CN117625508A - Expression of biomolecules with improved promoters and TIR - Google Patents

Abstract

The present disclosure relates to genetically modified host cells that produce serine that express or overexpress an operon comprising genes encoding one or more of: 3-phosphopyruvate dehydrogenase (serA) which converts D-3-Phosphopyruvate (PGA) to phosphohydroxypyruvate (PHP); a phosphoserine transaminase (serC) converting 3-phosphohydroxypyruvate to phosphoserine; and/or phosphoserine phosphatase (serB) that converts phosphoserine to L-serine, wherein the operon is operably linked to a constitutive promoter having a set of nucleotide sequences that is at least 95% (e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, e.g., 100%) identical to the constitutive promoter comprised in SEQ ID NO. 8.

Description

Expression of biomolecules with improved promoters and TIR

Technical Field

The present invention relates to the microbial industry, and in particular to the industrial production of L-serine or derivatives thereof by expressing or overexpressing an operon comprising genes encoding the serA, serB and serC enzymes of the L-serine pathway, said operon being operably linked to an improved constitutive promoter. Further disclosed are enzymes and polynucleotides encoding such enzymes of the pathway, polynucleotide constructs for expressing the enzymes, and cell cultures of host cells that produce L-serine or derivatives thereof when cultured in fermentation methods. Further disclosed are fermentation compositions, also referred to as biobased compositions, comprising host cells and/or L-serine or derivatives thereof produced by the methods.

Background

Genetically modified host cells which produce metabolites such as L-serine are known, for example from WO2016120326, which describe the production of L-serine using genetically engineered microorganisms which are deficient in the serine degradation pathway. WO2004/108894 discloses a method of producing an amino acid using a genetically modified bacterium comprising a polypeptide that hybridizes to SEQ ID NO:10, and a polypeptide similar thereto. WO2021/081185 describes microorganisms with increased availability of cofactors (e.g.NADPH) for increasing the production of various products. WO2020/0107626 describes a method for producing an L-amino acid comprising culturing an altered bacterial cell having an increased amount of NADPH compared to the unaltered bacterial cell, whereby the yield of the L-amino acid from the altered bacterial cell is greater than the yield from the unaltered bacterial cell. WO2021/195705 describes recombinant microorganisms for the production of biohydrogen and nucleic acid constructs and methods for modifying microorganisms to be able to produce hydrogen. CN103436504a describes a construction method and application of a corynebacterium glutamicum strain to be constructed against L-serine feedback inhibition by mutating the 3-phosphoglycerate dehydrogenase against L-serine feedback inhibition. 3-phosphoglycerate dehydrogenase and SEQ ID NO:10 have less similarity. PCT/EP2023/079297 (not disclosed) is incorporated herein by reference, describes L-serine producing host cells genetically modified to reduce intracellular accumulation of NADH and/or Hydroxyglutarate (HGA) and thereby reduce the negative effects of these compounds on L-serine and other metabolites.

Disclosure of Invention

It is an object of the invention described herein to provide a method which allows for a more efficient production and stabilization of L-serine and its derivatives. More specifically, it is an object of the invention described herein to provide a process which allows the production of L-serine with a higher nominal yield and an improved mass yield.

PGDH is the first key step in L-serine biosynthesis to convert 3-phosphopyruvate into 3-phosphohydroxypyruvate, and therefore efficient and stable expression of this enzyme is crucial for successful production of 3-phosphohydroxypyruvate and/or any metabolite thereof. Previous work used a T7 polymerase-based system to regulate expression of the L-serine pathway (Landberg et al, 2020;Rennig et al, 2019 b). However, this requires the use of an Inducer (IPTG) or low concentration of yeast extract in the medium to produce L-serine. However, IPTG induction adds cost and technical complexity to the process, and the use of low concentrations of yeast extract in the medium places an additional burden on the cells to produce all necessary metabolites and vitamins, resulting in reduced yields and titres.

Thus, different constitutive expression promoters were constructed and studied to avoid addition of IPTG while maintaining complex media addition levels most suitable for productivity, in particular to identify constitutive promoters providing similar or even exceeding expression as the T7-based induction system. Surprisingly, constitutive promoters were identified which not only resemble or even exceed the expression performance of the T7-based induction system, but which are also superior in terms of being very stable as observed by repeated fermentation of biomass from previous fermentation runs.

Thus, in a first aspect, there is provided a genetically modified host cell that produces L-serine or a derivative thereof and expresses or overexpresses an operon comprising genes encoding one or more of: 3-phosphopyruvate dehydrogenase (serA) which converts D-3-Phosphopyruvate (PGA) to phosphohydroxypyruvate (PHP); a phosphoserine transaminase (serC) converting 3-phosphohydroxypyruvate to phosphoserine; and/or phosphoserine phosphatase (serB) that converts phosphoserine to L-serine, wherein the operon is operably linked to a constitutive promoter having a set of nucleotide sequences that is at least 95% (e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, e.g., 100%) identical to the constitutive promoter comprised in SEQ ID NO. 8.

In another aspect, a polynucleotide construct is provided comprising an operon comprising genes encoding serA, serB and serC, wherein the operon is operably linked to a constitutive promoter having a set of nucleotide sequences that is at least 95% (e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, e.g., 100%) identical to the constitutive promoter comprised in SEQ ID NO. 8. In another aspect, a cell culture comprising a host cell of the present disclosure and a growth medium is provided.

In another aspect, there is provided a method for producing L-serine or a derivative thereof, comprising:

a) Culturing a cell culture described herein under conditions that allow the cell to produce L-serine or a derivative thereof; and

b) Optionally recovering and/or isolating L-serine or a derivative thereof.

In another aspect, a fermentation composition is provided comprising a cell culture as described herein and/or L-serine or a derivative thereof contained therein.

Drawings

FIGS. 1A to 1P show plasmid maps of vectors with different promoters.

FIG. 2 shows a plasmid map of the TIR-optimized TARSYN construct.

FIG. 3 shows the TIR region sequence of serA for ampicillin resistance selection.

FIG. 4 shows the TIR region sequence of serC for ampicillin resistance selection.

FIG. 5 shows serine production (in g/L) of pSEVA plasmids containing the constitutive promoters described above and comparison with the IPTG inducible promoter.

FIG. 6 shows a schematic of randomization and screening of TIR libraries.

FIG. 7 shows the fold decrease/increase in protein and serine concentrations of strains containing plasmids without bla cassette compared to control Ser 151. The strain was grown in M9 medium containing 0.5% (w/v) glucose, 2mM glycine and kanamycin at 37℃and 250 rpm. Proteomics and HPLC samples were collected after 24 hours.

FIG. 8 shows serine concentrations of the first 5 strains with optimized plasmid and Ser151 (positive control). Strains were grown in 250mL bioreactors using minimal medium at 37 ℃.

FIG. 9 shows the pathway for L-serine, including potential outlet split.

Detailed Description

Definition of the definition

The terms "heterologous" or "recombinant" or "genetically modified" and grammatical equivalents thereof, as used interchangeably herein with respect to nucleotides, polypeptides and cells, refer to "entities derived from different species or cells". For example, a heterologous or recombinant polynucleotide gene is a gene that does not naturally contain the gene in the host cell, i.e., the gene is from a different species or cell type than the host cell. A heterologous or recombinant polypeptide is a polypeptide that is produced in a host cell that does not naturally contain the polypeptide, i.e., the polypeptide is from a different species or cell type than the host cell. When used herein, the terms concerning host cells refer to host cells that contain and express a heterologous or polynucleotide.

The term "% identity" as used herein is in relation to the relatedness between two amino acid sequences or between two nucleotide sequences, using standard alignment software known in the art, and applying settings indicated by the software (including gaps) to obtain the maximum percent identity/similarity/homology, if necessary, taking into account any conservative substitutions (hftp:// www.chem.qmul.ac.uk/iubmb/misc/naseq. Html; NC-IUB, eur.J.Biochem. (1985)) as part of the sequence identity according to the NCIUB rules. Using such standard software, 5 'or 3' extension or insertion (for nucleic acids) or N 'or C' extension or insertion (for polypeptides) generally results in reduced identity, similarity or homology.

The terms "pathway" or "biosynthetic pathway" or "metabolic pathway" are used interchangeably herein to refer to one or more enzymes that act synergistically in living cells to convert one or more substrate precursors to a chemical product. The pathway may include one enzyme or multiple enzymes acting in sequence or in combination. The pathway comprising only one enzyme may also be referred to herein as "bioconversion", particularly in connection with embodiments in which host cells are fed with exogenous precursors or substrates for enzymatic conversion to the desired end product. The enzyme is characterized by catalytic activity and can change the chemical structure of the substrate. The enzyme may have more than one substrate and produce more than one product. Enzymes may also depend on cofactors, which may be inorganic or organic compounds (cofactors and/or coenzymes), which may or may not be considered part of a pathway.

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

As used herein, the term "in vitro" refers to the outside of a living cell or organism, including but not limited to, for example, in a microplate, tube, flask, beaker, tank, reactor, etc.

As used herein, the term "substrate" or "precursor" refers to any compound that can be converted to a different compound. For clarity, the substrate and/or precursor includes a compound produced in situ by an enzymatic reaction in the cell or an exogenously supplied compound, such as an exogenously supplied organic molecule that the host cell can metabolize to the desired compound.

The term "expression vector" refers to a single-or double-stranded, linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide and operably linked to control sequences that provide for its expression. Expression vectors include expression cassettes for integrating genes into host cells, plasmids and/or chromosomes comprising such genes.

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

The term "polynucleotide construct" refers to a single-or double-stranded polynucleotide that is isolated from a naturally occurring gene or that has been modified to contain a nucleic acid fragment in a manner that does not occur in nature, or that is synthetic, and that comprises a polynucleotide encoding a polypeptide and one or more control sequences.

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 the encoding polynucleotide such that the control sequence directs the expression of the encoding polynucleotide. More generally, a control sequence "operably linked" to a coding sequence is linked in a manner such 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 sufficiently close to the transcription initiation site of the gene to regulate transcription of the gene.

As used herein, "promoter" refers to a DNA sequence, typically located upstream (5') of the coding region of a structural gene, that controls expression of the coding region by providing recognition and binding sites for RNA polymerase and other factors that may be necessary for transcription initiation. The choice of promoter will depend on the nucleic acid sequence of interest. Suitable "promoters" are generally promoters capable of supporting transcription initiation in the bacteria of the invention, resulting in the production of mRNA molecules.

"polypeptide" and "protein" are used interchangeably herein to refer to a polymer of at least two amino acids covalently linked by an amide linkage, whether in length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristoylation, ubiquitination, etc.). Included in this definition are D-and L-amino acids, as well as mixtures of D-and L-amino acids.

As used herein, the term "biobased" is used to characterize a biobased product, wherein:

a) The total carbon content of the product is at least 30%, and

b) The renewable raw material (biobased) has a carbon content of at least 20%.

As acknowledged by the cyclic biobased european joint industry program (CBE joint industry program) established in 2021, the development of biobased materials is critical if the european union is to achieve the climatic objectives specified in the european green agreement. The present disclosure provides methods for efficiently providing fatty alcohols and fatty aldehydes having a high bio-based carbon content (%). Both fossil and renewable raw materials consist mainly of carbon (C). Carbon exists in a variety of isotopes. Isotope element ¹⁴ C is radioactive and naturally occurs in all organisms (plants, animals, etc.) at a fixed relative concentration, almost relative to the atmosphere ¹⁴ The C concentration was the same. At this concentration of the water, ¹⁴ the radioactivity level of C was 100%. This concentration, as well as the radioactivity, decays once the organism is no longer viable, with a half-life of approximately 5700 years. Thus, the radioactivity of the unknown substance ¹⁴ The level of C may help determine the age of the carbon contained in the substance. "young" carbon (0 to 10 years) relative isotopes from renewable raw materials (e.g., plants or animals) ¹⁴ The C concentration is almost relative to the atmosphere ¹⁴ C concentration is the same, thus, the radioactivity of the young carbon ¹⁴ The C level was about 100%. Isotopes in "old" carbon (millions of years) from synthetic or fossil (petrochemical) sources ¹⁴ C is greatly reduced because the age of this synthetic and fossil source far exceeds that of the isotope ¹⁴ Half-life of C (about 5700 years). Thus, carbon derived from synthetic or fossil sources has about 0% relative isotopes ¹⁴ C concentration, and the radioactivity of the old carbon ¹⁴ The C level is thus about 0%. In one embodiment, the term "radioactive ¹⁴ C level "refers to the total radioactivity of a given substance, product or composition ¹⁴ C level, as defined above. Isotope element ¹⁴ The C method can be used to determine a comparison of the concentration of young (renewable) materials to the concentration of old (fossil) resources. The carbon content of renewable raw materials is referred to as "biobased carbon content". Can be used forThe carbon content or "biobased carbon content" of the regenerated raw material may be determined as follows. When measuring biobased carbon content, the results can be reported as "% biobased carbon". This represents the percentage of carbon of "natural" (plant or animal by-products) origin to "synthetic" or "fossil" (petrochemical) origin. For reference, 100% biobased carbon means that the material is entirely derived from plant or animal by-products, and 0% biobased carbon means that the material does not contain any carbon from plant or animal by-products. Values between the two represent a mix of natural and fossil sources. For example: if the radioactivity of the product is ¹⁴ The C level is 80%, meaning that the product consists of 80% renewable carbon and 20% graphitized carbon (C). In other words, the product is 80% biobased. Analytical measurements may be referred to as "modern carbon percent (pMC)". This is measured in the sample ¹⁴ Percentage of C relative to the modern reference standard (NIST 4990C). The percent of biobased carbon content is by carbon dioxide in today's air ¹⁴ C was calculated from pMC using a small adjustment factor. Notably, all uses ¹⁴ The internationally accepted standards for C assume that plant or biomass feedstock is taken from the natural environment. pMC can be analyzed by standard test methods, such as "ASTM D6866".

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

The terms "comprises" and "comprising" as used throughout this specification and the appended claims should be construed as inclusive in such variations as "comprising", "including" and "comprising". These terms are intended to be construed to convey that the context may include other elements or integers not specifically recited.

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

No terms such as "preferably," "commonly," "particularly," and "typically" are used herein to limit the scope of the invention so referred to or suggest that certain features are critical, essential, or even important to the structure or function of the invention so referred to. Rather, these terms are merely intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment of the present invention.

The term "cell culture" as used herein refers to a culture medium comprising a plurality of host cells as described herein. The cell culture may comprise a single host cell strain or may comprise two or more different host cell strains. The medium may be any medium that may comprise a recombinant host, such as a liquid medium (i.e., a culture broth) or a semi-solid medium, and may comprise additional components, such as a carbon source; a nitrogen source; a phosphate source; a vitamin; trace elements; salts; amino acids; a nucleobase; etc.

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

The term "operon" as used herein refers to a functional unit of DNA comprising a cluster of genes under the control of a single regulatory signal or promoter. Operons are common in prokaryotes, such as bacteria, and they can effectively regulate gene expression.

The term "substantially" or "approximately" or "about" as used herein refers to a reasonable deviation of a value or parameter such that the value or parameter is not significantly changed. These terms of degree of deviation from the value should be interpreted to include a deviation of the value, where the deviation would not negate the meaning of the value it deviates from. For example, relative to a reference value, a degree term may include a range of values of plus or minus 10% of that value. For example, a deviation from a value can include the specified value being added or subtracted by a particular percentage of the value, such as adding or subtracting 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the specified value.

When numerical limits or ranges are specified herein, endpoints are included. Furthermore, all values and subranges within a numerical limitation or range are specifically included as if explicitly written out.

The term "and/or" as used herein is intended to mean an inclusive "or". The expression X and/or Y is intended to mean X or Y as well as X and Y. Furthermore, the expression X, Y and/or Z is intended to mean X, Y and Z alone or any combination of X, Y and Z.

The term "isolated" as used herein with respect to a compound refers to any compound that is placed in a form or environment by human intervention that is different from the form or environment in which it is found in nature. Isolated compounds include, but are not limited to, compounds of the present disclosure, wherein the ratio of the compounds relative to other components with which they are associated in nature is increased or decreased. In an important embodiment, the amount of the compound is increased relative to other ingredients associated with the compound in nature. In one embodiment, the compounds of the present disclosure may be isolated in 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 beginning of production or as it is produced during the manufacturing process. Such substantially pure compound preparations contain less than 10% by weight, such as less than 8% by weight, such as less than 6% by weight, such as less than 5% by weight, such as less than 4% by weight, such as less than 3% by weight, such as less than 2% by weight, such as less than 1% by weight, such as less than 0.5% by weight, of other extraneous or unwanted substances typically associated with naturally or recombinantly expressed compounds. In one 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).

The term "GAPDH" as used herein refers to a glyceraldehyde 3-phosphate dehydrogenase that converts glyceraldehyde 3-phosphate to 1, 3-diphosphoglycerate; in NAD ⁺ Or NADP ⁺ In the case of co-conversion to NADH or NADPH, respectively. GapA is in NAD ⁺ Examples of GAPDH which produces 1, 3-phosphoglycerate in the case of co-conversion to NADH, whereas GapC is a protein which is produced in NADP ⁺ Examples of GAPDH which produces 1, 3-phosphoglycerate when co-converted to NADPH.

The term "Nox" as used herein refers to the conversion of NADH to NAD ⁺ NADH oxidase of (C).

The term "PGDH" or "3-PGDH" or "PHGDH" as used herein refers to a 3-phosphopyruvate dehydrogenase that catalyzes the conversion of 3-phosphopyruvate to 3-phosphohydroxypyruvate with the simultaneous reduction of NAD+ to NADH. An example of a PGDH is SerA in the serine pathway.

The term "PSAT" as used herein refers to a phosphoserine transaminase that converts 3-phosphohydroxypyruvate to phosphoserine. An example of a PSAT is SerC in the serine pathway.

The term "PSPH" as used herein refers to phosphoserine phosphatase (PSPH) which converts phosphoserine to L-serine. An example of PSPH is SerB in the serine pathway.

The term "GDH" as used herein refers to a glutamate dehydrogenase that catalyzes the conversion of alpha-ketoglutarate to glutamate.

The term "deletion" as used herein in the context of polynucleotides and genes refers to manipulation of the gene such that it is no longer expressed in the host cell.

The term "disruption" as used herein refers to any mechanism that manipulates or participates in the expression of a gene such that it is no longer expressed in a host cell.

The term "attenuation" as used herein refers to manipulation of a gene or any mechanism involved in gene expression such that expression of the gene is reduced as compared to non-manipulated expression.

The term "operon" as used herein refers to a set of functionally related genes controlled by a single regulatory region called an operon. 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 into a single mRNA (messenger RNA) molecule.

All 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 as essential to the practice of the invention.

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

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

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, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting unless otherwise specified.

The 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 those skilled in the art. These techniques are well explained in the literature. See, e.g., current Protocols in Molecular Biology (Frederick M.AUSUBEL,2000,Wiley and son Inc,Library of Congress,USA); molecular Cloning: ALaboratory 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, APractical Guide To Molecular Cloning (1984); the services, methods In ENZYMOLOGY (j. Abelson and m. Simon, eds. -in-coef, academic Press, inc., new York), special, vols.154and 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 I-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 cells

As described above, in a first aspect, there is provided a genetically modified host cell whose expression or overexpression comprises a coding for serA; serC; and/or an operon of one or more genes of serB; wherein the operon is operably linked to a constitutive promoter having a set of nucleotide sequences at least 95% (e.g. at least 96%, e.g. at least 97%, e.g. at least 98%, e.g. at least 99%, e.g. 100%) identical to the constitutive promoter comprised in SEQ ID NO. 8.

In a preferred embodiment, the serA enzyme has an amino acid sequence which is at least 95% (e.g.at least 96%, e.g.at least 97%, e.g.at least 98%, e.g.at least 99%, e.g.100%) identical to the serA sequence comprised in any of SEQ ID NOS: 41 to 63, more preferably SEQ ID NOS: 46 to 63 and/or SEQ ID NOS: 46 to 55 and/or any of SEQ ID NOS: 54, 59 to 61 or 63. In another embodiment, the SerB enzyme has an amino acid sequence that is at least 95% (e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, e.g., 100%) identical to the serB sequence contained in SEQ ID NO: 65. In another embodiment, the serC enzyme has an amino acid sequence that is at least 95% (e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, e.g., 100%) identical to the serC sequence comprised in SEQ ID NO: 64.

In some embodiments, the operon and constitutive promoter are contained in SEQ ID NO: 18. 33 to 40 or 96 to 100.

The genetic operon of one or more genes or genes may be heterologous to the host cell.

In some embodiments, the heterologous serA can be a type I or type III PGDH, optionally a microbial type I or type III PGDH, and further it can be a serA enzyme.

Some exemplary serA enzymes are disclosed in SEQ ID NOS.41-63, and particularly useful serA enzymes are those comprising at least 20% (e.g., at least 40%, e.g., at least 50%, e.g., at least 60%, e.g., at least 70%, e.g., at least 80%, e.g., at least 90%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, e.g., 100%) of the same polypeptide as that comprised by any of SEQ ID NOS.41-63, more preferably that comprised by any of SEQ ID NOS.46-63 and/or SEQ ID NOS.46-55 and/or that comprised by any of SEQ ID NOS.54, 59-61 or 63.

The genetically modified host cells described herein preferably produce L-serine or a derivative thereof via a metabolic pathway. The pathway for L-serine is shown in FIG. 9.

In some embodiments, the serC enzyme comprises a polypeptide sequence that is at least 20% (e.g., at least 40%, e.g., at least 50%, e.g., at least 60%, e.g., at least 70%, e.g., at least 80%, e.g., at least 90%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, e.g., 100%) identical to a serC sequence comprised in SEQ ID NO. 64; and/or PSPH or serB and comprising a polypeptide sequence that is at least 20% (e.g. at least 40%, e.g. at least 50%, e.g. at least 60%, e.g. at least 70%, e.g. at least 80%, e.g. at least 90%, e.g. at least 95%, e.g. at least 96%, e.g. at least 97%, e.g. at least 98%, e.g. at least 99%, e.g. 100%) identical to the serB sequence comprised in SEQ ID NO: 65. Other genes of the L-serine pathway are known in the art.

In some embodiments, the operon further comprises the sequence set forth in SEQ ID NO: 101-126. The translation initiation region or TIR provided further enhances the expression of the operon. These particular TIRs have been found to further promote expression of genes in the operon, in particular serA and serC. The sequences of these TIRs are set forth in SEQ ID NOS: 101 to 126. Thus, in a separate aspect, there is provided a genetically modified host cell expressing an operon comprising genes encoding serA, serB and serC, said operon being operably linked to a gene comprising the sequence of SEQ ID NO: 101-126. For optimal effect, the TIR is preferably located upstream of the first, second and/or third codon of the gene to be transcribed, while the TIR is also preferably located downstream of a promoter operably linked to the gene to be expressed. Furthermore, in a separate embodiment, the host cell comprises a Translation Initiation Region (TIR) operably linked to a gene encoding a serA comprising the sequence set forth in any one of SEQ ID NOs 101 to 113. In other embodiments, the host cell comprises a Translation Initiation Region (TIR) operably linked to a gene serC comprising the sequence shown in any one of SEQ ID NOs 114 to 126.

Heterologous enzymes expressed by a host cell as described herein may be (i) enzymes from a different species than the host cell, (ii) mutated enzymes from a different species than the host cell, and/or (iii) mutated enzymes native to the host cell. In a further embodiment, the host cell further comprises at least one transporter molecule that facilitates transport of L-serine or a derivative or any precursor thereof. In further embodiments, one or more native or endogenous genes of the host cell may be attenuated, disrupted, and/or deleted. In further embodiments, the host cell further comprises at least 2 copies of one or more genes encoding serA, serB, and/or serC. In further embodiments, the host cell is further genetically modified to provide increased amounts of substrates for one or more L-serine pathway enzymes. In further embodiments, the host cell is further genetically modified to exhibit increased tolerance to one or more precursor, substrate, intermediate or product molecules from the L-serine pathway.

In further embodiments, the host cell is a prokaryotic cell, optionally a bacterium. The prokaryotic cell may be a Pseudomonas, optionally belonging to the class Gamma Proteus, optionally belonging to the family Enterobacteriaceae, optionally belonging to the genus Escherichia, optionally belonging to the species Escherichia coli. In some embodiments, the genetically engineered bacteria belong to the enterobacteriaceae family. In some embodiments, the genetically engineered bacterium belongs to the genus escherichia. In some embodiments, the genetically engineered bacterium is escherichia coli. Additionally or alternatively, the prokaryotic cell is of the phylum actinomycetes, optionally belonging to the class actinomycetes, optionally belonging to the family Corynebacteriaceae, optionally belonging to the genus Corynebacterium, optionally belonging to the species Corynebacterium glutamicum. In some embodiments, the genetically engineered bacterium is corynebacterium glutamicum. In a particular embodiment, the host cell described herein is a cell comprising SEQ ID NO: 18. 33 to 40 or 96 to 100 and producing L-serine.

In another aspect, a polynucleotide construct is provided comprising an operon comprising one or more genes encoding serA, serB and/or serC, wherein the operon is operably linked to a constitutive promoter having a set of nucleotide sequences that is at least 95% (e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, e.g., 100%) identical to the constitutive promoter comprised by SEQ ID NO. 8.

Alternatively, the constitutive promoter in the polynucleotide construct has a sequence identical to SEQ ID NO: 18. 33 to 40 or 96 to 100, comprising a constitutive promoter comprising a nucleotide sequence at least 95% to 100% identical.

The serA encoded by the gene in the polynucleotide construct comprises in particular a polypeptide sequence which is at least 20% (e.g. at least 40%, e.g. at least 50%, e.g. at least 60%, e.g. at least 70%, e.g. at least 80%, e.g. at least 90%, e.g. at least 95%, e.g. at least 96%, e.g. at least 97%, e.g. at least 98%, e.g. at least 99%, e.g. 100%) identical to a serA comprised by any of SEQ ID NOs 41 to 63, more preferably by any of SEQ ID NOs 46 to 63 and/or SEQ ID NOs 46 to 55 and/or by any of SEQ ID NOs 54, 59 to 61 or 63.

The serC encoded by a gene in the construct may suitably comprise a polypeptide sequence that is at least 70% (e.g. at least 80%, e.g. at least 90%, e.g. at least 95%, e.g. at least 96%, e.g. at least 97%, e.g. at least 98%, e.g. at least 99%, e.g. 100%) identical to a serC comprised in SEQ ID NO. 64. The serB encoded by the gene may suitably comprise a polypeptide sequence that is at least 70% (e.g. at least 80%, e.g. at least 90%, e.g. at least 95%, e.g. at least 96%, e.g. at least 97%, e.g. at least 98%, e.g. at least 99%, e.g. 100%) identical to the serB comprised in SEQ ID NO. 65.

The polynucleotide construct may also advantageously comprise a Translation Initiation Region (TIR) comprising the sequence shown in any one of SEQ ID NOS: 101 to 126 operably linked to an operon or a gene therein. More specifically, the Translation Initiation Region (TIR) may be operably linked to an operon in the construct or a gene therein, and may comprise the sequence shown in any one of SEQ ID NOS: 101 to 113 and/or the sequence shown in any one of SEQ ID NOS: 114 to 126.

In selected embodiments, the Translation Initiation Region (TIR) is located upstream of the operon or the gene therein. In a further alternative embodiment, TIR is located upstream of the first, second and/or third codons of the gene in the operon, said TIR comprising the set of sequences set forth in any one of SEQ ID NOs 101 to 126. In a separate aspect, it is provided that the TIR comprises the sequence as set forth in any one of SEQ ID NOs 101 to 126.

In some embodiments, the polynucleotide construct is an expression vector. In other embodiments, the polynucleotide construct comprises a plasmid comprised by any one of SEQ ID NOs 18, 33 to 40 or 96 to 100.

In preferred embodiments, the host cells described herein comprise a polynucleotide construct as described above.

In a further separate aspect, there is provided a cell culture comprising a host cell as described herein and a growth medium, and there is provided a cell culture produced by culturing the cell culture under conditions that allow the host cell to express an operon and an L-pathway to produce L-serine or a derivative thereof; and optionally recovering and/or isolating L-serine or a derivative thereof to produce L-serine or a derivative thereof.

Growth media suitable for use with prokaryotic cells are well known in the art. The cell culture may be cultured in a nutrient medium under conditions suitable for the production of L-serine and/or precursors thereof and/or for the enumeration of propagating cells using methods known in the art. For example, the culture may be cultured by shake flask culture or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentation) in a laboratory or industrial fermenter under suitable media and conditions, to allow host cells to grow and/or reproduce, optionally recovering and/or isolating the culture.

The culturing may be performed in a suitable nutrient medium comprising a carbon source and a nitrogen source, and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published methods (e.g., in catalogues of the American type culture Collection). The medium generally contains all nutrients required for the growth and survival of the respective bacteria, such as carbon sources, nitrogen sources and other inorganic salts. Suitable media, such as minimal media or complex media, are available from commercial suppliers or may be prepared according to published methods, e.g., in catalogues of the American Type Culture Collection (ATCC) strains. Non-limiting standard media well known to those skilled in the art include Luria Bertani (LB) broth, sabouraud Dextrose (SD) broth, MS broth, yeast peptone dextrose, BMMY, GMMY, or Yeast malt extract (YM) broth, all of which are commercially available. Non-limiting examples of suitable media for culturing bacterial cells, such as E.coli cells, include minimal media and rich media such as Luria Broth (LB), M9 media, M17 media, SA media, MOPS media, terrific Broth, YT, and others.

The selection of the appropriate medium may be based on the selection of the host cell and/or on regulatory requirements of the host cell. Such media are available in the art. If desired, the medium may contain additional components that favor the host cell over other potentially contaminating microorganisms. Thus, in one embodiment, a suitable nutrient medium comprises a carbon source (e.g., C6 sugar (e.g., glucose), maltose, molasses, starch, cellulose, xylan, pectin, lignocellulosic biomass hydrolysate, C5 sugar (e.g., arabinose or xylose), acetate, glycerol, vegetable oil, sucrose, yeast extract, peptone, casamino acids or mixtures thereof), a nitrogen source (e.g., ammonia, ammonium sulfate, ammonium nitrate, ammonium chloride, etc.), an organic nitrogen source (e.g., amine yeast extract, malt extract, peptone, soy hydrolysate or digested fermenting microorganisms, etc.), and an inorganic nutrient source (e.g., phosphate, magnesium, potassium, zinc, iron, etc.).

The culturing of the host cells may be performed for a period of about 0.5 to about 30 days. The cultivation process may be a batch process, a continuous or fed-batch process, suitably carried out at a temperature in the range of 0-100 ℃ or 10-80 ℃, e.g. about 20 ℃ to about 50 ℃ and/or at a pH of e.g. about 2 to about 10. Preferred fermentation conditions for the prokaryotic host cell are at a temperature in the range of about 25 ℃ to about 55 ℃ and at a pH of about 3 to about 9. The appropriate conditions are generally selected according to the choice of host cell. Thus, in one embodiment, the method may further comprise one or more elements/steps selected from the group consisting of:

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 between 25 and 50 ℃;

e) Culturing the cell culture at a pH between 3 and 9; and

f) The cell culture is cultured for 10 hours to 30 days.

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 L-serine or a derivative thereof. In particular, when L-serine is not the desired end product, further steps may be added to the process described herein to chemically or biologically/enzymatically modify L-serine or derivatives thereof.

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

Cell cultures of the present disclosure can be recovered and/or isolated using methods known in the art. For example, L-serine or derivatives thereof can be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, spray drying, or lyophilization. In a specific embodiment, the method comprises a recovery and/or isolation step comprising separating the liquid phase of the cells or cell culture from the solid phase of the cells or cell culture to obtain a supernatant comprising L-serine or a derivative thereof and/or subjecting the supernatant to one or more steps selected from the group consisting of:

a) Separating the supernatant from the solid phase of the cell culture, for example by filtration or gravity separation;

b) Contacting the supernatant with one or more adsorption resins to obtain at least a portion of the produced L-serine or derivatives thereof;

c) Contacting the supernatant with one or more ion exchange or reverse phase chromatography columns to obtain at least a portion of L-serine or a derivative thereof;

d) Extracting L-serine or a derivative thereof; and/or

e) Precipitating L-serine or a derivative thereof by crystallization or evaporation of a liquid phase solvent; and optionally isolating the L-serine or a derivative thereof by filtration or gravity separation;

thereby recovering and/or isolating L-serine or a derivative thereof.

In another aspect, a fermentation composition is provided comprising L-serine or a derivative obtained from the culture of a cell culture. In some embodiments, a majority of the solid cellular material/debris has been separated, e.g., at least 50%, e.g., at least 75%, e.g., at least 95%, e.g., at least 99%, of the solid cellular material has been separated from the composition.

The fermentation composition may further comprise one or more additional compounds or metabolites from the cell culture. Such compounds and/or metabolites of cell cultures include precursors of L-serine, and compounds selected from trace metals, vitamins, salts, yeast nitrogen bases, carbon sources, YNB and/or fermented amino acids. In particular, the composition comprises L-serine or a derivative thereof, in particular L-serine, in a concentration of at least 1mg/kg of the composition, such as at least 5mg/kg, such as at least 10mg/kg, such as at least 20mg/kg, such as at least 50mg/kg, such as at least 100mg/kg, such as at least 500mg/kg, such as at least 1000mg/kg, such as at least 5000mg/kg, such as at least 10000mg/kg, such as at least 50000 mg/kg. In other embodiments, the composition is substantially free of alpha-HGAs. The composition may further comprise one or more carriers, agents, additives and/or excipients.

Furthermore, 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 other embodiments, the composition comprises 20% to 100% biobased carbon, such as 30% to 100% biobased carbon, such as 40% to 100% biobased carbon, such as 50% to 100% biobased carbon, such as 60% to 100% biobased carbon, such as 70% to 100% biobased carbon, such as 75% to 100% biobased carbon, such as 80% to 100% biobased carbon, such as 85% to 100% biobased carbon, such as 90% to 100% biobased carbon, such as 95% to 100% biobased carbon, such as 100% biobased carbon. In 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 yet further embodiments, the composition comprises 90% biobased carbon, 91% biobased carbon, 92% biobased carbon, 93% biobased carbon, 94% biobased carbon, 95% biobased carbon, 96% biobased carbon, 97% biobased carbon, 98% biobased carbon, 99% biobased carbon, or 100% biobased carbon, e.g., 94% biobased carbon.

Having generally described this technical advancement, a further understanding may 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.

Example

Example 1-constitutive expression of the serine pathway.

In this example, several constitutive promoters were tested for their performance in serine pathway constitutive expression compared to the T7-based inducible expression system.

L-serine production was assessed for a panel of constitutive promoters of different strengths (SEQ ID NOS: 1 to 16). The constitutive promoter was cloned into a low copy serine producing plasmid (pSEVA derivative) with the Cg/serA (A285G, Y463A), ec/serB and Ec/serC genes. The component design included a promoter region MCD2 (Mutalik et al, 2013), followed by a USER site to facilitate gene exchange (Mutalik et al, 2013). The promoter is selected from two different sources a) Andersen library (http:// parts. Igem. Org/Promoters/catalyst/Anderson), including the J23119, J23102, J23108, J23104, J23106, J23115, J23107 and J23116 Promoters, and b) Mutalik et al 2013, including Promoters P1, P7, P2, P5, P6, P14, P11 and P9. Plasmids (pSER 10 through pSER 25) with 16 different constitutive promoters were constructed by two fragment USER clones (see Table 1.1) using the following PCR amplification and USER cloning protocols. The constitutive promoter primer sequences are shown in Table 1.2.

Table 1.1: information about the fragments used in the USER clone.

^a Both fragments were prepared using pSER5 as template.

^b Each primer carries a constitutive promoter followed by the MCD2, the USER site and the SerA binding site.

Table 1.2: primer sequences for constructing different constitutive promoters.

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PCR amplification and USER cloning protocol

All plasmid manipulations were performed using uracil-specific excision reagent (USER) clones. PCR was performed using Phusion UHot Start polymerase premix (Thermo Fisher Scientific, waltham, ma). Oligonucleotides were purchased from Integrated DNA Technologies (IDT, coleseveler, elsholtzia, usa) and listed in tables 1.1 and 1.2. PCR procedure: initial denaturation at 98℃for 40 seconds, denaturation at 98℃for 10 seconds, annealing at 65℃for 30 seconds, extension at 72℃for 3 minutes and 30 seconds, and cycle repeated 25 times. The sequence of all plasmids was then verified by sanger sequencing.

10. Mu.L of the USER reaction contained 1. Mu.L of the USER enzyme and 1. Mu.L of 10 Xcut smart buffer (New England Biolabs) and 200ng of each USER fragment. The reaction was incubated at 37℃for 30 minutes and then at 15℃for 30 minutes. The reaction mixture was transformed into chemically competent NEB5 Alpha cells, and the transformants were grown in SOC medium at 37℃and then plated on LB kanamycin plates and incubated overnight at 37 ℃.

The following day, single colonies were picked and incubated in 2XYT-kan medium and incubated at 37℃for 16 hours for plasmid preparation and Mulberry sequencing. The plasmid maps are shown in FIGS. 1A to 1P. The confirmed plasmid was transformed into E.coli strain to produce L-serine. The shake flask protocol described below was used for serine production.

Batch media and cultures for 24-well plate and shake flask screening.

L-serine production was examined in M9 minimal medium. Glucose M9 minimal medium consisted of 2 to 5g/L glucose, 2mM glycine, 0.1mM CaCl2, 2.0mM MgSO4, 1 Xtrace element solution and 1 XM 9 salt. 1,000Xmicroelement stock solution with 27g/L FeCl 3-6H 2O and 2g/L ZnCl ₂ *4H ₂ O、2g/L CoCl2*6H ₂ O、2g/L NaMoO ₄ *2H ₂ O、1g/L CaCl ₂ *H ₂ O、1.3g/L CuCl ₂ *6H ₂ O, 0.5g/L H3BO3 and concentrated HCl were dissolved in ddH2O and sterile filtered. The 10 XM 9 salt stock solution was composed of 68g/L anhydrous Na2HPO4, 30g/L KH2PO4, 5g/L NaCl and 10g/L NH4Cl dissolved in ddH2O and autoclaved.

The medium was filter sterilized and 3ml of M9 medium was used per well for the 24-well plate screening experiments. For shake flasks, 50ml was used in 250ml sterile baffled shake flasks. Unless otherwise indicated, overnight cultures were used as inoculums, starting o.d.0.1, 24 deep well plates were incubated at 37 ℃ and 300rpm, shake flasks were incubated at 37 ℃ and 250 rpm. For the IPTG induction system, 40 μm IPTG was added in mid-log phase (OD 0.5 to 0.7).

Analysis method

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

Use equipmentT Chiral (250 x2.1mm x 5 μm) column (Sigma-Aldrich, st.Louis, mitsugen, U.S.) and diodeThe serine concentration was measured by Dionex Ultimate 3000 HPLC (high Performance liquid chromatography) of an array detector (DAD-UV) detector. The mobile phase consisted of an aqueous milliQ solution of 60% acetonitrile (v/v) and 0.02% (v/v) formic acid. The mobile phase was delivered at a rate of 1.0mL/min, and the sample volumes of the standard and all samples were kept at 3. Mu.L. Detection of L-serine was monitored at 205 nm.

Results

All 16 strains were screened in 24 deep well plates. The first 6 strains were selected for shake flask selection. The data from the first six plasmids from shake flasks are shown in FIG. 5. As observed, J23119 not only provides an advantage as a constitutive promoter, but also performs better than T7-based induction systems. As observed by repeated fermentation of biomass from previous fermentation runs, very stable expression performance was provided as a constitutive J23119 and other promoters.

Example 2-optimization of translation Start region Using TARSYN System

To further increase gene expression, optimized Translation Initiation Regions (TIRs) were tested.

Construction, randomization and removal of TARSYN modules

The workflow is divided into three steps of building, randomizing and removing the TARSYN module. In the first step, the TARSYN module is cloned into plasmid pSER 10. The resulting plasmid was designated pSER_34. The map is shown in FIG. 2. Next, the TIR regions of serA and serC were randomized by the primers given in Table 2.1. Random sequences of serA and serC are shown in FIGS. 3 and 4, respectively. Through screening, the TARSYN module was removed from the backbones of the 12 best constructs. The PCR protocol, USER cloning and transformation protocol remained the same as mentioned in the examples above.

Screening of TARSYN library

For screening serine producing strains the aforementioned HPLC method (Rennig et al, 2019 a) was used. Briefly, five plates of different ampicillin concentrations of 5000, 6000, 7000, 8000, 10000ug/ml were received. 90 colonies from each plate were inoculated into 96 deep well plates. The culture medium and growth conditions were the same as described above. For 96-deep well plates, the screening volume per well was reduced to 500 μl instead of 3mL, including positive and negative controls at three different locations. Subsequently, positive strains were screened in 24 DWPs or flasks.

Plasmids of the ten best strains of the study were isolated. Plasmid purification was performed using the Machery Nagel kit (dillen, germany). Sequencing of the plasmid using primers gave TIR sequences for serA and serC, and Mix2Seq kit of Eurofins (ebeberg, germany) stored them as ser_160 to ser_171 after removal of the amp marker. Five strains were studied for serine production in fed-batch fermentation.

Table 2.1: primers for cloning the TARSYN module, generating the TIR library and removing the TARSYN module after screening:

table 2.2: sequencing results of test plasmid and control plasmid pSER10

As a result.

We further optimized the translation initiation region using the previously published approach (example 2). Interestingly, in addition to better L-serine production, the new mutants also showed an improvement in the expression of the serACB gene, as demonstrated by proteomic analysis and fed-batch fermentation example 4. Furthermore, the translation initiation region is different from the previously published work.

As shown in fig. 6, the TIR sequence was varied according to DNA topology, again repeatedly selected using the new construct.

EXAMPLE 3 proteomics Studies

To verify whether the serine pathway expression was enhanced, proteomic analysis was performed. Protocols for sampling and proteomic analysis are described below.

Medium and sampling

Unless otherwise indicated, M9 medium contained 0.5% (w/v) glucose, 1 XM 9 salts, 2mM MgSO4, 2mM glycine, 0.1mM CaCl2, 0.05 XYT, trace elements and vitamins. Precultures and strains for screening purposes were grown in 2 XYT, which consisted of 16g/L bacto tryptone, 10g/L yeast extract and 5g/L NaCl. The medium was supplemented with 1mM glycine and 0.1% (w/v) glucose. The strain was grown at 37℃and 250 rpm. Proteomics and HPLC samples were collected after 24 hours. Once the optical density of the culture was determined at 600nm, the volume corresponding to OD2 was removed for proteomic analysis and centrifuged at maximum speed at 4 ℃. After centrifugation, the supernatant was removed and the sample stored at-20 ℃.

Sample preparation for proteomic analysis

Frozen cells were stored at-80℃for up to 4 weeks, then thawed on ice and pelleted by centrifugation at 15,000g for 10 min. The supernatant was removed and 100. Mu.L of 95℃guanidine-HCl (6M guanidine hydrochloride (GuHCl), 5mM Tris (2-carboxyethyl) phosphine (TCEP), 10mM Chloroacetamide (CAA), 100mM Tris-HCl pH 8.5) was added to the sample along with two 3 mM zirconia beads (Greenmils, N.J., U.S.). Cells were disrupted at 25Hz for 5 min using a stirrer (MM 400Retsch, haen, germany). The sample was placed in a hot mixer at a temperature of 95℃and a rotational speed of 2000rpm for 10 minutes. Thereafter, the samples were centrifuged at 15,000g for 10 minutes, and 50. Mu.L of supernatant was collected and diluted with 50. Mu.L of 50mM ammonium bicarbonate. Protein concentration (BSA) was measured and trypsin digestion was performed using 100 μg. Trypsin was digested for 8 hours, then 10 μl of 10% TFA was added and the samples were Stage-Tip using C18 (Empore, 3M, usa).

After Stage-Tip of the samples, the samples were coupled to 15cm C18 easy spray columns (Pe using a CapLC system (Thermo Fisher Scientific, waltham, ma, usa)pMap RSLC C18 2μm，150. Mu.mx 15 cm). Initially, the sample is captured on a pre-column (μ -pre-column C18 PepMap 100,5 μm,/-for example)>) The peptide was then isolated at a constant flow rate of 1.2 μl/min over 60 minutes using a gradient from 4% acetonitrile in water to 76% acetonitrile in water. Samples were sprayed into an Orbitrap Q-exact HF-X mass spectrometer (Thermo Fisher Scientific, waltherm, ma, usa). MS level scanning is performed with the Orbitrap resolution set to 60,000; AGC target 3.0e6; maximum injection time 50ms; intensity threshold 5.0e3; dynamic exclusion was for 25 seconds. The data dependent MS2 selection was performed in Top20 speed mode with HCD collision energy set to 28% (AGC target 1.0e4, maximum injection time 22MS, isolation window 1.2 m/z).

Proteomic data analysis

The original file was analyzed using maxquant to obtain protein identification and quantification. In analyzing the data, the following settings were used: fixing and modifying: ureido methyl (C) and variable modification: oxidation of methionine residues. The mass tolerance was first searched for 20ppm and the MS/MS tolerance was 20ppm. Trypsin acts as an enzyme and allows one miss of cleavage. FDR was set to 0.1%. The matching window between runs was set to 0.7 minutes. For quantification, the LFQ value using maxquat only allows quantification based on unique peptides. The intensity is normalized within maxquant. For the search a protein database consisting of the reference E.coli proteome UP000000625 was used. For the case of the protein SerABC described, the data were checked manually to ensure correct quantification and identification. At the end of the shake flask experiment, proteomic samples were collected. These were analyzed and fold differences in the serine production pathway were determined for the enzyme compared to the positive control Ser 151.

As a result.

For almost all strains, the SerA concentration was higher than the control (fig. 7), and higher serine concentrations were observed. Restated, an increase in SerA concentration was observed from the data, resulting in a higher serine concentration compared to the positive control. The data obtained are expressed as fold increase compared to the wild-type strain ser_151.

Example 4-fermentation study.

From the experiments of example 2, the first 5 improved serine producing strains (ser_160, ser_162, ser_164, ser_165 and ser_166) were further characterized in fed-batch fermentation. These strains were grown on minimal medium with glucose or maltose as carbon source and used the same feed profile as the previous fed-batch fermentation.

Fed-batch serine production fermentation was performed in 1L bioreactor (Sartorius, huntington, germany), 250mL AMBR reactor (Sartorius) and 1L DASGIP bioreactor (Eppendorf, hamburg, germany), and the media for batch and fed are described below.

Precultures and strains for screening purposes were grown in 2 XYT, which consisted of 16g/L bacto tryptone, 10g/L yeast extract and 5g/L NaCl. According to the experiment, the medium was supplemented with 1mM glycine and 0.1% (w/v) glucose or other carbohydrates. The batch phase of the fermentation is carried out in a minimal medium containing 10g/L (NH 4) SO4, 2g/L (KH) 2PO4, 2g/L yeast extract, 2g/L MgSO4.7H2O, 0.6g/L glycine, trace elements and 10g/L glucose, 9.5g/L maltose, 10.6g/L glycerol or 10g/L sucrose. The feed used in the fed-batch experiment consisted of 6g/L glycine, 1mL/L defoamer and 590g/L glucose or 560g/L maltose. If necessary, appropriate antibiotics are added to the medium to ensure selection of the desired strain.

The reactor was inoculated with 10mL of inoculum in the morning. By increasing stirrer speed, gas flow into and through O in the gas stream ₂ The percentage maintains DO at 20% or higher and the cultures are allowed to grow under aerobic growth during the day. Once the DO peak is observed, indicating the end of the batch phase, the feed is started. The feed rate increased linearly for the first 20 hours, after which it remained constant. Samples were taken periodically, analyzed by HPLC and the optical density at 600nm was measured,as shown in table 4.1.

Table 4.1: summary of fold improvement in L-serine yields and titers for the first 5 optimized strains.

Results

The OD of all strains appeared to be similar to control Ser 151. Due to the technical problems of the fermenter, the strain ser_166 failed on maltose. Most strains have shown an increase in serine concentration from the beginning of fermentation (see FIG. 8). Ser151 is the only duplicate grown strain, although grown on glucose only. When the strain containing the optimized plasmid was grown on glucose, an increase in serine yield of 25% to 31% was observed. The average serine yield was determined from the previous fermentation of Ser151 on maltose. When this average yield was compared with serine yields obtained with the strain using the optimized plasmid, an increase of 1.13 to 2.13 fold was observed (see table 4.1).

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Sequence listing

The present application contains a list of sequences contained in the following table a, submitted electronically in ST26 format, the entire contents of which are incorporated herein by reference.

Table A

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Claims (14)

1. A genetically modified host cell that produces serine, which expresses or overexpresses an operon comprising genes encoding one or more of: 3-phosphopyruvate dehydrogenase (serA) which converts D-3-Phosphopyruvate (PGA) to phosphohydroxypyruvate (PHP); a phosphoserine transaminase (serC) converting 3-phosphohydroxypyruvate to phosphoserine; and/or phosphoserine phosphatase (serB) that converts phosphoserine to L-serine, wherein the operon is operably linked to a constitutive promoter having a set of nucleotide sequences that is 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. 8.

2. The genetically modified host cell of claim 1, wherein

a) The serA enzyme has an amino acid sequence that is at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to a serA sequence comprised in any one of SEQ ID NOs 41 to 63;

b) The serB enzyme has an amino acid sequence that is 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. 65;

c) The serC enzyme has an amino acid sequence that is 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. 64.

3. The host cell of claim 1 comprising a plasmid of any one of SEQ ID NOs 18, 33 to 40 or 96 to 100.

4. The host cell according to any one of the preceding claims, which expresses or overexpresses one or more additional genes of the L-serine pathway.

5. The host cell of any one of the preceding claims, wherein the gene or the operon of a gene further comprises a Translation Initiation Region (TIR) operably linked to the gene or the operon, said TIR comprising the sequences set forth in any one of SEQ ID NOs 101 to 126.

6. The host cell according to any one of the preceding claims, wherein the host cell is a prokaryotic cell, optionally an e.

7. A polynucleotide construct comprising an operon comprising genes encoding one or more of: 3-phosphopyruvate dehydrogenase (serA) which converts D-3-Phosphopyruvate (PGA) to phosphohydroxypyruvate (PHP); a phosphoserine transaminase (serC) converting 3-phosphohydroxypyruvate to phosphoserine; and/or phosphoserine phosphatase (serB) that converts phosphoserine to L-serine, wherein the operon is operably linked to a constitutive promoter having a set of nucleotide sequences that is 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. 8.

8. The polynucleotide construct of claim 7, wherein:

a) The serA enzyme has an amino acid sequence that is at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to a serA sequence comprised in any one of SEQ ID NOs 41 to 63;

b) The serB enzyme has an amino acid sequence that is 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. 65;

c) The serC enzyme has an amino acid sequence that is at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to a serA sequence comprised in any of SEQ ID NOs 64.

9. The polynucleotide construct of claims 7 to 8, further comprising a Translation Initiation Region (TIR) operably linked to the gene or operon, the TIR comprising the sequences set forth in any one of SEQ ID NOs 101 to 126.

10. The polynucleotide construct of claims 7 to 9, wherein the construct is an expression vector.

11. The polynucleotide construct according to any one of claims 7 to 10 comprising a plasmid of any one of SEQ ID NOs 18, 33 to 40 or 96 to 100.

12. The host cell according to claims 1 to 6 comprising the polynucleotide construct according to claims 7 to 11.

13. A cell culture comprising the host cell according to claim 1 to 6 or 12 and a growth medium.

14. A method of producing serine comprising:

a) Culturing the cell culture of claim 14 under conditions that allow the cells to produce L-serine; and

b) Optionally recovering and/or isolating L-serine.