Bioproduction of glycolic acid from glyoxal

Abstract

The present invention relates to a method for the biochemical production of glycollic acid or a derivative thereof, comprising a step of culturing a whole-cell biocatalyst in a suitable culture medium comprising glyoxal as a substrate and optionally recovering glycollic acid from the culture medium, the whole-cell biocatalyst comprises or consists of modified microbial cells that produce an enzyme that converts glyoxal to glycollic acid.

Description

Technical Field

[0001] The present invention belongs to the field of production of target molecules by engineered microorganisms, and more particularly relates to the bioconversion of glyoxal to glycolic acid by enzymatic catalysis in whole cells of microorganisms. Background Art

[0002] Glycolic acid (HOCH2COOH) belongs to the family of α-hydroxy acids and is a two-carbon organic acid with an alcohol functional group and a carboxylic acid functional group. In monomeric form, glycolic acid is mainly used in cosmetics and cleaning products. In polymeric form, it forms a polymer called polyglycolate, which exhibits significant mechanical properties and is suitable for packaging.

[0003] Commercially available glycolic acid is usually produced by plant extraction and chemical synthesis by the following three methods:

[0004] - Carbonylation of formaldehyde with carbon monoxide in the presence of an acid catalyst (H2SO4) at high temperature (200 - 250 °C) and high pressure (300 - 700 bar). However, the final product is colored due to high-temperature oxidation and contains trace amounts of formaldehyde, which is toxic, carcinogenic, potentially mutagenic, and restricted and prohibited in the cosmetics field (European regulation (CE) N 1223 / 2009);

[0005] - Neutralization and re-acidification of monochloroacetic acid (MCA). MCA is produced from chlorine and acetic acid. But the final product contains salts and chlorinated organic compounds;

[0006] - Enzymatic conversion of glyconitrile to glycolic acid by nitrilase. However, the main drawback of this method is the presence of glyconitrile, which is listed as an extremely dangerous substance because it polymerizes under the influence of trace amounts of acid or base and poses a risk of fire or explosion.

[0007] Some international publications have also reported the bioproduction of glycolic acid from ethylene glycol using yeasts or bacteria that naturally convert ethylene glycol to glycolic acid, as this method has the advantage of operating under physiological conditions. Ethylene glycol is not expensive, but bioproduction by fermentation has not yet led to industrial-scale production.

[0008] Metabolic engineering has made it possible to exceed the production capacity of organisms that naturally produce glycolic acid from ethylene glycol, but there is still a need to provide new methods for the bioproduction of glycolic acid under mild conditions (25 °C, pH 7.0) or even under acidic conditions and with improved performance.

[0009] As an acid, glycolic acid coexists with its conjugate base, glycolate, in solution, and the equilibrium between these two forms depends on the reaction pH, as determined by the dissociation curve of the acid. At the pKa (pH 3.83), 50% of the glycolic acid and 50% of the glycolate are present. Above the pKa, the glycolate form predominates. In the present specification, we intend to refer to the glycolic acid / glycolate combination as "glycolic acid". Thus, "glycolic acid production" and "glycolate production" can be used interchangeably, and the terms "glycolic acid", "glycolate", and "glycolic acid / glycolate" can also be used interchangeably, since these two forms coexist under the conditions of the present invention.

[0010] The present invention particularly provides a new method for producing glycolic acid, which involves a microbial strain modified to express glyoxalase in the cytoplasm or periplasm of a cell. In particular, the "biocatalyst" is a strain of Escherichia coli (E. coli) expressing glyoxalase, which effectively catalyzes the bioconversion of glyoxal to glycolic acid under mild conditions (25 °C, 1 atm, pH 7.5). In a preferred embodiment, the "biocatalyst" is a strain of E. coli expressing glyoxalase in the periplasm, which also effectively catalyzes the bioconversion of glyoxal to glycolic acid under acidic conditions (pH < 7) and uses a small amount of salt in the subsequent process (economic advantage, using less salt and simplified product purification).

[0011] In fact, the applicant has demonstrated that the periplasmic environment plays a crucial role in maintaining the functionality of the enzyme under acidic conditions, which is not observed in the purified enzyme form, and he has also demonstrated that the periplasmic whole-cell biocatalyst can achieve a 15% increase in yield compared to other systems. Summary of the Invention

[0012] A first object of the present invention is a method for the biocatalytic production of glycolic acid or its derivatives, which comprises the step of culturing a whole-cell biocatalyst in a suitable medium containing glyoxal as a substrate and optionally recovering glycolic acid from the medium, the whole-cell biocatalyst comprising or consisting of modified microbial cells that produce an enzyme capable of converting glyoxal into glycolic acid.

[0013] Another object of the present invention is a whole-cell biocatalyst comprising or consisting of modified microbial cells that produce an enzyme capable of converting glyoxal into glycolic acid as defined in the present invention.

[0014] The present invention also relates to a method for producing a whole-cell biocatalyst as defined in the present invention, which comprises the following steps:

[0015] a) Transforming a microbial cell with a plasmid containing an expression cassette, said expression cassette containing a gene encoding an enzyme (i.e., glyoxalase) that converts glyoxal to glycolic acid under the control of a constitutive or inducible promoter, and optionally containing a secretion signal gene for targeting the enzyme to the periplasmic space; or a') Modifying a microbial cell by integrating said expression cassette into the genome of the microbial cell;

[0016] b) Culturing the modified cell in a suitable medium for expressing said enzyme (glyoxalase);

[0017] c) Separating the cells from the supernatant by centrifugation; and

[0018] d) Optionally drying the cells and storing them at 4 °C or -20 °C or -80 °C.

[0019] Another object of the present invention is a method for producing a glycolic acid derivative or a product obtained by a reaction using glycolic acid as a substrate, which comprises at least one step in the method for the biochemical production of glycolic acid as defined in the present invention.

[0020] The present invention also relates to an expression cassette comprising a nucleotide sequence encoding an enzyme (i.e., glyoxalase) that converts glyoxal to glycolic acid, and optionally a secretion signal gene encoding a signal peptide as defined in the present invention, said secretion signal gene being used for targeting said enzyme (i.e., glyoxalase) to the periplasmic space of a microbial cell.

[0021] Another object of the present invention relates to a nucleotide sequence encoding a polypeptide or an expression cassette as defined in the present invention.

[0022] The present invention also relates to a vector comprising a nucleotide sequence as defined in the present invention. Detailed Description

[0023] Thus, a first object of the present invention is a method for the biochemical production of glycolic acid or its derivatives, which comprises culturing a whole cell biocatalyst in a suitable medium containing glyoxal as a substrate and optionally recovering glycolic acid from the medium, said whole cell biocatalyst comprising or consisting of modified microbial cells that produce an enzyme that converts glyoxal to glycolic acid.

[0024] "Biochemical production" in the present invention refers to production involving biological materials, preferably microbial cells, but not a fermentation process.

[0025] "Glycolic acid derivative" refers to a compound derived from glycolic acid used as an intermediate, particularly a compound obtained by chemical polymerization, methylation or esterification of glycolic acid.

[0026] Enzyme (glyoxalase)

[0027] "An enzyme that converts glyoxal to glycolic acid" refers to an enzyme that can convert glyoxal to glycolic acid according to the following reaction: glyoxal + H2O -> glycolic acid + H + .

[0028] The enzyme of the present invention converts glyoxal to glycolic acid and exhibits low activity towards glycolic acid, which is used to accumulate the glycolic acid in the reaction system. In particular, the activity of the enzyme of the present invention towards glycolic acid is preferably one-tenth or less, more preferably one-twentieth or less, and further preferably one-hundredth or less of its activity towards glyoxal.

[0029] In particular, the enzyme of the present invention for converting glyoxal to glycolic acid is glyoxalase, which is mainly characterized by the following physicochemical properties:

[0030] - Acts on glyoxal to produce the corresponding glycolic acid; and

[0031] - Shows activity towards glyoxal, but low or no activity towards glycolic acid.

[0032] The enzyme of the present invention, glyoxalase, may further have the following physicochemical properties:

[0033] - The molecular weight in gel filtration analysis is about 20 kDa;

[0034] - The optimal reaction temperature is 25 to 37 °C; and

[0035] - The optimal reaction is in the pH range of 1 to 8, especially 3.8 to 7 or 5 to 8, and preferably under acidic conditions with pH below 7, especially 3.8 to 7.

[0036] The enzyme of the present invention can be selected in the following in vitro tests:

[0037] - Mix the enzyme with glyoxal in a buffer solution (e.g., potassium phosphate buffer) at pH 7.5 for 0.10 to 60 minutes;

[0038] - After centrifugation and filtration, load the sample onto a high performance liquid chromatography (HPLC) column, and monitor the production of glycolic acid spectroscopically at 315 nm.

[0039] In a specific embodiment, the glyoxalase is selected from the group consisting of members of the DJ-1 superfamily of glyoxalases known in humans, worms, plants, and bacteria.

[0040] Preferably, the glyoxalase is selected from the group consisting of glyoxalases produced by Escherichia coli YajL, YhbO, and ElbB. In a preferred embodiment, the present invention uses Escherichia coli glyoxalase III (GLY III); this enzyme is known to protect cells from heat, oxidation, pH, and UV stress (Abdallah et al., 2007). In a more preferred embodiment, the present invention uses Escherichia coli glyoxalase III (Gene ID: NP_417622.2) encoded by the yhbO gene, as it has a high affinity and activity for glyoxal (Km: 0.38 mM; kcat: 118.44 min -1 ; kcat / Km: 3.11x105 min -1 M -1 , Lee et al., 2016).

[0041] Thus, in a preferred embodiment, the enzyme that converts the glyoxal substrate to glycolic acid is a glyoxalase having a catalytic triad of Cys-His-Asp / Glu, particularly advantageously a glyoxalase III (GLYIII) containing the conserved signature DJ-1_PfpA domain, preferably a glyoxalase III (SEQ ID NO:1) encoded by the yhbO gene (SEQ ID NO:2) from Escherichia coli or an amino acid sequence having at least 80% identity with SEQ ID NO:1.

[0042] Specifically, the amino acid-conserved DJ-1Pfpl domain of YhbO contains 168 amino acids, as shown in SEQ ID NO:3, which corresponds to the nucleotide sequence shown in SEQ ID NO:4.

[0043] The following table discloses several sequences illustrated in the examples of the present invention, but the present invention is not limited to the sequences.

[0044] Table 1

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051] The so-called "80% identity" refers to 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity.

[0052] The percentage of identity mentioned in the present invention is determined based on the global alignment of the sequences to be compared using any algorithm well known to those skilled in the art (such as the algorithm of Needleman and Wunsch 1970), that is, based on the sequence alignment performed globally over its entire length. This sequence comparison can be carried out using any software well known to those skilled in the art, for example, using the needle software, with a "gap open" parameter equal to 10.0, a "gap extension" parameter equal to 0.5, and a "BLOSUM 62" matrix. For example, the Needle software can be obtained under the name "Align" on the website ebi.ac.uk worldwide.

[0053] When the amino acid sequence of the glyoxalase according to the present invention is not 100% identical to one of the above sequences, but has at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with a such reference sequence, it may have insertions, deletions or substitutions with respect to the reference sequence. When it comes to substitutions, preferably "equivalent" amino acids are used for substitution, that is, any amino acid whose structure is similar to the original amino acid, so that it is impossible to change the biological activity of the antibody. Such substitutions are also referred to as "conservative mutations" or "conservative substitutions".

[0054] Examples of such substitutions are shown in Table 2 below:

[0055]

[0056]

[0057] Table 2. Substitution with equivalent amino acids

[0058] In particular, the present invention relates to DNA encoding the above-mentioned glyoxalase. Specifically, the present invention relates to DNA encoding the conserved signature DJ-1_PfpA domain (SEQ ID NO:3) of glyoxalase YhbO, which has the nucleotide sequence shown in SEQ ID NO:4.

[0059] The present invention also relates to DNA encoding Escherichia coli glyoxalase III, which has the nucleotide sequence shown in SEQ ID NO:2. In a specific embodiment, the amino acid sequence of the glyoxalase has 100% identity with SEQ ID NO:1.

[0060] In addition, the DNA encoding the protein is also included in the DNA of the present invention. The protein contains any amino acid sequence produced by deletion, substitution, or addition of one or several amino acids in the amino acid sequence shown by SEQ ID NO:1 (amino acid sequence of glyoxalase III YhbO) or SEQ ID NO:3 (amino acid sequence of the DJ1-1_PfpA domain of YhbO), as long as the protein containing the protein encoded by the above DNA SEQ ID NO:2 (nucleotide sequence of glyoxalase III YhbO) or SEQ ID NO:4 (nucleotide sequence of the DJ1-1_PfpA domain of YhbO) has the activity of converting glyoxal into glycolic acid.

[0061] Methods for deletion, substitution, or addition of specific amino acids are well-known to those skilled in the art. For example: PCR using synthetic DNA primers, which have nucleotide sequences containing deletions of specific amino acid codons, substitutions of other amino acid codons, or additions of other amino acid codons; and methods of ligating an enzyme gene produced by known methods (such as chemical DNA synthesis methods) to a gene expression vector and expressing it in a host (such as Escherichia coli) through gene recombination.

[0062] Genes of enzymes (such as glyoxalase) available in the present invention can be obtained by well-known methods, especially through the following methods:

[0063] - Purifying the enzyme protein from a microorganism or its culture broth, where the microorganism produces an enzyme that converts glyoxal into glycolic acid;

[0064] - Using peptides obtained by digesting the enzyme protein with a protease to determine a partial amino acid sequence;

[0065] - Using primers synthesized based on these partial amino acid sequences, and performing PCR with genomic DNA as a template according to well-known methods. Through such PCR, a part of the enzyme gene is amplified, and the nucleotide sequence of the gene can be determined. Reverse PCR is also performed using DNA primers synthesized from the N-terminal amino acid sequence and the amino acid sequence around the C-terminal to determine the signal sequence, N-terminal amino acid sequence, and C-terminal amino acid sequence.

[0066] In the present invention, these different nucleic acid sequences encoding the enzyme (glyoxalase) are integrated into a vector (plasmid), which is used to transform microbial cells (hosts) or to stably integrate the nucleic acid sequence encoding the enzyme into the microbial genome using well-known methods, as disclosed later in the specification.

[0067] Biocatalyst (modified microorganism expressing the enzyme)

[0068] "Biocatalyst" refers to a catalyst made of biological materials, especially microbial cells with catalytic activity.

[0069] The microorganism used in the present invention is a culture of a microorganism or microbial cells capable of producing an enzyme that converts glyoxal into glycolic acid.

[0070] The "whole-cell biocatalyst" in the present invention refers to a catalyst comprising or consisting of whole cells of a microorganism expressing an enzyme, which is completely different from a purified enzyme. In fact, in the present invention, the whole cells are not lysed for extracting and purifying the expressed enzyme, but are used as they are.

[0071] The "microorganism" in the present invention includes bacteria, yeast, fungi, actinomycetes, animal or plant cells, but preferably bacteria such as Escherichia coli, especially Gram-negative bacteria used for expressing enzymes in the periplasmic space.

[0072] "Modified microbial cells" refer to microbial cells that have been modified to express an enzyme that converts glyoxal into glycolic acid, preferably glyoxalase. In the present invention, the microbial cells are modified to express glyoxalase, which means that the level and / or activity of the enzyme is increased compared to unmodified microbial cells.

[0073] The expression "modified microorganism" includes "transformed microorganism", which refers to a microbial cell into which exogenous nucleic acid has been incorporated.

[0074] The microorganism capable of producing a specific enzyme that converts glyoxal into glycolic acid can be a wild-type strain, a mutant strain or a transformant (recombinant), and the transformant (recombinant) is obtained by using well-known methods as follows: (i) by integrating the DNA of the enzyme (glyoxalase) into a vector (plasmid) and introducing it into a microorganism (host), or (ii) by stably integrating the DNA encoding the enzyme into the microorganism genome.

[0075] In a specific embodiment, the microorganism, especially an Escherichia coli strain, such as the commercially available strain BL21(DE3) described in the following examples (Ref: C2527, ) is made competent for transformation under the supplier's protocol.

[0076] In another specific embodiment, Escherichia coli strains such as strain MG1655 are made competent for transformation according to the TSS protocol described by Chung and Miller (Chung et al., 1989). In fact, Chung and Miller developed a simple one-step procedure for preparing competent Escherichia coli, which uses a transformation and storage solution [TSS; 1×TSS is LB broth containing 10% (wt / vol) polyethylene glycol, 5% (vol / vol) dimethyl sulfoxide, and 50 mM Mg2+ at pH 6.5]. The cells are mixed with an equal volume of ice-cold 2x TSS and are immediately ready for use. Gene transformation is equally simple: plasmid DNA is added and the cells are incubated at 4 degrees Celsius for 5 - 60 min. No heat pulse is required, and the incubation time at 4 degrees Celsius is not critical, so there is no critical time step during the transformation process. The transformed bacteria are cultured and selected by standard methods.

[0077] In one specific embodiment, a microbial cell is modified by transformation with an expression cassette that is either on a circular DNA (plasmid) or integrated into the microbial genome.

[0078] In one specific embodiment, the expression cassette contains a gene encoding a glyoxalase as defined above under the control of a constitutive promoter or an inducible promoter.

[0079] As a "constitutive promoter", the proD promoter can be mentioned, promoters derived from the Anderson collection of constitutive promoters, such as J23100 to J23119 mentioned on the website http: / / parts.igem.org / Promoters / Catalog / Anderson.

[0080] As an "inducible promoter", the IPTG-inducible promoter, light-inducible promoter, T5 promoter, T7 promoter, lac promoter (pLac), lacT5 promoter, lacT7 promoter, tac promoter (pTac), rhaBAD promoter, araBAD promoter, tet promoter, penP promoter, cspA promoter, or a promoter containing the tetO or lacO operator as an operator sequence can be mentioned.

[0081] In one specific embodiment, the inducible promoter is selected from the group consisting of the rhaBAD promoter, the lac promoter, or the tac promoter.

[0082] In a specific embodiment, the enzyme that converts glyoxal into glycolic acid is produced in the cytoplasmic space or the periplasmic space of a microbial cell, preferably in the periplasmic space of the microbial cell. In other words, the enzyme that converts glyoxal into glycolic acid is produced in the cytoplasmic space or is transported into the periplasmic space of the microbial cell, preferably transported into the periplasmic space of the microbial cell.

[0083] In a specific embodiment, for example, in order to express glyoxalase in the periplasmic space of a microbe, the microbe is a Gram-negative strain, especially selected from the group consisting of Enterobacteriaceae, Alcaligenaceae, Vibrionaceae, and Pseudomonadaceae, especially Enterobacteriaceae, preferably Enterobacteriaceae belonging to the genus Salmonella, Yersinia, or Escherichia, more preferably Escherichia, and even more preferably Escherichia coli.

[0084] In another specific embodiment, especially in order to express glyoxalase in the cytoplasmic space of a microbe, other microbes can be used, such as Gram-positive bacteria (such as the genera Actinomyces, Clostridium, Mycobacterium, Streptococcus, Staphylococcus, and Nocardia), and fungi (such as species of the genus Saccharomyces and Candida).

[0085] In the case of an enzyme that is exported through the cytoplasmic membrane of a microbe (also known as being produced in the periplasm of the microbe), the signal peptide is usually encoded in a portion corresponding to dozens of amino acids starting from the gene start codon (meaning at the front of the gene sequence). In the step of the enzyme protein synthesized in the cell being exported through the cytoplasmic membrane of the cell, such a signal peptide sequence is further cleaved, thus becoming a mature enzyme.

[0086] In some cases, the native enzyme already contains its own signal peptide, but usually humans replace the original signal peptide sequence with another signal peptide sequence suitable for the host microbe, so chimeric genes can be constructed and used according to the method of the present invention.

[0087] Array proteins can be used as signal peptides. Soluble periplasmic proteins or those associated with the peripheral side of the periplasm of the inner or outer membrane can be mentioned. In a specific and preferred embodiment, soluble periplasmic proteins are used. In another specific and preferred embodiment, proteins associated with the periplasmic side of the inner or outer membrane, such as NlpA, which can be anchored to the membrane, are used.

[0088] As "signal peptides", DsbA, EOX, LamB, MglB, MmAp, OmpC, OmpT, SufI, SfmC, STII, TolB, TorA, TorT, GIII, MalE, OmpA, PelB, PhoA, and NlpA can be mentioned, preferably PelB.

[0089] In a specific embodiment, the PelB signal peptide amino acid sequence (SEQ ID NO: 5) encoded by the signal peptide nucleotide sequence (SEQ ID NO: 6) is used in the present invention.

[0090] Thus, in the case of an enzyme produced in the cytoplasm of a microbial cell used as a host, a human uses an enzyme gene without adding a nucleotide sequence encoding a signal peptide, or an enzyme gene from which the signal peptide sequence has been removed.

[0091] Moreover, in the preferred case of an enzyme produced in the periplasm of a microbial cell used as a host, a human uses an enzyme gene in which the nucleotide sequence encoding the signal peptide has replaced the original signal peptide, or an enzyme gene in which the nucleotide sequence encoding the signal peptide has been added in front of the enzyme gene.

[0092] Thus, in a specific embodiment, the present invention also provides an expression cassette containing an enzyme gene (yhbO gene, SEQ ID NO: 2) that does not have any signal peptide sequence for producing an enzyme in the cytoplasm of a microbial host.

[0093] Moreover, in another specific and preferred embodiment, the present invention also provides an expression cassette containing an enzyme gene (yhbO gene, SEQ ID NO: 2) and a signal peptide sequence (pelB, SEQ ID NO: 5) in front of the enzyme gene, forming a fusion nucleotide sequence pelB-yhbO (SEQ ID NO: 8) for producing an enzyme in the periplasm of a cell.

[0094] In this case, the fusion protein "signal peptide - enzyme" (PelB - YhbO) (SEQ ID NO: 7) is produced by the host microorganism, but the signal peptide is further cleaved so that the native glyoxalase III protein produced in the periplasm is soluble.

[0095] In a specific and preferred embodiment, glyoxalase is expressed in the periplasmic space of a microbial cell. Compared to cytoplasmic (cytosol) production, the transfer of the enzyme to the periplasm offers several advantages, such as avoiding protease attack and a better chance of correct protein folding in an oxidative subcellular environment. Many periplasmic proteins have been shown to be involved in protein folding and preventing aggregation. These include Dsb (disulfide bond formation) proteins that form and isomerize disulfide bonds, PPI enzymes that catalyze the trans → cis isomerization of peptidyl - prolyl bonds, and common chaperones such as FpkA (Ehrmann, 2007). The formation of disulfide bonds can result in a tightly folded structure that is resistant to proteases.

[0096] The Applicant has also demonstrated in the examples that when the whole-cell biocatalyst of the present invention is located in the periplasmic compartment according to the preferred embodiment, it effectively produces glycolic acid at a pH level as low as 4.5, while the whole-cell process located in the cytoplasmic compartment cannot operate effectively at such a low pH level. And the purified enzyme form is also not functional under acidic pH conditions.

[0097] The Applicant has demonstrated that the periplasmic environment plays a crucial role in maintaining the functionality of the enzyme under acidic conditions. Producing organic acids such as glycolic acid at acidic pH has the advantage of facilitating downstream processes.

[0098] In this particular embodiment, the enzyme is fused with a signal peptide to localize or transport the enzyme into the periplasmic space. Most proteins can successfully cross the cytoplasmic membrane and transfer to the periplasm. When the conditions for periplasmic accumulation are optimized, a large percentage of the total cellular proteins can be exported to the periplasm, with percentages in the range of 20% to 40% (Ehrmann, 2007).

[0099] Therefore, in a particular embodiment, the expression cassette further comprises a signal peptide for localizing or transporting glyoxalase to the periplasmic space, particularly selected from the group consisting of DsbA, EOX, LamB, MglB, MmAp, OmpC, OmpT, SufI, SfmC, STII, TolB, TorA, TorT, GIII, MalE, OmpA, PelB, PhoA, NlpA, preferably PelB.

[0100] Therefore, the present invention also relates to a whole-cell biocatalyst comprising or consisting of a modified microbial cell that produces an enzyme capable of converting glyoxal into glycolic acid as defined above.

[0101] The present invention also relates to a method for producing the whole-cell biocatalyst as defined above, which comprises the following steps:

[0102] a) Transforming a microbial cell with a plasmid comprising an expression cassette that contains a gene encoding an enzyme for converting glyoxal into glycolic acid under the control of a constitutive or inducible promoter, and optionally comprises a signal peptide for localizing the enzyme to the periplasmic space; or a') Engineering a microbial cell by integrating the expression cassette into the genome of the microbial cell;

[0103] b) Culturing the modified cell in a suitable medium for expressing the enzyme;

[0104] c) Separating the cells from the supernatant by centrifugation; and

[0105] d) Optionally drying the cells and storing them at 4°C or -20°C or -80°C.

[0106] In a specific and preferred embodiment, the microbial cell (host) is an Escherichia coli strain, preferably Escherichia coli BL21(DE3) strain or MG1655 strain, and the enzyme is glyoxalase III (GLYIII) encoded by the Escherichia coli yhbO gene (in particular, SEQ ID NO:1 is the amino acid sequence of Escherichia coli GLY III, and SEQ ID NO:2 is the nucleotide sequence of the yhbO gene).

[0107] Another object of the present invention is an expression cassette comprising a nucleotide sequence encoding the enzyme as defined above under the control of a promoter, preferably an inducible promoter (such as IPTG) as disclosed above.

[0108] In a specific embodiment, especially for expressing glyoxalase in the periplasm of the modified microbial cell, the expression cassette comprises a nucleotide sequence encoding the glyoxalase as defined above and a signal peptide sequence encoding the signal peptide as defined above, which is used to localize the glyoxalase to the periplasmic space of the microbial cell.

[0109] In a specific and preferred embodiment, the signal peptide is PelB (SEQ ID NO:5) encoded by the signal peptide pelB gene (SEQ ID NO:6).

[0110] Another object of the present invention relates to a nucleotide sequence encoding the polypeptide (enzyme) or fusion polypeptide (with a signal peptide) as defined above.

[0111] In a specific embodiment, the expression cassette comprises the fusion nucleotide sequence pelB-yhbO as shown in SEQ ID NO:8, which contains the pelB signal peptide and the yhbO gene, for producing the fusion polypeptide (or fusion protein PelB-YhbO) as shown in SEQ ID NO:7.

[0112] The present invention also relates to a vector comprising the nucleotide sequence as defined above.

[0113] Immobilization of the biocatalyst (optional)

[0114] The modified microbial cells can be free in the culture medium or advantageously immobilized. Immobilization can be carried out by methods well known to those skilled in the art (such as crosslinking method, physical adsorption method, embedding method, etc.).

[0115] In a specific embodiment, the microbial cells are immobilized on agar or carrageenan, or the microbial cells are crosslinked, especially crosslinked with glutaraldehyde (GA) and / or polyethyleneimine (PEI), or both immobilized and crosslinked.

[0116] In a specific embodiment, the biocatalyst is immobilized on agar or carrageenan, or crosslinked with glutaraldehyde and / or polyethyleneimine.

[0117] The immobilization of the whole-cell biocatalyst allows for the easy recovery of the biocatalyst for further use in another reaction.

[0118] In a specific embodiment, the microbial cells are encapsulated in carrageenan beads. Such an embodiment is particularly advantageous for bioproduction in the microbial periplasmic space.

[0119] In a specific embodiment, the microbial cells are immobilized with glutaraldehyde and / or polyethyleneimine.

[0120] The immobilization of the modified microbial cells facilitates the recovery of the modified microbial cells for another further reaction.

[0121] Conditions for the cultivation and conversion reaction

[0122] "Suitable medium" refers to a medium suitable for the proliferation of microorganisms. The medium is usually a liquid, gel or solid medium, which contains a carbon source (including sugars, alcohols), a nitrogen source and other compounds required for the proliferation of the microorganisms, and also the reaction of converting glyoxal present in the medium as a substrate into glycolic acid. Examples of suitable media (such as TB medium) will be disclosed later in the specification.

[0123] Culture conditions for glyoxalase production

[0124] The reaction conditions vary depending on the enzyme used, the microorganism used and its processed products.

[0125] In a specific embodiment, in order to produce glyoxalase III in the culture of Escherichia coli, the temperature is 20 °C to 37 °C or 25 °C to 37 °C, preferably 20 °C or 25 °C; the pH is pH 5 to 8, preferably between pH 5.5 and 8, more preferably pH 7.

[0126] After microbial culture and glyoxalase expression, the cells are harvested by a liquid / solid separation process. This includes separating the liquid medium containing the microbial cells from the rest of the culture. Different methods, such as centrifugation, filtration, are used based on the specific characteristics of the system.

[0127] Once the cells are recovered, they are used as biocatalysts for converting glyoxal into glycolic acid.

[0128] Conversion of glyoxal into glycolic acid

[0129] This enzymatic conversion can be achieved by introducing the harvested cells into a reaction system, in which glyoxal is converted into glycolic acid through the catalytic action of the expressed glyoxalase.

[0130] In a specific embodiment, glyoxal is directly added to the culture medium. For example, glyoxal is an aqueous solution of glyoxal (40%).

[0131] Glyoxal is the smallest dialdehyde. Glyoxal is soluble in water and forms an equilibrium between the monohydrate and the dihydrate. It can also form dimers and oligomers through (reversible) condensation. Glyoxal can be produced by the gas-phase oxidation of ethylene glycol with air in the presence of a copper or iron catalyst. BASF (Ludwigsafen, Germany) uses this method, and the reported production volume is 60 kt / year (BASF, 2016). Another method is based on the liquid-phase oxidation of acetaldehyde with nitric acid, and WeylChem produces glyoxal therefrom for the production of glyoxylic acid. Acetaldehyde is produced by the oxidation of ethylene or ethanol. The production of ethylene glycol and bio-based ethanol has attracted increasing attention, and glyoxal can now be synthesized from renewable resources.

[0132] The reaction involves mixing a set of reaction components under suitable reaction conditions, thereby producing glycolic acid in an aqueous solution or an organic solvent (such as methanol).

[0133] The production of glycolic acid in methanol exhibits some advantages:

[0134] - Reduced water consumption;

[0135] - Methanol evaporation consumes less energy than water evaporation

[0136] - Production of glycolic acid derivatives such as methyl glycolate: The esterification reaction may be limited by the presence of excess water.

[0137] The reaction can be carried out in a batch mode or a (repeated or non-repeated) fed-batch mode.

[0138] "Batch mode" is where no additional feed is used from the start to the end of the process.

[0139] "Fed-batch mode" is where feeding with the substrate (glyoxal) and supplements can extend the culture duration for higher cell densities to produce glycolic acid.

[0140] "Repeated fed-batch" is where all cells except for a small amount of residue in a complete (fed) batch are harvested, and the remaining cells are used as the inoculum for the next batch.

[0141] A continuous mode for biotransformation can also be mentioned, where the feeding rate matches the removal rate of the harvest, along with cell retention.

[0142] The biotransformation reaction is preferably carried out under conditions consisting of shaking and stirring (100 rpm to 600 rpm, preferably about 200 rpm). The temperature is from 25 °C to 37 °C, preferably 25 °C; the pH is from pH 3.8 to 8, preferably from pH 3.8 to 7. The reaction time generally ranges between one and fifty hours, preferably between one and three hours.

[0143] In a particular embodiment, in a variable reaction volume, the ratio of cell dry cell weight (dcw)*: substrate is from 1% to 30%, preferably from 1% to 10%, more preferably from 1% to 5%, and even more preferably 1%.

[0144] *dcw = dry cell weight.

[0145] In a particular embodiment considering a 40% glyoxal solution, the maximum concentration of glyoxal used is 8.7 M.

[0146] The biotransformation of glyoxal to glycolic acid involves the release of protons (H+) during the reaction due to the formation of glycolate- and H+ ions (glyoxal + H2O -> glycolate- + H+). Since enzyme activity is sensitive to pH, sodium hydroxide (NaOH) is added as a base to maintain a pH compatible with the enzyme. NaOH is a base that reacts with protons to form water, effectively neutralizing the acidic environment. The reaction involved is: Na+ + OH- + glycolate- + H+ -> Na+ + glycolate- + H2O. The addition of this base helps to maintain a controlled pH level throughout the biotransformation process, resulting in sodium glycolate (sodium salt of glycolic acid). In this embodiment, the purification process includes a step that requires the use of a cation exchange resin to replace sodium ions with H+ ions to ultimately obtain pure glycolic acid.

[0147] In another preferred embodiment using a "periplasmic whole cell biocatalyst", where the enzyme is protected from the acidic environment and a full pH control process using a base (NaOH) is not required, the reaction advantageously provides the direct production of glycolic acid without the sodium salt (sodium glycolate). This method greatly simplifies the downstream process, reduces costs, and minimizes wastewater. In this embodiment, pH adjustment and thus the addition of NaOH are minimized.

[0148] In fact, we have demonstrated that the mixture of glycolic acid and glycolate produced by the conversion reaction acts as a buffer system around the pKa (3.83). This means that when an acid or base is added, the solution can resist large pH changes, providing stability to the biotransformation process.

[0149] This buffering effect is typically achieved in the presence of a minimal amount of acid in the solution. In a particular embodiment, pH adjustment may initially be necessary to accumulate sufficient amounts of glycolic acid, stabilize the pH, and subsequently continue production without pH adjustment. As an example of the present invention, bioconversion begins with pH adjustment at pH 6.5 to produce a glycolic acid buffer, then glycolic acid and H+ are produced without a pH change, the pH stabilizes at 3.83, and no further pH adjustment is required. Compared to a process with full pH control, this two-step process aims to minimize the sodium content in the reactor.

[0150] The advantages of this method are the reduction in the amount of sodium hydroxide used and the amount of cation exchange resin required in the downstream process. Thus, it simplifies the overall process, reduces costs, and minimizes the generation of wastewater.

[0151] Product

[0152] The glycolic acid produced according to the method of the present invention can be recovered.

[0153] "Recovering glycolic acid from the culture medium" refers to the steps of separating, extracting, filtering, and / or purifying glycolic acid from the culture medium. Typically, this step includes centrifugation, microfiltration, cation and anion exchange, and evaporation.

[0154] In a particular embodiment, solid / liquid separation is carried out by centrifugation at 4000 to 5000 rpm, especially at 4500 rpm for 5 to 15 minutes, especially 10 minutes, and microfiltration is carried out using a filter with a porosity range of 0.1 to 0.2 μm, especially 0.2 μm.

[0155] In a particular embodiment, the extraction of glycolic acid is carried out by anion exchange chromatography.

[0156] In a particular embodiment, the extraction of glycolic acid from the supernatant is carried out by anion exchange chromatography using an ion exchange column such as a Dowex resin column (e.g., Dowex 1x8 resin OH form).

[0157] As an illustrative example, first, the sample is added to the ion exchange column using a peristaltic pump at a rate of 1 to 5 mL / min, especially 2.5 mL / min. Then, the ion exchange resin column is washed with deionized water. A sequential elution process is carried out using NaOH at different concentrations (60, 120, 200 mM). For subsequent use, the stored ion exchange resin is washed with 1 M NaOH.

[0158] The target fraction is then eluted on a cation exchange resin (e.g., DOWEX 50WX8 H form) to convert sodium glycolate (sodium glycolate salt) to glycolic acid.

[0159] Finally, water is evaporated under reduced pressure (below 50 °C) to obtain a 70% glycolic acid solution. In a specific embodiment, the evaporation is carried out using an evaporator (Buchi R-215) at about 40 °C and about 40 mbar, and then completed by freeze-drying (Epsilon 2-4LSC).

[0160] In a preferred purification method, to convert sodium glycolate (sodium glycolate salt) to glycolic acid, the purification of glycolic acid from the supernatant is carried out by cation exchange chromatography using a cation exchange column such as a Dowex resin column (e.g., DOWEX 50WX8 H form).

[0161] In fact, as described above, the production of glycolic acid from glyoxal results in a decrease in pH. Thus, according to one embodiment, especially for "periplasmic whole cell biocatalyst", sodium hydroxide is used to control the pH, and glycolic acid is converted to sodium glycolate (sodium glycolate). Subsequently, glycolic acid is recovered using cation exchange by replacing Na+ with H+. General cation exchangers, such as cation exchange resins, need to be regenerated with an acid such as sulfuric acid (H2SO4) after use. Those skilled in the art will closely adjust the resin volume for sodium removal, the amount of sulfuric acid for regeneration, and the resulting wastewater containing sodium sulfate (Na2SO4) according to the sodium content at the end of production.

[0162] Optionally, impurity removal is carried out using granular charcoal adsorption on the column.

[0163] Finally, water is evaporated under reduced pressure (below 50 °C) to obtain a 70% glycolic acid solution. In a specific embodiment, the evaporation is carried out using an evaporator (Buchi R-215) at about 40 °C and about 40 mbar.

[0164] Such a biological production process involves the use of salts (e.g., NaOH) for pH adjustment and purification steps to recover glycolic acid.

[0165] Therefore, the applicant has demonstrated that the use of a periplasmic whole cell biocatalyst resistant to acidic conditions according to the present invention allows for the direct production of glycolic acid without sodium. This strategy greatly simplifies the downstream process.

[0166] The quantification of glyoxal and glycolic acid can be carried out by high performance liquid chromatography (HPLC). In a particular embodiment, the concentrations of glyoxal (substrate) and glycolic acid (product) are measured by UHPLC (Dionex UltimateTM 3000) equipped with a Phenomenex ROA - organic acid H+(8%) column (300x7.8 mm) and a pre - column (50x7.8 mm), and 5 mM H2SO4 is used as the mobile phase at 0.5 mL / min for 35 minutes. Detection is carried out by a refractometer (Shodex RI - 101) and a UV detector (Dionex UltiMate 3000 Diode Array Detectors 3000 (RS)). The samples are filtered at 0.2 μm. Under these conditions, as Figure 1 shown, glyoxal elutes at 15 minutes and glycolic acid elutes at 18 minutes.

[0167] After extracting glycolic acid, the quality and properties of the resulting glycolic acid can be further analyzed.

[0168] In particular, NMR (nuclear magnetic resonance spectroscopy) techniques allow the observation of the local magnetic field around atomic nuclei and the use of this analysis to obtain high - resolution information about the product.

[0169] In particular, NMR analysis applied to glycolic acid produced by the method of the present invention shows that the main product is glycolic acid. Only 0.05% impurities are found in the proton NMR.

[0170] On the other hand, IR (infrared spectroscopy or vibrational spectroscopy) techniques are used to study and identify compounds by measuring the interaction of infrared radiation with matter through absorption, emission, or reflection.

[0171] In the present invention, IR analysis applied to glycolic acid produced by the method of the present invention shows a 95% correlation with the commercial standard of glycolic acid.

[0172] Mass spectrometry (MS) is also used to confirm the molecular weight and molecular formula of glycolic acid.

[0173] Thus, in a particular embodiment, the method of the present invention further comprises the step of recovering glycolic acid from the culture medium and, optionally, the step of analyzing the resulting glycolic acid, preferably by NMR, MS, and / or IR analysis.

[0174] And in a particular embodiment, glycolic acid produced according to the present invention is characterized by the following properties:

[0175] - According to proton NMR, it contains less than 30% impurities, preferably less than 0.05% impurities;

[0176] - In the IR, its structure and quality show a correlation of greater than 90%, preferably 95%, with the commercial standard of glycolic acid;

[0177] - The molecular weight of the substance [M-H] in the mass spectrum in negative mode is 75,0082 (+ / -1 mDa), and the expected molecular formula is C2H3O3.

[0178] The produced glycolic acid can be used as it is, or can be used as an intermediate in further chemical reactions such as esterification or polymerization.

[0179] Therefore, the present invention also relates to a method for producing a glycolic acid derivative or a product obtained by a reaction using glycolic acid as a substrate, including at least one step of the method for the biochemical production of glycolic acid as defined above.

[0180] The method may further include, at the end of the biotransformation reaction, the step of recovering the modified microbial cells (biocatalyst) that produce the enzyme that converts glyoxal to glycolic acid.

[0181] Generally, the modified microbial cells can be recycled 2 to 4 times.

[0182] Recovery generally includes the steps of washing the cells with PBS and harvesting them for further use. Description of the Drawings

[0183] Figure 1 : (A) HPLC-RI chromatogram of a 10 g / L glyoxal standard solution; (B) HPLC-RI chromatogram of a 10 g / L glycolic acid standard solution.

[0184] Figure 2 : Glycolic acid production in different volumes (2 to 2000 ml).

[0185] Figure 3 : Glycolic acid production kinetics according to the production volume.

[0186] Figure 4 : Kinetics of glycolic acid production and glyoxal consumption.

[0187] Figure 5 : Glycolate production in a 700 mL reactor with continuous substrate supply.

[0188] Figure 6 : Glycolate production in a 2000 mL reactor with substrate feeding.

[0189] Figure 7 : Glycolate production using free resting cells and crosslinked resting cells

[0190] Figure 8: % of bioconversion after 2 h in batch mode including free and cross-linked cell systems.

[0191] Figure 9 : Glycolate production by carrageenan-immobilized cells.

[0192] Figure 10 : % of bioconversion after 2 h in batch mode including free cells and cells treated with glutaraldehyde (GA) and / or polyethylenimine (PEI).

[0193] Figure 11 : Glycolate production over time using periplasmic whole-cell biocatalyst and cytoplasmic whole-cell biocatalyst at pH 7.5 (·), 6.5 (■), 5.5 (●), 4.5 ( ) and 3.5 (×).

[0194] Figure 12 : Glycolate production over time using purified glyoxalase (cytoplasmic) and the periplasmic fraction from periplasmic whole-cell biocatalyst containing glyoxalase at pH 7.5 and 4.5.

[0195] Figure 13 : Glycolate production using periplasmic whole-cell biocatalyst and cytoplasmic whole-cell biocatalyst in medium buffered with glycolic acid and without pH adjustment at acidic pH.

[0196] The present invention will now be illustrated by the following non-limiting examples.

[0197] Examples

[0198] Example 1: Materials and methods

[0199] 1.1 Materials

[0200] 1.1.1 Plasmids for expressing glyoxalase into the periplasmic space or cytoplasmic space

[0201] Prepare different plasmids:

[0202] 1) pET28b-yhbO, a plasmid designed for cytoplasmic production of YhbO enzyme and IPTG-inducible T7 promoter, was prepared as follows: from The pET28b plasmid (ori pBR322, kanamycin, T7 promoter) of (Ref 69864) was used to express the yhbO gene (SEQ ID NO:2) encoding a deglycase (glyoxalase) from Escherichia coli into the cytoplasm of the cells. The yhbO gene was extracted from Escherichia coli MG1655 (ATCC700926) using a genomic DNA purification kit (ThermoFisher Ref:K0721), then amplified by PCR (polymerase chain reaction) using appropriate conditions and primers, and then inserted into the linearized plasmid by homologous recombination. The primers used for gene amplification are generally of known types. Those skilled in the art know how to select and use them in PCR technology.

[0203] 2) pET22b-yhbO, a plasmid designed for the periplasmic production of the YhbO enzyme, carrying an IPTG-inducible T7 promoter, was prepared as follows: from (Ref 69744) The pET22b plasmid (ori pBR322, ampicillin, T7 promoter, pelB) carries a signal sequence pelB at the N-terminus and a His-tag sequence at the C-terminus. This plasmid was used to express the yhbO gene encoding a deglycase (glyoxalase) from Escherichia coli. The yhbO gene was extracted from Escherichia coli MG1655 (ATCC 700926) using a genomic DNA purification kit (ThermoFisherRef:K0721), and then amplified by PCR (polymerase chain reaction) using appropriate conditions and primers (Taq 2X Master Mix (M0270) and then inserted into the linearized plasmid by homologous recombination. The primers used for gene amplification are generally of known types. Those skilled in the art know how to select and use them in PCR technology.

[0204] 3) pET28b is a plasmid similar to pET28b-yhbO but without the yhbO gene and was used as a negative control.

[0205] 4) pET28b-yhbO-His is similar to pET28b-yhbO disclosed above but has a His-tag for the purification of cytoplasmic proteins in Example 2.9.

[0206] 5) pZa3-proD-yhbO is derived from the pZA33 plasmid A plasmid of (p15A, pA1lac0-1, chloramphenicol Chm), in which the PA1lac0-1 promoter has been replaced by the constitutive proD promoter. This plasmid is used to express the yhbO gene encoding a deglycase (glyoxalase) from Escherichia coli for the production of YhbO protein in the cytoplasm. The yhbO gene was extracted from Escherichia coli MG1655 (ATCC 700926) using a genomic DNA purification kit (ThermoFisher Ref:

[0207] K0721), and then amplified by PCR (polymerase chain reaction) using appropriate conditions and primers (Taq 2X Master Mix (M0270) and inserted into the linearized plasmid by homologous recombination. Primers for gene amplification are usually of known types. Those skilled in the art know how to select and use them in PCR technology.

[0208] 1.1.2 Construction of Strains (Biocatalysts)

[0209] Different types of biocatalysts (transformed Escherichia coli strains) were produced and used in the following examples.

[0210] Four Escherichia coli BL21(DE3) (Ref: C2527, ) strains were prepared, each carrying one of the above plasmids:

[0211] 1) pET28b-yhbO, a plasmid designed for cytoplasmic production of the YhbO enzyme, IPTG-inducible;

[0212] 2) pET22b-yhbO, a plasmid designed for periplasmic production of the YhbO enzyme, IPTG-inducible; or

[0213] 3) pET28b empty, as a negative control;

[0214] 4) pET28b-yhbO-His, a plasmid designed for cytoplasmic production of the YhbO-His enzyme, IPTG-inducible, and purified as described in Example 2.9;

[0215] An Escherichia coli MG1655 (ATCC 700926) strain carrying the above plasmid pZa3-proD-yhbO (a plasmid designed for constitutive expression of cytoplasmic production of the YhbO enzyme) was prepared. The prod constitutive promoter enables the use of the common laboratory strain Escherichia coli MG1655 that does not express T7 RNA polymerase, which is necessary when using the T7 promoter, and reduces the production cost of the catalyst by eliminating the use of IPTG.

[0216] Transform commercial competent cells such as BL21(DE3)(Réf:C2527, ) according to the supplier's protocol. Transform the MG1655 strain using the TSS protocol described by Chung and Miller (Chung et al., 1989). The strains used are given in Table 3 below.

[0217] Table 3: Escherichia coli strains used

[0218]

[0219] The glyoxalase-producing strain (“biocatalyst”) was obtained in TB medium with the following components (per liter) through a two-step enrichment culture procedure: 24 g yeast extract, 20 g tryptone, 17 mmol KH2PO4, 72 mmol K2HPO4, 4 g glycerol, 10 mg FeSO4, 2 mmol MgSO4, 1 mmol CaCl2, and the corresponding antibiotics (100 mg / L ampicillin for the BL21-yhbO-periplasmic strain, 50 mg / L kanamycin for the BL21-YhbO-His cytoplasmic strain, and 30 mg / L chloramphenicol for the MG1655-YhbO-cytoplasmic strain). After growing overnight on solid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L agar) at 37 °C, the strain was first pre-cultured in 10 ml TB medium at 37 °C and 200 rpm for 4 h, then inoculated into a 500 mL culture in a 2 L conical flask and shaken at 37 °C and 200 rpm. The expression of glyoxalase was induced by 0.1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) in the mid-exponential growth phase (0.4 < DO600nm < 0.6) and shaken overnight at 20 °C and 200 rpm for about 16 h, except for the constitutive strain which was directly incubated at 30 °C without IPTG. The cell pellet was harvested by centrifugation at 4500 rpm for 10 minutes. According to the standard protocol, the standard relationship established between the dry cell mass per mL and the optical density at 600 nm is as follows: dry weight cell (dwc) (mg / ml) = 0.6897 * OD 600nm

[0220] Dowex resins and chemicals used for production, purification and analysis, such as NaOH, HCl, H2SO4, NaCl, KCl, glyoxal, glycolic acid, were all obtained from Sigma Co. Other solutions of glutaraldehyde (GA), polyethyleneimine (PEI) and carrageenan are also commercially available

[0221] 1.2 Whole-cell catalytic reaction

[0222] The whole cells prepared as above (E. coli strains transformed with plasmids and constituting the "biocatalyst") were centrifuged at 4500 rpm for 10 minutes and resuspended in water.

[0223] As disclosed in some other embodiments below, the whole cells prepared as above can also be fixed with glutaraldehyde and / or polyethyleneimine to facilitate further recovery of the biocatalyst.

[0224] Cells carrying the yhbO gene (used as the "biocatalyst") were introduced into the reaction mixture. This included a buffer for stabilizing the pH and a glyoxal substrate. To ensure optimal enzyme activity and minimize production costs, the composition was deliberately made as simple as possible.

[0225] The glycolic acid production reaction was placed at 25 °C with stirring (200 rpm). The components of the reaction mixture were: 100 mM HEPES buffer pH 7.5, 100 mM glyoxal. The production was carried out in volumes of 2 mL, 20 mL, 200 mL, and 2000 mL in 2 mL Eppendorf tubes, 50 mL, 500 mL, and 5000 mL flasks respectively. A negative control without substrate (glyoxal) and a negative control (cells transformed with an empty plasmid) were carried out. Samples were collected by taking 300 μl of the reaction medium, centrifuged at 20,000 g for 2 minutes, and then filtered (0.2 μm).

[0226] The fed-batch catalytic technique for the bioconversion of glyoxal to glycolic acid was further tested. The biological production of glycolic acid was carried out in a 700 mL reactor with a catalytic broth containing phosphate buffer at an initial concentration of 200 mM at pH 7.5 and 30 °C in a fed-batch manner, stirred with a single Rushton-type stirrer with 6 blades at 1000 rpm, and an air flow of 150 mL / min. The initial volume of the reaction mixture was set to 300 mL to have sufficient volume for feeding and pH adjustment. Glyoxal (6.2 M) was continuously added at 0.59 mL / min by a peristaltic pump. The pH value was adjusted to 7.5 with 3 M NaOH.

[0227] Another biological production of glycolic acid was carried out in a 2000 mL reactor and was carried out in a phosphate buffer at an initial concentration of 200 mM at pH 7.5 and 25 °C, stirred with two 6-blade Rushton-type stirrers at 600 rpm, and an air flow of 100 mL / min. The initial volume of the reaction mixture was set to 500 mL. Glyoxal (8.7 M) was continuously added by a peristaltic pump. The flow rate was adjusted according to the amount of glycolate produced within 1 hour. The pH was adjusted to 7.5 with 8.3 M NaOH.

[0228] The reaction was carried out in a phosphate buffer at an initial concentration of 200 mM at pH 7.5 and 25 °C, stirred with two 6-blade Rushton-type stirrers at 600 rpm, and an air flow of 100 mL / min. The initial volume of the reaction mixture was set to 500 mL. Glyoxal (8.7 M) was continuously added by a peristaltic pump. The flow rate was adjusted according to the amount of glycolate produced within 1 hour. The pH was adjusted to 7.5 with 8.3 M NaOH.

[0229] After a batch cycle, the catalytic broth and Escherichia coli cells (biocatalyst) were separated by centrifugation at 10,000 g for 15 minutes.

[0230] Free enzyme-catalyzed reaction

[0231] To demonstrate the stability provided by the cellular environment, bioconversion of glyoxal to glycolic acid was carried out at pH 4.5 and 7.5 using purified enzymes with his-tag from the cytoplasm and without tag from the periplasm under the same conditions as above.

[0232] YhbO-His cytoplasmic glyoxalase was purified from 1000 mL of culture of BL21-YhbO-His using affinity purification due to the C-terminal His-tag.

[0233] YhbO periplasmic glyoxalase was isolated from 1000 mL of culture of BL21-pET22b by osmotic shock as further disclosed in point 1.8. The purified protein or periplasmic fraction was resuspended in 500 mL of 0.9% NaCl and directly transferred to a 2 L fermenter. The temperature was maintained at 25 °C and stirring was carried out at 200 rpm using a single Rushton impeller with 6 blades. The reaction was initiated by adding 12 mL of 8.7 M glyoxal (final concentration 200 mM). When the glyoxal was completely bioconverted, 12 mL of 8.7 M glyoxal was added again to the bioreactor. The pH was adjusted using 8.3 M NaOH and 3 M H2SO4. To determine the activity limit of each catalyst, different pH values were evaluated: pH 4.5 and pH 7.5.

[0234] 1.3 Analytical methods

[0235] The concentrations of glyoxal (substrate) and glycolic acid (product) were measured by UHPLC (Dionex UltimateTM 3000) equipped with a Phenomenex ROA-organic acid H+ (8%) column (300 x 7.8 mm) and a pre-column (50 x 7.8 mm), and using 5 mM H2SO4 as the mobile phase at 0.5 mL / min for 35 minutes. Detection was carried out by a refractometer (Shodex RI-101) and a UV detector (Dionex UltiMate 3000 Diode Array Detectors 3000 (RS)). Samples were filtered at 0.2 μm. The concentrations of glycolic acid and glyoxal were monitored using HPLC-RI / UV analysis. External calibration curves for glyoxal and glycolic acid were prepared as Figure 1 shown.

[0236] The structural confirmation analysis was carried out by the Toulouse Institute of Chemistry (ICT) using nuclear magnetic resonance (NMR), infrared (IR), and mass spectrometry (MS).

[0237] 1.4 Ion exchange chromatography

[0238] The extraction of glycolic acid was carried out by anion exchange chromatography using Dowex 1x8 resin in the OH- form with a capacity of 1.2 mEq / mL. An 8 mL or 120 mL column connected to a peristaltic pump was used to hold the resin. First, the sample was added to the ion exchange column at a rate of 2.5 mL / min using the peristaltic pump. Then, the ion exchange resin column was washed with deionized water.

[0239] Sequential elution was carried out with NaOH at different concentrations (60, 120, 200 mM). For subsequent use, the stored ion exchange resin was washed with 1 M NaOH. Then the target fraction was eluted on a cation exchange resin (DOWEX 50WX8 H+ form) to convert the sodium salt of glycolic acid to glycolic acid.

[0240] 1.5 Drying and evaporation of water

[0241] Evaporation was first carried out using a rotary evaporator (Buchi R-215) at 40 °C and 40 mbar, and then completed by freeze-drying (Epsilon 2-4LSC).

[0242] 1.6 Full mass spectrometry analysis of the product (glycolic acid)

[0243] The equipment used was a "Xevo G2 Q-Tof" mass spectrometer from Waters, equipped with an electrospray ionization (ESI) source and a time-of-flight (TOF) analyzer, capable of determining the mass accurate to the fourth decimal place and proposing the original molecular formula.

[0244] The exact mass of the substance was detected in the negative ion mode [M-H]−, i.e., 75.0082 when less than 1 mDa.

[0245] 1.7 Purification of the enzyme on nickel resin

[0246] a) Cell lysis

[0247] 15 OD 600The bacterial pellet from 1000 mL was resuspended in 100 mL of 50 mM Tris / HCl buffer, 300 mM NaCl, 20 mM imidazole (pH 8.0), the temperature was adjusted to 4 °C and the suspension was disrupted using a high-pressure homogenizer by passing through the homogenizer three times at 1100 bar. After each pass, the suspension was cooled to 4 °C before starting the next disruption cycle. The system was rinsed with 50 mL of 50 mM Tris / HCl buffer, 300 mM NaCl, 20 mM imidazole (pH 8.0). Then 150 mL of the suspension was centrifuged at 10000×g for 15 min at 4 °C to obtain a clarified solution. The clarified supernatant (crude extract, 150 mL) was directly applied to the IMAC column.

[0248] b) Affinity purification

[0249] The His-tag enzyme was purified by IMAC using the following conditions. Medium: Streamline Chelating, Ni-NTA; Column: XK 16 / 20; CV (column volume): 15 mL; Flow rate for sample application: 3 mL / min; Flow rate for washing and elution: 3 mL / min; Binding buffer: 50 mM Tris / HCl buffer and 300 mM NaCl, 20 mM imidazole (pH 8.0); Washing buffer: 50 mM Tris / HCl buffer and 300 mM NaCl, 40 mM imidazole (pH 8.0); Elution buffer: 50 mM Tris / HCl buffer and 300 mM NaCl, 300 mM imidazole (pH 8.0); Sample: Clarified lysate (crude extract) containing His-fusion protein from E. coli after high-pressure homogenization and centrifugation; Sample volume: 150 mL; Fraction volume: 1.5 mL. After loading the sample and washing the column with 10 CV of the binding buffer and washing buffer, stepwise elution was carried out using the elution buffer at 500 mM imidazole. The fractions containing the His-tag enzyme were analyzed by SDS-PAGE. The fractions were stored at 4 °C. The fractions containing the His-tag enzyme were combined and directly used for biotransformation analysis.

[0250] 1.8 Osmotic shock

[0251] The pellet from 1000 mL of the culture was resuspended in 200 mL of osmotic shock buffer 1 (20 mM Tris-HCl, 0.25 mM EDTA, 200 g / L sucrose, pH 8) and incubated on ice for 10 min. After centrifugation (16,000×g, 10 min, 4 °C), the pellet was resuspended in 200 mL of osmotic shock buffer 2 (20 mM Tris-Hcl, 0.25 mM EDTA, pH 8) and incubated as above. After further centrifugation as above, the supernatant containing the periplasmic protein fraction was transferred to a new reaction tube and stored at 4 °C (Eichmann et al., 2019).

[0252] Take 10 μL samples of each supernatant, add them to 30 μL of loading buffer 4X + 50 mM DTT, and denature at 95 °C for 10 minutes. Load the samples onto an SDS-PAGE gel.

[0253] Example 2: Results of bioproduction of glycolic acid from glyoxal using whole-cell biocatalyst

[0254] The reaction scheme for the production of glycolic acid from glyoxal is as follows:

[0255]

[0256] 2.1 Production of glycolic acid from glyoxal using a biocatalyst in the periplasmic space in volumes ranging from 2 mL to 2000 mL

[0257] The production of glycolic acid from glyoxal was carried out in volumes of 2 mL, 20 mL, 200 mL, and 2000 mL. The reaction mixture consisted of:

[0258] - Biocatalyst: 2DO / ml BL21(DE3)pET22b-YhbO cells

[0259] - Substrate: Glyoxal (100 mM)

[0260] - Buffer solution: HEPES pH 7.5 (100 mM)

[0261] - Temperature: 30 °C

[0262] Within 20 h, 74 mM of glycolic acid was produced with a conversion rate of 75%. UPLC analysis showed that regardless of the production scale, glyoxal was converted to glycolic acid without the formation of by-products ( Figure 2 ), in addition, the kinetic curves of glycolic acid production were consistent in the four tested volumes ( Figure 3 ). Therefore, it is possible to increase the volume by 1000-fold without loss of efficiency.

[0263] Production was carried out under the following conditions with a higher amount of catalyst (16 DO / ml instead of 2 DO / ml):

[0264] - Biocatalyst: 16 DO / ml BL21(DE3)pET22b - YhbO cells

[0265] - Substrate: Glyoxal (100 mM)

[0266] - Buffer solution: HEPES pH 7.5 (100 mM)

[0267] - Volume: 2 mL

[0268] We observed the production of 100 mM or 7.6 g / L of glycolic acid with a 100% yield within 3 hours ( Figure 4 ).

[0269] Thus, the whole - cell biocatalyst containing BL21 - YhbO cells is an effective tool for the production of glycolic acid from glyoxal. We demonstrated a stable glycolic acid production of 5.7 g / L with a conversion rate of 75% in the volume range from 2 mL to 2000 mL. By optimizing the amount of biocatalyst used, the production of 7.6 g / L of glycolic acid from 5.8 g / L of glyoxal with a 100% yield within 3 hours has been achieved.

[0270] 2.2 Production of glycolic acid from glyoxal in fed - batch catalysis

[0271] - Biocatalyst: 128 DO / ml BL21(DE3)pET22b - YhbO cells

[0272] - Substrate: Glyoxal (6.2 M, flow rate of 800 mM / h)

[0273] - Buffer solution: Phosphate buffer pH 7.5 (200 mM)

[0274] - pH regulation: NaOH (3 M) and HCl (3 M)

[0275] - Volume: 547 mL in a 700 - mL reactor

[0276] - Temperature: 30 °C

[0277] The fed - batch catalysis technique for the bioconversion of glyoxal to glycolic acid was also tested. The first fed - batch reaction was carried out in a reactor with a capacity of 700 mL ( Figure 5 ). The parameters are described in section 1.2. Fed - batch catalysis with limited substrate accumulation was implemented, so glyoxal was consumed immediately.

[0278] The production of 120 g / L glycollate with a 99% yield was observed within 7 hours, with no residual glyoxal (Table 4). The reaction could not proceed beyond this point as the maximum volume of the reactor had been reached. Adjusting the pH with 3M NaOH accounted for nearly 60% of the volume added during the reaction process.

[0279] The production process allows for maximizing the yield through pH adjustment, but optimization is required to minimize product dilution due to the addition of sodium hydroxide.

[0280] Table 4. Performance of biocatalyst for glycollate production in a 700 mL capacity reactor

[0281]

[0282] The production of glycollate in the reactor is expected to reach a productivity of 23 g / L / h, and several improvement points can even optimize the performance:

[0283] - The NaOH concentration can be increased to limit the volume increase;

[0284] - A reactor with a larger capacity can be used to allow for a higher volume yield and finer control of the base, acid, and feed rate;

[0285] - The temperature can be lowered to reduce the process cost; and

[0286] - The amount of cells can be optimized to reduce costs.

[0287] Therefore, the following fed-batch biotransformation with the described improvement points was carried out, namely an increased NaOH concentration of 8.3M, a reactor capacity 2.5 times larger, the temperature reduced to 25 °C, and the final catalyst concentration reduced by 19%.

[0288] Therefore, the second fed-batch reaction was carried out in a 2000 mL bioreactor (fully stirred open reactor) ( Figure 6 )

[0289] - Biocatalyst: 128 DO / ml BL21(DE3)pET22b-YhbO cells

[0290] - Substrate: Glyoxal (6.2M, flow rate 800 mM / h)

[0291] - Buffer solution: Phosphate buffer pH 7.5 (200 mM)

[0292] - pH adjustment: NaOH (8.3M) and H2SO4 (4M)

[0293] - Volume: 1086 mL in a 2000 mL reactor

[0294] - Temperature: 25 °C

[0295] The main performance data are shown in Table 5. Finally, glycolic acid was obtained at a yield of 98% (equivalent to 1.29 g of glycolate per g of glyoxal), with a concentration of 247 g / L, and a volumetric productivity of 5.7 g / L / h over 43 h, which is the highest reported level of glycolic acid production. 334 g of glycolate was produced from 259 g of glyoxal and 44.6 g of biocatalyst. No residual glyoxal was observed at the end of the reaction.

[0296] The specific activity of the biocatalyst was 0.37 g 乙二醛 / g 生物质 / h at the start of the reaction and 0.28 g 乙二醛 / g 生物质 / h at the end of the reaction. This may be due to the accumulation of glyoxal at the start of the experiment, which may have damaged the cells or blocked the fixed part of the reactor with a large amount of cells, such that despite strong stirring, part of the biomass could not be used for the reaction.

[0297] Table 5. Performance of the biocatalyst in a 2 L reactor with glyoxal feed and pH adjusted to 7.5 over a 43 h reaction

[0298]

[0299] 2.3 Structural confirmation by nuclear magnetic resonance (NMR), infrared (IR) and mass spectrometry (MS)

[0300] Purified glycolic acid obtained by ion exchange chromatography was used for structural analysis. NMR (proton, carbon, 2D HSBC and 2D HSQC), IR and MS were performed by the Institute of Chemistry of Toulouse.

[0301] - NMR analysis showed that the main product was glycolic acid. Only 0.05% impurities were found in the proton NMR.

[0302] - IR analysis showed 95% correlation with the commercial standard of glycolic acid. The difference could be explained by the different crystalline forms.

[0303] - Full mass spectrometry (MS) analysis showed that the original molecular formula obtained by mass spectrometry of the glycolic acid produced by the method of the present invention corresponded to the expected original molecular formula, i.e., C2H3O3, which means a perfect correlation between the commercial standard and the produced glycolic acid.

[0304] The production of glycolic acid was 247 g / L, the yield was 99%, and the volumetric productivity was 5.7 g / l / h, exceeding the performance of all published fermentation processes. The process temperature was also successfully reduced from 30 °C to 25 °C; making the operating conditions competitive from an energy perspective. After anion exchange chromatography, the purified product was recovered with an HPLC purity > 99%. Three structural analyses confirmed the high purity of glycolic acid, with only 0.05% impurities found in the NMR. The purified glycolic acid is a white crystalline powder particularly suitable for cosmetic grade.

[0305] The following examples illustrate the production of glycolic acid from glyoxal using a biocatalyst (whole cell) that produces glyoxalase into the cytoplasm and periplasm of the biocatalyst (with or without immobilization).

[0306] 2.4 - Influence of periplasmic and cytoplasmic biocatalysts and biocatalyst immobilization on the biotransformation reaction

[0307] Three types of catalysts were produced as disclosed in Example 1.2, all being Escherichia coli BL21(DE3) strains carrying different plasmids, the plasmids being as follows: 1) pET28b - yhbO, a plasmid designed for cytoplasmic production of the YhbO enzyme, IPTG - inducible, 2) pET22b - yhbO, a plasmid designed for periplasmic production of the YhbO enzyme, IPTG - inducible, and 3) pET28b empty, as a negative control.

[0308] The BL21(DE3) strains were grown in 100 mL of TB (37 °C, 200 rpm); after induction with 0.1 mM IPTG, the protein production phase was then carried out overnight at 20 °C, and the catalysts were harvested by centrifugation at 4000 rpm for 10 minutes. For each strain, half of the cells were resuspended in 25 mL of water (these cells constituted the "free biocatalyst"), and the other half of the cells were resuspended in 25 mL of 0.1% glutaraldehyde solution at room temperature for 1 h (these cells constituted the "immobilized or cross - linked biocatalyst"). The harvested cells were washed with 25 mL of PBS1X and recovered by centrifugation.

[0309] The reaction was carried out at 25 °C and 200 rpm and consisted of 67 mg dcw catalyst / ml in a solution of 5 ml of glyoxal (250 mM) and HEPES pH 7.5 (500 mM).

[0310] The bioproduction process was monitored by high - performance liquid chromatography (HPLC) by taking 0.5 mL samples at fixed time intervals. The glyoxalase activity was the same regardless of whether the glyoxalase was located in the periplasmic or cytoplasmic space ( Figure 7), the free catalyst has the same specific activity, equal to or greater than 42.6 μmol glyoxylate.min -1 .g -1 dcw catalyst. Treatment with glutaraldehyde seems to affect the activity, yet about 75% of the activity is retained: the reaction remains efficient and is completed within 1 h.

[0311] These results demonstrate that glyoxylase activity and glyoxylate production are the same whether the glyoxylase is located in the periplasmic or cytoplasmic space. Immobilization of the biocatalyst seems to slow down the reaction and / or affect the glyoxylase activity, but the maximum level of glyoxylate production is obtained after 1 h of treatment.

[0312] 2.5 - Biocatalyst recycling

[0313] Two BL21(DE3) strains were produced as disclosed in Example 1.2, carrying plasmids 1) pET28b - yhbO, a plasmid designed for cytoplasmic production of the YhbO enzyme, IPTG - inducible, or 2) pET22b - yhbO, a plasmid designed for periplasmic production of the YhbO enzyme, IPTG - inducible, and cultured as described in Example 2.4.

[0314] As described in Example 2.4, the two strains were cross - linked with glutaraldehyde. To study the effect of recycling on the extent of glyoxal conversion, biotransformation reactions were carried out in batch mode with free cells and cross - linked cells. After 4 cycles, complete conversion of glyoxal was observed within less than 2 h for six types of catalysts ( Figure 8 ).

[0315] These results indicate that the biocatalysts can be recycled 2 to 3 times (as in the 4 - cycle case above) without losing their catalytic efficacy, as we obtained complete conversion of glyoxal within less than 2 h. Immobilization facilitates harvesting of the biocatalyst at the end of one cycle and its continuous or delayed reuse in the next cycle.

[0316] 2.6 Preparation of carrageenan beads with Escherichia coli BL21(DE3) / pET22b - yhbO and Escherichia coli BL21(DE3) / pET28b - yhbO respectively

[0317] Two catalysts were produced as disclosed in Example 1.2: 1) Escherichia coli BL21(DE3) / pET22b - yhbO produces glyoxylase III from Escherichia coli into the periplasmic space, and 2) Escherichia coli BL21(DE3) / pET28b - yhbO produces the enzyme into the cytoplasmic space.

[0318] Both catalysts were immobilized with carrageenan. The strain was cultured in TB at 37 °C and 200 rpm until the OD600nm reached 0.6, where 0.1 mM IPTG was added to the medium to induce YhbO expression overnight at 20 °C. The harvested cells were resuspended in water and diluted twice in a 2.5% carrageenan solution.

[0319] The suspension was kept in a water bath at 60 °C and was dripped dropwise into a 0.3 M KCL solution at 4 °C using a syringe. The reaction was carried out at 30 °C and 200 rpm and consisted of a mixture of all the beads (carrageenan beads encapsulating the biocatalyst) in 5 mL of a solution containing 250 mM glyoxal and 500 mM HEPES pH 7.5. For both catalysts, 100% conversion was reached after 3 h.

[0320] BL21(DE3) / pET22b-yhbO immobilized cells showed a specific activity of 60.7 μmol glyoxylate.min -1 .g -1 -1 dcw of the catalyst, while BL21(DE3) / pET28b-yhbO immobilized cells had an activity of 51.8 μmol glyoxylate.min -1 .g -1 -1 dcw of the catalyst ( Figure 9 ).

[0321] When the cells are encapsulated (in carrageenan beads in this example), the periplasmic secretion of glyoxalase III is favored, which is of course due to the better transport of the molecule across the periplasm.

[0322] 2.7 Preparation of glutaraldehyde (GA) / polyethyleneimine (PEI)-crosslinked BL21(DE3) / pET22b-yhbO transformants expressing glyoxalase III from Escherichia coli

[0323] The BL21(DE3) / pET22b-yhbO strain was used to compare different treatments for stabilizing glyoxalase activity, flocculating the cells with polyethyleneimine (PEI) and crosslinking the cells with glutaraldehyde to provide reaction products that can be used multiple times in biocatalytic processes.

[0324] Cells were produced as described in Example 2.4. The volume equivalent to 435 mg dcw cells was centrifuged at 4000 rpm for 10 min. The harvested cells were washed with 50 mL of PBS1X and recovered by centrifugation. Table 6 provides different test conditions.

[0325] Table 6: Conditions for treating the BL21(DE3) / pET22b-yhbO strain with glutaraldehyde (GA) and / or polyethyleneimine (PEI).

[0326]

[0327] The reaction was carried out at 30 °C and 200 rpm and consisted of 90 mg / ml of free or treated cells in a solution of 5 ml of glyoxal (250 mM) and HEPES pH 7.5 (50 mM). The bioproduction process was monitored by high performance liquid chromatography (HPLC) by taking 0.5 mL samples at fixed time intervals. The pH of each sample was measured and, if necessary, the pH was readjusted to 7.5 using 3 M NaOH in the reaction. After 18 h, the cells were harvested and reused in a fresh mixture containing glyoxal (250 mM) and HEPES pH 7.5 (50 mM). Three reactions were carried out in batch mode and, starting from the second reuse of free cells, the bioconversion decreased by 50% ( Figure 10 ). The difference from Example 2.5 can be explained by the lower concentration of HEPES buffer and the exposure of the cells to acidic pH between samples. GA and PEI seem to protect the treated cells from pH variations and their catalytic efficiency remained stable at about 90%. The use of cationic flocculants and crosslinkers to stabilize the microorganism is suitable for the optimized bioconversion of glyoxal.

[0328] 2.8 Bioconversion of glyoxal to glycolic acid in methanol

[0329] The BL21(DE3)pET28b-yhbO strain was prepared as described in Example 1.2. The reaction consisting of 67 mg dcw catalyst in 1 ml of methanol solution containing glyoxal (200 mM) was carried out at 25 °C and 200 rpm. The reaction was monitored by high performance chromatography (HPLC) by taking 0.2 mL samples after 3 h. Complete conversion of glyoxal to glycolic acid was observed, indicating the formation of glycolic acid in the organic solvent.

[0330] 2.9: Effect of pH on the conversion of glyoxal to glycolic acid

[0331] The production of glycolic acid at different acidic pH levels was compared using periplasmic whole cell biocatalysts and cytoplasmic whole cell biocatalysts.

[0332] In a 2 L reactor Whole - cell cytoplasmic glyoxalase (MG1655 - YhbO cytoplasmic) and whole - cell periplasmic glyoxalase (BL21 - YhbO periplasmic) were compared in batch mode for the bioconversion of glyoxal to glycolic acid. After production, the cells were resuspended at 30 DO / mL in 500 mL of 0.9% NaCl and transferred directly to a 2 - L fermenter. The temperature was maintained at 25 °C and stirring was carried out at 200 rpm using a single Rushton impeller with 6 blades. The reaction was initiated by adding 12 mL of 8.7 M glyoxal (final concentration 200 mM). When the glyoxal was completely bioconverted, 12 mL of 8.7 M glyoxal was added again to the bioreactor. The pH was adjusted using 8.3 M NaOH and 3 M H2SO4. To determine the activity limits of each catalyst, different pH values (pH 6.5, pH 5.5, pH 4.5, and pH 3.5) were evaluated.

[0333] As Figure 11 shown, compared with the cytoplasmic whole - cell biocatalyst, the periplasmic whole - cell biocatalyst effectively produced glycolic acid at pH levels as low as 4.5.

[0334] The inventors explored whether this result was due to the enzyme structure in the periplasmic compartment that allows disulfide bond formation, or whether it was a result of the compartment itself. Therefore, additional production experiments were carried out using purified enzymes from the cytoplasmic whole - cell biocatalyst and the periplasmic whole - cell biocatalyst.

[0335] As Figure 12 shown, both enzymes showed the expected activity levels at pH 7.5, consistent with the previous results. However, under acidic conditions, especially at pH 4.5, they tended to precipitate, making them ineffective for the synthesis of glycolic acid. This phenomenon emphasizes the significant effect of the cell periplasmic compartment on the acid tolerance of the enzyme, which enables it to function effectively at pH levels above 4.5. Thus, the periplasmic environment plays a crucial role in maintaining the functionality of the enzyme under acidic conditions, which is not observed in the purified enzyme form.

[0336] 2.10 Effect of glycolic acid buffer on the bioconversion of glyoxal to glycolic acid

[0337] A buffer solution consists of an aqueous solution containing a combination of a weak acid and its conjugate base, or conversely, a weak base and its conjugate acid. When a small amount of strong acid or strong base is introduced into the buffer solution, its pH value remains relatively stable. Buffer solutions are widely used in various chemical processes to maintain a constant pH value.

[0338] The purpose of this experiment was to demonstrate the potential of glycolic acid (glycolic acid / glycolate) as a buffer. Specifically, the inventors intended to initiate the bioconversion process with pH regulation and then stop this regulation.

[0339] In a 2 L reactor the whole cell cytoplasmic and periplasmic glyoxalase activities were compared in batch mode.

[0340] After production, the BL21 - YhbO periplasmic or MG1655 - YhbO cytoplasmic cells were resuspended at 30 OD / ml in 500 mL of 0.9% NaCl and directly transferred to a 2 L fermenter. The temperature was maintained at 25 °C and stirring was carried out at 200 rpm using a single Rushton impeller with 6 blades. The reaction was initiated by adding 12 mL of 8.7 M glyoxal. When the glyoxal was completely biotransformed, 12 mL of 8.7 M glyoxal was added again to the bioreactor.

[0341] In the first step, the biotransformation was initiated with the pH adjusted to 6.5, using 8.3 M NaOH and 3 M H2SO4 to adjust the pH to 6.5. When the glycolic acid concentration reached the desired concentration (in the examples > 400 mM), the pH adjustment was stopped and the reaction was monitored. In the second step, glycolic acid was produced at a stable pH of 3.8 without the addition of salts. In the case of biocatalyst inactivation at acidic pH, the pH was temporarily readjusted to pH 6.5 to evaluate whether the inactivation was reversible and depended only on pH.

[0342] When using the cytoplasmic whole - cell biocatalyst, the pH dropped to 4.6 and only 120 mM of glycolic acid was produced. At the end of the reaction, glyoxal remained in the medium. This result was consistent with previous results, demonstrating that the cytoplasmic enzyme was not active at pH 4.5. When using the periplasmic whole - cell biocatalyst, the pH rapidly dropped and stabilized at 3.83( Figure 13 ). Under these conditions, we started the process with 462 mM of sodium glycolate and obtained a glycolic acid yield of 250 mM without pH adjustment. This result first confirmed the functional viability of the periplasmic whole - cell biocatalyst at pH 4.5, especially at pH 4. In addition, it demonstrated the effectiveness of the glycolate buffer, which led to a glycolic acid yield of approximately 35% without pH adjustment.

[0343] At the pKa of glycolic acid (pH = 3.8), the concentrations of the acid and the conjugate base are equivalent, as is known in the art (glycolic acid predominates at pH < pKa and glycolate predominates at pH > pKa). So theoretically, if we start with 460 mM of glycolate, we could obtain a glycolic acid yield of 460 mM without pH adjustment. Therefore, it should be possible to achieve a yield increase of 15% or more, since the enzyme seems to be active near the pKa.

[0344] These results demonstrate the significant advantages of periplasmic whole-cell biocatalysts for the production of organic acids at acidic pH levels.

[0345] This method offers several advantages, including reducing the amount of sodium hydroxide required. Additionally, it reduces the amount of cation-exchange resin needed to remove sodium in downstream process (DSP) steps.

[0346] All these results demonstrate that, in biochemical production according to the present invention, using whole cells of transformed microorganisms as biocatalysts, such as Escherichia coli strains transformed with plasmids containing the yhbO gene with / without signal sequences and constitutive / inducible promoters (i.e., BL21(DE3) and MG1655 strains), allows for the rapid (within a few hours) production of glycolic acid from glyoxal, with high yields, and mass and volume productivities exceeding the performance of all published fermentation methods.

[0347] These biocatalysts can be used as free biocatalysts or, advantageously, as immobilized biocatalysts, cross-linked with glutaraldehyde (GA) and / or polyethyleneimine (PEI), or encapsulated in carrageenan beads to facilitate their recovery and reuse in multiple reactions (2 to 4 cycles).

[0348] Finally, using biocatalysts that express glyoxalase in the periplasmic space of the microorganism offers several advantages in addition to producing active proteins that are properly folded in an oxidative environment. Small molecule solutes such as glyoxal and glycolic acid can freely equilibrate between the external solution and the periplasm, unlike cytoplasmic exchange that can be strictly regulated by membrane active transporters. Periplasmic enzymes have easy access to substrates, and products can immediately diffuse into the external medium for optimal reaction rates. Additionally, the separation of the periplasm from the metabolic complexity of the cytoplasmic environment is beneficial for bioproduction: in this subcellular compartment, side reactions are limited, resulting in little by-product formation and high yields. Finally, periplasmic whole-cell biocatalysts are advantageously tolerant of acidic conditions, which allows for the production of glycolic acid even at low pH, with reduced salt content and a simplified process (producing glycolic acid directly without adding sodium (NaOH)).

[0349] References

[0350] -Abdallah,J.,Caldas,T.,Kthiri,F.,Kern,R.,&Richarme,G.(2007).YhbOprotects cells against multiple stresses.Journal of Bacteriology,189(24),9140–9144.

[0351] -BASF.(2016).Glyoxal More Sustainable Solutions for Your Business.4.

[0352] -Chung, C.T., Niemela, S.L., & Miller, R.H (1989). One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proceedings of the National Academy of Sciences, 86(7), 2172–2175.

[0353] -Ehrmann M. (2007). 《The Periplasm-ASM Press, Washington DC》

[0354] -He, Y.C., Xu, J.H., Su, J.H., & Zhou, L. (2010). Bioproduction of Glycolic acid from glycolonitrile with a new bacterial isolate of alcaligenes sp. ECU0401. Applied Biochemistry and Biotechnology, 160(5), 1428–1440. https: / / doi.org / 10.1007 / s12010-009-8607-y

[0355] -KATAOKA, M., SASAKI, M., HIDALGO, A.-RGD, NAKANO, M., & SHIMIZU, S. (2001). Glycolic Acid Production Using Ethylene Glycol-Oxidizing Microorganisms. In Bioscience, Biotechnology, and Biochemistry (Vol.65, Issue 10, pp.2265–2270). https: / / doi.org / 10.1271 / bbb.65.2265

[0356] -Lee, C., Lee, J., Lee, JY, & Park, C. (2015). Characterization of the Escherichia coli yajl, yhbo and elbb glyoxalases. FEMS Microbiology Letters, 363(3), 1–7. https: / / doi.org / 10.1093 / femsle / fnv239

[0357] -Panova, A., Mersinger, LJ, Liu, Q., Foo, T., Roe, DC, Spillan, WL, Sigmund, AE, Ben-Bassat, A., Wagner, LW, O'Keefe, DP, Wu, S., Petrillo, KL, Payne, MS, Breske, ST, Gallagher, FG, & DiCosimo, R. (2007). Chemoenzymatic Synthesis of Glycolic Acid. Advanced Synthesis & Catalysis, 349(8–9), 1462–1474. https: / / doi.org / 10.1002 / adsc.200700061

[0358] -Wei, G., Yang, X., Gan, T., Zhou, W., Lin, J., & Wei, D. (2009). High cell density fermentation of Gluconobacter oxydans DSM 2003 for glycolic acid production. Journal of Industrial Microbiology and Biotechnology, 36(8), 1029–1034. https: / / doi.org / 10.1007 / s10295-009-0584-1

Claims

1. A method for the biochemically production of glyoxylic acid or its derivatives, which comprises the step of culturing a whole-cell biocatalyst in a suitable medium containing glyoxal as a substrate and optionally recovering glyoxylic acid from the medium, wherein the whole-cell biocatalyst comprises or consists of modified microbial cells that produce an enzyme capable of converting glyoxal into glyoxylic acid.

2. The method according to claim 1, wherein the enzyme capable of converting glyoxal into glyoxylic acid is produced into the cytoplasmic space or the periplasmic space of the microbial cell, preferably into the periplasmic space of the microbial cell.

3. The method according to claim 1 or 2, wherein the enzyme capable of converting the glyoxal substrate into glyoxylic acid is a glyoxalase having a catalytic triad Cys-His-Asp / Glu, in particular glyoxalase III (GLYIII), which advantageously comprises an amino acid sequence having a conserved signature DJ-1_PfpI domain, preferably glyoxalase III (SEQ ID NO:1) encoded by the YhbO gene from Escherichia coli or an amino acid sequence having at least 80% identity with SEQ ID NO:

1.

4. The method according to any one of claims 1 to 3, wherein the microorganism is a Gram-negative strain selected from the group consisting of the families Enterobacteriaceae, Alcaligenaceae, Vibrionaceae, and Pseudomonadaceae, in particular the family Enterobacteriaceae, preferably the Enterobacteriaceae belonging to the genus Salmonella, Yersinia, or Escherichia, more preferably the genus Escherichia, and even more preferably Escherichia coli.

5. The method according to any one of claims 1 to 4, wherein the microbial cell is modified by transformation with an expression cassette, which is on a circular DNA (plasmid) or integrated into the microbial genome.

6. The method according to claim 5, wherein the expression cassette comprises a gene encoding a glyoxalase as defined in claim 3 under the control of a constitutive promoter or an inducible promoter.

7. The method according to claim 5 or 6, wherein the expression cassette further comprises a secretion signal gene for targeting the glyoxalase to the periplasmic space, in particular selected from the group consisting of DsbA, EOX, LamB, MglB, MmAp, OmpC, OmpT, SufI, SfmC, STII, TolB, TorA, TorT, GIII, MalE, OmpA, PelB, PphoA, NlpA, preferably PelB.

8. The method according to any one of claims 1 to 7, wherein the modified microbial cells are immobilized, in particular immobilized on agar or carrageenan, or the microbial cells are crosslinked, in particular crosslinked with glutaraldehyde and / or polyethyleneimine (PEI), or both immobilized and crosslinked.

9. The method according to claim 8, which further comprises, at the end of the biotransformation reaction, the step of recovering the modified microbial cells that produce the enzyme capable of converting glyoxal into glyoxylic acid.

10. A whole-cell biocatalyst comprising or consisting of a modified microbial cell that produces an enzyme capable of converting glyoxal into glycolic acid as defined in any one of claims 3 to 6.

11. The whole-cell biocatalyst according to claim 10, wherein the biocatalyst is immobilized on agar or carrageenan, or cross-linked with glutaraldehyde and / or polyethyleneimine.

12. A method for producing a whole-cell biocatalyst as defined in claim 10, comprising the following steps: a) transforming a microbial cell with a plasmid containing an expression cassette comprising a gene encoding glyoxalase GLYIII under the control of a constitutive or inducible promoter, and optionally comprising a secretion signal gene for targeting the glyoxalase to the periplasmic space; or a’) modifying the microbial cell by integrating the expression cassette into the genome of the microbial cell; b) culturing the modified cell in a suitable medium for expressing glyoxalase; c) separating the cells from the supernatant by centrifugation; and d) optionally drying the cells and storing them at 4 °C or -20 °C or -80 °C.

13. A method for producing a glycolic acid derivative or a product obtained by a reaction using glycolic acid as a substrate, comprising at least one step in the method for the biocatalytic production of glycolic acid as defined in any one of claims 1 to 9.

14. An expression cassette comprising a nucleotide sequence encoding a glyoxalase as defined in claim 3, and optionally a secretion signal gene encoding a signal peptide as defined in claim 7, the secretion signal gene being used for targeting the glyoxalase to the periplasmic space of a microbial cell.

15. A nucleotide sequence encoding the expression cassette according to claim 14.

16. A vector comprising the nucleotide sequence according to claim 15.

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