Field

[0001] The present disclosure describes methods for producing L-cysteine and/or L-cystine comprising converting L-serine in a fermentation medium produced by a genetically engineered host cell into L-cysteine and/or L-cystine by contacting the L-serine with a pyridoxal 5-phosphate (PLP)- dependent enzyme in the presence of the source of sulfur. Further described herein are the resulting fermentation media, compositions comprising said medium, microbial cells genetically modified to produce the L-cysteine and/or L-cystine and cell cultures of such genetically modified microbail cells.

Background

[0002] The amino acid L-cysteine is widely used as a pre-cursor in the food, pharmaceutical and personal-care industries, and the estimated annual production of L-cysteine in 2015 was approx. 14000 tons. At present, L-cysteine is primarily produced by acid hydrolysis of feathers and hair (Hashim et al., 2014). This process employs 32 L of concentrated HCI per kg of L-cysteine and is therefore not only expensive, but also creates a lot of acidic wastewater (Hashim et al., 2014). Furthermore, the end-product is known to contain impurities of animal and even human origin known to cause allergies. As an alternative production method, companies such as Wacker Chemie AG, use a microbial fermentation process, which unfortunately produces low yields (Reutter-Maier et al., 2014). Therefore, identification of new L-cysteine production methods that are economically feasible and sustainable is highly desirable.

[0003] Previous research has shown that L-cysteine can be produced via an enzymatic reaction using the enzyme tryptophan synthase (TrpS) (Ishiwata et al., 1989). TrpS is a pyridoxal 5-phosphate (PLP)- dependent enzyme found in many domains of life as an appa heterodimeric complex. Physiologically, it is known to catalyze the synthesis of tryptophan and glyceraldehyde-3-phosphate from L-serine and indole 3-glycerol phosphate (Raboni et al., 2009). The a-subunit of tryptophan synthase converts indole-3-glycerol phosphate to indole and D-glyceraldehyde-3-phosphate. The indole is transported to the p-subunit via a substrate tunnel, where it reacts with L-serine in the presence of the cofactor PLP, to generate tryptophan and water (Raboni et al., 2009).

[0004] E. coli strains capable of producing L-serine titers >50 g/L via fermentation have previously bee described for example in US20210095245 or US10513682. Summary

[0005] Over this background art the present inventors have found and/or confirmed that the betasubunit of TrpS is highly promiscuous and accept an array of different nucleophilic substrates and that amongst the nucleophiles accepted are the hydrogen sulfide ion which, in the presence of TrpS, can react with L-serine to produce L-cysteine. Using this approach, for example TrpS form E. coli can be shown to effectively produce L-cysteine from purified L-serine in the presence of sodium hydrogen sulfide and the PLP enzyme co-factor. These findings suggest that TrpS can produce L-cysteine efficiently, but the relatively high cost of L-serine has prohibited development of commercially attractive production of L-cysteine from L-serine and sodium hydrogen sulfide.

[0006] While production of L-serine via fermentation reduces the production cost, one of the primary cost contributors to L-serine production is the extensive downstream processing required to obtain pure L-serine (see eg. D'Este et al., 2018; Hermann, 2003; Kumar et al., 2014). To resolve the drawbacks of using the technically complicated and therefore expensive process in the art for making purified L-serine, the present inventors have found a method using impure L-serine from a fermentation directly as a substrate for TrpS in the making of cysteine and/or cystine.

[0007] These results are indeed unexpected, as production of cysteine from L-serine in fermentation broth or even crude serine preparations has not previously been demonstrated, and because the presence of inhibitors in the broth were expected to inhibit or block the reaction. Moreover, the methods for cysteine biocatalysis described herein were, at the onset of developing the method, presumed to be challenging, because it required sulfur containing compounds, such as hydrogen sulfide, because such compounds are strong bases, and highly ionic molecules, which are known to considerably affect and impede enzyme stability and activity (Yang, 2009). In addition, fermentation broths of high cell density of for example E. coli cultures are known to comprise many inhibiting compounds that affects and impede enzyme stability and activity. Therefore it was unexpected to find that fermentation broths comprising L-serine produced by fermenting a microorganism genetically modified to produce L-serine, said broths having an optical density (OD 600 nm) of up to and above 40 and a serine concentration of up to and above >50 g/L, could be used for making enzymatically converted L-cysteine and/or L-cystine and that the method was, in efficiency, comparable to using pure L-serine as a substrate. In particular it was unexpected that TrpS catalysed the conversion of L- serine in the fermentation media at a rate comparable to the conversion of pure L-serine.

[0008] This suggests that L-serine fermentation broths can advantageously be used directly as a substrate for L-cysteine/L-cystine production, without any prior technically complicated, expensive and yield reducing purification steps.

[0009] Accordingly, in a first aspect the disclosure describes a method for producing L-cysteine and/or L-cystine comprising: a) providing a fermentation medium comprising serine from of a genetically engineered host cell producing said serine; b) providing one or more sources of sulfur; c) providing one or more pyridoxal 5-phosphate (PLP)-dependent enzymes capable of converting serine into cysteine and/or cystine in the presence of the source of sulfur; d) contacting the serine in the fermentation medium with the pyridoxal 5-phosphate (PLP)- dependent enzyme in the presence of PLP and the source of sulfur under conditions allowing the serine to be converted into cysteine and/or cystine; and optionally e) recovering and/or isolating the cysteine and/or cystine

[0010] In a further aspect the disclosure describes a fermentation medium comprising L-cysteine and/or L-cystine, obtainable from the method as described herein.

[0011] In a further aspect the disclosure describes a composition comprising the fermentation medium described herein and one or more agents, additives and/or excipients.

[0012] In a further aspect the disclosure describes a microbial host cell genetically modified to produce L-cysteine and/or L-cystine, wherein the host cell expresses a heterologous gene encoding a pyridoxal 5-phosphate (PLP)-dependent enzyme and comprises an operative biosynthetic pathway capable of producing L-serine, said pathway comprising one or more pathway polypeptides selected from: a) glucokinase (glk) phosphorylating glucose to glucose-6-phosphate; b) phosphoglucose isomerase (pgi) isomerizing glucose-6-phosphate to fructose-6-phosphate; c) phosphofructokinase 2(pfkB) phosphorylating fructose-6-phosphate to fructose-1.6- bisphosphate; d) fructose-bisphosphate aldolase class 1 (fbaB) or fructose-bisphosphate aldolase class 2 (fbaA) splitting fructose 1,6-bisphosphate into dihydroxyacetone phosphate and glyceraldehyde 3- phosphate; e) triosephosphate isomerase (tpiA) converting dihydroxyacetone phosphate to D-glyceraldehyde- 3-phosphate; f) glyceraldehyde-3-phosphate dehydrogenase A (gapA) oxidatively phosphorylating D- glyceraldehyde 3-phosphate (G3P) to 1,3-bisphosphoglycerate (BPG) using the cofactor NAD; g) glyceraldehyde-3-phosphate dehydrogenase (gapC) converting D-glyceraldehyde 3-phosphate into 1,3-bisphosphoglyceric acid; h) phosphoglycerate kinase (pgk) dephosphorylating 1,3-bisphosphoglyceric acid to generate 3- phospho-D-glycerate; i) D-3-phosphoglycerate dehydrogenase (serA) oxidizing 3-phospho-D-glycerate to 3- phosphohydroxypyruvate; j) phosphoserine aminotransferase (serC) converting 3-phosphohydroxypyruvate to phosphoserine; or k) phosphoserine phosphatase (serB) dephosphorylating phosphoserine to L-serine.

Brief description of drawings and figures

Figure 1 shows a reaction scheme for the R-subunit of tryptophan synthase, (top) The natural reaction catalysed by the R-subunit of tryptophan synthase where the indole is transported to the p-subunit via a substrate tunnel, where it reacts with L-serine in the presence of the cofactor PLP, to generate tryptophan and water. (Bottom) The reaction of the present disclosure for producing L-cysteine from L-serine and sodium hydrogen sulfide in the presence of PLP. Upon oxidation, cysteine may subsequently be converted to its dimeric form cystine.

Figure 2 shows the production of L-serine by fermentation by a strain of E. coli. (top) E. coli growth as a function of time for a representative serine producing fermentation. (Bottom) Serine titers as a function of time for a representative serine producing fermentation.

Figure 3 shows the expression of E. coli tryptophan synthase. (Top) Plasmid map of the plasmid used to express the trpS complex (trpA and trpB) from E. coli. (Bottom) SDS-PAGE analysis of TrpS expression. Distinct bands of overexpressed proteins are visible at approx. 30 and 43 kDa, which correlates well with the expected mass of nHis-trpA (29.8 kDa) and trpB (43 kDa), respectively.

Figure 4 shows the fermentation of Cys_39 producing TrpS. (Top) The feed volume scheme resulting from a standard DO-sat controlled fermentation of Cys_39 to generate biomass for cysteine production. (Bottom) Cys_39 growth as a function of time for a representative Cys_39 DO-stat controlled fermentation.

Figure 5 to 7 shows the conversion of L-serine to L-cysteine and L-cystine using pure L-serine and L- serine fermentation broth as substrates. Results from pure serine are shown in gray, and results from the serine fermentation broth are shown in black.

Figure 8 shows a blok flow diagram of the designed serine downstream purification process. The serine broth used as a TrpS substrate in the examples is also indicated in the diagram. Figure 9 shows the metabolic pathway from glucose to cysteine.

Incorporation by reference

[0013] All publications, patents, and patent applications referred to herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between a term herein and a term in an incorporated reference, the term herein prevails and controls.

Details

Definitions

[0014] Any EC numbers used herein refers to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, San Diego, California, including 30 supplements 1-5 published in Eur. J. Bio-chem. 1994, 223, 1- 5; Eur. J. Biochem. 1995, 232, 1-6; Eur. J. Biochem. 1996, 237, 1-5; Eur. J. Biochem. 1997, 250, 1-6; and Eur. J. Biochem. 1999, 264, 610-650; respectively. The nomenclature is regularly supplemented and updated; see e.g. http://enzyme.expasy.org/. The term "PEP" as used herein refers to phosphoenol pyruvate.

[0015] The term "pyridoxal 5-phosphate (PLP)-dependent enzymes" as used herein refers to any PLP dependent enzyme catalyzing the reaction of L-serine and sulfur containing reactant into L-cysteine.

[0016] The term "glucokinase or glk" as used herein refers to an enzyme catalyzing the reaction of phosphorylating glucose into glucose-6-phosphate.

[0017] The term "phosphoglucose isomerase or pgi" as used herein refers to an enzyme catalyzing the reaction of isomerizing glucose-6-phosphate into fructose-6-phosphate

[0018] The term "phosphofructokinase or pfkB" as used herein refers to an enzyme catalyzing the reaction of phosphorylating fructose-6-phosphate into fructose-1.6-bisphosphate.

[0019] The term "fructose-bisphosphate aldolase calss 1 or 2 or fbaB or fbaA" as used herein refers to enzymes splitting fructose 1,6-bisphosphate into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate.

[0020] The term "triosephosphate isomerase or tpiA" as used herein refers to an enzyme catalyzing the reaction of converting dihydroxyacetone phosphate into D-glyceraldehyde-3-phosphate.

[0021] The term "glyceraldehyde-3-phosphate dehydrogenase A or gapA" as used herein refers to an enzyme catalyzing the reaction of oxidatively phosphorylating D-glyceraldehyde 3-phosphate (G3P) into 1,3-bisphosphoglycerate (BPG) using the cofactor NAD.

[0022] The term "glyceraldehyde-3-phosphate dehydrogenase or gapC" as used herein refers to an enzyme catalyzing the reaction of converting D-glyceraldehyde 3-phosphate into 1,3- bisphosphoglyceric acid.

[0023] The term "phosphoglycerate kinase or pgk" as used herein refers to an enzyme catalyzing the reaction of dephosphorylating 1,3-bisphosphoglyceric acid into generate 3-phospho-D-glycerate.

[0024] The term "D-3-phosphoglycerate dehydrogenase or serA" as used herein refers to an enzyme catalyzing the reaction of oxidizing 3-phospho-D-glycerate into 3-phosphohydroxypyruvate.

[0025] The term "phosphoserine aminotransferase or serC" as used herein refers to an enzyme catalyzing the reaction of converting 3-phosphohydroxypyruvate into phosphoserine.

[0026] The term "phosphoserine phosphatase (serB)" as used herein refers to an enzyme catalyzing the reaction of dephosphorylating phosphoserine into L-serine.

[0027] The terms "heterologous" or "recombinant" or "genetically modified" and their grammatical equivalents as used herein interchangeably 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. The terms as used herein about microbial host cells refers to microbial host cells comprising and expressing heterologous or recombinant polynucleotide genes.

[0028] The term "biosynthetic pathway" as used herein is intended to mean two or more enzymes acting sequentially in a live cell to convert chemical substrate(s) into chemical product(s). 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 such as proteins for example enzymes (co-enzymes).

[0029] The term "operative biosynthetic pathway" refers to a metabolic pathway that occurs in a live recombinant host, as described herein.

[0030] The term "in vivo", as used herein refers to within a living cell or organism, including, for example animal, a plant or a microorganism.

[0031] 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.

[0032] 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 a 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.

[0033] Term "endogenous" or "native" as used herein refers to a gene or a polypepetide in a host cell which originates from the same host cell.

[0034] The term "deletion" as used herein refers to manipulation of a gene so that it is no longer expressed in a host cell.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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 or environment in which it is found in nature. Isolated compounds include but is no 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.

[0040] The term "% identity" is used herein about the relatedness between two amino acid sequences or between two nucleotide sequences. The term "% identity" as used herein about amino acid sequences refers to the degree of identity in percent between two amino acid sequences obtained when using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443- 453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: identical amino acid residues - x 100 Length of alignment — total number of gaps in alignment

The term "% identity" as used herein about nucleotide sequences refers to the degree of identity in percent between two nucleotide sequences obtained when using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: identical deoxyribonucleotides - - - x 100 Length of alignment — total number of gaps in alignment

The protein sequences of the present disclosure can further be used as a "query sequence" to perform a search against sequence databases, for example to identify other family members or related sequences. Such searches can be performed using the BLAST programs. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). BLASTP is used for amino acid sequences and BLASTN for nucleotide sequences. The BLAST program uses as defaults: Cost to open gap: default= 5 for nucleotides/ 11 for proteins

Cost to extend gap: default = 2 for nucleotides/ 1 for proteins

Penalty for nucleotide mismatch: default = -3

Reward for nucleotide match: default= 1

Expect value: default = 10

Wordsize: default = 11 for nucleotides/ 28 for megablast/ 3 for proteins.

Furthermore, the degree of local identity between the amino acid sequence query or nucleic acid sequence query and the retrieved homologous sequences is determined by the BLAST program. However only those sequence segments are compared that give a match above a certain threshold. Accordingly, the program calculates the identity only for these matching segments. Therefore, the identity calculated in this way is referred to as local identity.

[0041] The term "expression" includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification, and secretion.

[0042] 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.

[0043] 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 of the present disclosure. Host cell encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0044] The term "polynucleotide construct" or "nucleic acid construct" refers to a polynucleotide, either single- or double stranded, which is separated from its naturally occurring environment and/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.

[0045] The term "operably linked" refers to a configuration in which a control sequence is placed at an appropriate position relative to the coding polynucleotide such that the control sequence directs expression of the coding polynucleotide.

[0046] The terms "nucleotide sequence and "polynucleotide" are used herein interchangeably.

[0047] 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.

[0048] The articles "a" and "an" are used herein refers to one or to more than one (i.e. to one or at least one) of the grammatical object of the article. By way of example, "an element" may mean one element or more than one element.

[0049] Terms like "preferably", "commonly", "particularly", and "typically" are not utilized herein to limit the scope of the itemed disclosure or to imply that certain features are critical, essential, or even important to the structure or function of the itemed disclosure. 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 disclosure.

[0050] The term "cell culture" as used herein refers to a culture medium comprising a plurality of host cells of the disclosure. 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 such as dextrose, sucrose, glycerol, or acetate; a nitrogen source such as ammonium sulfate, urea, or amino acids; a phosphate source; vitamins; trace elements; salts; amino acids; nucleobases; and yeast extract.

[0051] The term "fermentation medium" as used herein refers to any medium in which a genetically modified host is producing or has produced L-serine. A fermentation medium may contain solid cellular material (broths) or may be partially or wholly free of solid cellular material.

[0052] The term "sources of sulfur" as used herein refers to any compound comprising sulfur in a form which the pyridoxal 5-phosphate (PLP)-dependent enzyme can covalently bind to serine to from cysteine.

Method for producing cysteine and/or cystine comprising

[0053] As described, supra, the first aspect of the present disclosure concerns a method for producing cysteine and/or cystine comprising a) providing a fermentation medium and/or broth comprising serine from of a genetically engineered host cell producing said serine, and optionally comprising metabolites and/or cells and/or cell debris from the serine producing host cell; b) providing one or more sources of sulfur; c) providing one or more pyridoxal 5-phosphate (PLP)-dependent enzymes; d) contacting the serine in the fermentation medium with the pyridoxal 5-phosphate (PLP)- dependent enzyme in the presence of the source of sulfur under conditions allowing the serine to be converted into cysteine and/or cystine; and optionally e) recovering and/or isolating the cysteine and/or cystine.

[0054] In some aspects, the method for producing cysteine and/or cystine is provided comprising a) providing a fermentation medium comprising serine from of a genetically engineered host cell producing said serine; b) providing one or more sulfur sources which are NaSH; NajS; and/or H2S c) providing a pyridoxal 5-phosphate (PLP)-dependent enzyme TrpS comprising i) an enzyme subunit having an amino acid sequence comprised in SEQ ID NO: 13 (trpB) and ii) an enzyme subunit having an amino acid sequence comprised in SEQ. ID NO: 14 (trpA), wherein the subunits, alone and together, are capable of converting serine into cysteine and/or cystine in the presence of the one or more sulfur sources; d) contacting the serine in the fermentation medium with the pyridoxal 5-phosphate (PLP)- dependent enzyme TrpS in the presence of PLP and the one or more sulfur sources under conditions allowing the serine to be converted into cysteine and/or cystine; and e) optionally recovering and/or isolating the cysteine and/or cystine.

[0055] In the said method it is particularly attractive to produce L-cysteine and/or L-cystine. Cysteine is usually in equilibrium with cystine through oxidation, but in further embodiment the method can also comprise steps with the aim of converting the monomeric cysteine to the dimeric cystine or shifting the equilibriums in either direction depending on which of cysteine or cystine that are most desirable.

[0056] In some preferred embodiements the fermentation medium used in the methods described herein is a broth which comprises components of the host cells and/or components produced by the host cells from the extracellular or intracellular matrix or both, components produced by the host cells includes particularly metabolites of the host cell production of serine such as those selected from glucose-6-phosphate, fructose-l,6-phosphate, glyceraldehyde-3-phosphate, dihydroxyacetone phosphate, 1,3-diphosphoglycerate, 3-phosphoglycerate, 3-phosphopyrovate, and/or 3- phosphoserine. In additional or alternative embodiments such components of the fermentation mediums can also comprise by one or more components selected from: a) a carbon source, optionally selected from glycerol; carbohydrates (such as monosaccharides and/or disaccharides; complex sources (such as molasses, fats and oils, and/or starch biomass; b) a nitrogen source, optionally selected from ammonium compounds, nitrates, Urea and/or Amino acids such as alanine arginine, Leucine, and/or D,L-aspartate; c) a phosphate source, optionally selected from potassium phosphate; d) a sulfate source, optionally selected from ammonium sulfate and/or magnesium sulfate; e) trace elements, optionally selected from Fe, Zn, Cu, Mn, Mo, Co, and/or Ni.

[0057] In further embodiments the fermentation medium comprises ammonium ions, optionally between 0,1 g/L to 20 g/L, optionally between 0,5 g/L to 15 g/L, optionally between 1 g/L to 10 g/L, optionally between 2 g/L to 5 g/L of ammonium ions.

[0058] In further embodiments the fermentation medium comprises phosphate ions, optionally between 0,01 g/L to 10 g/L, optionally between 0,02 g/L to 5 g/L, optionally between 0,05 g/L to 2 g/L of phosphate ions.

[0059] In further embodiments the fermentation medium comprises proteins, DNA and RNA liberated by the host cells.

[0060] In further embodiments the fermentation medium comprises live cells and/or cell debris, optionally between 0,1 %wt to 75 %wt, optionally between 0,5 %wt to 50 %wt, optionally between 1 %wt to 40 %wt, optionally between 2 %wt to 35 %wt, optionally between 3 %wt to 30 %wt, optionally between 5 %wt to 25 %wt, optionally between 10 %wt to 20 %wt.

[0061] In further embodiments the fermentation medium comprises N-acetyl serine, optionally between 0,05 g/L to 20 g/L, optionally between 0,1 g/L to 10 g/L, optionally between 0,5 g/L to 2 g/L of N-acetyl serine.

[0062] The fermentation medium comprises pyridoxal 5-phosphate (PLP) cofactor, suitable in amounts of at least 0,01 pM, such as at least 0,05 pM, such as at least 0,1 pM, such as at least 0,5 pM, such as at least 1 pM, such as at least 5 pM, such as at least 10 pM, such as at least 50 pM, such as at least 100 pM, such as at least 250 pM, such as at least 500 pM, such as at least 1 mM but suitably not higher than 10 mM.

[0063] The fermentation medium comprises serine, in amounts which suitably priorto any conversion of serine into cysteine is at least 10 g/L serine, such as at least 20 g/L, such as at least 30 g/L, such as at least 40 g/L, such as at least 50 g/L, such as at least 60 g/L, such as at least 70 g/L, such as at least 70 g/L, such as at least 80 g/L, such as at least 90 g/L, but suitably below 500 g/L, such as below 250 g/L, such as below 200 g/L, such as below 150 g/L, such as below 125 g/L, such as below 100 g/L. A particular amount is between 50 to 100 g/L of serine which is obtainable in high yield fermentations. In some embodiments during and after the conversion of serine to cysteine, the concentration of serine in the fermentation medium is lowered, while in other embodiments where a host cell continuously produces new serine, concentrations of serine may reach a stady state within the aforementions ranges. An additional or alternative embodiments the fermentation providing the fermentation medium is advantageously progressed to a stage where the optical density (OD) at 600 nm is at least 1, such as at least 2, such as at least 4, such as at least 6, such as at least 8, such as at least 10, such as at least 15, such as at least 20, inducating the concentration of cells or cellular debris is so high as to also provide for a high concentration of serine.

[0064] The the one or more sources of sulfur can be any compound comprising sufur and which the pyridoxal 5-phosphate (PLP)-dependent enzyme is capable of using for covalendly binding hydrogen sulfide (-SH) to serine. Such sulfur compounds include any compound which in solution can generate such as H2S, S ^2- , and/or NaHS. In a particular embodiment the one or more sources of sulfur comprises hydrogen sulfide. Additionally or alternatively, the source of sulfur comprises a basic compound, such as a strongly basic compound. Additionally or alternatively, the source of sulfur comprises an acidic compound, such as a strongly acidic compound. In some embodiments it is desired to maintain a certain minimum level of the the sulfur source during at least the major period of conversion of serine to cysteine. In particular it is desired to maintain a concentration of the source of sulfur of at least 0,1 % wt, such as least 0,5 % wt, such as least 1 % wt, such as least 2 % wt, such as least 4 % wt, such as least 6 % wt, such as least 8 % wt, such as least 10 % wt, but suitably less than 15 % wt.

[0065] The pyridoxal 5-phosphate (PLP)-dependent enzyme used the method described can be derived from a bacterium, such as a strain of the genus Eschericia, particularly of the species Eschericia coli. In some embodiemnts the pyridoxal 5-phosphate (PLP)-dependent enzyme is selected from lyases (EC 4.-.-.-), carbon oxygen lyases (EC 4.2.-.-), and/or hydro-lyases (EC 4.2.1.-). In other additional or alternative embodiments the pyridoxal 5-phosphate (PLP)-dependent enzyme comprises a fold type II configuration such as described in (Percudani 2009, B6 database). In some embodiments the pyridoxal 5-phosphate (PLP)-dependent enzyme is a Tryptophan synthase (TrpS), especially an E. coli TrpS, optionally comprising an amino acid sequence which has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to one or more of SEQ ID NO: 13 (trpB), 14 (trpA), 15 (trpB) or 16 (trpA).

[0066] [0060] [0061] In further additional or alternative embodiments, the pyridoxal 5-phosphate (PLP)-dependent enzyme comprises one or more mutations outside the PLP binding site and/or outside the substrate binding site. The PLP binding site of the pyridoxal 5-phosphate (PLP)-dependent enzyme suitably comprises one or more amino acid moieties corresponding to position H86, K87, Q114, T190, C230, G232, G233, G234, S235, N236, A237, G303, E350, S377, or G378 of SEQ. ID NO: 15 or 16, while the substrate binding site of the pyridoxal 5-phosphate (PLP)-dependent enzyme suitably comprises one or more amino acid moieties corresponding to position N305, A112, Gill, T110, Q114, H115, G113, or E109 of SEQ ID NO: 15 or 16.

[0067] In further additional or alternative embodiments, the pyridoxal 5-phosphate (PLP)-dependent enzyme comprises one or more mutations selected from D47S and L81V in the corresponding amino acid sequence of SEQ. ID NO: 15 or 16 or conservative substitutions thereof.

[0068] The method produces cysteine/cystine with high efficiency and in some embodiments at least 50%, such as at least 75%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% of the serine in the fermentation medium is converted to cysteine and/or cystine.

[0069] The method can further comprise steps for generating serine from one or more precursors including one or more steps selected from a) Phosphorylation of glucose by Glucokinase (glk) to generate glucose-6-phosphate; b) Isomerisation of glucose-6-phosphate by phosphoglucose isomerase (pgi) to generate fructose- 6-phosphate; c) Phosphorylation of fructose-6-phosphate by phosphofructokinase 2(pfkB) to generate fructose- 1.6-bisphosphate; d) Splitting of fructose 1,6-bisphosphate by fructose-bisphosphate aldolase class 1 (fbaB) or fructose-bisphosphate aldolase class 2 (fbaA) into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate; e) Conversion of dihydroxyacetone phosphate by triosephosphate isomerase (tpiA) to D- glyceraldehyde-3-phosphate; f) oxidative phosphorylation of D-glyceraldehyde 3-phosphate (G3P) by glyceraldehyde-3- phosphate dehydrogenase A (gapA) to 1,3-bisphosphoglycerate (BPG) using the cofactor NAD; g) conversion of D-glyceraldehyde 3-phosphate by glyceraldehyde-3-phosphate dehydrogenase (gapC) into 1,3-bisphosphoglyceric acid; h) Dephosphorylation of 1,3-bisphosphoglyceric acid by phosphoglycerate kinase (pgk) to generate 3-phospho-D-glycerate; i) oxidation of 3-phospho-D-glycerate by D-3-phosphoglycerate dehydrogenase (serA) to 3- phosphohydroxypyruvate; j) conversion of 3-phosphohydroxypyruvate by phosphoserine aminotransferase (serC) to phosphoserine; k) dephosphorylation of phosphoserine by phosphoserine phosphatase (serB) to serine; and/or l) conversion of formaldehyde and glycine by serine hydroxymethyl transferase (glyA) to serine.

[0070] In a special embodiment the method comprises: a) oxidation of 3-phospho-D-glycerate by D-3-phosphoglycerate dehydrogenase (serA) to 3- phosphohydroxypyruvate; b) conversion of 3-phosphohydroxypyruvate by phosphoserine aminotransferase (serC) to phosphoserine; or c) dephosphorylation of phosphoserine by phosphoserine phosphatase (serB) to serine. [0071] In the method described herein in some embodiments the glucokinase (glk) has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the Glucokinase comprised in SEQ ID NO: 1. In other embodiments the phosphoglucose isomerase (pgi) has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the phosphoglucose isomerase (pgi) comprised in SEQ. ID NO: 2. In other embodiments the phosphofructokinase 2 (pfkB) has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the phosphofructokinase 2 (pfkB) comprised in SEQ ID NO: 3. In other embodiments the fructose-bisphosphate aldolase class 1 (fbaB) has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the fructose-bisphosphate aldolase comprised in SEQ ID NO: 4. In other embodiments the fructose-bisphosphate aldolase class 2 (fbaA) has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the fructose-bisphosphate aldolase comprised in SEQ ID NO: 5. In other embodiments the triosephosphate isomerase (tpiA) has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the triosephosphate isomerase comprised in SEQ ID NO: 6. In other embodiments the glyceraldehyde-3-phosphate dehydrogenase A (gapA) has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the glyceraldehyde-3-phosphate dehydrogenase A comprised in SEQ ID NO: 7. In other embodiments the glyceraldehyde-3-phosphate dehydrogenase (gapC) has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the glyceraldehyde-3-phosphate dehydrogenase comprised in SEQ ID NO: 8. In other embodiments the phosphoglycerate kinase (pgk) has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the phosphoglycerate kinase comprised in SEQ ID NO: 9. In other embodiments the D-3- phosphoglycerate dehydrogenase (serA) has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the D-3-phosphoglycerate dehydrogenase comprised in a sequence selected from the group consisting of: SEQ ID NO: 10, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47. In one embodiment, the D-3-phosphoglycerate dehydrogenase (serA) has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the D-3- phosphoglycerate dehydrogenase comprised in SEQ ID NO: 10. In other embodiments the phosphoserine aminotransferase (serC) has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the phosphoserine aminotransferase comprised in SEQ ID NO: 11. In other embodiments the phosphoserine phosphatase (serB) has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the phosphoserine phosphatase comprised in SEQ ID NO: 12. In the method described herein in some embodiments the serine hydroxymethyl transferase (glyA) has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the serine hydroxymethyl transferase comprised in SEQ ID NO: 26.

[0072] In further optimizing the method described herein the pH in the fermentation medium during conversion of serine to cysteine is suitably kept from about 6 to about 10, such as from about 7 to about 9, allowing the pH to fluctuate around these end points with 0,1 pH.

[0073] In further optimizing the method described herein the temperature in the fermentation medium is kept from about 25°C to about 60°C, such as from about about 30°C to about 50°C, such as from about about 35°C to about 40°C, such as about 37°C allowing the temperature to fluctuate around these end points with 1 °C.

[0074] The recovery and/or isolation of cysteine and/or cystine as described herein suitable comprise one or more steps selected from: a) Separation of any biomass from the fermentation medium b) contacting the fermentation medium with one or more adsorbent resins to obtain at least a portion of the produced cysteine and/or cystine; c) contacting the fermentation medium with one or more ion exchange or reversed-phase chromatography columns in order to obtain at least a portion of the cysteine and/or cystine; d) extracting the cysteine and/or cystine from the fermentation medium; and e) precipitating the cysteine and/or cystine from the fermentation medium by crystallization or evaporating the solvent of the liquid phase; and optionally isolating cysteine and/or cystine by filtration or gravity separation; thereby recovering and/or isolating the cysteine and/or cystine.

Fermentation mediums and compositions

[0075] As described, supra, a further aspect of the present disclosure concerns a fermentation medium comprising cysteine and/or cystine, obtainable from the method described herein. Apart from cysteine/cystine the fermentation medium can include one or more component selected from unspent PLP, unspent sulfur source; unspent serine, pyridoxal 5-phosphate (PLP)-dependent enzyme; as well as metabolites of the host cell production of serine such as those selected from glucose-6- phosphate, fructose-l,6-phosphate, glyceraldehyde-3-phosphate, dihydroxyacetone phosphate, 1,3- diphosphoglycerate, 3-phosphoglycerate, 3-phosphopyrovate, and/or 3-phosphoserine. The fermentation medium can also include unspent components selected from: f) a carbon source, optionally selected from glycerol; carbohydrates (such as monosaccharides and/or disaccharides; complex sources (such as molasses, fats and oils, and/or starch biomass; g) a nitrogen source, optionally selected from ammonium compounds, nitrates, Urea and/or Amino acids such as alanine arginine, Leucine, and/or D,L-aspartate; h) a phosphate source, optionally selected from potassium phosphate; i) a sulfate source, optionally selected from ammonium sulfate and/or magnesium sulfate; or j) trace elements, optionally selected from Fe, Zn, Cu, Mn, Mo, Co, and/or Ni;

[0076] In the fermentation after conversion of serine to cysteine/cystine the concentration of cysteine and/or cystine at least 0,1 % by weight, such as at least 0,5 % by weight, such as at least 1 % by weight, such as at least 5 % by weight, such as at least 10 % by weight, such as at least 15 % by weight.

[0077] As described, supra, a further aspect of the present disclosure concerns a composition comprising the fermentation medium described herein and one or more agents, additives and/or excipients. In such as composition the fermentation medium is suitably further processed into a dry solid form or a stabilized liquid form, but removing water or other solvents

[0078] In some embodiments, a fermentation compound is a constituent of a fermentation medium or a compound produced by a fermentation process. In some embodiments, non-limiting exemplary fermentation compounds are selected from glucose-6-phosphate, fructose-l,6-phosphate, glyceraldehyde-3-phosphate, dihydroxyacetone phosphate, 1,3-diphosphoglycerate, 3- phosphoglycerate, 3-phosphopyrovate, 3-phosphoserine, glycerol, monosaccharides, disaccharides, ammonium compounds, nitrates, urea, alanine, arginine, leucine, D-aspartate, potassium phosphate, ammonium sulfate, magnesium sulfate, Fe, Zn, Cu, Mn, Mo, Co, and/or Ni.

Genetically modified host cells

[0079] As described, supra, a further aspect of the present disclosure concerns a microbial host cell genetically modified to produce cysteine and/or cystine, wherein the host cell expresses a heterologous gene encoding a pyridoxal 5-phosphate (PLP)-dependent enzyme and comprises an operative biosynthetic pathway capable of producing serine, said pathway comprising one or more pathway polypeptides selected from: a) glucokinase (glk) phosphorylating glucose to glucose-6-phosphate; b) phosphoglucose isomerase (pgi) isomerizing glucose-6-phosphate to fructose-6-phosphate; c) phosphofructokinase 2(pfkB) phosphorylating fructose-6-phosphate to fructose-1.6- bisphosphate; d) fructose-bisphosphate aldolase class 1 (fbaB) or fructose-bisphosphate aldolase class 2 (fbaA) splitting fructose 1,6-bisphosphate into dihydroxyacetone phosphate and glyceraldehyde 3- phosphate; e) triosephosphate isomerase (tpiA) converting dihydroxyacetone phosphate to D-glyceraldehyde- 3-phosphate; f) glyceraldehyde-3-phosphate dehydrogenase A (gapA) oxidatively phosphorylating D- glyceraldehyde 3-phosphate (G3P) to 1,3-bisphosphoglycerate (BPG) using the cofactor NAD; g) glyceraldehyde-3-phosphate dehydrogenase (gapC) converting D-glyceraldehyde 3-phosphate into 1,3-bisphosphoglyceric acid; h) phosphoglycerate kinase (pgk) dephosphorylating 1,3-bisphosphoglyceric acid to generate 3- phospho-D-glycerate; i) D-3-phosphoglycerate dehydrogenase (serA) oxidizing 3-phospho-D-glycerate to 3- phosphohydroxypyruvate; j) phosphoserine aminotransferase (serC) converting 3-phosphohydroxypyruvate to phosphoserine; or k) phosphoserine phosphatase (serB) dephosphorylating phosphoserine to serine.

[0080] In some aspects, a microbial host cell genetically modified to produce cysteine and/or cystine is provided, wherein the host cell expresses heterologous genes encoding a pyridoxal 5-phosphate (PLP)-dependent enzyme TrpS comprising i) an enzyme subunit having an amino acid sequence comprised in SEQ ID NO: 13 (trpB) and ii) an enzyme subunit having an amino acid sequence comprised in SEQ. ID NO: 14 (trpA), further comprising an operative biosynthetic pathway capable of producing serine, said pathway comprising one or more pathway polypeptides selected from: a) glucokinase (glk) comprised in SEQ ID NO: 1 phosphorylating glucose to glucose-6-phosphate; b) phosphoglucose isomerase (pgi) comprised in SEQ. ID NO: 2 isomerizing glucose-6-phosphate to fructose-6-phosphate; c) phosphofructokinase 2 (pfkB) comprised in SEQ ID NO: 3 phosphorylating fructose-6-phosphate to fructose-1.6-bisphosphate; d) fructose-bisphosphate aldolase class 1 (fbaB) comprised in SEQ ID NO: 4 or fructose-bisphosphate aldolase class 2 (fbaA) comprised in SEQ ID NO: 5 splitting fructose 1,6-bisphosphate into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate; e) triosephosphate isomerase (tpiA) comprised in SEQ ID NO: 6 converting dihydroxyacetone phosphate to D-glyceraldehyde-3-phosphate; f) glyceraldehyde-3-phosphate dehydrogenase A (gapA) comprised in SEQ ID NO: 7 oxidatively phosphorylating D-glyceraldehyde 3-phosphate (G3P) to 1,3-bisphosphoglycerate (BPG) using the cofactor NAD; g) glyceraldehyde-3-phosphate dehydrogenase (gapC) comprised in SEQ ID NO: 8 converting D- glyceraldehyde 3-phosphate into 1,3-bisphosphoglyceric acid; h) phosphoglycerate kinase (pgk) comprised in SEQ ID NO: 9 dephosphorylating 1,3- bisphosphoglyceric acid to generate 3-phospho-D-glycerate; i) D-3-phosphoglycerate dehydrogenase (serA) comprised in SEQ ID NO: 10 oxidizing 3-phospho-D- glycerate to 3-phosphohydroxypyruvate; j) phosphoserine aminotransferase (serC) comprised in SEQ ID NO: 11 converting 3- phosphohydroxypyruvate to phosphoserine; and/or k) phosphoserine phosphatase (serB) comprised in SEQ ID NO: 12 dephosphorylating phosphoserine to serine.

[0081] Among the said pathway polypeptides the pyridoxal 5-phosphate (PLP)-dependent enzyme is in some embodiments Tryptophan synthase comprising an amino acid sequence which has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the Tryptophan synthase units comprised in SEQ ID NO: 13, 14, 15 or 16. In other embodiments the glucokinase (glk) has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the Glucokinase comprised in SEQ ID NO: 1. In other embodiments the phosphoglucose isomerase (pgi) has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the phosphoglucose isomerase (pgi) comprised in SEQ ID NO: 2. In other embodiments the phosphofructokinase 2 (pfkB) has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the phosphofructokinase 2 (pfkB) comprised in SEQ. ID NO: 3. In other embodiments the fructose-bisphosphate aldolase class 1 (fbaB) has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the fructose- bisphosphate aldolase comprised in SEQ ID NO: 4. In other embodiments the fructose-bisphosphate aldolase class 2 (fbaA) has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the fructose-bisphosphate aldolase comprised in SEQ ID NO: 5. In other embodiments the triosephosphate isomerase (tpiA) has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the triosephosphate isomerase comprised in SEQ ID NO: 6. In other embodiments the glyceraldehyde-3-phosphate dehydrogenase A (gapA) has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the glyceraldehyde-3-phosphate dehydrogenase A comprised in SEQ ID NO: 7. In other embodiments the glyceraldehyde-3-phosphate dehydrogenase (gapC) has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the glyceraldehyde-3-phosphate dehydrogenase comprised in SEQ ID NO: 8. In other embodiments the phosphoglycerate kinase (pgk) has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the phosphoglycerate kinase comprised in SEQ ID NO: 9. In other embodiments the D-3- phosphoglycerate dehydrogenase (serA) has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the D-3-phosphoglycerate dehydrogenase comprised in a sequence selected from the group consisting of: SEQ ID NO: 10, SEQ ID NO: 27 , SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47. In one embodiment, the the D-3-phosphoglycerate dehydrogenase (serA) has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the D-3- phosphoglycerate dehydrogenase comprised in: SEQ ID NO: 10. In other embodiments the phosphoserine aminotransferase (serC) has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the phosphoserine aminotransferase comprised in SEQ. ID NO: 11. In other embodiments the phosphoserine phosphatase (serB) has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the phosphoserine phosphatase comprised in SEQ ID NO: 12.

[0082] Additionally or alternatively, the genetically modified host cell comprises and expresses one or more genes encoding the one or more pathway polypeptides, which genes are selected from the group of: a) a gene which has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the gene encoding the Tryptophan synthase comprised in SEQ ID NO: 13, 14, 15 or 16; b) a gene which has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the gene encoding the glucokinase comprised in SEQ ID NO: 1; c) a gene which has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the gene encoding the phosphoglucose isomerase (pgi) comprised in SEQ ID NO: 2; d) a gene which has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the gene encoding the phosphofructokinase 2 (pfkB) comprised in SEQ ID NO: 3; e) a gene which has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the gene encoding the fructose-bisphosphate aldolase comprised in SEQ ID NO: 4; f) a gene which has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the gene encoding the fructose-bisphosphate aldolase comprised in SEQ ID NO: 5; g) a gene which has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the gene encoding the triosephosphate isomerase comprised in SEQ. ID NO: 6; h) a gene which has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the gene encoding the glyceraldehyde-3-phosphate dehydrogenase A comprised in SEQ ID NO: 7; i) a gene which has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the gene encoding the glyceraldehyde-3-phosphate dehydrogenase comprised in SEQ ID NO: 8; j) a gene which has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the gene encoding the phosphoglycerate kinase comprised in SEQ ID NO: 9; k) a gene which has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the gene encoding D-3-phosphoglycerate dehydrogenase comprised in SEQ ID NO: 10; l) a gene which has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the gene encoding the phosphoserine aminotransferase comprised in SEQ ID NO: 11; and m) a gene which has at least 70% identity, such at least 75% identity, such at least 80% identity, such at least 85% identity, such at least 90% identity, such at least 95%, such at least 99% identity, such as 100% identity to the gene encoding the phosphoserine phosphatase comprised in SEQ ID NO: 12.

[0083] In some embodiments one or more of the said genes and/or pathway polypeptides are heterologous to the host cell and moreover the host cell can comprise two or more copies of one or more of these genes or otherwise overexpressed. In further embodiments the host cell can comprise a transporter molecule facilitating transport of a precursor for or a product of the serine pathway. The host cell can also be further genetically modified to provide an increased amount of a substrate in the serine pathway, and/or one or more native or endogenous genes of the host cell can be deleted, disrupted and/or attenuated to increase production of serine. Still further the host cell can be further genetically modified to exhibit increased tolerance towards one or more substrates, intermediates, or products in the serine pathway. In some embodiments, the one or more genes to be deleted, disrupted and/or attenuated to increase production of serine are selected from the group consisting of: sdaA (SEQ ID NO: 23), sdaB (SEQ ID NO: 24), tdcG (SEQ ID NO: 25), and glyA (SEQ ID NO: 26) or any homologs or paralogs thereof having at least 90% sequence identity thereto. In some embodiments, the host cell according to the present disclosure is provided wherein one or more native or endogenous genes of the host cell are deleted, disrupted and/or attenuated, wherein the one or more genes are selected from the group consisting of: sdaA, sdaB, tdcG, and glyA.

[0084] The host cell described herein is suitably prokaryotic or eukaryotic such as of the genus Escherichia, optionally of the species Escherichia coli or C. glutamicum.

[0085] In some embodiments, the host cell described herein is a Gram-positive or Gram-negative bacterium.

[0086] Examples of bacteria which can be used according to the present disclosure belong to the Enterobacteriaceae family, such as bacteria belonging to a genus selected from the group consisting of Escherichia, Arsenophonus, Biostraticola, Brenneria, Buchnera, Budvicia, Buttiauxella, Cedecea, Citrobacter, Cosenzaea, Cronobacter, Dickeya, Edwardsiella, Enterobacter, Erwinia, Ewingella, Gibbsiella, Hafnia, Klebsiella, Leclercia, Leminorella, Lonsdalea, Mangrovibacter, Moellerella, Morganella, Obesumbacterium, Pantoea, Pectobacterium, Phaseolibacter, Photorhabdus, Plesiomonas, Proteus, Rahnella, Raoultella, Saccharobacter, Salmonella, Samsonia, Serratia, Shimwellia, Sodalis, Tatumella, Thorsellia, Trabulsiella, Wigglesworthia, Yersinia and Yokenella.

[0087] According to certain other embodiments, the bacterium belongs to a genus selected from the group selected from Escherichia, Bacillus, Lactococcus, Lactobacillus, Clostridium, Corynebacterium, Geobacillus, Streptococcus, Pseudomonas, Streptomyces, Shigella, Acinetobacter, Citrobacter, Salmonella, Klebsiella, Enterobacter, Erwinia, Kluyvera, Serratia, Cedecea, Morganella, Hafnia, Edwardsiella, Providencia, Proteus and Yersinia.

[0088] According to particular embodiments, the bacterium belongs to the genus Escherichia. According to particular embodiments, the bacterium is Escherichia coli. Non-limiting examples of a bacterium belonging to the genus Escherichia, which can be used to derive a host cell of the present disclosure are Escherichia coli K-12 (especially substrain MG1655 or W3110), BL21, W, or Crooks. According to more particular embodiments, the bacterium is Escherichia coli K-12.

[0089] According to other particular embodiments, the bacterium belongs to the genus Corynebacterium. A non-limiting example of a bacterium of the genus Corynebacterium is Corynebacterium glutamicum. According to other particular embodiments, the bacterium is Corynebacterium glutamicum.

[0090] According to other particular embodiments, the bacterium belongs to the genus Bacillus. Nonlimiting examples of a bacterium of the genus Bacillus are Bacillus subtitlis, Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus mojavensis. According to more particular embodiments, the bacterium is Bacillus subtitlis. According to other particular embodiments, the bacterium is Bacillus licheniformis.

[0091] According to other particular embodiments, the bacterium belongs to the genus Lactococcus. A non-limiting example of a bacterium of the genus Lactococcus is Lactococcus lactis. According to other particular embodiments, the bacterium is Lactococcus lactis.

[0092] According to other particular embodiments, the bacterium belongs to the genus Streptomyces. A non-limiting examples of a bacterium of the genus Streptomyces are Streptomyces lividans, Streptomyces coelicolor, or Streptomyces griseus. According to other particular embodiments, the bacterium is Streptomyces lividans. According to other particular embodiments, the bacterium is Streptomyces coelicolor. According to other particular embodiments, the bacterium is Streptomyces griseus.

[0093] According to other particular embodiments, the bacterium belongs to the genus Pseudomonas. A non-limiting example of a bacterium of the genus Pseudomonas is Pseudomonas putida. According to more particular embodiments, the bacterium is Pseudomonas putida.

[0094] As described, supra, an additional aspect of the present disclosure concerns a cell culture, comprising host cell described herein and a fermentation medium and in some embodiments the methods described herein also include steps of: a) culturing the said cell culture at conditions allowing the host cell to produce serine and the pyridoxal 5-phosphate (PLP)-dependent enzyme; and b) feeding the source of sulfur to the fermentation medium.

[0095] The said method can further comprise one or more steps selected from: a) culturing the cell culture under aerobic and/or anaerobic conditions b) culturing the cell culture under agitation; c) culturing the cell culture at a temperature of between 25 to 70 °C; d) culturing the cell culture at a pH of between 6-10; e) culturing the cell culture for between 10 hours to 30 days; and f) culturing the cell culture under fed-batch, repeated fed-batch, continuous, or semi-continuous conditions.

[0096] The methods described herein can also include feeding one or more exogenous serine precursors to the cell culture.

Sequence listings

[0097] The present application contains a Sequence Listing prepared in Patentin included below but also submitted electronically in ST26 format which is hereby incorporated by reference in its entirety.

Table A

Examples

[0098] The following examples further demonstrates the method for producing cysteine/cystine and related aspects.

Materials and methods

[0099] Chemicals used in the examples herein, e.g. for buffers and substrates, are commercial products of at least reagent grade.

Example 1: Generation of L-serine fermentation broth

[0100] The strain and fermentation method described in US20210095245, US10513682, and in Rennig et al., 2019 were used to produce fermentation mediums with high L-serine titers. Briefly, the seed cultures of the strain were grown aerobically in 2xYT medium containing 0.2% glucose, ImM glycine and supplemented with required antibiotics, or in fermentation media as detailed below, until at least an optical density at 600nm (OD600) above 5. Fed batch fermentation was performed at 37°C using a starting batch volume of 40% of the total working reactor volume. The seed inoculation was 2,5% of the batch volume. Dissolved oxygen tension (DO) was maintained at 20% and pH controlled at pH 7,0 +/-0,l using ammonia liquid (15-25% w/v). The batch media contained 2 g/L MgSO4-7H2O, 2 g/L KH2PO4, 10 g/L ammonium sulfate, 60mM glucose or carbohydrate equivalent based on C-mol basis, 2 g/L yeast extract, 0.6 g/L glycine, lx trace elements and 50 mg/L kanamycin. Onset of the fed- batch feeding was initiated by increase of the DO upon depletion of batch glucose. The feed solution contained 650 g/L glucose, 6 g/L glycine and 50 pg/mL kanamycin. Fed-batch fermentation was performed until a L-serine concentration 50 g/L or more was reached (see figure 2). Upon completion of the fermentation, the fermentation liquid was centrifuged at 4200xg for 20 min at 4°C and the supernatant collected and stored at -80°C until later use. Using this approach, it was possible to generate L-serine fermentation broth with L-serine concentrations >50 g/L without any purification step except removal of biomass. The resulting L-serine fermentation broth was subsequently used as a substrate for tryptophan synthase to produce L-cysteine (see example 3 and 4).

Example 2: Generation of an E. coli BL21 (DE3) strain expressing tryptophan synthase

[0101] A pSEVA27 vector backbone containing the two genes (trpA and trpB) encoding E. coli TrpS was constructed using uracil excision cloning as described in Cavaleiro et al., 2015. Briefly, a pSEVA27 vector backbone fragment was PCR amplified using the oCys_50 and oCys_51 primers (table 1), and TrpS was amplified from the genome of E. coli BI21(DE3). trpB was amplified using primers oCys_48 and oCys_53 (table 1) and trpA using primers oCys_49 and oCys_52 (table 1). Amplification of trpA using these primers generated an n-terminal hexahistidine tag. Each PCR reaction contained 200 nM of forward and reverse primer, 1 X Phusion U mastermix (Thermoscientific, Waltham, MA, USA) and approx. 1 ng of DNA template. The following PCR protocol was used for amplification: An initial denaturation step at 98°C for 40 seconds, followed by 20 cycles of denaturation at 98°C for 12 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 60 seconds. The resulting PCR products were column purified (Nucleospin PCR clean up kit) and subjected to Dpnl (New England Biolabs, Ipswitch, MA, USA) digestion overnight. The digested products were column purified (Nucleospin PCR clean up kit) and concentration was measured using a Nanodrop instrument (Thermoscientific, Waltham, MA, USA). The resulting fragments were assembled using USER enzyme (New England Biolabs, Ipswitch, MA, USA) by incubation at 37°C for 30 min followed by 16°C for 25 min, using a molar ratio of vector to insert of 1:1.5. 2.5 pL of the USER mix was transformed into chemically competent E. coli DH5a cells (New England Biolabs, Ipswitch, MA, USA) using a standard heat shock protocol and 100 pL of the transformed cells plated on LB-kan plates. A single colony was used to inoculate 10 ml of 2xYT media with 50 pg/ml Kanamycin, which was incubated O/N at 37°C and shaking at 250 rpm. Plasmid from the resulting culture was purified (Nucleospin plasmid purification kit) and the plasmid sequence verified by sanger sequencing (Eurofins Scientific, Luxembourg). 100 ng of verified plasmid was transformed into chemically competent E. coli BL21(DE3) using a standard heat shock protocol and 100 pL of the culture plated on LB-Kan agar plates. A single colony from the plate was used to inoculate 10 ml of 2xYT media with 50 pg/ml Kanamycin to generate the final strain expressing tryptophan synthase, subsequently referred to as Cys_39. Table 1: Primers used for generation of a pSEVA27 vector backbone encoding trpB and trpA with an n-terminal hexahistidine tag.

[0102] This experiment demonstrated the generation of an E. coli BL21(DE3) strain overexpressing E. coli tryptophan synthase (see figure 3) and this strain was subsequently tested for its L-cysteine production capabilities as outlined in example 3.

Example 3: Production of L-cysteine from L-serine and sodium hydrogen sulfide using tryptophan synthase

[0103] To test the L-cysteine production capabilities of Cys_39, a two-step approach was developed. First, bacteria expressing tryptophan synthase were grown by fermentation of Cys_39, followed by whole cell biocatalysis using L-serine and sodium hydrogen sulfide as substate for TrpS.

[0104] The Cys_39 biomass was generated as follows: Seed cultures were grown aerobically in 2xYT medium containing 0.2% glucose, ImM glycine and supplemented with required antibiotics, or in fermentation media as detailed below, until at least an optical density at 600nm (OD600) above 5 was reached. Fed batch fermentation was performed at 32°C using a starting batch volume of 40% of the total working reactor volume. The seed inoculation was 2,5% of the batch volume. Dissolved oxygen tension (DO) was maintained at 30% and pH controlled at pH 7,0 +/-0,l using ammonia liquid (15-25% w/v). The batch media contained 2 g/L MgSO4-7H2O, 2 g/L KH2PO4, 5 g/L ammonium sulfate, 0.5 % Glucose and 0.5 % Glycerol glucose or carbohydrate equivalent based on C-mol basis, 5 g/L yeast extract, 2x trace elements and 50 mg/L kanamycin. Onset of the fed-batch feeding was initiated by increase of the DO upon depletion of batch glycerol, thus the second observed DO increased based on carbohydrate depletion. Fed-batch feeding was controlled as a typical DO-stat fermentation. Upon each DO increase, addition of feed solution was used equal to 2,9% relative to starting batch volume. The feed solution contained 350 g/L glycerol and 10 g/L of Lactose and yeast extract, 50 mg/L kanamycin. Fed-batch fermentation was typically performed for 24 hours with either exponential feeding profile or DO stat or until a biomass concentration of at least OD at 600 nm of 50 was reached (see figure 4). Upon completion of the fermentation, the fermentation liquid was centrifuged at 4200xg for 20 min at 4°C, the supernatant discarded, and the biomass stored at -80°C until further use.

[0105] The biocatalytic conversion of L-serine to L-cysteine was done by resuspending the previously prepared Cys_39 biomass to an OD at 600 nm between 10 and 40, in 25-100 ml of 99% L-serine (Lake Avenue Nutrition, CA, USA) solution with an L-serine concentration >50 g/L. PLP was added to a final concentration of 0.25 mM. The conversion process was started by addition of 600 g/L sodium hydrogen sulfide (4% of total reaction volume) and incubation of the reaction solution at 37°C and 250 rpm shaking. To supply sufficient hydrogen sulfide ions, a volume corresponding to 4% of the total reaction volume of 600 g/L sodium hydrogen sulfide was added every 30 minutes for the first 2.5 hours of the conversion. The conversion was allowed to proceed for up to 24 hours and the L-serine and L- cysteine followed by HPLC analysis. At high concentrations, L-cysteine was oxidised to its dimeric form, cystine, therefore this was also quantified using HPLC. Samples for HPLC analysis were prepared by taking a sample from the bioconversion vessel and diluting it 1:1 with 1 M H2SO4. This solution was then further diluted 1:50 with deionised water, resulting in a 100-fold final sample dilution. The L- serine, L-cysteine and cystine concentrations was measured using a Dionex Ultimate 3000 HPLC (high- performance liquid chromatography) equipped with a CHIROBIOTIC T Chiral (250 x 2.1 mm x 5 pm) column (Sigma-Aldrich, St. Louis, MO, USA) and a Diode Array Detector (DAD-UV). The mobile phase comprised of 60% acetonitrile (v/v) and 0.02% (v/v) formic acid in Milli-Q. water. The mobile phase was delivered at a rate of 1.0 mL/min. The injection volume was 2.5 pL and the standard curve varied from 0.05 to 2 g/L for all compounds.

[0106] Using this approach, biomass from Cys_39 converted 10 grams of serine to 9.03 grams of L- cysteine and 0.8 grams of cystine (see figure 5). These results confirmed that TrpS could be used to produce L-cysteine from serine and sodium hydrogen sulfide and show that the bioconversion could be followed by HPLC analysis. This method was subsequently used to compare pure L-serine and L- serine fermentation broth as substrates for L-cysteine production (example 4).

Example 4: Conversion of L-serine to L-cysteine using pure L-serine and L-serine fermentation broth as substrates

[0107] To investigate if broth from the L-serine fermentation described in example 1 could be used directly as a substrate for TrpS, the production of L-cysteine from either 99 % pure L-serine (Sigma) or L-serine fermentation broth was compared (see figures 5 to 7). The experiment was conducted as described in example 3, except the Cys_39 biomass was either resuspended in 100 g/L of pure L-serine or L-serine fermentation broth with a L-serine concentration of 100 g/L. The L-serine, L-cysteine, and cystine concentrations were followed by HPLC for five hours after which all the added L-serine, both pure and in the fermentation broth, had been consumed (figure 5). L-serine consumption rates did not vary with the L-serine source, with all L-serine being consumed after four hours in both the pure L-serine and L-serine fermentation broth sample. Correspondingly, L-cysteine and cystine production rates were similar and independent of the L-serine being pure or from fermentation broth (figures 6 and 7). These results clearly showed that a complex L-serine fermentation broth, in combination with a hydrogen sulfide donor such as sodium hydrogen sulfide, can be used as a substrate for TrpS to efficiently produce L-cysteine. Furthermore, unexpectedly the L-serine fermentation broth is comparable to pure L-serine as a substrate for TrpS. Using L-serine fermentation broth as opposed to pure L-serine avoids expensive downstream processing steps associated with L-serine purification.

Example 5: Downstream purification steps that can be avoided when using serine fermentation broth as a substrate for TrpS.

[0108] The separation of amino acids from the fermentation broth is usually done by centrifugation or filtration followed by a purification steps using chromatographic techniques chosen according to the product properties such as solubility, isoelectric point, and affinity to adsorbent (Hermann, 2003). In this instance the SuperPro Designer (Intelligen inc) software was used to design an in silica downstream purification process for L-serine. The designed process was based on the downstream process currently used to purify the amino acid L-threonine from fermentation broth. L-threonine and L-serine are chemically almost identical, with the only difference being that L-threonine has an extra methyl substituent on the R-carbon. Considering their similarities, including an identical functional hydroxyl group, the downstream process for L-serine purification was expected to be similar to the process used for L-threonine.

[0109] The designed in silica L-serine downstream purification process consists of five main sections: separation, primary purification, concentration, secondary purification, and product recovery. Briefly described the process was as follows: After the serine fermentation has been stopped, the biomass was separated from the fermentation broth by centrifugation. In the primary purification step, the resulting supernatant was loaded onto an ion exchange column, removing the target product from the aqueous feed. After elution, the product solution is pumped into an activated carbon bed to remove any coloured compounds. Next, ammonia was removed, and the resulting product concentrated using a multi-stage evaporator to remove any excess water. The concentrated L-serine liquor was then crystallised, and the crystals recovered and passed through a rotary vacuum filter to generate product of greater than 95% purity (see figure 8).

[0110] The downstream process described above is expected to take approximately 30 hours and requires the purchase, maintenance, and operation of a selection of expensive equipment. In addition to this, the numerous removal operations necessary to obtain the required serine purity is expected to result in a significant loss of product as this has been observed for several other amino acids produced via fermentation (D'Este et al., 2018; Hermann, 2003; Kumar et al., 2014). Considering these observations, it is highly desirable to eliminate the need for the expensive and tedious downstream purification process. This is achieved by the disclosed invention, where serine fermentation broth as opposed to pure serine is used as a substrate for TrpS when producing L-cysteine.

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