Bioproduction of glycolic acid from glyoxal

Abstract

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

Description

[Technical Field]

[0001] The present invention is in the field of the production of molecules of interest by engineered microorganisms, and in particular the enzymatically catalyzed bioconversion of glyoxal to glycolic acid carried out in whole microbial cells. [Background technology]

[0002] Glycolic acid (HOCH2COOH) belongs to the family of alpha-hydroxy acids and is a two-carbon organic acid containing alcohol and carboxyl functional groups. In its monomeric form, glycolic acid is used primarily in cosmetics and cleansers. In its polymerized form, it forms polymers called polyglycolates, which exhibit remarkable mechanical properties suitable for packaging applications.

[0003] Commercially available glycolic acid is generally obtained by plant extraction and through three steps: - carbonylation of formaldehyde with carbon monoxide in the presence of an acid catalyst (H2SO4) at high temperature (200-250°C) and pressure (300-700 bar) (however, the final product is coloured by high-temperature oxidation and contains traces of formaldehyde, which is toxic, carcinogenic and potentially mutagenic, and is therefore restricted and prohibited in the cosmetics sector (European Regulation (CE) No. 1223 / 2009)); - Neutralization and re-acidification of monochloroacetic acid (MCA). MCA is produced from chlorine gas and acetic acid. However, the final product contains salts and chlorinated organic compounds; - enzymatic conversion of glycolonitrile to glycolic acid by nitrilase (however, the main drawback of this process is the presence of glycolonitrile, which is listed as a highly hazardous substance since it can polymerize under the influence of traces of acid or base, with the risk of fire or explosion). It is produced by chemical synthesis from

[0004] Several international publications have also reported the bioproduction of glycolic acid from ethylene glycol using yeast or bacteria that naturally convert ethylene glycol to glycolic acid, as this method has the advantage of operating under physiological conditions. Although ethylene glycol is inexpensive, bioproduction by fermentation has not, to date, led to industrial-scale production.

[0005] Although metabolic engineering has made it possible to exceed the production capacity of organisms that naturally produce glycolic acid from ethylene glycol, there remains a need to provide new bioproduction methods for glycolic acid under mild conditions (25° C., pH 7.0) or even under acidic conditions, and with improved performance.

[0006] The acid glycolic acid coexists in solution with its conjugate base glycolate, and the balance between these two forms depends on the reaction pH, as affected by the dissociation curve of the acid. At pKa (pH 3.83), 50% glycolic acid and 50% glycolate are present, and above pKa, the glycolate form predominates. In this description, the glycolic acid / glycolate combination is intended to be referred to as "glycolic acid." Therefore, since both forms coexist in the conditions of the present invention, the terms "glycolic acid production" and "glycolate production" are used interchangeably, and the terms "glycolic acid," "glycolate," and "glycolic acid / glycolate" are also used interchangeably.

[0007] Specifically, the present invention proposes a novel method for producing glycolic acid, comprising a microbial strain engineered to express glyoxalase either in the cytoplasm or periplasm of the cell. Specifically, the "biocatalyst" is a strain of Escherichia coli (E. coli) expressing a glyoxalase that effectively catalyzes the bioconversion of glyoxal to glycolic acid under mild conditions (25°C, 1 atmosphere, pH 7.5). In a preferred embodiment, the "biocatalyst" is a strain of E. coli expressing a glyoxalase in the periplasm that effectively catalyzes the bioconversion of glyoxal to glycolic acid under mild conditions (25°C, 1 atmosphere, pH 7.5) and even under acidic conditions (pH < 7), resulting in reduced salt usage in downstream steps (economic benefits, less salt usage, and simplified product purification).

[0008] Indeed, applicants have demonstrated that the periplasmic environment plays a key role in preserving the enzyme's functionality under acidic conditions, which is not observed in the purified enzyme form, and further demonstrated that periplasmic whole-cell biocatalysis allows for a 15% increase in production to be achieved compared to other systems. Summary of the Invention

[0009] A first object of the present invention is a method for the biochemical production of glycolic acid or a derivative thereof, comprising culturing a whole cell biocatalyst comprising or consisting of modified microbial cells producing an enzyme that converts glyoxal to glycolic acid in a suitable culture medium containing glyoxal as a substrate, and optionally recovering glycolic acid from the culture medium.

[0010] 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 to glycolic acid as defined in the present invention.

[0011] The present invention further comprises: a) transforming a microbial cell with a plasmid containing an expression cassette comprising a gene encoding an enzyme that converts glyoxal to glycolic acid (i.e., glyoxalase) under the control of a constitutive or inducible promoter, and optionally a secretion signal gene for delivering the enzyme to the periplasmic space; or a') engineering the microbial cell by integrating the expression cassette into its genome; b) culturing the modified cells in a culture medium suitable for expression of said enzyme (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. The present invention relates to a method for producing a whole cell biocatalyst as defined in the present invention, comprising:

[0012] Another object of the present invention is a method for producing glycolic acid derivatives or products obtained by reactions using glycolic acid as substrate, comprising at least one step of the method for the biochemical production of glycolic acid as defined in the present invention.

[0013] The present invention further relates to an expression cassette comprising a nucleotide sequence encoding an enzyme that converts glyoxal to glycolic acid (i.e., a glyoxalase), and optionally a secretion signal gene encoding a signal peptide as defined in the present invention for delivering said enzyme (i.e., a glyoxalase) to the periplasmic space of a microbial cell.

[0014] Another object of the present invention relates to a nucleotide sequence that codes for a polypeptide or an expression cassette as defined in the present invention.

[0015] The present invention further relates to a vector comprising a nucleotide sequence as defined in the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0016] Therefore, a first object of the present invention is a method for the biochemical production of glycolic acid or a derivative thereof, comprising culturing a whole cell biocatalyst comprising or consisting of modified microbial cells producing an enzyme that converts glyoxal to glycolic acid in a suitable culture medium containing glyoxal as a substrate, and optionally recovering glycolic acid from the culture medium.

[0017] By "biochemical production" in the present invention is meant production involving biological substances, preferably microbial cells, but which is not a fermentation process.

[0018] By "glycolic acid derivatives" is meant compounds derived from glycolic acid used as an intermediate, in particular compounds resulting from chemical polymerization, methylation or esterification of glycolic acid.

[0019] Enzyme (glyoxalase) "An enzyme that converts glyoxal to glycolic acid" is the enzyme that converts glyoxal to glycolic acid by the reaction: glyoxal + HO → glycolic acid + H + The enzyme of the present invention refers to an enzyme that can convert glyoxal to glycolic acid by the above reaction. The enzyme of the present invention converts glyoxal to glycolic acid, but also exhibits low activity toward glycolic acid, which accumulates in the reaction system. In particular, the activity of the enzyme of the present invention toward glycolic acid is preferably at least 1 / 10, more preferably at least 1 / 20, and even more preferably at least 1 / 100 of its activity toward glyoxal.

[0020] In particular, the enzyme of the present invention for converting glyoxal to glycolic acid has mainly the following physicochemical properties: - Acts on glyoxal to produce the corresponding glycolic acid; and - Active against glyoxal, but poor or no activity against glycolic acid It is a glyoxalase characterized by the following.

[0021] The enzyme glyoxalase of the present invention further has the following physicochemical properties: - molecular weight of approximately 20 kDa in gel filtration analysis; - an optimal reaction temperature between 25 and 37°C; and - optimum reaction under acidic conditions with a pH ranging from 1 to 8, in particular from 3.8 to 7 or from 5 to 8, preferably below 7, in particular from 3.8 to 7; may have:

[0022] The enzyme of the present invention has been demonstrated in the following in vitro tests: - mixing the enzyme with glyoxal in a buffer (e.g., potassium phosphate buffer) at pH 7.5 for 0.10 to 60 minutes; After centrifugation and filtration, the sample is loaded onto a high performance liquid chromatography (HPLC) column and glycolic acid production is monitored spectrophotometrically at 315 nm. can be selected in

[0023] In one particular embodiment, the glyoxalase is selected in the group consisting of members of the DJ-1 superfamily of glyoxalases known in humans, helminths, plants and bacteria. Preferably, the glyoxalase is selected from the group consisting of glyoxalases produced by E. coli YajL, YhbO, and ElbB. In a preferred embodiment, the present invention uses E. coli glyoxalase III (GLY III), which is known to protect cells from thermal, oxidative, pH, and UV stress (Abdallah et al., 2007). In a further preferred embodiment, the present invention uses E. coli glyoxalase III encoded by the yhbO gene (gene ID: NP_417622.2), because this enzyme has high affinity and activity for glyoxal (Km: 0.38 mM; kcat: 118.44 min -1 ;kcat / Km:3.11×10 5 min -1 M -1 , Lee et al., 2016). Thus, in a preferred embodiment, the enzyme that converts the glyoxal substrate to glycolic acid is a glyoxalase having the catalytic triad Cys-His-Asp / Glu, in particular a glyoxalase III (GLYIII) advantageously comprising the conserved hallmark DJ-1_PfpA domain, preferably the glyoxalase III (SEQ ID NO: 1) encoded by the yhbO gene from Escherichia coli (SEQ ID NO: 2), or an amino acid sequence having at least 80% identity to SEQ ID NO: 1.

[0024] In particular, the amino acid conserved DJ-1 PfpI domain of YhbO comprises the 168 amino acids set forth in SEQ ID NO:3, which corresponds to the nucleotide sequence represented by SEQ ID NO:4.

[0025] The following table discloses some sequences that are exemplified in the examples of the present invention, but the present invention is not limited to said sequences.

[0026] [Table 1A]

[0027] [Table 1B]

[0028] [Table 1C]

[0029] By "80% identity" is meant 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity. The percentage of identity referred to in the present invention is determined based on a global alignment of the sequences to be compared, i.e., by aligning the sequences in their entirety over their entire length, using any algorithm known to those skilled in the art, for example, the algorithm of Needleman and Wunsch 1970. This sequence comparison can be performed using any software known to those skilled in the art, for example, using the Needle software, with the "Gap Open" parameter equal to 10.0, the "Gap Extend" parameter equal to 0.5, and the "BLOSUM 62" matrix. The Needle software is available, for example, from the worldwide website ebi.ac.uk under the name "Align".

[0030] While the glyoxalase according to the present invention is not 100% identical to one of the above sequences, it has an amino acid sequence that is at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to such a reference sequence, and the sequence may have insertions, deletions, or substitutions with respect to the reference sequence. If it is a substitution, the substitution is preferably made with an "equivalent" amino acid, i.e., any amino acid whose structure is similar to that of the original amino acid and is therefore unlikely to alter the biological activity of the antibody. Such substitutions are also called "conservative mutations" or "conservative substitutions."

[0031] Examples of such substitutions are provided in Table 2 below.

[0032] [Table 2]

[0033] In particular, the present invention relates to a DNA encoding the glyoxalase. Specifically, the present invention relates to a DNA encoding the conserved hallmark DJ-1_PfpA domain (SEQ ID NO: 3) of glyoxalase YhbO, which DNA has the nucleotide sequence represented by SEQ ID NO: 4.

[0034] The present invention further relates to a DNA encoding glyoxalase III of E. coli, having the nucleotide sequence represented by SEQ ID NO: 2. In one particular embodiment, the amino acid sequence of the glyoxalase has 100% identity with SEQ ID NO: 1.

[0035] Furthermore, DNA encoding a protein containing any amino acid sequence resulting from the deletion, substitution, or addition of one or several amino acids in the amino acid sequence represented by SEQ ID NO: 1 (amino acid sequence of glyoxalase III YhbO) or SEQ ID NO: 3 (amino acid sequence of the hallmark DJ1-1_PfpA domain of YhbO) is also included in the DNA of the present invention, as long as the protein encoded by the DNA SEQ ID NO: 2 (nucleotide sequence of glyoxalase III YhbO) or SEQ ID NO: 4 (nucleotide sequence of the hallmark DJ1-1_PfpA domain of YhbO) has the activity of converting glyoxal to glycolic acid.

[0036] Methods for deleting, substituting, or adding a specific amino acid are well known to those skilled in the art, and include, for example, PCR using synthetic DNA primers having a nucleotide sequence that includes the deletion of a codon for a specific amino acid, substitution with a codon for another amino acid, or addition of a codon for another amino acid; and a method in which an enzyme gene produced by a known method, for example, chemical DNA synthesis, is ligated into a gene expression vector and expressed in a host such as E. coli by genetic recombination.

[0037] Genes for enzymes (e.g., glyoxalase) that can be used in the present invention can be generated by well-known methods, particularly by the following: - purifying the enzyme protein from a microorganism producing the enzyme that converts glyoxal to glycolic acid or from its culture broth; - determining a partial amino acid sequence using peptides obtained by digestion of the enzymatic protein with a protease; - performing PCR using primers synthesized based on the partial amino acid sequence and genomic DNA as a template by well-known methods (such PCR amplifies a portion of the enzyme gene and allows the nucleotide sequence of the gene to be determined. Inverse PCR can also be performed using DNA primers synthesized from the N-terminal amino acid sequence and the amino acid sequence near the C-terminus so that the signal sequence, N-terminal amino acid sequence, and C-terminal amino acid sequence can be determined). can be obtained as follows.

[0038] These different nucleic acid sequences encoding the enzymes (glyoxalase) are used in the present invention, as disclosed later in this description, using well-known methods to incorporate them into vectors (plasmids) used to transform microbial cells (hosts) or to stably integrate the nucleic acid sequences encoding the enzymes into the genome of the microbial organisms.

[0039] Biocatalyst (modified microorganism expressing said enzyme) By "biocatalyst" is meant a catalyst consisting of biological material with catalytic activity, in particular microbial cells. The microorganisms used in the present invention are microbial cultures or microbial cells capable of producing the enzyme that converts glyoxal to glycolic acid.

[0040] By "whole cell biocatalyst" in the present invention is meant that the catalyst comprises or consists of the whole cells of the microorganism that expresses the enzyme, in contrast to a purified enzyme. Indeed, in the present invention, the whole cells are used as such, rather than being lysed to extract and purify the expressed enzyme.

[0041] "Microorganisms" in the present invention include bacteria, yeasts, fungi, actinomycetes, animal or plant cells, but preferably bacteria, such as E. coli, particularly gram-negative bacteria for expression of enzymes in the periplasmic space.

[0042] By "modified microbial cells" is meant microbial cells that have been modified to express an enzyme, preferably a glyoxalase, that converts glyoxal to glycolic acid. In the present invention, the microbial cells are modified to express a glyoxalase, meaning that the level and / or activity of said enzyme is increased compared to the unmodified microbial cells. The expression "modified microorganism" includes "transformed microorganism," which refers to a microbial cell into which an exogenous nucleic acid has been incorporated. A microorganism capable of producing a specific enzyme that converts glyoxal to glycolic acid can be a wild-type strain, a mutant strain, or a transformant (recombinant) obtained by (i) incorporating the DNA of the enzyme (glyoxalase) into a vector (plasmid) and introducing it into a microorganism (host), or (ii) stably integrating the DNA encoding the enzyme into the genome of the microorganism, using well-known methods.

[0043] In one particular embodiment, the microorganism, in particular the E. coli strain exemplified later in the examples, such as the BL21(DE3) commercial strain (Ref: C2527, NEB®), is transformable according to the supplier's protocol. In another specific embodiment, an E. coli strain, e.g., strain MG1655, is rendered competent according to the TSS protocol described by Chung and Miller (Chung et al., 1989). Indeed, Chung and Miller developed a simple, one-step method for preparing competent E. coli using transformation and storage solution [TSS; 1xTSS is LB broth (pH 6.5) containing 10% (wt / vol) polyethylene glycol, 5% (vol / vol) dimethyl sulfoxide, and 50 mM Mg2+]. Cells are immediately ready for use after mixing with an equal volume of ice-cold 2xTSS. Genetic transformation is similarly straightforward: plasmid DNA is added and the cells are incubated at 4°C for 5–60 minutes. Because no heat pulse is required and the incubation time at 4°C is not critical, there are no critical timing steps in the transformation method. Transformed bacteria are grown and selected by standard methods.

[0044] In one particular embodiment, the microbial cells are modified by transformation with an expression cassette that is on a circular DNA (plasmid) or integrated into the microbial genome.

[0045] In one particular embodiment, the expression cassette comprises a gene encoding a glyoxalase as defined above under the control of a constitutive or inducible promoter. By "constitutive promoter" one may refer to the Anderson constitutive promoter collection, for example the proD promoter, which is a promoter derived from J23100 to J23119, as referenced on the website: http: / / parts.igem.org / Promoters / Catalog / Anderson. "Inducible promoter" may refer to an IPTG-inducible promoter, a light-inducible promoter, a T5 promoter, a T7 promoter, a lac promoter (pLac), a lacT5 promoter, a lacT7 promoter, a tac promoter (pTac), a rhaBAD promoter, an araBAD promoter, a tet promoter, a penP promoter, a cspA promoter, or a promoter containing the tetO or lacO operator as the operator sequence. In one particular embodiment, the inducible promoter is selected in the group consisting of the rhaBAD promoter, the lac promoter or the tac promoter.

[0046] In a particular embodiment, the enzyme that converts glyoxal to glycolic acid is produced in the cytoplasmic space or in the periplasmic space of the microbial cell, preferably in the periplasmic space of the microbial cell, In other words, the enzyme that converts glyoxal to glycolic acid is produced in the cytoplasmic space of the microbial cell or is transported into the periplasmic space, preferably in the periplasmic space of the microbial cell. In one particular embodiment, for example with regard to the expression of a glyoxalase enzyme into the periplasmic space of a microorganism, the microorganism is a Gram-negative bacterial strain, in particular selected from the group consisting of the families Enterobacteriaceae, Alcaligenaceae, Vibrionaceae and Pseudomonadaceae, in particular Enterobacteriaceae, preferably Enterobacteriaceae belonging to the genus Salmonella, Yersinia or Escherichia coli, more preferably the genus Escherichia, and even more preferably Escherichia coli.

[0047] In another specific embodiment, other microorganisms may be used, such as gram-positive bacteria, e.g., Actinomyces, Clostridium, Mycobacterium, Streptococci, Staphylococci, and Nocardia, fungi, e.g., Saccharomyces species and Candida species, particularly with regard to expression of the glyoxalase enzyme into the cytoplasmic space of the microorganism.

[0048] In the case of enzymes that are exported across the cytoplasmic membrane of a microorganism (also referred to as being produced in the periplasm of a microorganism), a signal peptide is generally encoded in a portion of the gene corresponding to several dozen amino acids from the initiation codon (meaning the upfront of the gene sequence). Such a signal peptide sequence is further cleaved to produce a mature enzyme during the process of exporting the enzyme protein synthesized within the cell across the cytoplasmic membrane of the cell.

[0049] In some cases, the native enzyme already contains its own signal peptide, but generally the native signal peptide sequence is replaced with another signal peptide sequence that is appropriate for the host microorganism, so that chimeric genes can be constructed and used according to the methods of the present invention.

[0050] Several groups of proteins can be used as signal peptides. Reference can be made to soluble periplasmic proteins or proteins peripherally associated with the periplasmic side of the inner or outer membrane. In a particularly preferred embodiment, a soluble periplasmic protein is used. In another particularly preferred embodiment, a protein associated with the periplasmic side of the inner or outer membrane, such as NlpA, is used, which allows for membrane anchoring.

[0051] By "signal peptide" there may be mentioned DsbA, EOX, LamB, MglB, MmAp, OmpC, OmpT, SufI, SfmC, STII, TolB, TorA, TorT, GIII, MalE, OmpA, PelB, PhoA and NlpA, preferentially PelB. In one particular 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.

[0052] Therefore, in the case of an enzyme produced in the cytoplasm of a cell of a microorganism used as a host, the enzyme gene is used without adding a nucleotide sequence encoding a signal peptide, or the enzyme gene from which the signal peptide sequence has been removed. In the preferred case of an enzyme produced in the periplasm of the cells of a microorganism used as a host, an enzyme gene is used in which a nucleotide sequence encoding a signal peptide replaces the original signal peptide or is added to the upfront of the enzyme gene.

[0053] Therefore, in a particular embodiment, the present invention further provides an expression cassette comprising an enzyme gene (yhbO gene, SEQ ID NO: 2) without any signal peptide sequence for producing the enzyme in the cytoplasm of a microorganism used as a host. In another particularly preferred embodiment, the present invention further provides an expression cassette comprising an enzyme gene (yhbO gene, SEQ ID NO: 2) and a signal peptide sequence (pelB, SEQ ID NO: 5) upfront of the enzyme gene, forming a fusion nucleotide sequence pelB-yhbO (SEQ ID NO: 8) for producing the enzyme in the periplasm of a cell. 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.

[0054] In a particularly preferred embodiment, the glyoxalase is expressed in the periplasmic space of a microbial cell. Translocation of the enzyme to the periplasm offers several advantages over cytosolic production, such as avoiding protease attack and increasing the likelihood of correct protein folding in an oxidizing subcellular environment. A number of periplasmic proteins have been shown to be involved in protein folding and preventing aggregation. These include Dsb (disulfide bond formation (Dsb)), which forms and isomerizes disulfide bonds. D i s sulfide b These include disulfide bond forming proteins, PPIases that catalyze the trans-to-cis isomerization of peptidyl-prolyl bonds, and general chaperones such as FpkA (Ehrmann, 2007). Disulfide bond formation can result in tightly folded structures that are resistant to proteases. Applicant further demonstrated in the Examples that the whole-cell biocatalyst of the present invention, when located in the periplasmic compartment according to a preferred embodiment, efficiently produces glycolic acid at a pH level as low as 4.5, while the whole-cell process located in the cytoplasmic compartment cannot function effectively at such a low pH level. The purified enzyme form is also not functional under acidic pH conditions. Applicant demonstrated that the periplasmic environment plays an important role in preserving the functionality of the enzyme under acidic conditions. Production of organic acids, such as glycolic acid, at an acidic pH offers the advantage of facilitating downstream processing. In this particular embodiment, the enzyme is fused to a signal peptide to direct or transport the enzyme to the periplasmic space. Most proteins can successfully translocate across the cytoplasmic membrane to the periplasm. When conditions are optimized for periplasmic accumulation, a large fraction of total cellular protein can be exported to the periplasm, with percentages ranging from 20% to 40% being achievable (Ehrmann, 2007).

[0055] Thus, in one particular embodiment, the expression cassette further comprises a signal peptide for delivering or transporting 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, PhoA, NlpA, preferably PelB.

[0056] Therefore, the present invention further relates to a whole cell biocatalyst comprising or consisting of modified microbial cells as defined above that produce an enzyme capable of converting glyoxal to glycolic acid.

[0057] The present invention further comprises: a) transforming a microbial cell with a plasmid containing an expression cassette comprising a gene encoding an enzyme that converts glyoxal to glycolic acid under the control of a constitutive or inducible promoter, and optionally a signal peptide for targeting said enzyme to the periplasmic space; or a') engineering the microbial cell by integrating said expression cassette into its genome; b) culturing the modified cells in a culture medium suitable for expression of said enzyme; 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. The present invention also relates to a method for producing a whole cell biocatalyst as defined above, comprising:

[0058] In a particularly preferred embodiment, the microbial cell (host) is an E. coli strain, preferably the E. coli strain BL21(DE3) or MG1655, and the enzyme is glyoxalase III (GLY III) encoded by the yhbO gene of E. coli (in particular, SEQ ID NO: 1 is the amino acid sequence of GLY III of E. coli, and SEQ ID NO: 2 is the nucleotide sequence of the yhbO gene).

[0059] Another object of the present invention is an expression cassette comprising a nucleotide sequence encoding an enzyme as defined above under the control of a promoter, preferably an inducible promoter as disclosed above (for example IPTG).

[0060] In one particular embodiment, particularly for expressing a glyoxalase in the periplasm of an engineered microbial cell, the expression cassette comprises a nucleotide sequence encoding a glyoxalase as defined above, and also a signal peptide sequence encoding a signal peptide as defined above for targeting said glyoxalase into the periplasmic space of the microbial cell.

[0061] In a particularly preferred embodiment, the signal peptide is PelB (SEQ ID NO: 5) encoded by the signal peptide pelB gene (SEQ ID NO: 6).

[0062] Another object of the invention relates to a nucleotide sequence encoding a polypeptide (enzyme) or a fusion polypeptide (including a signal peptide) as defined above.

[0063] In one particular embodiment, the expression cassette comprises the fusion nucleotide sequence pelB-yhbO set forth in SEQ ID NO:8, which comprises the pelB signal peptide and the yhbo gene, for producing the fusion polypeptide (or fusion protein PelB-YhbO) set forth in SEQ ID NO:7.

[0064] The present invention further relates to a vector comprising a nucleotide sequence as defined above.

[0065] Biocatalyst immobilization (optional) The modified microbial cells may be free in the culture medium or, advantageously, immobilized. Immobilization may be performed by methods well known to those skilled in the art (e.g., cross-linking, physical adsorption, entrapment, etc.). In a particular embodiment, the microbial cells are immobilized on agar or carrageenan, or the microbial cells are cross-linked, especially with glutaraldehyde (GA) and / or polyethyleneimine (PEI), or both immobilized and cross-linked.

[0066] In one particular embodiment, the biocatalyst is immobilized on agar or carrageenan or cross-linked with glutaraldehyde and / or polyethyleneimine.

[0067] Immobilization of whole cell biocatalysts allows for easy recycling of the biocatalyst for further use in other reactions.

[0068] In one particular embodiment, the microbial cells are encapsulated in carrageenan beads, which is particularly advantageous for bioproduction within the periplasmic space of microorganisms.

[0069] In one particular embodiment, the microbial cells are fixed with glutaraldehyde and / or polyethyleneimine.

[0070] Immobilization of the modified microbial cells is advantageous for recycling the modified microbial cells for another further reaction.

[0071] Culture and transformation conditions "Suitable culture medium" means a medium suitable for the growth of a microorganism. The medium is generally a liquid, gelled, or solid medium and contains a carbon source including sugars, alcohols, a nitrogen source, and other compounds required for the growth of the microorganism and for the conversion reaction of glyoxal to glycolic acid, which is also present in the medium as a substrate. Examples of suitable media (e.g., TB medium) are disclosed later in this description.

[0072] Culture conditions for glyoxalase production The reaction conditions vary depending on the enzyme used, the microorganism used, and the product being treated.

[0073] In one particular embodiment, for producing glyoxalase III in the culture of E. coli, the temperature is between 20 and 37° C., or between 25 and 37° C., preferably 20 or 25° C. The pH is between 5 and 8, preferably between 5.5 and 8, more preferably 7. After microbial cultivation and expression of glyoxalase, the cells are harvested by a liquid / solid separation method, which involves separating the liquid medium containing the microbial cells from the remainder of the culture. Depending on the specific characteristics of the system, various methods are used, such as centrifugation and filtration.

[0074] Once the cells are harvested, they are used as a biocatalyst for the conversion of glyoxal to glycolic acid.

[0075] Conversion of glyoxal to glycolic acid This enzymatic conversion can be achieved by introducing the harvested cells into a reaction system where glyoxal is converted to glycolic acid catalyzed by the expressed glyoxalase enzyme.

[0076] In one particular embodiment, glyoxal is added directly to the culture medium, for example, the glyoxal is an aqueous solution of glyoxal (40%).

[0077] Glyoxal is the smallest dialdehyde. It is water-soluble and forms an equilibrium between the monohydrate and dihydrate forms. Glyoxal can also form dimers and oligomers by condensation (reversible). Glyoxal can be produced by the gas-phase oxidation of ethylene glycol with air in the presence of a copper or iron catalyst. This process is used by BASF (Ludwigsafen, Germany), with a reported production of 60 kt / year (BASF, 2016). Another process is based on the liquid-phase oxidation of acetaldehyde with nitric acid; WeylChem produces glyoxal in this way for use in the production of glyoxylic acid. Acetaldehyde is produced from the oxidation of ethylene or ethanol. The production of ethylene glycol and bio-based ethanol has generated growing interest, and glyoxal can now be synthesized from renewable resources.

[0078] The reaction involves a combined set of reaction components under suitable reaction conditions, whereby glycolic acid is produced in an aqueous solution or an organic solvent (e.g., methanol). Producing glycolic acid in methanol has several advantages: - Reduced water use; - Evaporating methanol requires less energy than evaporating water - Production of glycolate derivatives, e.g., methyl glycolate. The esterification reaction may be limited by the presence of excess water. Present the following.

[0079] The reaction may be run in batch or fed-batch mode and may or may not be repeated. "Batch mode" uses no additional feed from start to finish of the process. In the "fed-batch mode," the supply of substrate (glyoxal) and supplements allows for an extended period of cultivation towards higher cell densities to produce glycolic acid. A "repeated fed-batch" harvests all but a small residue of a completed (fed) batch, and the available residual cells are provided for use as inoculum for the next batch. It can also be referred to as a continuous mode of bioconversion, where the input feed rate is equal to the recovery removal rate, with cell retention.

[0080] The bioversion reaction is preferably carried out under conditions consisting of shaking and stirring (100 to 600 rpm, preferably approximately 200 rpm). The temperature is between 25°C and 37°C, preferably 25°C, and the pH is between 3.8 and 8, preferably between 3.8 and 7. The reaction time is generally comprised between 1 and 50 hours, preferably between 1 and 3 hours. In one particular embodiment, the cell dcw*:substrate ratio is between 1 and 30%, preferably between 1 and 10%, more preferably between 1 and 5%, and even more preferably 1% in various reaction volumes. *dcw=dry cell weight. In one particular embodiment, considering a 40% glyoxal solution, the highest concentration of glyoxal used is 8.7M.

[0081] The bioconversion of glyoxal to glycolic acid results from the formation of glycolic acid ions and H+ ions, including the release of protons (H+) during the reaction (glyoxal + HO → glycolic acid + H+). Because enzyme activity is pH-sensitive, sodium hydroxide (NaOH) is added as a base to maintain an enzyme-compatible pH. NaOH is a base that reacts with protons to form water, effectively neutralizing the acidic environment. The reaction involved is Na+ + OH− + glycolic acid + H+ → Na+ + glycolic acid + HO. This addition of base helps maintain a controlled pH level throughout the bioconversion process, resulting in the production of glycolic acid sodium salt (sodium glycolate). In this embodiment, to ultimately obtain pure glycolic acid, a purification step is required, involving the use of a cation exchange resin, to replace the sodium ions with H+ ions.

[0082] In another preferred embodiment using a "periplasmic whole-cell biocatalysis," the enzyme is protected from the acidic environment and does not require a fully controlled pH process using a base (NaOH), and the reaction advantageously provides direct production of glycolic acid without the sodium salt (sodium glycolate). This approach significantly simplifies downstream processing, reduces costs, and minimizes wastewater. In this embodiment, pH adjustment, and therefore the addition of NaOH, is minimized.

[0083] In fact, the inventors have demonstrated that the mixture of glycolic acid and glycolate produced by the conversion reaction acts as a buffer system with a pKa of near 3.83, meaning that the solution can withstand large changes in pH when either acid or base is added, providing stability to the bioconversion process.

[0084] This buffering effect is generally achieved when a minimal amount of acid is present in the solution. In a particular embodiment, initial pH adjustment may be necessary to accumulate sufficient glycolic acid and stabilize the pH, followed by continued production without pH adjustment. As an example of the present invention, bioconversion was initiated with pH adjustment at pH 6.5 to produce a glycolic acid buffer; then, glycolic acid and H+ were produced without pH change, the pH stabilized at 3.83, and no further pH adjustment was required. This two-step process aims to minimize the sodium content in the reactor compared to a fully controlled pH process.

[0085] This method offers the advantage of reducing sodium hydroxide usage and lowering the amount of cation exchange resin required in downstream processes, thus streamlining the overall process, cutting costs, and minimizing wastewater production.

[0086] product The glycolic acid produced by the methods of the present invention can be recovered.

[0087] "Recovery of glycolic acid from the medium" refers to the process of separating, extracting, filtering, and / or purifying glycolic acid from the medium. Generally, this process includes centrifugation, microfiltration, cation and anion exchange, and evaporation.

[0088] In one particular embodiment, the solid / liquid separation is carried out by centrifugation at between 4000 and 5000 rpm, in particular at 4500 rpm, for a time ranging from 5 to 15 minutes, in particular 10 minutes, and the microfiltration is carried out using a filter with a porosity ranging from 0.1 to 0.2 μm, in particular 0.2 μm.

[0089] In one particular embodiment, extraction of glycolic acid is carried out by anion exchange chromatography.

[0090] In one particular embodiment, extraction of glycolic acid from the supernatant was performed by anion exchange chromatography using an ion exchange column, for example a Dowex resin column (eg Dowex 1x8 resin OH form). As an illustrative example, first, the sample was loaded onto an ion exchange column using a peristaltic pump at a rate of 1 to 5 mL / min, specifically 2.5 mL / min. The ion exchange resin column was then cleaned with deionized water. A subsequent elution step with NaOH at different concentrations (60, 120, 200 mM) was performed. The ion exchange resin, which was stored for later use, was cleaned with 1 M NaOH.

[0091] The fraction of interest is then eluted on a cation exchange resin (eg, DOWEX 50WX8 H type) to convert sodium glycolate (glycolic acid sodium salt) to glycolic acid. Finally, evaporation of water under reduced pressure (below 50°C) results in glycolic acid at a concentration of 70% in solution. In one particular embodiment, evaporation is carried out in an evaporator (Buchi R-215) at approximately 40°C and approximately 40 mbar, followed by completion by freeze-drying (Epsilon 2-4 LSC).

[0092] In a preferred purification step, glycolic acid was purified from the supernatant by cation exchange chromatography using a cation exchange column, for example, a Dowex resin column (e.g., DOWEX 50WX8 H type), to convert sodium glycolate (glycolic acid sodium salt) to glycolic acid.

[0093] Indeed, as disclosed above, the production of glycolic acid from glyoxal results in a decrease in pH. Therefore, according to one embodiment, particularly when using a "cytoplasmic whole-cell biocatalyst," sodium hydroxide is used to control the pH, and glycolic acid is converted to glycolic acid sodium salt (sodium glycolate). Subsequently, cation exchange is used to recover glycolic acid by replacing Na+ with H+. Typical cation exchangers, such as cation exchange resins, require regeneration with an acid such as sulfuric acid (H2SO4) after use. Those skilled in the art will closely adapt the volume of resin for sodium removal, the amount of sulfuric acid for regeneration, and the resulting wastewater containing sodium sulfate (Na2SO4) to the sodium content at the end of production.

[0094] Optionally, granular charcoal adsorption on a column is used to remove impurities.

[0095] Finally, evaporation of water under reduced pressure (below 50°C) results in glycolic acid at a concentration of 70% in solution. In one particular embodiment, evaporation is carried out in an evaporator (Buchi R-215) at approximately 40°C and approximately 40 mbar.

[0096] Such bioproduction steps include the use of salts (e.g., NaOH) to adjust the pH and purification steps to recover glycolic acid. Thus, Applicants have demonstrated that the use of a periplasmic whole-cell biocatalyst according to the present invention, which is tolerant to acidic conditions, enables the direct production of glycolic acid without sodium. This strategy significantly streamlines downstream processing.

[0097] Quantitation of glyoxal and glycolic acid can be performed by high-performance liquid chromatography (HPLC). In one specific embodiment, the concentrations of glyoxal (substrate) and glycolic acid (product) were measured by UHPLC (Dionex Ultimate™ 3000) equipped with a Phenomenex ROA-organic acid H+ (8%) column (300 × 7.8 mm) and a precolumn (50 × 7.8 mm). 5 mM H2SO4 was used as the mobile phase at 0.5 mL / min for 35 min. Detection was performed using a refractometer (Shodex RI-101) and a UV detector (Dionex UltiMate 3000 Diode Array Detector 3000 (RS)). Samples were filtered to 0.2 μm. Under these conditions, glyoxal eluted at 15 min, and glycolic acid eluted at 18 min, as shown in Figure 1.

[0098] Extraction of glycolic acid can be further followed by analysis of the quality and properties of the glycolic acid produced.

[0099] In particular, NMR (nuclear magnetic resonance spectroscopy) techniques allow the observation of local magnetic fields near atomic nuclei, and such analysis is used to obtain high-resolution information of the products. In particular, NMR analysis applied to glycolic acid produced by the method of the present invention shows that the majority of the product is glycolic acid. Only 0.05% impurity was found in proton NMR. IR (infrared spectroscopy or vibrational spectroscopy) techniques, on the other hand, are used to study and identify compounds by measuring the interaction of infrared radiation with materials by absorption, emission or reflection.

[0100] In the present invention, IR analysis applied to glycolic acid produced by the method of the present invention shows 95% correlation with a commercial standard of glycolic acid. Mass spectrometry (MS) was also used to confirm the molecular weight and formula of glycolic acid.

[0101] Thus, in one particular embodiment, the method of the invention further comprises the step of recovering glycolic acid from the culture medium and, optionally, analyzing the glycolic acid produced, preferably by NMR, MS and / or IR analysis.

[0102] In one particular embodiment, the glycolic acid produced by the present invention has the following characteristics: - contains less than 30% impurities, preferably less than 0.05% impurities by proton NMR; - its structure and quality show a correlation in IR of more than 90%, preferably 95%, with the commercial standard of glycolic acid; - Mass spectrometry in negative mode for the [MH] species gave a molecular weight of 75.0082 (+ / - 1 mDa) and the expected formula C2H3O3 It is characterized by:

[0103] The glycolic acid produced may be used as such or as an intermediate in further chemical reactions, such as esterification or polymerization.

[0104] The present invention therefore further relates to 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 of the method for the biochemical production of glycolic acid as defined above.

[0105] The method may further include recycling the engineered microbial cells (biocatalyst) that produce the enzyme that converts glyoxal to glycolic acid at the end of the bioconversion reaction.

[0106] Generally, the modified microbial cells can be recycled two to four times.

[0107] Recycling generally involves washing the cells with PBS and recovering the cells for further use. [Brief explanation of the drawings]

[0108] [Figure 1] HPLC-RI chromatograms of standard solutions of (A) glyoxal 10 g / L; (B) glycolic acid 10 g / L. [Figure 2] Production of glycolic acid in various volumes (2 to 2000 mL). [Figure 3] Kinetics of glycolic acid production according to volume of production. [Figure 4] Kinetics of glycolic acid production and glyoxal consumption. [Figure 5] Glycolate production in a 700 mL reactor with continuous substrate feeding. [Figure 6] Glycolate production in a 2000 mL reactor with substrate feeding. [Figure 7] Glycolate production by free and cross-linked resting cells. [Figure 8] Percent bioconversion after 2 hours in batch mode with both free and cross-linked cell lines. [Figure 9] Glycolate production by carrageenan-immobilized cells. [Figure 10] Percent bioconversion after 2 hours in batch mode including both free cells and cells treated with glutaraldehyde (GA) and / or polyethyleneimine (PEI). [Figure 11] Time course of glycolate production by periplasmic and cytoplasmic whole-cell biocatalysts at pH 7.5 (·), 6.5 (■), 5.5 (●), 4.5 (▲), and 3.5 (×). [Figure 12] Glycolic acid production over time at pH 7.5 and 4.5 by purified glyoxalase (cytoplasmic) and periplasmic fractions from periplasmic whole-cell biocatalysts containing glyoxalase. [Figure 13] Production of glycolic acid by periplasmic and cytoplasmic whole-cell biocatalysts at acidic pH in glycolic acid-buffered unadjusted medium. [Example]

[0109] The invention is illustrated by the following non-limiting examples.

[0110] Example 1 Materials and Methods 1.1 Materials 1.1.1 Plasmids for expressing glyoxalase into the periplasmic or cytoplasmic space Different plasmids were prepared.

[0111] 1) The plasmid pET28b-yhbO, designed for cytoplasmic production of the YhbO enzyme and using an IPTG-inducible T7 promoter, is prepared as follows: Novagen's pET28b plasmid (ori pBR322, kanamycin, T7 promoter)® (Ref. 69864) is used to express the yhbO gene (SEQ ID NO: 2), encoding deglycase (glyoxalase), from E. coli into the cytoplasm of the cells. The yhbO gene is extracted from E. coli MG1655 (ATCC 700926) using a genomic DNA purification kit (ThermoFisher Ref: K0721), 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 of a commonly known type. Those skilled in the art will understand how to select and use primers in the context of PCR technology.

[0112] 2) Plasmid pET22b-yhbO, designed for periplasmic production of the YhbO enzyme and containing an IPTG-inducible T7 promoter, is prepared as follows: Novagen's pET22b plasmid (ori pBR322, ampicillin, T7 promoter, pelB)® (Ref 69744) contains the N-terminal signal sequence pelB and a C-terminal His-tag sequence. This plasmid is used to express the yhbO gene encoding deglycase (glyoxalase) from E. coli. The yhbO gene is extracted from E. coli MG1655 (ATCC 700926) using a genomic DNA purification kit (ThermoFisher Ref: K0721), then amplified by PCR (polymerase chain reaction) using appropriate conditions and primers (Taq2X Master Mix (M0270) (NEB®)) and then inserted into the linearized plasmid by homologous recombination. The primers used for gene amplification are of the generally known type. Those skilled in the art will know how to select and use primers in the context of PCR technology.

[0113] 3) pET28b is a plasmid similar to pET28b-yhbO but without the yhbO gene and is used as a negative control.

[0114] 4) pET28b-yhbO-His is similar to pET28b-yhbO disclosed above, but with a His tag for cytoplasmic protein purification, as used in Example 2.9.

[0115] 5) pZa3-proD-yhbO is a plasmid derived from the pZA33 plasmid (Expressys®) (p15A, pA1lacO-1, chloramphenicol Chm), in which the PA1lacO-1 promoter has been replaced with the constitutive proD promoter. This plasmid is used to express the yhbO gene, encoding deglycase (glyoxalase), from E. coli to produce the YhbO protein in the cytoplasm. The yhbO gene is extracted from E. coli MG1655 (ATCC 700926) using a genomic DNA purification kit (ThermoFisher Ref: K0721), then amplified by PCR (polymerase chain reaction) using appropriate conditions and primers (Taq2X Master Mix (M0270) (NEB®)) and then inserted into the linearized plasmid by homologous recombination. The primers used for gene amplification are of the generally known type. Those skilled in the art will know how to select and use primers in the context of PCR technology.

[0116] 1.1.2 Strain (biocatalyst) construction Different types of biocatalysts (transformed E. coli strains) were produced and used in the following examples.

[0117] Four E. coli BL21(DE3) (Ref: C2527, NEB®) strains were prepared, each carrying the above-mentioned plasmid. 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); or 3) pET28b empty as a negative control; 4) pET28b-yhbO-His, a plasmid designed for cytoplasmic production of the YhbO-His enzyme (IPTG inducible) and purification as illustrated in Example 2.9;

[0118] E. coli MG1655 (ATCC 700926) strain was prepared harboring the plasmid pZa3-proD-yhbO, a plasmid designed for constitutive expression for cytoplasmic production of the YhbO enzyme. The prod constitutive promoter allows the use of E. coli MG1655, a common laboratory strain that does not express T7 RNA polymerase, as required when using a T7 promoter, thereby reducing the cost of catalyst production by eliminating the need for IPTG.

[0119] Commercially available competent cells, such as BL21(DE3) (Ref: C2527, NEB®), were transformed according to the supplier's protocol, and the MG1655 strain was transformed using the TSS protocol described by Chung and Miller (Chung et al., 1989). The strains used are shown in Table 3 below.

[0120] [Table 3]

[0121] The glyoxalase-producing strain ("biocatalyst") was obtained by a two-step enrichment culture method on TB medium with the following composition (per liter): 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). The strain was grown overnight at 37 °C on solid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L agar), and then first precultured in 10 ml of TB medium at 37 °C and 200 rpm for 4 hours, and then inoculated into 500 mL of culture in a 2 L Erlenmeyer 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) at the mid-logarithmic phase (0.4 < DO600nm < 0.6), except for the constitutive strain directly incubated at 30 °C without adding IPTG, and shaken at 20 °C and 200 rpm overnight for approximately 16 hours. The cell pellet was recovered by centrifugation at 4500 rpm for 10 minutes. The established standard relationship between the dry cell mass per mL and the optical density at 600 nm according to the standard protocol is as follows: Dry weight cell (dwc) (mg / ml) = 0.6897 * OD 600nm

[0122] The Dowex resin and chemicals used for production, purification, and analysis standards, such as NaOH, HCl, H2SO4, NaCl, KCl, glyoxal, and glycolic acid, were obtained from Sigma Co. Other solutions of glutaraldehyde (GA), polyethyleneimine (PEI), and carrageenan were commercially available (Sigma-Aldrich (registered trademark)).

[0123] 1.2 Whole-cell catalytic reaction The whole cells prepared as above (the E. coli strain transformed with the plasmid constituting the "biocatalyst") are centrifuged at 4500 rpm for 10 minutes and resuspended in water. As disclosed in some other examples below, the whole cells prepared as described above can also be immobilized using glutaraldehyde and / or polyethyleneimine to facilitate further recycling of the biocatalyst. Cells carrying the yhbO gene (used as a "biocatalyst") are introduced into a reaction mixture that includes a pH-stabilizing buffer and a glyoxal substrate. The composition is intentionally kept as simple as possible to ensure optimal enzyme activity and minimal production costs. The glycolic acid production reaction is placed at 25°C under stirring (200 rpm). The reaction mixture consists of 100 mM HEPES buffer pH 7.5 and 100 mM glyoxal. Production is carried out in 2 mL Eppendorf tubes, 50 mL, 500 mL, and 5000 mL flasks at volumes of 2 mL, 20 mL, 200 mL, and 2000 mL, respectively. Negative controls without substrate (glyoxal) and negative controls (cells transformed with an empty plasmid) are run. Samples are collected by removing 300 μl of the reaction medium, centrifuging at 20,000 g for 2 minutes, and then filtering (0.2 μm).

[0124] The fed-batch catalytic technology was further tested for the bioconversion of glyoxal to glycolic acid. Bioproduction of glycolic acid was carried out in a fed-batch manner in a 700 mL reactor (Solaris®) at pH 7.5 and 30°C with catalytic broth containing an initial concentration of 200 mM phosphate buffer, agitated with a Rushton-type single-blade, six-blade agitator at 1000 rpm and an airflow of 150 mL / min. The initial volume of the reaction mixture was set to 300 mL to ensure sufficient volume was available for feeding and pH adjustment. Glyoxal (6.2 M) was continuously added at 0.59 mL / min via a peristaltic pump. The pH was adjusted to 7.5 with 3 M NaOH.

[0125] Another bioproduction of glycolic acid was carried out in a 2000 mL reactor (Sartorius®). The reaction was carried out in phosphate buffer with an initial concentration of 200 mM, pH 7.5, and 25°C, and was stirred with two six-blade Rushton-type agitators at 600 rpm and an airflow 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 in 1 hour. The pH was adjusted to 7.5 with 8.3 M NaOH. After one batch cycle, the catalytic broth and E. coli cells (biocatalyst) were separated by centrifugation at 10,000 g for 15 min.

[0126] Free enzyme-catalyzed reactions To demonstrate the stabilization provided by the cellular environment, bioconversion of glyoxal to glycolate was carried out at pH 4.5 and 7.5 under the same conditions as above, using purified enzyme from the cytoplasm with a his-tagged tag and purified enzyme from the periplasm without the tag.

[0127] YhbO-His cytosolic glyoxalase was purified from a 1000 mL culture of BL21-YhbO-His using affinity purification via the C-terminal His tag. YhbO periplasmic glyoxalase was further isolated from a 1000 mL culture of BL21-pET22b by osmotic shock as described in Section 1.8. The purified protein or periplasmic fraction was resuspended in 500 mL of 0.9% NaCl and transferred directly to a 2 L fermenter. The temperature was maintained at 25 °C, and agitation was performed at 200 rpm using a single six-blade Rushton impeller. The reaction was initiated with the addition of 12 mL of glyoxal (8.7 M) to a final concentration of 200 mM. When glyoxal was completely bioconverted, another 12 mL of glyoxal (8.7 M) was added to the bioreactor. The pH was adjusted using 8.3 M NaOH and 3 M H2SO4. Different pHs, i.e., pH 4.5 and pH 7.5, were evaluated to determine the activity limit for each catalyst.

[0128] 1.3 Analysis method The concentrations of glyoxal (substrate) and glycolic acid (product) were measured by UHPLC (Dionex Ultimate™ 3000) equipped with a Phenomenex ROA-organic acid H+ (8%) column (300 × 7.8 mm) and a precolumn (50 × 7.8 mm). 5 mM H2SO4 was used as the mobile phase at 0.5 mL / min for 35 min. Detection was performed using a refractometer (Shodex RI-101) and a UV detector (Dionex UltiMate 3000 Diode Array Detector 3000 (RS)). Samples were filtered to 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 and are shown in Figure 1. Structural confirmation analyses were performed by the Toulouse Institute of Chemistry (ICT) using nuclear magnetic resonance spectroscopy (NMR), infrared spectroscopy (IR), and mass spectrometry (MS).

[0129] 1.4 Ion-exchange chromatography Extraction of glycolic acid was performed by anion exchange chromatography using Dowex 1x8 resin (OH-form) with a capacity of 1.2 mEq / mL. An 8 mL or 120 mL column containing the resin was used, connected to a peristaltic pump. The sample was first loaded onto the ion exchange column at a rate of 2.5 mL / min using a peristaltic pump. The ion exchange resin column was then sanitized with deionized water. Subsequent elution steps with NaOH at different concentrations (60, 120, 200 mM) were performed. The ion exchange resin, which was stored for later use, was cleaned with 1 M NaOH. The fractions of interest were then eluted on a cation exchange resin (DOWEX 50WX8 H type) to convert the glycolic acid sodium salt to glycolic acid.

[0130] 1.5 Drying and Water Evaporation Evaporation was first carried out using a rotary evaporator (Buchi R-215) at 40° C. and 40 mbar, then completed by freeze-drying (Epsilon 2-4 LSC).

[0131] 1.6 Complete mass analysis of the product (glycolic acid) The instrument used was a Waters "XevoG2QTof" mass spectrometer equipped with an electrospray ionization (ESI) source and a time-of-flight (Tof) analyzer, allowing the determination of accurate masses to four decimal places and the proposal of raw formulas. The exact mass of the species was detected in negative ion mode [MH] − , ie, 75.0082 at less than 1 mDa.

[0132] 1.7 Purification of enzymes on nickel resin a) Cell lysis 15OD 600 The bacterial pellet from 1000 mL of PBS was resuspended in 100 mL of 50 mM Tris / HCl buffer, 300 mM NaCl, 20 mM imidazole, pH 8.0, thermostated to 4°C, and disrupted using a high-pressure homogenizer with three passages at 1100 bar. After each passage, 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. 150 mL of the suspension was then centrifuged at 10,000 × g for 15 minutes at 4°C to obtain a clear solution. The clear supernatant (crude extract, 150 mL) was applied directly to an IMAC column.

[0133] b) Affinity purification The His-tagged enzyme was purified by IMAC using the following conditions: Support: Streamline Chelate, Ni-NTA; Column: XK 16 / 20; CV (Column Volume): 15 mL; Sample Application Flow Rate: 3 mL / min; Wash and Elution Flow Rate: 3 mL / min; Binding Buffer: 50 mM Tris / HCl buffer (containing 300 mM NaCl, 20 mM imidazole, pH 8.0); Wash Buffer: 50 mM Tris / HCl buffer (containing 300 mM NaCl, 40 mM imidazole, pH 8.0); Elution Buffer: 50 mM Tris / HCl buffer (containing 300 mM NaCl, 300 mM imidazole, pH 8.0); Sample: Clarified lysate (crude extract) from E. coli containing the His-fusion protein after high-pressure homogenization and centrifugation; Sample Volume: 150 mL; Fraction Volume: 1.5 mL. After loading and washing the column with 10 CV of binding buffer and wash buffer, an elution step was performed in 500 mM imidazole using elution buffer. His-tagged enzyme-containing fractions were analyzed by SDS-PAGE. Fractions were stored at 4°C. His-tagged enzyme-containing fractions were pooled and used directly in the biotransformation assay.

[0134] 1.8 Osmotic shock The pellet from a 1000 mL 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 minutes. After centrifugation (16,000 × g, 10 minutes, 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 described above. After further centrifugation as described above, the supernatant containing the periplasmic protein fraction was transferred to a new reaction tube and stored at 4°C (Eichmann et al., 2019). A 10 μL sample is removed from each supernatant and denatured with 30 μL of loading buffer 4X+50 mM DTT for 10 minutes at 95° C. The samples are loaded onto an SDS-PAGE gel.

[0135] Example 2 Results of bioproduction of glycolic acid from glyoxal using whole-cell biocatalysts The reaction scheme for the production of glycolic acid from glyoxal is as follows:

[0136] [ka]

[0137] 2.1 Production of glycolic acid from glyoxal in volumes ranging from 2 mL to 2000 mL using a biocatalyst in the periplasmic space The production of glycolic acid from glyoxal is carried out in volumes of 2 mL, 20 mL, 200 mL, and 2000 mL. The reaction mixture contains the following components: - Biocatalyst: 2 DO / ml BL21(DE3)pET22b-YhbO cells - Substrate: Glyoxal (100mM) - Buffer: HEPES pH7.5 (100mM) - Temperature: 30℃ It consists of: In 20 hours, 74 mM glycolic acid was produced, a conversion rate of 75%. UPLC analysis shows that glyoxal is converted to glycolic acid without co-product formation at all production scales (Figure 2), and furthermore, the kinetic profile of glycolic acid production remains identical for the four volumes tested (Figure 3). Thus, it is possible to increase the volume by 1000 times without a loss of efficiency.

[0138] Production with a higher amount of catalyst (16 DO / ml instead of 2 DO / ml) was achieved under the following conditions: - Biocatalyst: 16 DO / ml BL21(DE3)pET22b-YhbO cells - Substrate: Glyoxal (100mM) - Buffer: HEPES pH7.5 (100mM) - Volume: 2mL Do below. The production of 100 mM or 7.6 g / L glycolic acid was observed in 3 hours with a 100% yield (FIG. 4). Therefore, the whole-cell biocatalyst containing BL21-YhbO cells is an effective tool for the production of glycolic acid from glyoxal. We demonstrated stable production of 5.7 g / L of glycolic acid at 75% conversion for volumes ranging from 2 ml to 2000 ml. By optimizing the amount of biocatalyst used, production of 7.6 g / L of glycolic acid from 5.8 g / L of glyoxal was achieved in 3 hours with a 100% yield.

[0139] 2.2 Fed-batch catalytic production of glycolic acid from glyoxal - Biocatalyst: 128DO / ml BL21(DE3)pET22b-YhbO cells - Substrate: glyoxal (6.2 M at a flow rate of 800 mM / h) - Buffer solution: Phosphate buffer solution pH 7.5 (200mM) - pH adjustment: NaOH (3M) and HCl (3M) - Volume: 547 mL in a 700 mL reactor - Temperature: 30℃

[0140] Fed-batch catalysis was further tested for the bioconversion of glyoxal to glycolic acid. The first fed-batch reaction was carried out in a 700 mL reactor (Figure 5). The parameters are described in Section 1.2. Because fed-batch catalysis was performed with limited substrate accumulation, glyoxal was consumed immediately. Glycolate production of 120 g / L was observed in 7 hours with a 99% yield and no residual glyoxal (Table 4). The reaction could not be continued beyond this point, as the maximum reactor capacity had been reached. pH adjustment with 3 M NaOH accounted for approximately 60% of the volume addition during the reaction. This production process requires optimization to allow maximum yield by adjusting the pH but minimize product dilution by adding sodium hydroxide.

[0141] [Table 4]

[0142] The production of glycolate in the reactor is promising with a productivity of 23 g / L / h, and some improvements would even optimize the performance. - The NaOH concentration can be increased to limit the volume increase; - Larger capacity reactors can be used to allow for higher volumetric production and more precise control of base, acid and feed rates; - the temperature can be reduced to reduce the cost of the process; and - The amount of cells can be optimized to reduce costs. As a result, the following fed-batch bioconversion was carried out using the above improvements: increased NaOH concentration to 8.3 M, 2.5-fold larger reactor volume, reduced temperature to 25°C, and a 19% reduction in final catalyst concentration.

[0143] Therefore, a second fed-batch reaction was carried out in a bioreactor (fully stirred open reactor) with a volume of 2000 mL (Figure 6). - Biocatalyst: 128DO / ml BL21(DE3)pET22b-YhbO cells - Substrate: glyoxal (6.2 M at a flow rate of 800 mM / h) - Buffer solution: Phosphate buffer solution pH 7.5 (200mM) - pH adjustment: NaOH (8.3M) and H2SO4 (4M) - Volume: 1086 mL in a 2000 mL reactor - Temperature: 25℃

[0144] Key performance data are listed in Table 5. Finally, 247 g / L of glycolic acid was obtained in 43 h with a 98% yield (equivalent to 1.29 g glycolate / g glyoxal) and a volumetric productivity of 5.7 g / L / h, the highest level of glycolic acid production reported to date. A quantity of 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. The specific activity of the biocatalyst was 0.28 g at the end of the reaction. グリオキサール / g バイオマス / h, 0.37g at the start of the reaction グリオキサール / g バイオマス / h. This could be due to the accumulation of glyoxal at the start of the experiment, which could have damaged the cells, or due to clogging of the reactor stationary part with cell mass, which made part of the biomass unavailable for reaction despite vigorous stirring.

[0145] [Table 5]

[0146] 2.3 Structural confirmation by nuclear magnetic resonance spectroscopy (NMR), infrared spectroscopy (IR), and mass spectrometry (MS) The purified glycolic acid obtained by ion exchange chromatography was used for structural analysis. NMR (proton, carbon, 2D HSBC and 2D HSQC), IR, and MS analyses were performed by the Toulouse Institute of Chemistry. - NMR analysis shows that the majority of the product is glycolic acid. Only 0.05% impurity was found in proton NMR. - IR analysis shows 95% correlation with a commercial standard of glycolic acid. The difference may be explained by the difference in crystalline form. - Full mass spectrometry (MS) shows that the crude formula obtained by mass spectrometry of glycolic acid produced by the process of the present invention corresponds to the expected crude formula, i.e., C2H3O3, indicating a perfect correlation between the commercial standard and the glycolic acid produced.

[0147] The production of 247 g / L glycolic acid at a 99% yield with a volumetric productivity of 5.7 g / L / h exceeds the performance of any published fermentation process. The process temperature was also effectively reduced from 30°C to 25°C, making the operating conditions competitive from an energy standpoint. After anion exchange chromatography, the purified product was recovered with an HPLC purity of >99%. Three structural analyses confirmed that highly pure glycolic acid was obtained, containing only 0.05% impurities as found by NMR. The purified glycolic acid is a white crystalline powder, particularly suitable for cosmetic grades.

[0148] The following example illustrates the production of glycolic acid from glyoxal using biocatalysts producing glyoxalase in the cytoplasm versus the periplasm of the biocatalyst (whole cell), with or without immobilization.

[0149] 2.4 - The effects of periplasmic versus cytoplasmic biocatalysis and the effect of immobilizing biocatalysts on biotransformation reactions As disclosed in Example 1.2, three catalysts were produced, all in E. coli BL21(DE3) strain harboring different plasmids: 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. The BL21(DE3) strain was grown in 100 mL of TB (37°C, 200 rpm) and induced with 0.1 mM IPTG, followed by an overnight protein production phase at 20°C, after which the catalyst was 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 was resuspended in 25 mL of 0.1% glutaraldehyde solution for 1 hour at room temperature (these cells constituted the "immobilized or crosslinked biocatalyst"). The harvested cells were washed with 25 mL of PBS 1X and collected by centrifugation. The reaction consisted of 67 mg dcw catalyst / ml in a 5 ml solution of glyoxal (250 mM) and HEPES pH 7.5 (500 mM) and was carried out at 25° C. and 200 rpm. The bioproduction process was monitored using high-performance liquid chromatography (HPLC) by withdrawing 0.5 mL samples at regular time intervals. Although the glyoxalase was located in the periplasmic or cytoplasmic space, the glyoxalase activity was identical (Figure 7). The free catalyst had the same specific activity, 42.6 μmol glycolate.min -1 .g -1 Treatment with glutaraldehyde appears to affect activity, but approximately 75% of the activity remains, and the reaction is still very efficient, reaching completion in under an hour.

[0150] These results demonstrated that glyoxalase activity and glycolate production were identical whether the glyoxalase was located in the periplasmic or cytoplasmic space. Although immobilization of the biocatalyst appears to slow the reaction and / or affect the activity of glyoxalase, the highest level of glycolate production was obtained after 1 hour of treatment.

[0151] 2.5 - Biocatalytic Recycling Two BL21(DE3) strains 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), respectively, were generated as disclosed in Example 1.2 and cultured as described in Example 2.4. The two strains were cross-linked using glutaraldehyde as described in Example 2.4. To investigate the effect of recycling on the extent of glyoxal conversion, biotransformation reactions were performed in batch mode using both free and cross-linked cells. After four cycles, complete conversion of glyoxal was observed in less than two hours for six catalysts (Figure 8). These results demonstrate that the biocatalyst can be recycled two to three times (four cycles as described above) without losing its catalytic effectiveness, since complete conversion of glyoxal was obtained in less than two hours. Immobilization is advantageous for recovering the biocatalyst at the end of a cycle and reusing it in further cycles, either continuously or delayed.

[0152] 2.6 Preparation of carrageenan beads containing E. coli BL21(DE3) / pET22b-yhbO and E. coli BL21(DE3) / pET28b-yhbO, respectively Two catalysts: 1) E. coli BL21(DE3) / pET22b-yhbO, which produces glyoxalase III from E. coli in the periplasmic space, and 2) E. coli BL21(DE3) / pET28b-yhbO, which produces the enzyme in the cytoplasmic space, were produced as described in Example 1.2. Carrageenan was used to immobilize both catalysts. The strains were grown on TB at 37°C and 200 rpm until an OD600nm of 0.6 was reached, at which point 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% solution of carrageenan. The suspension was kept in a water bath at 60° C. and poured dropwise using a syringe into a 0.3 M KCL solution at 4° C. The reaction consisted of a mixture of all the beads (carrageenan beads encapsulating the biocatalyst) formed into a 5 mL solution containing 250 mM glyoxal and 500 mM HEPES pH 7.5 and was carried out at 30° C. and 200 rpm. 100% conversion was achieved after 3 hours for both catalysts. Immobilized BL21(DE3) / pET22b-yhbO cells were incubated at 60.7 μmol glycolate.min -1 .g -1 The specific activity of the dcw catalyst was 51.8 μmol glycolate.min -1 .g -1 It has the activity of a dcw catalyst (Figure 9). Periplasmic secretion of glyoxalase III is advantageous when the cells are encapsulated (in this example in carrageenan beads), no doubt due to better transport of the molecule across the periplasm.

[0153] 2.7 Preparation of glutaraldehyde (GA) / polyethyleneimine (PEI) cross-linked BL21(DE3) / pET22b-yhbO transformants expressing glyoxalase III from E. coli To prepare a reaction product that can be used multiple times in biocatalytic processes, the BL21(DE3) / pET22b-yhbO strain was used to compare different treatments to stabilize glyoxalase activity, using polyethyleneimine (PEI) to aggregate the cells and glutaraldehyde to cross-link the cells. Cells were produced as described in Example 2.4. A volume equivalent to 435 mg of DCW cells was centrifuged at 4000 rpm for 10 minutes. The collected cells were washed with 50 mL PBS 1X and collected by centrifugation. The different conditions tested are presented in Table 6.

[0154] [Table 6]

[0155] Reactions consisting of 90 mg / ml free or treated cells in a 5 ml solution of glyoxal (250 mM) and HEPES pH 7.5 (50 mM) were performed at 30°C and 200 rpm. The bioproduction process was monitored using high-performance liquid chromatography (HPLC) by withdrawing 0.5 mL samples at regular intervals. The pH of each sample was measured and, if necessary, readjusted to pH 7.5 during the reaction using 3 M NaOH. 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 batch-mode reactions were performed, and for free cells, bioconversion decreased by 50% from the second reuse (Figure 10). The difference from Example 2.5 can be explained by the lower concentration of HEPES buffer, and the cells were exposed to an acidic pH between samples. The treated cells appeared to be protected from pH changes by GA and PEI, and their catalytic efficiency remained stable at approximately 90%. Microbial stabilization using cationic flocculants and cross-linking agents is suitable for optimizing glyoxal bioconversion.

[0156] 2.8 Bioconversion of glyoxal to glycolic acid in methanol The BL21(DE3) pET28b-yhbO strain was prepared as described in Example 1.2. The reaction consisted of 67 mg of dcw catalyst in 1 ml of methanol solution containing glyoxal (200 mM) and was carried out at 25 °C and 200 rpm. The reaction was monitored using high-performance liquid chromatography (HPLC) by withdrawing a 0.2 mL sample after 3 hours. Total conversion of glyoxal to glycolic acid was observed, demonstrating the production of glycolic acid in an organic solvent.

[0157] 2.9: Effect of pH on the conversion of glyoxal to glycolic acid The production of glycolic acid at different acidic pH levels was compared using periplasmic and cytoplasmic whole-cell biocatalysts. The bioconversion of glyoxal to glycolic acid was compared between whole-cell cytoplasmic glyoxalase (MG1655-YhbO cytoplasm) and whole-cell periplasmic glyoxalase (BL21-YhbO periplasm) in a 2-L reactor (Sartorius®) in batch mode. After production, cells were resuspended in 500 mL of 0.9% NaCl at 30 DO / mL and transferred directly to a 2-L fermentor. The temperature was maintained at 25°C, and agitation was performed at 200 rpm using a single six-blade Rushton impeller. The reaction was initiated with the addition of 12 mL of 8.7 M glyoxal to a final concentration of 200 mM. When 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 limit of activity for each catalyst, different pHs were evaluated (pH 6.5, pH 5.5, pH 4.5, and pH 3.5).

[0158] As shown in Figure 11, the periplasmic whole-cell biocatalyst efficiently produced glycolic acid at a pH level as low as 4.5 compared to the cytoplasmic whole-cell biocatalyst. We investigated whether this was due to the structure of the enzyme in the periplasmic compartment that allows disulfide bond formation or whether it was a result of the compartment itself. Therefore, further production experiments were performed using purified enzymes from both the cytoplasmic and periplasmic whole-cell biocatalysts. As shown in Figure 12, both enzymes exhibited the expected activity levels at pH 7.5, consistent with previous results. However, under acidic conditions, specifically at pH 4.5, the enzymes tended to precipitate, rendering them ineffective with respect to glycolic acid synthesis. This phenomenon highlighted the important influence of the cellular periplasmic compartment on the acid resistance of the enzymes, allowing them to function efficiently at pH levels above 4.5. Thus, the periplasmic environment plays a key role in preserving the enzyme's functionality under acidic conditions, which is not observed in the purified enzyme form.

[0159] 2.10 Effect of glycolic acid buffer on the bioconversion of glyoxal to glycolic acid A buffer solution comprises 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 a strong acid or strong base is introduced into a buffer solution, its pH remains relatively stable. Buffer solutions are widely used in various chemical processes to maintain a consistent pH. The purpose of this experiment was to demonstrate the potential of glycolic acid (glycolic acid / glycolate) as a buffering agent. Specifically, we intended to start the bioconversion process while adjusting the pH, and then stop the adjustment. Whole cell cytoplasmic glyoxalase and whole cell periplasmic glyoxalase activities were compared in batch mode in a 2 L reactor (Sartorius®). After production, BL21-YhbO periplasmic cells or MG1655-YhbO cytoplasmic cells were resuspended in 500 mL of 0.9% NaCl at 30 DO / mL and transferred directly to a 2 L fermenter. The temperature was maintained at 25°C, and agitation was performed at 200 rpm using a single six-blade Rushton impeller. The reaction was initiated by the addition of 12 mL of glyoxal (8.7 M). When glyoxal was completely bioconverted, 12 mL of glyoxal (8.7 M) was added again to the bioreactor. In the first step, bioconversion was initiated with pH adjustment to pH 6.5, using 8.3 M NaOH and 3 M H2SO4 to adjust the pH to 6.5. Once the glycolic acid concentration reached the desired concentration (>400 mM in the examples), the pH adjustment was stopped and the reaction was monitored. In this second step, glycolic acid was produced without added salt at a stable pH of 3.8. In the event of biocatalyst inactivation at acidic pH, the pH was temporarily readjusted to pH 6.5 to assess whether the inactivation was reversible and solely pH dependent. When using cytoplasmic whole-cell biocatalysts, the pH decreased 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 the inactivity of cytoplasmic enzymes at pH 4.5. When using periplasmic whole-cell biocatalysts, the pH decreased rapidly and stabilized at 3.83 (Figure 13). Under this condition, the process was initiated with 462 mM of sodium glycolate, and the production of 250 mM of glycolic acid was achieved without pH adjustment. This result initially confirmed the functional viability of periplasmic whole-cell biocatalysts at pH 4.5, especially at pH 4. Furthermore, this result demonstrated the effectiveness of the glycolate buffer, which resulted in approximately 35% glycolic acid production without pH adjustment. At the pKa of glycolic acid (pH = 3.8), the concentrations of the acid and the conjugate base are equivalent to those known from the art (glycolic acid is dominant at pH < pKa, and glycolate is dominant at pH > pKa). Thus, theoretically, starting with 460 mM of glycolate, the production of 460 mM of glycolic acid could be achieved without pH adjustment. Therefore, since the enzyme appears to be active near the pKa, an increase in production of more than 15% should be achievable. This result demonstrated a significant advantage of periplasmic whole-cell biocatalysts regarding the production of organic acids at acidic pH levels. This approach offers several advantages, including a reduction in the amount of sodium hydroxide required. Furthermore, it reduces the amount of cation exchange resin required in downstream processing (DSP) steps for sodium removal. All of these results demonstrate that the use of whole cells of transformed microorganisms as biocatalysts, e.g., Escherichia coli strains transformed with plasmids containing the yhbO gene with or without signal sequences and constitutive / inducible promoters (i.e., BL21(DE3) and MG1655 strains), enables the rapid (within hours), high-yield, and quantitative and volumetric production of glycolic acid from glyoxal, exceeding the performance of any published fermentation process in biochemical production according to the present invention. These biocatalysts can be used as free biocatalysts or, advantageously, as immobilized biocatalysts cross-linked with glutaraldehyde (GA) and / or polyethyleneimine (PEI) or encapsulated within carrageenan beads to facilitate their recycling and reuse in multiple reactions (2 to 4 cycles). Finally, biocatalysts expressing glyoxalase within the periplasmic space of the microorganism offer several advantages in addition to the production of properly folded, active proteins in an oxidizing environment. Unlike cytoplasmic exchange, which can be tightly regulated via membrane-active transporters, small solutes, such as glyoxal and glycolic acid, can freely equilibrate between the external solution and the periplasm. Substrates are readily accessible to periplasmic enzymes, and products can readily diffuse into the external medium for optimal reaction rates. Furthermore, the isolation of the periplasm from the metabolic complexity of the cytoplasmic environment is advantageous for bioproduction. In this subcellular compartment, side reactions are limited, resulting in minimal by-product production and high production yields. Finally, periplasmic whole-cell biocatalysts advantageously tolerate acidic conditions, which allows for the production of glycolic acid through a simplified process (direct production of glycolic acid without sodium addition (NaOH)) even at low pH and with reduced salt amounts.

[0160] References -Abdallah, J., Caldas, T., Kthiri, F., Kern, R., & Richarme, G. (2007). YhbO protects cells against multiple stresses. Journal of Bacteriology , 189 (24), 9140-9144. https: / / doi.org / 10.1128 / JB.01208-07 -BASF. (2016). Glyoxal More Sustainable Solutions for Your Business. 4. -Chung, CT, Niemela, SL, & Miller, RH (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. https: / / doi.org / 10.1073 / pnas.86.7.2172 -Ehrmann M. (2007). << The Periplasm - ASM Press, Washington DC >> -He, YC, Xu, JH, Su, JH, & 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 -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 -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 -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 -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 biochemical production of glycolic acid or a derivative thereof, comprising culturing a whole cell biocatalyst comprising or consisting of modified microbial cells that produce an enzyme that converts glyoxal to glycolic acid in a suitable culture medium containing glyoxal as a substrate, and optionally recovering glycolic acid from the culture medium.

2. 2. The method of claim 1, wherein the enzyme that converts glyoxal to glycolic acid is produced into the cytoplasmic space or into the periplasmic space of the microbial cell, preferably into the periplasmic space of the microbial cell.

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

1.

4. 4. The method according to claim 1, wherein the microorganism is a Gram-negative bacterial strain selected from the group consisting of Enterobacteriaceae, Alcaligenes, Vibrionaceae and Pseudomonadaceae, in particular Enterobacteriaceae, preferably belonging to the genus Salmonella, Yersinia or Escherichia, more preferably the genus Escherichia, and even more preferably Escherichia coli.

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

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

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

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

9. 10. The method of claim 8, further comprising recycling the modified microbial cells that produce the enzyme that converts glyoxal to glycolic acid upon completion of the bioconversion reaction.

10. 7. A whole cell biocatalyst comprising or consisting of modified microbial cells producing an enzyme capable of converting glyoxal to glycolic acid as defined in any one of claims 3 to 6.

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

12. a) transforming said 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 a secretion signal gene for delivering said glyoxalase to the periplasmic space; or a') engineering said microbial cell by integrating said expression cassette into its genome; b) culturing the modified cells in a culture medium adapted for the expression of 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. Including, 11. A method for producing the whole cell biocatalyst of claim 10.

13. 10. A method for producing a glycolic acid derivative or a product obtained by a reaction using glycolic acid as a substrate, the method comprising at least one step of the method for biochemical production of glycolic acid according to 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 for delivering the glyoxalase to the periplasmic space of a microbial cell.

15. 15. A nucleotide sequence encoding the expression cassette of claim 14.

16. A vector comprising the nucleotide sequence of claim 15.

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