one has been attenuated, disrupted and / or deleted, in particular genes selected from the group of sdaA, having the amino acid sequence set forth in SEQ ID NO: 177, sdaB, , having the amino acid sequence set forth in SEQ ID NO: 178, tdcG, having the amino acid sequence set forth in SEQ ID NO: 179, and glyA, having the amino acid sequence set forth in SEQ ID NO: 181, or any variant thereof having at least 70% identity to said sequences, or any paralog, ortholog or combinations thereof. Said sequence identity can preferably be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%.

[0091] In further embodiments the host cell expresses a mutant aspartate kinase l / homoserine dehydrogenase I (ThrA) mutant having one or more amino acid substitutions which increase tolerance towards L-serine, such as the ThrA of SEQ ID NO: 181, having a mutation which which increases the tolerance towards L-serine. Such mutation can include mutation at positions corresponding to positions Y356, S357 and S359, including substitutions or deletions. Such mutation can in some embodiments be selected from the group of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G,Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R, Y356L, S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M, S359R, S359G, S359M, S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L or any combination thereof. A particularly preferred mutation in the ThrA is corresponding to Y356C.

[0092] In further embodiments the host cell overexpresses a serine transporter, such as the transporter encoded by the gene ydeD in E. coli or a paralog or ortholog thereof. Further information regarding ydeD of, e.g., Escherichia coli is available at EcoCyc (www.biocyc.org) under Accession numbers EG11639. A representative amino acid sequence of such exporter protein is set forth in SEQ ID NO: 182 (9).

[0093] In further embodiments the host cell expresses a mutant Irp gene or a paralog or ortholog thereof encoding a protein having an amino acid substitution at a position corresponding to D143 of the Irp sequence of SEQ ID NO: 183, preferably D143G. Further information regarding Irp of, e.g., Escherichia coli is available at EcoCyc (www.biocyc.org) under Accession number EG10547.

[0094] In further embodiments the host cell expresses a mutant rho gene or a paralog or ortholog thereof encoding a protein having an amino acid substitution at a position corresponding to R87 of the rho sequence of SEQ ID NO: 184, preferably R87L. Further information regarding rho of, e.g., Escherichia coli such as nucleotide sequence of the gene or amino acid sequence of the encoded polypeptide is available at EcoCyc (www.biocyc.org) under Accession number EG10845.

[0095] In further embodiments the host cell expresses a mutant eno gene or a paralog or ortholog thereof encoding a protein having an amino acid substitution at a position corresponding to V164 of the eno sequence of SEQ ID NO: 185, preferably V164L. Further information regarding eno of, e.g., Escherichia coli is available at EcoCyc (www.biocyc.org) under Accession number EG10258.

[0096] In further embodiments the host cell expresses a mutant argP gene or a paralog or ortholog thereof encoding a protein having an amino acid substitution at a position corresponding to Q132 of the argP sequence of SEQ ID NO: 186 preferably Q132K. Further information regarding argP of, e.g., Escherichia coli is available at EcoCyc (www.biocyc.org) under Accession number EG10490.

[0097] In further embodiments the host cell expresses a mutant tufA gene or a paralog or ortholog thereof encoding a protein having an amino acid substitution at a position corresponding to G19 of the tufA sequence of SEQ ID NO: 187 preferably G19V. Further information regarding tufA of, e.g., Escherichia coli is available at EcoCyc (www.biocyc.org) under Accession number EG11036.

[0098] In further embodiments the host cell expresses a mutant cycA gene or a paralog or ortholog thereof encoding a protein having an amino acid substitution at a position corresponding to 1220 of the cycA sequence of SEQ ID NO: 188 preferably 1220V. Further information regarding cycA of, e.g., Escherichia coli is available at EcoCyc (www.biocyc.org) under Accession numbers EG12504.

[0099] In further embodiments the host cell expresses a mutant rpe gene or a paralog or ortholog thereof encoding a protein having an amino acid substitution at a position corresponding to 1202 of the rpe sequence of SEQ ID NO: 189 preferably I202T. Further information regarding rpe of, e.g., Escherichia coli is available at EcoCyc (www.biocyc.org) under Accession numbers M004.

[0100] In further embodiments the host cell expresses a mutant yojl gene or a paralog or ortholog thereof encoding a protein having an amino acid substitution at a position corresponding to D334 of the yojl sequence of SEQ ID NO: 190 preferably D334H. Further information regarding yojl of, e.g., Escherichia coli is available at EcoCyc (www.biocyc.org) under Accession numbers EG12070.

[0101] In further embodiments the host cell expresses a mutant hyaF gene or a paralog or ortholog thereof encoding a protein having an amino acid substitution at a position corresponding to V120 of the hyaF sequence of SEQ ID NO: 191 preferably V120G. Further information regarding hyaF of, e.g., Escherichia coli is available at EcoCyc (www.biocyc.org) under Accession numbers EG10473.

[0102] In further embodiments the host cells comprise genes corresponding to the pykF gene (SEQ ID NO: 192), the malT gene (SEQ ID NO: 193), and / or the a lamB gene (SEQ ID NO: 194) of E. coli or paralogs or orthologs thereof which have been modified to attenuate, disrupt and / or delete the genes. Further information regarding pykF, malT and lamB of, e.g., Escherichia coli is available at EcoCyc (www.biocyc.org) under Accession numbers EG10804, EG10562 and EG10528 respectively.

[0103] In further embodiments the host cell expresses a mutant rpoB gene or a paralog or ortholog thereof encoding a protein having an amino acid substitution at a position corresponding to P520 of the rpoB sequence of SEQ ID NO: 195 preferably P520L. Further information regarding rpoB of, e.g., Escherichia coli is available at EcoCyc (www.biocyc.org) under Accession numbers EG10894.

[0104] In further embodiments the host cell expresses a mutant fumB gene or a paralog or ortholog thereof encoding a protein having an amino acid substitution at a position corresponding to T218 of the fumB sequence of SEQ ID NO: 196 preferably T218P. Further information regarding rpoB of, e.g., Escherichia coli is available at EcoCyc (www.biocyc.org) under Accession number EG10357.

[0105] In further embodiments the host cell expresses a mutant gshA gene or a paralog or ortholog thereof encoding a protein having an amino acid substitution at a position corresponding to A178 of the gshA sequence of SEQ ID NO: 197, preferably A178V. Further information regarding rpoB of, e.g., Escherichia coli is available at EcoCyc (www.biocyc.org) under Accession number EG10418.

[0106] In further embodiments the host cell is a prokaryotic cells, optionally a bacterium. The prokaryotic cell can be a Pseudomonadota, optionally of the class g ammaproteobacteria, optionally of the family Enterobacteriaceae, optionally of the genus Escherichia, optionally of the species Escherichia coli. In some embodiments the genetically engineered bacterium belongs to the Enterobacteriaceae family. In some embodiments the genetically engineered bacterium belongs tothe genus Escherichia. In some embodiments the genetically engineered bacterium is Escherichia coli. Additionally or alternatively the prokaryotic cell is an Actinomycetota, optionally of the class Actinobacteria, optionally of the family Corynebacteriaceae, optionally of the genus Corynebacterium, optionally of the species Corynebacterium glutamicum. In some embodiments the genetically engineered bacterium is Corynebacterium glutamicum.

[0107] In some embodiments the host cell comprises a plasmid having a sequence comprised in any one of SEQ ID NO: 18, 38 or 39. In a particular embodiment the host cell described herein is a strain of E. coli comprising a plasmid of anyone of SEQ ID NO: 17 to 40 or 140 to 144 and which produces L- serine. In still further embodiments the host cell is a strain of E.coli comprising a plasmid having a sequence of anyone of SEQ ID NO: 18, 38 or 39. In still further embodiments the host cell is a genetically modified E. coli host cell producing L-serine expressing or overexpressing an operon comprising: a) a gene encoding a 3-phosphoglycerate dehydrogenase (serA) converting D-3- phosphopyruvate (PGA) into phosphohydroxypyruvate (PHP) and having an amino acid sequence set forth in SEQ ID NO: 60, operably linked to a Translation Initiation Region (TIR) having a sequence set forth in SEQ ID NO: 145; and a gene encoding a phosphoserine aminotransferase (serC) converting 3- phosphohydroxypyruvate into phosphoserine and having an amino acid sequence set forth in SEQ ID NO: 108, operably linked to a Translation Initiation Region (TIR) having a sequence set forth in SEQ ID NO: 158; and a gene encoding a phosphoserine phosphatase (serB) converting phosphoserine into L-serine and having an amino acid sequence set forth in SEQ ID NO: 109; OR b) a gene encoding a 3-phosphoglycerate dehydrogenase (serA) converting D-3- phosphopyruvate (PGA) into phosphohydroxypyruvate (PHP) and having an amino acid sequence set forth in SEQ ID NO: 60, operably linked to a Translation Initiation Region (TIR) having a sequence set forth in SEQ ID NO: 150; and a gene encoding a phosphoserine aminotransferase (serC) converting 3- phosphohydroxypyruvate into phosphoserine and having an amino acid sequence set forth in SEQ ID NO: 108, operably linked to a Translation Initiation Region (TIR) having a sequence set forth in SEQ ID NO: 163; and a gene encoding a phosphoserine phosphatase (serB) converting phosphoserine into L-serine and having an amino acid sequence set forth in SEQ ID NO: 109; or c) a gene encoding a 3-phosphoglycerate dehydrogenase (serA) converting D-3- phosphopyruvate (PGA) into phosphohydroxypyruvate (PHP) and having an amino acidsequence set forth in SEQ ID NO: 60, operably linked to a Translation Initiation Region (TIR) having a sequence set forth in SEQ ID NO: 151; and a gene encoding a phosphoserine aminotransferase (serC) converting 3- phosphohydroxypyruvate into phosphoserine and having an amino acid sequence set forth in SEQ ID NO: 108, operably linked to a Translation Initiation Region (TIR) having a sequence set forth in SEQ ID NO: 164; and a gene encoding a phosphoserine phosphatase (serB) converting phosphoserine into L-serine and having an amino acid sequence set forth in SEQ ID NO: 109;

[0108] wherein the operon is operably linked to a constitutive promoter having a nucleotide sequence set forth in SEQ ID NO: 8. In a further aspect a polynucleotide construct is provided which comprises one or more genes of an L-serine pathway, or an operon comprising such one or more genes, operably linked to a constitutive promoter which has a nucleotide sequence which is at least 70 % identity, 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 constitutive promoter comprised in SEQ ID NO: 1 to 16. In particular these genes or operon of genes includes genes encoding a PGDH or serA; a PSAT or serC and / or PSPH or serB. More particularly these genes or operon of genes includes genes encoding a PGDH or serA; a PSAT or serC and PSPH or serB. Particularly, the constitutive promoter has a nucleotide sequence which is at least 95 % to 100% identical to the constitutive promoter comprised in SEQ ID NO: 8. Alternatively, the constitutive promoter in the polynucleotide construct has a nucleotide sequence which is at least 95 % to 100% identical to the constitutive promoter for SerA comprised in comprised in any of the plasmids of SEQ ID NO: 17 to 40 or 140 to 144, more particularly in comprised in any of the plasmids of SEQ ID NO: SEQ ID NO: 18, 33 to 40 or 140 to 144.

[0109] The PGDH or SerA encoded by the gene in the polynucleotide construct particularly comprises a polypeptide sequence 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: 41 to 63, more preferably comprised in any one of SEQ ID NO: 46 to 63 and / or SEQ ID NO: 46 to 55 and / or SEQ ID NO: 54, 59 to 61, or 63.

[0110] The PSAT or SerC encoded by genes in the construct can suitably comprise a polypeptide sequence which is at least 70% identity, 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 SerC comprised in SEQ ID NO: 108. The PSPH or serB encoded by genes in can suitably comprise a polypeptide sequence which is at least 70% identity, such as at least 80%, such asat 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 SerC comprised in SEQ ID NO: 109.

[0111] The polynucleotide construct may further advantageously comprise a Translation Initiation Region (TIR) operably linked to the genes or operon of genes, said TIR comprising a sequence set forth in any of SEQ ID NO: 145 to 170. More specifically the Translation Initiation Region (TIR) operably linked to the genes or operon of genes in the construct, may comprise a sequence set forth in any of SEQ ID NO: 145 to 157 and / or a sequence set forth in any of SEQ ID NO: 158 to 170.

[0112] In select embodiments the Translation Initiation Region (TIR) is positioned upstream of the genes or operon of genes. In further select embodiments the TIR is positioned upstream of the first, second and / or third codons of the genes or operon of genes, said TIR comprising a sequence set forth in any of SEQ ID NO: 145 to 170. Especially, the Translation Initiation Region (TIR) may be operably linked to an operon comprising the genes encoding serA, serB and serC as described above, said TIR comprising a sequence set forth in any of SEQ ID NO: 145 to 157, or SEQ ID NO: 158 to 170. In a separate aspect a TIR is provided which comprises a sequence as set forth in anyone of SEQ ID NO 145 to 170.

[0113] In some embodiments the polynucleotide construct is an expression vector. In other embodiments the polynucleotide construct is a plasmid comprised in any one of SEQ ID NO: 17 to 40 or 140 to 144, more preferably comprised in any one of SEQ ID NO: 18, 33 to 40 or 140 to 144. In still further embodiments the polynucleotide construct comprises the plasmid of any one of SEQ ID NO: 18, 38 or 39. In still further embodiments the polynucleotide construct comprises an operon comprising. a) a gene encoding a 3-phosphoglycerate dehydrogenase (serA) converting D-3- phosphopyruvate (PGA) into phosphohydroxypyruvate (PHP) and having an amino acid sequence set forth in SEQ ID NO: 60, operably linked to a Translation Initiation Region (TIR) having a sequence set forth in SEQ ID NO: 145; and a phosphoserine aminotransferase (serC) converting 3-phosphohydroxypyruvate into phosphoserine and having an amino acid sequence set forth in SEQ ID NO: 108, operably linked to a Translation Initiation Region (TIR) having a sequence set forth in SEQ ID NO: 158; and a phosphoserine phosphatase (serB) converting phosphoserine into L-serine and having an amino acid sequence set forth in SEQ ID NO: 109; OR b) a gene encoding a 3-phosphoglycerate dehydrogenase (serA) converting D-3- phosphopyruvate (PGA) into phosphohydroxypyruvate (PHP) and having an amino acid sequence set forth in SEQ ID NO: 60, operably linked to a Translation Initiation Region (TIR)having a sequence set forth in SEQ ID NO: 150; and a phosphoserine aminotransferase (serC) converting 3-phosphohydroxypyruvate into phosphoserine and having an amino acid sequence set forth in SEQ ID NO: 108, operably linked to a Translation Initiation Region (TIR) having a sequence set forth in SEQ ID NO: 163; and a phosphoserine phosphatase (serB) converting phosphoserine into L-serine and having an amino acid sequence set forth in SEQ ID NO: 109; OR c) a gene encoding a 3-phosphoglycerate dehydrogenase (serA) converting D-3- phosphopyruvate (PGA) into phosphohydroxypyruvate (PHP) and having an amino acid sequence set forth in SEQ ID NO: 60, operably linked to a Translation Initiation Region (TIR) having a sequence set forth in SEQ ID NO: 151; and a phosphoserine aminotransferase (serC) converting 3-phosphohydroxypyruvate into phosphoserine and having an amino acid sequence set forth in SEQ ID NO: 108, operably linked to a Translation Initiation Region (TIR) having a sequence set forth in SEQ ID NO: 164; and a phosphoserine phosphatase (serB) converting phosphoserine into L-serine and having an amino acid sequence set forth in SEQ ID NO: 109; wherein the operon is operably linked to a constitutive promoter having a nucleotide sequence set forth in SEQ ID NO: 8.

[0114] In a preferred embodiment the host cell described herein comprises the polynucleotide construct described, supra. In further separate aspects a cell culture is provided which comprises the host cell described herein and a growth medium, as well as methods are provided for producing L- serine or a derivative thereof by culturing the cell culture at conditions allowing the host cell to produce the L-serine or a derivative thereof; and optionally recovering and / or isolating the L-serine or a derivative thereof.

[0115] Suitable growth mediums for prokaryotic cell are well known in the art. The cell culture can be cultivated in a nutrient medium using methods known in the art at conditions suitable for production of the L-serine and / or its precursors and / or for propagating cell count. For example, the culture may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentations) in laboratory or industrial fermenters in a suitable medium and under conditions allowing the host cells to grow and / or propagate, optionally to be recovered and / or isolated.

[0116] 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 fromcommercial suppliers or may be prepared according to published recipes (e.g. from catalogues of the American Type Culture Collection). 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. 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. A non-limiting example of 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.

[0117] 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 favoring the host cells over other potentially contaminating microorganisms. Accordingly, in an embodiment a suitable nutrient medium comprises a carbon source (e.g. C6 sugars (such as glucose), maltose, molasses, starch, cellulose, xylan, pectin, lignocellolytic biomass hydrolysate, C5 sugars (such as arabinose or xylose), acetate, glycerol, plant oils, sucrose, yeast extract, peptone, casamino acids or mixtures thereof), a nitrogen source (e.g. ammonia, ammonium sulphate, ammonium nitrate, ammonium chloride, etc.), an organic nitrogen source (e.g. amines yeast extract, malt extract, peptone, soybean-hydrolysate, or digested fermentative microorganisms etc.) and inorganic nutrient sources (e.g. phosphate, magnesium, potassium, zinc, iron, etc.).

[0118] Culturing of the host cell may be performed over a period of about 0.5 to about 30 days. 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. Accordingly, in an embodiment the method may further comprise one or more elements / steps 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.

[0119] In some embodiments the method further comprises feeding the cell culture with one or more L-serine precursors or substrates in the L-serine pathway. In other embodiments the method comprises one or more in vitro steps in the process of producing the L-serine or the derivative thereof. In particular where the L-serine is not the desired end product further steps may be added to the method described herein either chemically or biologically / enzymatically modifying the L-serine or the derivative thereof.

[0120] The method can further comprise recovering the L-serine or the derivative thereof and mixing it with one or more carriers, agents, additives, adjuvants and / or excipients.

[0121] The cell culture of the disclosure may be recovered and or isolated using methods known in the art. For example, the L-serine or the derivative thereof may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, spraydrying, or lyophilization. In a particular embodiment 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 L-serine or the derivative thereof and / or subjecting the supernatant to one or more steps selected from: a) separating the supernatant from the solid phase of the cell culture, such as by filtration or gravity separation; b) contacting the supernatant with one or more adsorbent resins to obtain at least a portion of the produced L-serine or the derivative thereof; c) contacting the supernatant with one or more ion exchange or reversed-phase chromatography columns in order to obtain at least a portion of the L-serine or the derivative thereof; d) extracting the L-serine or the derivative thereof; and / or e) precipitating the L-serine or the derivative thereof by crystallization or evaporating the solvent of the liquid phase; and optionally isolating the L-serine or the derivative thereof by filtration or gravity separation; thereby recovering and / or isolating the L-serine or the derivative thereof.

[0122] In a further aspect a fermentation composition is provided which comprises the L-serine or the derivative obtained from the culturing of the cell culture. In some embodiments a majority of the solid cellular material / debris has been separated, such as at least 50%, such as at least 75%, such as at least 95%, such as at least 99% of solid cellular material has been separated from the composition.

[0123] 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 includesprecursors for the L-serine as well as 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 the L-serine or derivative thereof, in particular L-serine, 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. In other embodiments the composition is substantially free of a-HGA. The composition may also further comprise one or more carriers, agents, additives and / or excipients.

[0124] Further at least 20% by weight of the carbon in the composition is biobased. In some embodiments, 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%. In other embodiments, 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. In still other embodiments 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. In still other embodiments 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. 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, for example 94% biobased carbon.

[0125] Having generally described this technical advancement, a further understanding can be obtained by reference to certain specific examples, which are provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified.ExamplesExample 1 - Constitutive expression of serine pathway.

[0126] In this example several constitutive promoters were tested for performance in constitutive expression in the serine pathway compared with the T7 based induction expression system.

[0127] A set of constitutive promoters (SEQ. ID NO: 1 to 16) of different strengths were evaluated for L-serine production. Constitutive promoters were cloned into a low copy serine production plasmid (pSEVA derivative) with Cg / serA (A285G, Y463A), Ec / serB and Ec / serC genes. The denotation Cg refers to Corynebacterium glutamicum, while Ec refers to Escherichia coli. The parts design included a promoter region, MCD2 (Mutalik et al., 2013) followed by USER site to facilitate exchange of genes (Mutalik et al., 2013). The promoters were selected from two different sources a) Andersen library (http: / / parts.igem.org / Promoters / Catalog / Anderson) including the J23119, J23102, J23108, J23104, J23106, J23115, J23107 and J23116 promoters, and b) research work of Mutalik et al., 2013, including promotors Pl, P7, P2, P5, P6, P14, Pll and P9. Plasmids (pSERlO to pSER25) with 16 different constitutive promoters were constructed by two fragment USER cloning (see table 1.1), using the PCR amplification and USER cloning protocol described below. The constitutive promoter primer sequences are shown in table 1.2.Table 1.1: Information on fragments used in USER cloning.aBoth fragments were made using pSER5 as template.bEach primer carries a constitutive promoter, followed by MCD2, USER site and a SerA binding site.Table 1.2: Primer sequences for constructing different constitutive promoters.PCR amplification and USER cloning protocol

[0128] All plasmid manipulations were performed using Uracil-Specific Excision Reagent (USER) cloning. PCRs were carried out with 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 1.1 and 1.2. PCR program: initial denaturation at 98°C for 40 sec, denaturation at 98°C for 10 sec, annealing 65°C for 30 sec, extension 72°C for 3 min and 30 sec the cycle was repeated 25 times. All the plasmids were then sequence verified by Sanger sequencing.

[0129] 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 kanamycin plates and incubated at 37°C overnight.

[0130] Next day, single colonies were picked and incubated in 2xYT-kan medium and incubated at 37°C for 16 h for plasmid prep and sanger sequencing. The plasmid maps are given in Figures 1A to IP.Confirmed plasmids were transformed in the E. coli strain for L-serine production. The shake flask protocol described below was used for serine production.Batch media and cultivations used for 24 Deep well plate and shake flask screening.

[0131] L-serine production was checked in M9 minimal media. Glucose M9 minimal media consisted of 2 to 5 g / L glucose, 2mM Glycine, 0.1 mM CaCI2, 2.0 mM MgSO4, lx trace element solution, and lx M9 salts. The l,000x trace element stock solution consisted of 27 g / L FeCI3*6H2O, 2 g / L ZnCI2*4H2O, 2 g / L CoCI2*6H2O, 2 g / L NaMoO4*2H2O, 1 g / L CaCI2*H2O, 1.3 g / L CuCI2*6H2O, 0.5 g / L H3BO3, and concentrated HCI dissolved in ddH2O and sterile filtered. The 10x M9 salts stock solution consisted of 68 g / L Na2HPO4 anhydrous, 30 g / L KH2PO4, 5 g / L NaCI, and 10 g / L NH4CI dissolved in ddH2O and autoclaved.

[0132] Media was filter sterilized, for 24 well plate screening experiments 3 ml of M9 media was used in each well. While for shake flask 50 ml was used in 250 ml sterile baffled shake flask. Unless otherwise stated, overnight cultures were used as inoculum, with starting O.D. 0.1 and the incubation was at 37°C and 300 rpm for 24 deep well plate and 37°C and 250 rpm for shake flask. For IPTG inducible system 40 pM of IPTG was added at mid log phase (OD 0.5 to 0.7).Analytical Methods

[0133] Glucose, HGA and other organic acids were quantified using previously published HPLC method (Rennig et al., 2019a).

[0134] 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 m ill iQ. 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.Results

[0135] All 16 strains were subjected to screening in 24 deep well plate. Top 6 strains were selected for screening in shake flask. The data from shake flask for top six plasmids is shown in figure 5. As observed J23119 not only provided the benefits of being a constitutive promoter it also performed better than T7 based induction system. Being constitutive J23119 and the other promoters provided very stable expression performance as observed by repetitive fermentation from the biomass of previous fermentation run.Example 2 - Optimization of translation initiation region using TARSYN system

[0136] To further improve the expression of genes optimized translation initiation region (TIR) were tested.Construction, randomization and removal of the TARSYN modules

[0137] The workflow was divided into the three steps of construction, randomization, and removal of TARSYN modules. In the first step TARSYN module was cloned into the plasmid pSERlO. The resulting plasmid was named pSER_34. The map is shown in Figure 2. In the next step the TIR region of serA and serC was randomized by the primer given in Table 2.1. The randomized sequences for serA (SEQ ID NO: 173 and 174) and serC (SEQ ID NO:175 and 176) are shown in Figure 3 and Figure 4. respectively. After screening, the TARSYN module was removed from the backbone of 12 best constructs. The PCR protocol, USER cloning and transformation protocol remains same as mentioned in above example.Screening of the TARSYN library

[0138] To screen strains for serine production the HPLC method mentioned previously (Rennig et al., 2019a) was used. Briefly, five plates of different ampicillin concentrations of 5000, 6000, 7000, 8000, 10000 ug / ml were received. 90 colonies from each plate were inoculated into 96 deep well plates. The media and growth conditions remain the same as above. For 96 deep well plate screening volume per well was reduced to 500 pL instead of 3 mL including positive and negative controls in three different locations. Subsequently, positive strains were screened in 24 DWP or flasks.

[0139] Plasmids of the ten best strains of this study were isolated. Plasmid purifications were done by using Machery Nagel kits (Duren, Germany). Sequencing of the plasmids was done using primers, which gave the sequences of the TIRs of serA and serC, and the Mix2Seq kit of Eurofins (Ebersberg, Germany) after removal of amp marker they were stocked as SER_160 to ser_171. Five strains were studied for serine production in fed batch fermentation.Table 2.1: Primer used for cloning TARSYN module, TIR library generation and removing of TARSYN module after screening:Table 2.2: Sequencing results of the test plasmids and the control plasmid pSERlOResults.

[0140] We further optimized the translation initiation region (Example 2) using the method published before. Interestingly, in addition to a better L-serine production the new mutants also exhibit improved expression of the serACB genes, confirmed by proteomics analysis and fed batch fermentation example 4. Also, the translation initiation region differs from the previous published work.

[0141] As shown in figure 6 the TIR sequence varies based on the DNA topology, the selection was repeated with the new constructs again.Example 3 - Proteomics study

[0142] To verify whether the expression of serine pathway is enhanced, proteomics analysis was performed. The protocols for sampling and proteomics analysis were as described below.Media and Sampling

[0143] M9 media contained 0.5% (w / v) glucose, lx M9salts, 2mM MgSO4, 2mM Glycine, O.lmM CaCI2, 0.05X YT, Trace elements and vitamins, unless stated otherwise. Pre-cultures and strains for screening purposes were grown in 2X YT, which consisted of 16 g / L bacto-tryptone, 10 g / L yeast extract and 5 g / L NaCI. The media was supplemented with ImM glycine and 0.1% (w / v) glucose. Strains were grown at 37 °C and 250 rpm. Proteomic and HPLC samples were taken after 24 hours. Once the optical density of a culture was determined at 600nm, the volume corresponding to OD 2 was taken for the proteomics analysis and spun down at max speed at 4 °C. Once spun down, the supernatant was removed, and samples were stored at -20 °C.Sample preparation for proteomic analysis

[0144] Frozen cells were kept at -80 °C for up to 4 weeks, after which they were thawed on ice and pelleted by centrifugation at 15,000g for 10 min. The supernatant was removed, and 100 pL 95 °C Guanidinium-HCI (6 M Guanidinium hydrochloride (GuHCI), 5 mM tris-2-carboxyethyl)phosphine (TCEP), 10 mM chloroacetamide (CAA), 100 mM Tris-HCI pH 8.5) was added to the samples together with two 3-mm zirconium oxide beads (Glen Mills, NJ, USA). Cells were disrupted using a Mixer Mill (MM 400 Retsch, Haan, Germany) for 5 min at 25 Hz. The samples were placed in a thermo mixer at 95 °C for 10 min at 2000 rpm. After this the samples were centrifuged at 15,000g for 10 min, and 50 pL of supernatant was collected and diluted with 50 pL of 50 mM ammonium bicarbonate. Protein concentrations were measured (BSA), and 100 pg were used for tryptic digestion. Tryptic digestion was carried out for 8 h, after which 10 pL of 10% TFA was added and samples were Stage-Tipped using C18 (Empore, 3M, USA).

[0145] After stage-tipping the samples were analyzed using a CapLC system (Thermo Fisher Scientific, Waltham, MA, USA) coupled to a 15 cm C18 easy spray column (PepMap RSLC C18 2 pm, 100 A, 150 pmxl5cm). Initially the samples were trapped on a precolumn (p-precolumn C18 PepMap 100, 5pm, 100A) after which the peptides were separated using a gradient going from 4% acetonitrile in water to 76% over 60 minutes at a constant flowrate of 1.2 pl / min. The samples were sprayed into anOrbitrap Q-exactive HF-X mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). MS-level scans were performed with Orbitrap resolution set to 60,000; AGC Target 3.0e6; maximum injection time 50 ms; intensity threshold 5.0e3; dynamic exclusion 25 sec. Data dependent MS2 selection was performed in Top 20 Speed mode with HCD collision energy set to 28% (AGC target 1.0e4, maximum injection time 22 ms, Isolation window 1.2 m / z).Proteomics data analysis

[0146] The raw files were analyzed using maxquant to obtain protein identifications and quantification. While analyzing the data the following settings were used: Fixed modifications: Carbamidomethyl (C) and Varible modifications: oxidation of methionine residues. First search mass tolerance 20 ppm and a MS / MS tolerance of 20 ppm. Trypsin as enzyme and allowing one missed cleavage. FDR was set at 0.1%. The Match between runs window was set to 0.7 min. For quantification, the LFQ. values from maxquant was used only allowing quantification based on unique peptides. Intensities were normalized within maxquant. For the searches a protein database consisting of the reference E. coli proteome UP000000625 was used. In the case of the depicted proteins SerABC, the data was manually inspected to ensure correct quantification and identification. At the end of the shake flask experiment, proteomic samples were taken. These were analyzed and the fold differences of the enzymes in the serine production pathway were determined compared to the positive control, Serl51.Results.

[0147] For almost all strains, the concentration of SerA was higher than in the control (Figure 7), and higher concentrations of serine were observed. Reiterating, from the data an increase in the concentration of SerA was observed resulting in higher serine concentrations compared to the positive control. The data obtained was represented as fold increase as compared to the strain ser_151.Example 4 - Fermentation studies.

[0148] From the experiment of example 2, the top 5 improved serine producing strains (Ser_160, Ser_162, Ser_164, Ser_165 and Ser_166) were further characterized in fed-batch fermentations. The strains were grown on minimal media with either glucose or maltose as carbon source and the same feed profile was used as in previous fed-batch fermentations.

[0149] The fed-batch serine productions were performed as fermentations in IL bioreactors (Sartorius, Gottingen, Germany), 250mL AMBR reactors (Sartorius) and IL DASGIP bioreactors (Eppendorf, Hamburg, Germany) and the medias used for the batch and the feed are described below.

[0150] Pre-cultures and strains for screening purposes were grown in 2X YT, which consisted of 16 g / L bacto-tryptone, 10 g / L yeast extract and 5 g / L NaCI. The media was supplemented with ImM glycine and 0.1% (w / v) glucose or other carbohydrates, depending on the experiment. The batch phase of fermentations was carried out on minimal media containing 10 g / L (NH4)SO4, 2 g / L KH2PO4, 2 g / L yeast extract, 2 g / L MgSO4 • 7 H2O, 0.6 g / L glycine, trace elements and 10 g / L glucose, 9.5 g / L maltose, 10.6 g / L glycerol or 10 g / L sucrose. The feed used in fed-batch experiments consisted of 6 g / L glycine, ImL / L antifoam and 590 g / L glucose, or 560 g / L maltose. When needed, the appropriate antibiotics were added to the media to ensure the selection of the desired strain.

[0151] Reactors were inoculated with lOmL of inoculum in the morning. The cultures were left to grow during the day under aerobic growth as the DO was maintained at 20% or higher by increasing the stirrer speed, the gas inflow, and the C -percentage in the gassing stream. Once a DO spike was observed, indicating the end of the batch phase, the feed was started. The feed increased in a linear manner for the first 20 hours and was there after maintained constant. Samples were taken at regular intervals, which were subjected to HPLC analysis and measurement of the optical density at 600nm as shown in table 4.1.Table 4.1: Summary of fold improvement in yield and titre of L-serine for the top-5 optimized strains.Results

[0152] The OD of all the strains seemed to be similar to the control, Serl51. Strain ser_166 failed on maltose due to technical issue of the fermenter. Most of the strains already showed an increase in serine concentration from the start of the fermentation (see figure 8). Serl51 was the only strain grown in duplicate, although only on glucose. When the strains containing the optimized plasmid weregrown on glucose, an increase of 25% to 31% in serine yield was observed. From previous fermentations of Serl51 on maltose, a mean serine yield was determined. When comparing this mean yield to the serine yield acquired for the strains with the optimized plasmids, a 1.13 to 2.13 fold increase was observed (see table 4.1).

[0153] These results showed that not only the promoter of SEQ ID NO: 8 greatly improved expression but also optimization of the TIR sequences improved expression significantly (see figure 7 and 8). These favourable results also applied to E. coli host cells transformed with plasmids pSER-10, pSER_43 and pSER_44, ie. E. coli hosts expressing a) the SEQ ID NO: 60 serA, operably linked to the SEQ ID NO: 145 TIR, and the SEQ ID NO: 108 serC operably linked to the SEQ ID NO: 158 TIR, and the SEQ ID NO: 109 serB; OR b) the SEQ ID NO: 60 serA, operably linked to the SEQ ID NO: 150 TIR, and the SEQ ID NO: 108 serC operably linked to the SEQ ID NO: 163 TIR, and the SEQ ID NO: 109 serB; OR c) the SEQ ID NO: 60 serA, operably linked to the SEQ ID NO: 151 TIR, and the SEQ ID NO: 108 serC, operably linked to the SEQ ID NO: 164 TIR, and the SEQ ID NO: 109 serB; wherein the operon is operably linked to a constitutive promoter having a nucleotide sequence set forth in SEQ ID NO: 8.

[0154] Several studies such as shake flask data (Table 4.1) and fed batch fermentation data (Figure 8) demonstrated increased serine production due to increased expression of pathway enzymes as validated by proteomics (Figure 8).List of references1. Grant, G. A. D-3-phosphoglycerate3-phosphoglycerate dehydrogenase. Front. Mol. Biosci. 5, 1-18 (2018).2. Wei Xu, Hui Yang, Ying Liu, Ying Yang, Ping Wang, Se-Hee Kim, S. Ito, Chen Yang, Pu Wang, Meng-Tao Xiao, Li-xia Liu, Wen-qing Jiang, J. Liu, Jin-ye Zhang, Bin Wang, Stephen Frye, Yi Zhang, Yan-hui Xu, Q. Lei, Kun-Liang Guan, Shi-min Zhao, and Yue Xiong; Control of Embryonic Stem Cell State Richard. Cancer Cell 29, 997-1003 (2012).3. Kalliri, E., Mulrooney, S. B. & Hausinger, R. P. 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 forhuman 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 I -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 in Corynebacterium glutamicum to 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 a-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- Tai 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-Tai N. (2009). The ConSurf-DB: Pre-calculated evolutionary conservation profiles of protein structures. Nucleic Acids Research (Database issue), 37:D323-D327; PMID: 18971256.Sequence listings

[0155] The present application contains a listing of sequences included in the below table A submitted electronically in ST26 format which is hereby incorporated by reference in its entirety.Table A

Claims

Claims1. A genetically modified host cell expressing or overexpressing one or more genes of an L-serine pathway, or an operon comprising said one or more genes, wherein the genes or operon are operably linked to a constitutive promoter having a nucleotide sequence which is 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 constitutive promoter comprised in anyone of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

2. The host cell of claims 1 wherein the one or more genes encode: a) a 3-phosphoglycerate dehydrogenase (PGDH or serA) enzyme producing 3- phosphopyruvate; b) a Phosphoserine aminotransferase (PSAT or serC) converting 3-phosphohydroxypyruvate into phosphoserine; and / or c) a Phosphoserine phosphatase (PSPH or serB) converting phosphoserine into L-serine.

3. The host cell of claims 2 wherein the one or more genes encode: a) a 3-phosphoglycerate dehydrogenase (PGDH or serA) enzyme producing 3- phosphopyruvate; b) a Phosphoserine aminotransferase (PSAT or serC) converting 3-phosphohydroxypyruvate into phosphoserine; and c) a Phosphoserine phosphatase (PSPH or serB) converting phosphoserine into L-serine.

4. The host cell of any preceding claim wherein the constitutive promoter has a nucleotide sequence which is at least 95 % identical to the constitutive promoter comprised in SEQ ID NO: 8.

5. The host cell of any preceding claim wherein the gene encoding the 3-phosphoglycerate dehydrogenase (PGDH or SerA) enzyme producing 3-phosphopyruvate operably linked to the constitutive promoter is comprised in any of the plasmids of SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 140, 141, 142, 143, or 144.

6. The host cell of claim 5 wherein the gene encoding the 3-phosphoglycerate dehydrogenase (PGDH or SerA) enzyme producing 3-phosphopyruvate operably linked to the constitutive promoter is comprised in any of the plasmids of SEQ ID NO: 18, 33, 34, 35, 36, 37, 38, 39, 40, 140, 141, 142, 143, or 144.

7. The host cell of any preceding claims wherein one or more of the genes are heterologous to the host cell.

8. The host cell of any preceding claim wherein PGDH or SerA is a heterologous enzyme and has a reduced or eliminated side activity converting a-ketoglutarate (a-KGA) into a-hydroxyglutarate (a- HGA) compared to a native PGDH.

9. The host cell of any preceding claim wherein the heterologous PGDH is overexpressed compared to a native PGDH having a side activity of converting a-ketoglutarate (a-KGA) into a- hydroxyglutarate.

10. The host cell of claim 9 wherein the heterologous PGDH is overexpressed compared to a native PGDH by 10 to 10.000%.

11. The host cell of any one of claim 7 to 10 wherein the heterologous PGDH comprise a conserved region facing the active site which comprises the motif G / AXAGV, wherein the underlined X is a cationic residue positioned corresponding to position 129 of the PGDH from Mycobacterium tuberculosis (SEQ ID NO: 50).

12. The host cell of claim 11 wherein the underlined X is selected from Arginine, Leucine or Histidine.

13. The host cell of claim 12 wherein the underlined X is Arginine.

14. The host cell of any one of claim 7 to 10 wherein the heterologous PGDH 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: 50).

15. The host cell of any one of claim 7 to 14 wherein 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.

16. The host cell of any one of claim 7 to 15 wherein the heterologous PGDH is a type I or type III PGDH, optionally a microbial type I or a type III PGDH.

17. The host cell of any one of claim 7 to 16 wherein the heterologous PGDH enzymes is a SerA enzyme.

18. The host cell of any one of claim 7 to 17 wherein the PGDH comprises a polypeptide sequence 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 sequence comprised in any one of SEQ ID NO: 41 to 63.

19. The host cell of claim 18 wherein the PGDH comprises a polypeptide sequence 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 sequence comprised in any one of SEQ ID NO: 46 to 63.

20. The host cell of claim 19 wherein the PGDH comprises a polypeptide sequence 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 sequence comprised in any one of SEQ ID NO: 46 to 55.

21. The host cell of claim 19 wherein the PGDH comprises a polypeptide sequence 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 sequence comprised in SEQ ID NO: 54, 59 to 61, or 63.

22. The host cell of any one of claim 2 to 21 wherein: a)the PSAT or serC comprises a polypeptide sequence 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 least90%, 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 serC sequence comprised in SEQ ID NO: 108; and b)the PSPH or serB and comprises a polypeptide sequence 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 sequence comprised in SEQ ID NO: 109.

23. The host cell of any preceding claim expressing or overexpressing one or more further genes of the L-serine pathway and to produce L-serine or a derivative thereof.

24. The host cell of claim 23 wherein the one or more further genes are heterologous to the host cell.

25. The host cell of any one of claim 23 to 24 comprising expression or overexpression of one or more enzymes selected from: a) a first heterologous NADPH generating enzyme converting glyceraldehyde 3-phosphate into 1,3-bisphosphoglycerate or a downstream precursor in the L-serine pathway; b) a heterologous enzyme converting NADH to NAD+; c) a heterologous enzyme converting a side product of an enzyme in the L-serine pathway into a substrate of a L-serine pathway enzyme consuming NADH or NADPH; d) a native enzyme converting a side product of an enzyme in the L-serine pathway into a substrate of a L-serine pathway enzyme consuming NADH or NADPH; e) a second heterologous NADPH producing enzyme, which is not comprised in the L-serine pathway; and / or f) a native NADPH producing enzyme, which is not comprised in the L-serine pathway.

26. The host cell of claim 25 wherein the expression of genes of the L-serine pathway comprises expression of the first heterologous NADPH generating enzyme producing a precursor for the L- serine pathway.

27. The host cell of any one of claim 25 to 26 wherein the expression of genes of the L-serine pathway comprises: a) expression of the first heterologous NADPH generating enzyme converting glyceraldehyde 3- phosphate into 1,3-bisphosphoglycerate or a downstream precursor in the L-serine pathway; andb) expression of the heterologous enzyme converting NADH to NAD+.

28. The host cell of claim 27 wherein the expression of genes of the L-serine pathway comprises: a) expression of the first heterologous NADPH generating enzyme converting glyceraldehyde 3- phosphate into 1,3-bisphosphoglycerate or a downstream precursor in the L-serine pathway; b) expression of the heterologous enzyme converting NADH to NAD+; and c) expression of the heterologous enzyme and / or overexpression of the native enzyme converting the side product of the enzyme in the L-serine pathway into the substrate of the L-serine pathway enzyme consuming NADH or NADPH.

29. The host cell of claim 28 wherein the expression of genes of the L-serine pathway comprises: a) expression of the first heterologous NADPH generating enzyme converting glyceraldehyde 3- phosphate into 1,3-bisphosphoglycerate or a downstream precursor in the L-serine pathway; b) expression of the heterologous enzyme converting NADH to NAD+; c) expression of the heterologous enzyme and / or overexpression of the native enzyme converting the side product of the enzyme in the L-serine pathway into a substrate of the L-serine pathway enzyme consuming NADH or NADPH; and d) expression of the second heterologous NADPH producing enzyme and / or overexpression of the native NADPH producing enzyme, any of which are not comprised in the L-serine pathway.

30. The host cell of any one of claim 25 to 29 wherein: a) the first heterologous NADPH generating enzyme converting glyceraldehyde 3-phosphate into 1,3-bisphosphoglycerate or a downstream precursor in the L-serine pathway partially or completely replaces a NADH producing enzyme native to the host cell; b) the heterologous enzyme converting the side product of the enzyme in the L-serine pathway into the substrate of the L-serine pathway enzyme consuming NADH or NADPH partially or completely replaces a native enzyme converting the side product of the enzyme in the L-serine pathway into the substrate of the L-serine pathway enzyme consuming NADH or NADPH; and / or c) the second heterologous enzyme producing NADPH partially or completely replaces a native NADPH producing enzyme, which is not comprised in the L-serine pathway.

31. The host cell of any one of claim 25 to 30 wherein: a) the first heterologous NADPH generating enzyme converting glyceraldehyde 3-phosphate into 1,3-bisphosphoglycerate or a downstream precursor in the L-serine pathway isbisphoshoglycerate 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; b) the heterologous enzyme converting NADH to NAD+is a NADH oxidase (Nox); c) the enzyme converting the side product of the enzyme in the L-serine pathway into the substrate of a L-serine pathway enzyme is a glutamate dehydrogenase (GDH), the side product is a- ketoglutarate, and the substrate is glutamate; and / or d) the second heterologous or the native NADPH producing enzyme, which are not comprised in the L-serine pathway is a Glucose-6-phosphate dehydrogenase and / or a 6- phosphogluconolactonase.

32. The host cell of claim 31 wherein: a) the GAPDH enzyme comprises a polypeptide sequence 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 sequence comprised in any one of SEQ ID NO: 64 to 87; b) the Nox comprises a polypeptide sequence 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 sequence comprised in any one of SEQ ID NO: 90 to 97; c) the GDH comprises a polypeptide sequence 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 GDH sequence comprised in any one of SEQ ID NO: 98 to 107; d) the Glucose-6-phosphate dehydrogenase comprises a polypeptide sequence 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 Glucose-6-phosphate dehydrogenase sequence comprised in SEQ ID NO: 88; and / or e) the 6-phosphogluconolactonase comprises a polypeptide sequence 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 atleast 98%, such as at least 99%, such as 100% identical to the 6-phosphogluconolactonase sequence comprised in SEQ ID NO: 89.

33. The host cell of claim 32 wherein further comprising GAPDH comprising a polypeptide sequence 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 sequence comprised in any one of SEQ ID NO: 79 to 87.

34. The host cell of claim 33 wherein the GAPDH comprises a polypeptide sequence 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 sequence comprised in SEQ ID NO: 84.

35. The host cell of any one of claim 18 to 34 comprising a PGDH comprises a polypeptide sequence 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 anyone of SEQ ID NO: 54, 59 to 61, or 63 and a GAPDH comprising 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 sequence comprised in SEQ ID NO: 84.

36. The host cell of any preceding claims wherein the gene or operon of genes further comprises a Translation Initiation Region (TIR) operably linked to the genes or operon or genes, said TIR comprising the sequences set forth in any of SEQ ID NO: 145 to 170.

37. The host cell of claim 36 wherein the TIR comprises the sequences set forth in any of SEQ ID NO: 145 to 157.

38. The host cell of claim 36 wherein the TIR comprises the sequences set forth in any of SEQ ID NO: 158 to 170.

39. The host cell of any one of claim 7 to 38 wherein the heterologous genes or enzymes are (i) genes or enzymes from a species different from the host cell, (ii) mutated genes or enzymes from a species different from the host cell, and / or (iii) mutated genes or enzyme native to the host cell.

40. The host cell of any preceding claim, further comprising at least one transporter molecule facilitating transport of metabolite or any of its precursors.

41. The host cell of any preceding claim, wherein one or more native or endogenous genes of the cell is attenuated, disrupted and / or deleted.

42. The host cell of claim 41 wherein the native gene encodes a NADH dependent GAPDH or a a-HGA producing PGDH.

43. The host cell of any preceding claim, further comprising at least 2 copies of one or more polynucleotides encoding one or more L-serine pathway enzymes.

44. The host cell of any preceding claim further genetically modified to provide an increased amount of a substrate for one or more L-serine pathway enzymes.

45. The host cell of any preceding claim further genetically modified to exhibit increased tolerance towards one or more substrates, intermediates, or product molecules from the L-serine pathway.

46. The host cell of any preceding claim wherein the host cell is a prokaryotic cells, optionally a bacterium.

47. The host cell of claim 46 wherein the prokaryotic cell is a cell of Pseudomonadota, optionally of the class gammaproteobacteria, optionally of the family Enterobacteriaceae, optionally of the genus Escherichia, optionally of the species Escherichia coli.

48. The host cell of claim 46 wherein the prokaryotic cell is an Actinomycetota, optionally of the class Actinobacteria, optionally of the family Corynebacteriaceae, optionally of the genus Corynebacterium, optionally of the species Corynebacterium glutamicum.

49. The host cell of any preceding claim comprising a plasmid having a sequence comprised in any one of SEQ ID NO: 18, 38 or 39.

50. The host cell of claims 49 wherein the host cell is a genetically modified E. coli host cell.

51. The host cell of any preceding claim wherein the host cell is a genetically modified E. coli host cell producing L-serine expressing or overexpressing an operon comprising: a) a gene encoding a 3-phosphoglycerate dehydrogenase (serA) converting D-3- phosphopyruvate (PGA) into phosphohydroxypyruvate (PHP) and having an amino acid sequence set forth in SEQ ID NO: 60, operably linked to a Translation Initiation Region (TIR) having a sequence set forth in SEQ ID NO: 145; and a gene encoding a phosphoserine aminotransferase (serC) converting 3-phosphohydroxypyruvate into phosphoserine and having an amino acid sequence set forth in SEQ ID NO: 108, operably linked to a Translation Initiation Region (TIR) having a sequence set forth in SEQ ID NO: 158; and a gene encoding a phosphoserine phosphatase (serB) converting phosphoserine into L-serine and having an amino acid sequence set forth in SEQ ID NO: 109; OR b) a gene encoding a 3-phosphoglycerate dehydrogenase (serA) converting D-3-phosphopyruvate (PGA) into phosphohydroxypyruvate (PHP) and having an amino acid sequence set forth in SEQ ID NO: 60, operably linked to a Translation Initiation Region (TIR) having a sequence set forth in SEQ ID NO: 150; and a gene encoding a phosphoserine aminotransferase (serC) converting 3-phosphohydroxypyruvate into phosphoserine and having an amino acid sequence set forth in SEQ ID NO: 108, operably linked to a Translation Initiation Region (TIR) having a sequence set forth in SEQ ID NO: 163; and a gene encoding a phosphoserine phosphatase (serB) converting phosphoserine into L-serine and having an amino acid sequence set forth in SEQ ID NO: 109; OR c) a gene encoding a 3-phosphoglycerate dehydrogenase (serA) converting D-3-phosphopyruvate (PGA) into phosphohydroxypyruvate (PHP) and having an amino acid sequence set forth in SEQ ID NO: 60, operably linked to a Translation Initiation Region (TIR) having a sequence set forth in SEQ ID NO: 151; and a gene encoding a phosphoserine aminotransferase (serC) converting 3-phosphohydroxypyruvate into phosphoserine and having an amino acid sequence set forth in SEQ ID NO: 108, operably linked to a Translation Initiation Region (TIR) having a sequence set forth in SEQ ID NO: 164; and a gene encoding a phosphoserine phosphatase (serB) converting phosphoserine into L-serine and having an amino acid sequence set forth in SEQ ID NO: 109;wherein the operon is operably linked to a constitutive promoter having a nucleotide sequence set forth in SEQ ID NO: 8.

52. A polynucleotide construct comprising one or more genes of an L-serine pathway, or an operon comprising such one or more genes, operably linked to a constitutive promoter which has a nucleotide sequence which is at least 70 % identity, 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 constitutive promoter comprised in any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 .

53. The polynucleotide construct of claim 52 wherein the one or more genes or operon of genes encodes: a) a 3-phosphoglycerate dehydrogenase (PGDH or serA) enzyme producing 3- phosphopyruvate; b) a Phosphoserine aminotransferase (PSAT or serC) converting 3-phosphohydroxypyruvate into phosphoserine; and / or c) a Phosphoserine phosphatase (PSPH or serB) converting phosphoserine into L-serine.

54. The polynucleotide construct of claim 53 wherein the one or more genes or operon of genes encodes: a) a 3-phosphoglycerate dehydrogenase (PGDH or serA) enzyme producing 3- phosphopyruvate; b) a Phosphoserine aminotransferase (PSAT or serC) converting 3-phosphohydroxypyruvate into phosphoserine; and c) a Phosphoserine phosphatase (PSPH or serB) converting phosphoserine into L-serine.

55. The polynucleotide construct of any one of claim 52 to 54 wherein the constitutive promoter has a nucleotide sequence which is at least 95 % to 100% identical to the constitutive promoter comprised in SEQ ID NO: 8.

56. The polynucleotide construct of any one of claim 52 to 55 wherein the constitutive promoter has a nucleotide sequence which is at least 95 % to 100% identical to the constitutive promoter comprised in comprised in any of the plasmids of SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 140, 141, 142, 143, or 144.

57. The polynucleotide construct of claim 56 wherein the constitutive promoter has a nucleotide sequence which is at least 95 % to 100% identical to the constitutive promoter comprised in any of the plasmids of SEQ ID NO: SEQ ID NO: 18, 33 to 40 or 140 to 144.

58. The polynucleotide construct of any one of claim 52 to 57 wherein the PGDH or SerA comprises a polypeptide sequence 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: 41 to 63.

59. The polynucleotide construct of claim 58 wherein the PGDH or serA comprising 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: 46 to 63.

60. The polynucleotide construct of claim 59 wherein the PGDH or SerA 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: 46 to 55.

61. The polynucleotide construct of claim 59 wherein the PGDH or SerA 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 SEQ ID NO: 54, 59 to 61, or 63.

62. The polynucleotide construct of any one of claim 53 to 61 wherein the PSAT or SerC comprises a polypeptide sequence which is at least 70% identity, 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 SerC comprised in SEQ ID NO: 108.

63. The polynucleotide construct of any one of claim 53 to 61 wherein the PSPH or serB comprises a polypeptide sequence which is at least 70% identity, 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 SerC comprised in SEQ ID NO: 109.

64. The polynucleotide construct of any one of claim 52 to 62 further comprising a Translation Initiation Region (TIR) operably linked to the genes or operon of genes, said TIR comprising a sequence set forth in any of SEQ ID NO: 145 to 170.

65. The polynucleotide construct of any one of claim 52 to 64 comprising a Translation Initiation Region (TIR) operably linked to the genes or operon of genes, said TIR comprising a sequence set forth in any of SEQ ID NO: 145 to 157.

66. The polynucleotide construct of claim 65 comprising a Translation Initiation Region (TIR) operably linked to the genes or operon of genes, said TIR comprising the sequences set forth in any of SEQ ID NO: 158 to 170.

67. The polynucleotide construct of claim 66 wherein the TIR is positioned upstream of the genes or operon of genes, optionally upstream of the first, second and / or third codon of the genes or operon of genes.

68. The polynucleotide construct of any one of claim 66 to 67 comprising an operon comprising genes encoding a serA, a serB and a SerC operably linked to a TIR comprising a sequence set forth in any of SEQ ID NO: 145 to 170 positioned upstream of the first, second and / or third codon of operon.

69. The polynucleotide construct of any one of claim 52 to 68, wherein the construct is an expression vector.

70. The polynucleotide construct of any one of claim 52 to 69 comprising the plasmid of any one of SEQ ID NO: 17 to 40 or 140 to 144.

71. The polynucleotide construct of claim 70 comprising the plasmid of any one of SEQ ID NO: 18, 33, 34, 35, 36, 37, 38, 39, 40, 140, 141, 142, 143, or 144.

72. The polynucleotide construct of claim 71 comprising the plasmid of any one of SEQ ID NO: 18, 38 or 39.

73. The polynucleotide construct of any one of claim 52 to 71 comprising an operon comprising. a) a gene encoding a 3-phosphoglycerate dehydrogenase (serA) converting D-3- phosphopyruvate (PGA) into phosphohydroxypyruvate (PHP) and having an amino acid sequence set forth in SEQ ID NO: 60, operably linked to a Translation Initiation Region (TIR) having a sequence set forth in SEQ ID NO: 145; and a phosphoserine aminotransferase (serC) converting 3-phosphohydroxypyruvate into phosphoserine and having an amino acid sequence set forth in SEQ ID NO: 108, operably linked to a Translation Initiation Region (TIR) having a sequence set forth in SEQ ID NO: 158; and a phosphoserine phosphatase (serB) converting phosphoserine into L-serine and having an amino acid sequence set forth in SEQ ID NO: 109; OR b) a gene encoding a 3-phosphoglycerate dehydrogenase (serA) converting D-3-phosphopyruvate (PGA) into phosphohydroxypyruvate (PHP) and having an amino acid sequence set forth in SEQ ID NO: 60, operably linked to a Translation Initiation Region (TIR) having a sequence set forth in SEQ ID NO: 150; and a phosphoserine aminotransferase (serC) converting 3-phosphohydroxypyruvate into phosphoserine and having an amino acid sequence set forth in SEQ ID NO: 108, operably linked to a Translation Initiation Region (TIR) having a sequence set forth in SEQ ID NO: 163; and a phosphoserine phosphatase (serB) converting phosphoserine into L-serine and having an amino acid sequence set forth in SEQ ID NO: 109; OR c) a gene encoding a 3-phosphoglycerate dehydrogenase (serA) converting D-3-phosphopyruvate (PGA) into phosphohydroxypyruvate (PHP) and having an amino acid sequence set forth in SEQ ID NO: 60, operably linked to a Translation Initiation Region (TIR) having a sequence set forth in SEQ ID NO: 151; and a phosphoserine aminotransferase (serC) converting 3-phosphohydroxypyruvate into phosphoserine and having an amino acid sequence set forth in SEQ ID NO: 108, operably linked to a Translation Initiation Region (TIR) having a sequence set forth in SEQ ID NO: 164; and a phosphoserine phosphatase (serB) converting phosphoserine into L-serine and having an amino acid sequence set forth in SEQ ID NO: 109; wherein the operon is operably linked to a constitutive promoter having a nucleotide sequence set forth in SEQ ID NO: 8.

74. The host cell of any one of claim 1 to 51 comprising the polynucleotide construct of any one of claims 52 to 71.

75. A genetically modified TIR comprising a sequence as set forth in anyone of SEQ. ID NO 145 to 170.

76. A cell culture, comprising the host cell of any one of claim 1 to 51 or 74 and a growth medium.

77. A method for producing L-serine or a derivative thereof comprising: a) culturing the cell culture of claim 76 at conditions allowing the cell to produce the L-serine or the derivative thereof; and b) optionally recovering and / or isolating the L-serine or the derivative thereof.

78. The method of claims 77, further comprising 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.

79. The method of any one of claim 77 to 78, wherein one or more steps of producing the L-serine or a derivative thereof is performed in vitro.

80. The method of any one of claim 77 to 79, comprising feeding the cell culture exogenously with one or more L-serine precursors.

81. The method of claim any one of 77 to 80 wherein the recovery and / or isolation step comprises 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 L-serine or the derivative thereof and / or subjecting the supernatant to one or more steps selected from: a) separating the supernatant from the solid phase of the cell culture, such as by filtration or gravity separation; b) contacting the supernatant with one or more adsorbent resins to obtain at least a portion of the produced L-serine or the derivative thereof;c) contacting the supernatant with one or more ion exchange or reversed-phase chromatography columns in order to obtain at least a portion of the L-serine or the derivative thereof; d) extracting the L-serine or the derivative thereof; and / or e) precipitating the L-serine or the derivative thereof by crystallization or evaporating the solvent of the liquid phase; and optionally isolating the L-serine or the derivative thereof by filtration or gravity separation; thereby recovering and / or isolating the L-serine or the derivative thereof.

82. A fermentation composition comprising the cell culture of claim 76 and / or the L-serine or the derivative thereof comprised therein.

83. The fermentation composition of claims 82, wherein at least 50%, such as at least 75%, such as at least 95%, such as at least 99% of solid cellular material has been separated from the composition.

84. The fermentation composition of any one of claim 82 to 83, further comprising one or more compounds selected from trace metals, vitamins, salts, yeast nitrogen base, carbon source, YNB, and / or amino acids of the fermentation; wherein the concentration of the L-serine or the derivative thereof is at least 1 mg / kg composition.

85. The fermentation composition of any one of claim 82 to 84 wherein at least 20% by weight of the carbon is biobased.

86. The fermentation composition of any one of claim 82 to 85, being substantially free of a-HGA.

87. The fermentation composition of any one of claim 82 to 86 further comprising one or more carriers, agents, additives and / or excipients.* * ** * *

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