GENETICALLY MODIFIED CELLS OF METHYLOBACTERIACEAE FOR FERMENTATIVE PRODUCTION OF GLYCOLIC ACID AND LACTIC ACID FROM CX COMPOUNDS

Abstract

The present invention relates to a genetically modified Methylobacteriaceae cell comprising at least one exogenous nucleic acid sequence encoding a glyoxylate reductase from the bacterium Escherichia, to a process for producing the genetically modified Methylobacteriaceae cell, to a biocatalyst comprising the genetically modified Methylobacteriaceae cell, to a bioreactor comprising the genetically modified Methylobacteriaceae cell, to a process for producing a product containing glycolic acid and lactic acid, and to a process for producing polyglycolic acid, polylactic acid or polylactide-co-glycolide.

Claims

1. A genetically modified Methylobacteriaceae cell comprising at least one exogenous nucleic acid sequence encoding a glyoxylate reductase from the bacterium Escherichia.

2. The genetically modified cell of claim 1, wherein the Methylobacteriaceae cell is a Methylorubrum cell, in particular a cell of Methylorubrum extorquens, in particular Methylorubrum extorquens AM1, Methylorubrum extorquens TK 0001, Methylorubrum extorquens PA1, Methylorubrum rhodesianum or Methylorubrum zatmanii, or a Methylobacterium cell, in particular a cell of Methylobacterium organophilum or Methylobacterium radiotolerans.

3. The genetically modified Methylobacteriaceae cell of claim 1, wherein the bacterium is Escherichia coli, in particular E. coli K-12 MG1655.

4. The genetically modified Methylobacteriaceae cell of claim 1, wherein the glyoxylate reductase from the bacterium Escherichia is encoded by a nucleic acid sequence according to SEQ ID No. 3 or a functional equivalent thereof, wherein the functional nucleic acid sequence equivalent has a nucleic acid sequence identity of 30.0 to 99.9% to the nucleic acid sequence according to SEQ ID No. 3, or wherein the glyoxylate reductase has an amino acid sequence according to SEQ ID No. 2 or a functional amino acid sequence equivalent thereof, wherein the functional amino acid sequence equivalent has an amino acid sequence identity of 30.0 to 99.9% to the amino acid sequence of SEQ ID No. 2.

5. The genetically modified Methylobacteriaceae cell of claim 1, comprising at least one exogenous nucleic acid sequence that encodes an ethylmalonyl-CoA mutase, in particular from at least one bacterium selected from the group consisting of Methylorubrum extorquens, in particular Methylorubrum extorquens TK 0001 DSM 1337, and Rhodobacter sphaeroides, in particular Rhodobacter sphaeroides ATCC 17029.

6. The genetically modified Methylobacteriaceae cell of claim 5, wherein the ethylmalonyl-CoA mutase is encoded by a nucleic acid sequence according to SEQ ID No. 8 or 13 or a functional equivalent thereof, wherein the functional nucleic acid sequence equivalent has a nucleic acid sequence identity of 30.0 to 99.9% to the nucleic acid sequence according to SEQ ID No. 8 or 13, or wherein the ethylmalonyl-CoA mutase has an amino acid sequence according to SEQ ID No. 5 or 7, or a functional equivalent thereof, wherein the functional amino acid sequence equivalent has an amino acid sequence identity of 30.0 to 99.9% to the amino acid sequence according to SEQ ID No. 5 or 7.

7. The genetically modified Methylobacteriaceae cell of claim 1, wherein the at least one exogenous nucleic acid sequence encoding the glyoxylate reductase and/or encoding the ethylmalonyl-CoA mutase is integrated into the chromosome of the Methylobacteriaceae cell or is present extrachromosomally, in particular is present in the cell integrated in an episomal expression vector.

8. The genetically modified Methylobacteriaceae cell of claim 1, wherein the genetically modified Methylobacteriaceae cell is a cell of the Methylorubrum strain Methylorubrum extorquens Mea-GA1, (DSM 34286), Methylorubrum extorquens Mea-GA2, (DSM 34287) or Methylorubrum extorquens Mea-GA3 (DSM 34288), each deposited on 10 Jun. 2022 at the DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany, or a derivative thereof, or wherein the genetically modified Methylobacteriaceae cell is a cell of the Methylorubrum strain Methylorubrum rhodesianum Mrh-GA4 (DSM 34697), Methylorubrum rhodesianum Mrh-GA5 (DSM 34698), Methylorubrum zatmanii Mza-GA14 (DSM 34701), Methylorubrum extorquens Mea-GA17 (DSM 34702) or a cell of the Methylobacterium strain Methylobacterium radiotolerans Mra-GA12 (DSM 34700) or Methylobacterium organophilum Mor-GA8 (DSM 34699), each deposited on 19 Jul. 2023 at the DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany, or a derivative thereof.

9. The genetically modified Methylobacteriaceae cell of o claim 1, wherein the at least one exogenous nucleic acid sequence encoding glyoxylate reductase and/or encoding ethylmalonyl-CoA mutase is functionally connected to additionally at least one regulatory unit by forming an expression cassette, in particular a promoter, in particular an inducible, derepressible or constitutive promoter, an enhancer, a ribosomal binding site and/or a terminator.

10. A process for producing a genetically modified Methylobacteriaceae cell of claim 1, comprising the process steps of: a) providing a Methylobacteriaceae cell, in particular a wild-type cell, and an expression vector or a genome editing system comprising at least one exogenous nucleic acid sequence encoding a glyoxylate reductase from the bacterium Escherichia, in particular an expression cassette comprising this nucleic acid sequence, b) transforming the Methylobacteriaceae cell with the expression vector or the genome editing system under conditions that enable the uptake and, optionally stable, integration of the at least one exogenous nucleic acid sequence into the Methylobacteriaceae cell, and c) obtaining the genetically modified Methylobacteriaceae cell having at least one exogenous, glyoxylate reductase.

11. The process of claim 10, wherein in process step a) at least one exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase, in particular from at least one bacterium selected from the group consisting of Methylorubrum extorquens, in particular Methylorubrum extorquens TK 0001 DSM 1337, and Rhodobacter sphaeroides, in particular Rhodobacter sphaeroides ATCC 17029, in particular an expression cassette comprising this nucleic acid sequence, is provided, in process step b) the Methylobacteriaceae cell is transformed with the exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase, in particular the expression cassette comprising it, and in process step c) a genetically modified Methylobacteriaceae cell having at least one exogenous, glyoxylate reductase, which additionally has at least one exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase, is obtained.

12. A genetically modified Methylobacteriaceae cell of claim 1, wherein the cell is present alive or dead or lyophilised or in the form of a cell lysate or cell extract recovered from a genetically modified Methylobacteriaceae cell.

13. A biocatalyst comprising a genetically modified Methylobacteriaceae cell of claim 1, wherein it is arranged on a carrier.

14. A bioreactor comprising a genetically modified Methylobacteriaceae cell of claim 1.

15. A process for producing a product containing glycolic acid from a reactant containing at least one Cx compound, comprising the process steps of: x) providing a genetically modified Methylobacteriaceae cell of claim 1, a reaction medium and the reactant containing at least one Cx compound, y) converting the reactant under conditions that enable the formation of glycolic acid from the Cx compound, and z) obtaining the product, containing glycolic acid, from the reaction medium.

16. The process of claim 15, wherein the Cx compound is a Cx compound with x=1, 2 or 4, in particular formic acid, methanol, methane, methylamine, acetic acid or succinic acid.

17. The process of claim 15, wherein the product containing glycolic acid is a product containing glycolic acid and lactic acid.

18. The process of claim 15, wherein the Cx compound is produced from CO.sub.2, in particular synthesis gas comprising a mixture of CO.sub.2, CO and H.sub.2, in particular by means of a heterogeneous catalytic chemical process.

19. The process of claim 18, wherein the CO.sub.2, in particular synthesis gas, is produced by chemical conversion of organic compounds or materials, in particular of sewage sludge and other biogenic residual and waste materials.

20. A process for producing polyglycolic acid, polylactic acid or polylactide-co-glycolide, comprising carrying out a process of claim 15 and subsequently polymerising the glycolic acid, lactic acid or mixture of glycolic acid and lactic acid obtained from these processes.

Description

[0236] The figures show:

[0237] FIG. 1: the screening result for glycolic acid production in recombinant, thus genetically modified, M. extorquens TK 0001 strains that have and express codon-optimised genes of the glyoxylate reductases (A), screening results according to 1A in (B and C), wherein enzyme activities of the glyoxylate reductases from the biomass used according to 1(A) are expressed with the expression vector pTE1887 in the strain background M. extorquens TK 0001 with NADH (B) and NADPH (C) as cofactor are represented,

[0238] FIG. 2: an HPLC-chromatogram comparison of the cultivation samples (22 to 24 h after induction) of the genetically modified Methylobacteriaceae cells M. extorquens TK 0001 glyoxylate reductase strains, which have and express codon-optimised genes of the glyoxylate reductases,

[0239] FIG. 3: a GC-MS chromatogram and mass spectra of the glycolic acid peak (retention time: 7.22 min) of a 100 mg L.sup.1 glycolic acid standard, a sample of the reaction medium at t=0 h, a sample of M. extorquens TK 0001+pTE1887 empty vector cultivation after induction, and a sample according to the invention of +pTE1887-ghrA.sub.eco-c-optimised (M. extorquens GA1) cultivation after induction,

[0240] FIG. 4: a GC-MS chromatogram and mass spectra of the lactic acid peak (retention time: 6.88 min) of a 100 mg L.sup.1 lactic acid standard, a sample of the reaction medium at t=0 h, a sample of M. extorquens TK 0001+pTE1887 empty vector cultivation 22-24 h after induction, and a sample of M. extorquens TK 0001+pTE1887-ghrA.sub.eco-c-optimised (M. extorquens GA1) cultivation 22-24 h after induction,

[0241] FIG. 5: a detailed view of the mass spectra of the glycolic acid peak (A) and the lactic acid peak (B) of a sample of M. extorquens GA1 cultivation 22 to 24 h after induction and database verification of the glycolic acid identity (A) and lactic acid identity (B) in the M. extorquens GA1 sample,

[0242] FIG. 6: the growth curve (OD600), the pH value and the methanol, glyoxylate, glycolic acid and lactic acid concentrations of M. extorquens TK 0001+pTE1887 (A+C) and M. extorquens TK 0001+pTE1887-ghrA.sub.eco-c-optimised (M. extorquens GA1) according to the invention (B+D) in the reaction medium, namely minimal medium, wherein as carbon source 8 g L.sup.1 methanol (A+B) or 9 g L.sup.1 methanol+1.5 g L.sup.1 glyoxylic acid (C+D) was added,

[0243] FIG. 7: the growth (OD600), pH value, methanol and the glycolic and lactic acid concentrations of M. extorquens TK 0001+pTE1887 (A), M. extorquens TK 0001+pTE1887-ghrA.sub.eco-c-optimised (M. extorquens GA1) according to the invention (B), M. extorquens TK 0001+pTE1887-ghrA.sub.eco-c-optimised-ecm.sub.mea (M. extorquens GA 2) according to the invention (C) and M. extorquens TK 0001+pTE1887-ghrA.sub.eco-c-optimised-ecm.sub.rsh (M. extorquens GA3) according to the invention (D) in a reaction medium with 9 g L.sup.1 methanol as sole substrate,

[0244] FIG. 8: the plasmid map of the expression vector pTE1887,

[0245] FIG. 9: the plasmid map of the expression vector pTE1887-ghrA.sub.eco-c-optimised,

[0246] FIG. 10: the plasmid map of the expression vector pTE1887-ghrA.sub.eco-c-optimised-ecm.sub.mea,

[0247] FIG. 11: the plasmid map of the expression vector pTE1887-ghrA.sub.eco-c-optimised-ecm.sub.rsh,

[0248] FIG. 12: the results of the glyoxylate reductase enzyme activity tests of ghrA.sub.eco and ghrB.sub.eco in native and codon-optimised DNA sequence expressed with the expression vector pTE1887 in the strain background M. extorquens TK 0001,

[0249] FIG. 13: the taxonomic classification of the methylotrophic microorganisms tested with the expression vectors according to the invention,

[0250] FIG. 14: the screening result for glycolic acid and lactic acid production 22 h to 28 h after induction of gene expression in recombinant, thus genetically modified, M. rhodesianum DSM 5687 strains that have and express codon-optimised genes of the glyoxylate reductases and, in some strains, additionally ethylmalonyl-CoA mutases,

[0251] FIG. 15: the screening result for glycolic acid and lactic acid production 22 h to 28 h after induction of gene expression in recombinant, thus genetically modified, M. zatmanii DSM 5688 strains that have and express the codon-optimised gene of glyoxylate reductase from Escherichia according to the invention and in one strain additionally a codon-optimised gene of ethylmalonyl-CoA mutase from Rhodobacter sphaeroides ATCC 17029,

[0252] FIG. 16: the screening result for glycolic acid and lactic acid production 22 h to 28 h after induction of gene expression in a recombinant, thus genetically modified, M. radiotolerans DSM 760 strain that has and expresses the combination according to the invention of the codon-optimised gene of glyoxylate reductase from Escherichia and additionally a native gene of the ethylmalonyl-CoA mutase from M. extorquens TK 0001 DSM 1337, and

[0253] FIG. 17: the screening result for glycolic acid and lactic acid production 22 h to 28 h after induction of gene expression in recombinant, thus genetically modified, M. organophilum DSM 18172 strains that have and express codon-optimised genes of the glyoxylate reductases and in some strains additionally ethylmalonyl-CoA mutases,

[0254] FIG. 18: the screening result for glycolic acid and lactic acid production 22 h to 28 h after induction of the gene expression of a recombinant, thus genetically modified, M. extorquens PA1 DSM 23939 strain, that has and expresses according to the invention the codon-optimised gene of the glyoxylate reductase from Escherichia coli K12 1655,

[0255] FIG. 19: the screening result for glycolic acid and lactic acid production 22 h to 28 h after induction of gene expression in recombinant, thus genetically modified, M. extorquens AM1cel (based on the strain DSM 1338) strains that have and express codon-optimised genes of the glyoxylate reductases.

EXAMPLES

Example 1: Production of Genetically Modified Methylobacteriaceae Cells

[0256] 12 different exogenous glyoxylate reductases were identified and the associated DNA and amino acid sequences were extracted using bioinformatics methods using the KEGG database (www.genome.jp/kegg/) and the Brenda Enzymes database (https://www.brenda-enzymes.org/). Only glyoxylate reductases that occur in prokaryotes or Saccharomyces cerevisiae were considered. The native glyoxylate reductase from M. extorquens TK 0001 was also selected. An overview of the 13 selected glyoxylate reductases is summarised in Table 2. In particular, the glyoxylate reductase from Thermococcus litoralis was identified as an NADH-dependent enzyme (Ohshima, et al., European Journal of Biochemistry, 2001, 268(17): p. 4740-4747). The influence of the specific redox equivalent on glycolic acid production can be substantial, depending on the availability of the specific redox equivalent in the cytosol and the adaptation of the metabolic network to the intervention (overexpression of glyoxylate reductase).

TABLE-US-00002 TABLE 2 Summary of the tested enzymes Length of DNA Length of amino KEGG- sequence acid sequence Name Enzyme entry Origin [base pairs] [amino acids] PfGoxRed_1 2-Ketogluconate Pfl01_0936 Pseudomonas 981 326 reductase fluorescens Pf0-1 PfGoxRed_2 Putative D-isomer specific Pf101_2771 Pseudomonas 966 321 2-hydroxyacid fluorescens Pf0-1 dehydrogenase family protein PfGoxRed_3 Putative 2-hydroxyacid Pf101_3899 Pseudomonas 930 309 dehydrogenase fluorescens Pf0-1 TlitGoxRed_1 Glyoxylate reductase OCC_02245 Thermococcus litoralis 996 331 TlitGoxRed_2 2-Hydroxyacid dehydrogenase OCC_08355 Thermococcus litoralis 1002 333 PfuGoxRed Putative PF0319 Pyrococcus furiosus 1011 336 phosphoglycerate DSM 3638 dehydrogenase SceGoxRed Glyoxylate reductase YNL274C Saccharomyces cerevisiae 1053 350 TthGoxRed Glycerate dehydrogenase/ TT_C0431 Thermus 1017 338 glyoxylate reductase thermophilus HB27 ghrA.sub.eco Glyoxylate/reductase b1033 Escherichia coli K- 939 312 (invention) 12 MG1655 ghrB.sub.eco Hydro-xypyruvate b3553 Escherichia coli K- 975 324 reductase 12 MG1655 MeaGoxRed Putative 2-hydroxyacid Methylorubrum 996 331 dehydrogenase extorquens TK 0001 AaceGoxRed_1 Glyoxylate reductase AOU92_03200 Acetobacter aceti 987 328 (NADP(+)) AaceGoxRed_2 Glyoxylate/hydroxypyruvate AOU92_11415 Acetobacter aceti 942 313 reductase A

[0257] The heterologous enzymes from Pseudomonas fluorescens Pf0-1, Thermococcus litoralis, Pyrococcus furiosus DSM 3638, Saccharomyces cerevisiae, Thermus thermophilus HB27, Escherichia coli K-12 MG1655 and Acetobacter aceti were encoded by synthetic genes in a codon-optimised form for Methylobacteriaceae (BioCat GmbH, Heidelberg, Germany, Table 1) to support the best possible gene expression. Since the homologous gene from M. extorquens (SEQ ID NO: 1) had the start codon TTG, the start codon was changed to ATG by PCR (Kozak, M., Gene, 1999, 234(2): p. 187-208). In further steps, both gene variants of SEQ ID NO: 1 and 3 were tested. This results in 14 variants of the tested glyoxylate reductases.

[0258] The synthetic genes were cloned in a codon-optimised form using Gibson assembly on the episomal expression vector pTE1887 (Carrillo, M. et al., ACS Synthetic Biology, 2019, 8(11): p. 2451-2456) under the control of the P.sub.L/O4/A1 promoter (IPTG-inducible) (FIG. 8). FIG. 8 shows the vector with the following elements: lacI gene, lacI promoter, PL/O4/A1 promoter,.fwdarw.33 region.fwdarw.10 region.fwdarw.transcription start, PL/O4/A1 promoter ribosomal binding site (RBS), lambda T0 terminator, kanamycin resistance, mobilisation genes mobS and mobL, regulatory protein RepA, origin of replication colE1.

[0259] For this cloning, the expression vector was cut with the restriction enzyme NcoI. The sequence identity and correctness of the constructs were ensured by sequencing. Subsequently, the produced constructs and a wild-type strain Methylobacteriaceae cell, in particular M. extorquens TK 0001 cells and in particular M. extorquens PA1, were according to process step a) provided and according to process step b) transformed into each of the Methylobacteriaceae cells by means of electroporation and a genetically modified Methylobacteriaceae cell is obtained according to process step c). Clones of the Methylobacteriaceae cells, thus genetically modified Methylobacteriaceae cells, which carry the individually produced constructs containing the synthetic genes in codon-optimised form, were selected on minimal medium agar plates with kanamycin as a selection marker. The presence of the expression vectors and the respective expected sequence size of the PCR product which represents the cloned gene were verified by colony PCR in the individual clones obtained. The verified strains were stored at 80 C. as cryocultures.

[0260] To test the ability of the genetically modified Methylobacteriaceae strains to produce glycolic acid, the strains, a minimal medium as a reaction medium, in particular also referred to as a culture medium, and a Cx compound with x=1, namely methanol (Cui, L.-Y. et al., Biochemical Engineering Journal, 2017, 119: p. 67-73) were provided (process step x) according to the invention), cultivated in baffled shake flasks (250 mL flask volume, 50 mL culture volume) at 30 C., 150 RPM (revolutions per minute) and in a water vapour-saturated atmosphere (process step y) according to the invention) (New Brunswick Innova 44, Eppendorf AG, Hamburg, Germany) and a product containing glycolic acid is obtained in the reaction medium (process step z)). Similarly, cultivation with formic acid, among other things, is also possible which can likewise be used as a reactant for glycolic acid production.

[0261] The inoculation of the main cultures was carried out from precultures grown under the same conditions (final OD.sub.600 between 3 and 5) to a starting OD.sub.600 of 0.05. After the cultures had reached an OD.sub.600 of 1.0, the gene expression of the codon-optimised glyoxylate reductase genes was induced with 1 mM IPTG (final concentration in the culture volume). In order to verify the production of glycolic acid, a sample volume of 1 mL of the minimal medium was withdrawn from the culture volume before inoculation and a sample volume of 1 mL respectively from all cultures was withdrawn before induction, directly after induction and about 20 hours after induction. After the biomass was separated from the reaction medium by centrifugation, the samples were analysed using high-performance liquid chromatography (HPLC) and refractive index detection (RID) with regard to the contained concentrations of methanol, formic acid, glyoxylate, glycolic acid and lactic acid. The HPLC measurement was carried out using a Synergi 4 m Hydro-RP 80A, LC column 2504.6 mm (Phenomenex Inc., Torrance, CA, USA) and 20 mM K.sub.2HPO.sub.4 (pH 1.5) as the eluent at 30 C. and a flow rate of 0.5 mL min.sup.1 for 20 minutes per sample. The identification and quantification of the analytes were carried out using external standards of known concentration.

[0262] Glycolic acid was clearly detected in the culture samples by gas chromatography coupled with mass spectrometry (GC-MS) using a glycolic acid standard (100 mg L.sup.1). For this purpose, the OH and NH groups contained in the culture samples and standard were converted into the corresponding tert-butyldimethylsilyl ethers (TBDMS) by derivatisation. For this purpose, a volume of 50 L of standard or 50 L of sample was lyophilised and subsequently resuspended in 50 L DMF+0.1% (v/v) pyridine. Derivatisation was carried out with 50 L N-methyl-N-tert-butyldimethylsilyltrifluoracetamide (MBDSTFA, Macherey-Nagel) and incubation at 80 C. for 30 minutes. Any precipitates that formed were removed by centrifugation and the samples were subsequently analysed using GC-MS. The GC method was set with a carrier gas flow of 1.7 mL min.sup.1, an inlet temperature of 250 C., an interface temperature of 230 C. and a quadrupole temperature of 150 C. The separation of the analytes was carried out using a temperature gradient: 120 C. (2 min), ramp 8 C. min.sup.1 to 200 C. and 10 C. min.sup.1 to 325 C. The analytes were identified in scan mode (m/z 50 to 750) using MS.

[0263] Genetically modified Methylobacteriaceae cells comprising an exogenous, codon-optimised nucleic acid sequence (SEQ ID NO: 3) encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MG1655 of the strain Methylorubrum extorquens Mea-GA1 were deposited on 10 Jun. 2022 at the DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany under the deposit number DSM 34286.

Example 2: Screening of Functional Glyoxylate Reductases in M. extorquens TK 0001

[0264] The glyoxylate reductase-encoding nucleic acid sequences in codon-optimised form listed in Table 2 were cloned into the pTE1887 expression vector as described in example 1 and the corresponding genetically modified Methylobacteriaceae strains were constructed. M. extorquens TK 0001 strain, which contains the pTE1887 vector, was used as a reference strain, which does not carry a recombinant plasmid, but the pTE1887 empty vector.

[0265] In a first experimental step, these initially constructed strains, as described in example 1, were examined for their ability to produce glycolic acid. The results are summarised in FIG. 1.

[0266] FIG. 1A shows a bar chart, wherein the genetically modified Methylobacteriaceae cells are represented on the x-axis and the y-axis shows the concentration of glycolic acid (black filled bar) in g L.sup.1 in the reaction medium. All sample collection times are 22-24 hours after induction of gene expression with 1 mM IPTG. To determine the amount of methanol consumed, the reaction medium was measured at time t=0 h. All concentrations are given in g L.sup.1, determined by HPLC, refractive index detection and external standards.

[0267] FIG. 1A shows the screening result of glycolic acid production in recombinant M. extorquens TK 0001 strains expressing glyoxylate reductases based on the corresponding codon-optimised genes. pTE1887 was used as expression vector, which also serves as a negative control in the form of the empty vector in the reference strain M. extorquens TK 0001+pTE1887 (first entry from the left on the x-axis).

[0268] Surprisingly, neither the reference strain M. extorquens TK 0001+pTE1887 (first entry from the left) nor the genetically modified Methylobacteriaceae cells showed any glycolic acid production (entries from the left: 2 and 3 and 5 to 15), with the exception of the genetically modified Methylobacteriaceae cell according to the invention, comprising M. extorquens TK 0001+pTE1887-ghrA.sub.eco (in codon-optimised nucleic acid form according to SEQ ID NO: 3), thus a genetically modified Methylobacteriaceae cell according to the invention comprising at least one exogenous nucleic acid sequence encoding a glyoxylate reductase from the bacterium Escherichia (entry from the left: 4, is the only entry to show a black bar). FIG. 9 shows the map of the vector used to generate these Methylorubrum cells with the following elements: lacI gene, lacI promoter, PL/O4/A1 promoter,.fwdarw.33 region.fwdarw.10 region.fwdarw.transcription start, PL/O4/A1 promoter ribosomal binding site (RBS), ghrA.sub.eco-c-optimised, Lambda T0 terminator, kanamycin resistance, mobilisation genes mobS and mob, regulatory protein RepA. Origin of replication colE1.

[0269] FIG. 1B shows a bar chart, wherein the genetically modified Methylobacteriaceae cells are represented on the x-axis and the y-axis represents the enzyme activity in mU mg.sup.1 (white, unfilled bar: NADH as cofactor).

[0270] FIG. 1C shows a bar chart wherein the genetically modified Methylobacteriaceae cells are represented on the x-axis and the y-axis represents the enzyme activity in mU mg.sup.1 (grey bar: NADPH as cofactor).

[0271] FIGS. 1B and 1C show the screening result of an enzyme assay with recombinant M. extorquens TK 0001 strains that express glyoxylate reductases, based on the corresponding codon-optimised genes. The enzyme assay was carried out in the same way as in example 5. The biomass used was that used in 1A. In the case of 1B, the enzyme assay was carried out with NADH as redox cofactor. In the case of 1C, the enzyme assay was carried out with NADPH as redox cofactor.

[0272] In the case of 1B, all tested Methylobacteriaceae cells containing recombinant glyoxylate reductases show no measurable glyoxylate reductase enzyme activity with NADH as cofactor, with the exception of the Methylobacteriaceae cell containing the glyoxylate reductase ghrB.sub.eco not according to the invention (entry from the left: 5).

[0273] In FIG. 1C, the reference strain M. extorquens TK 0001+pTE1887 (first entry from the left), as well as several tested Methylobacteriaceae cells containing recombinant glyoxylate reductases (entries from the left: 2, 8 and 9, 11 to 15) showed no measurable glyoxylate reductase enzyme activity with NADPH as cofactor. Only the genetically modified Methylobacteriaceae cells (entries from the left: 3 to 7 and 10) showed increased glyoxylate reductase enzyme activity, wherein the genetically modified Methylobacteriaceae cells of M. extorquens TK 0001+pTE1887-ghrA.sub.eco (in codon-optimised nucleic acid form according to SEQ ID NO: 3), thus a genetically modified Methylobacteriaceae cell comprising at least one exogenous nucleic acid sequence encoding a glyoxylate reductase from the bacterium Escherichia, had a high glyoxylate reductase enzyme activity (entry from the left: 4).

[0274] No enzyme activity of the glyoxylate reductase Tlit could be measured and neither was it associated with a glycolic acid production. Only the enzyme activity of ghrA.sub.eco, thus the glyoxylate reductase from E. coli according to the invention, is connected with a glycolic acid production.

[0275] Accordingly, the genetically modified Methylobacteriaceae cell comprising M. extorquens TK 0001+pTE1887-ghrA.sub.eco-c-optimised according to the invention has NADPH dependence but not NADH dependence.

[0276] FIG. 2 shows HPLC chromatograms of the cultivation samples according to FIG. 1 (22 to 24 hours after induction) of the genetically modified Methylobacteriaceae cells M. extorquens TK 0001 glyoxylate reductase strains. It can be clearly seen that only the genetically modified Methylobacteriaceae cell M. extorquens TK 0001+pTE1887-ghrA.sub.eco according to the invention (referred to in FIG. 2 as M. extorquens GA1, containing the codon-optimised form of the ghrA.sub.eco gene) is the only strain to produce the mixture of glycolic acid and lactic (glycolic acid retention time=6.20 min and lactic acid retention time=9.1 min) (comparison of standardslane 1 and lane 2with M. extorquens TK 0001+pTE1887-ghrA.sub.eco, M. extorquens GA1lane 7 in 1A and lane 5 in 1B). The lack of glycolic acid (and lactic acid production when using glyoxylate reductases not according to the invention) indicates a lack of functionality. These enzymes could be hydroxypyruvate reductases, which reduce the hydroxypyruvate that also accumulates in the serine cycle, depending on NAD(P)H, to D-glycerate. In this case, as shown in FIG. 1 and FIG. 2, no accumulation of glycolic acid would be observed.

[0277] This sample was analysed by GC-MS in comparison with external standards to confirm the presence of glycolic acid and lactic acid in the sample and thus verify the production of glycolic acid and the surprising production of lactic acid by expression of the ghrA.sub.eco enzyme. Further peaks: glyoxylate (retention time=5.40 min), methanol (retention time=7.6 min), peaks not described in further detail here (retention time=6.50 min and 8.00 min).

[0278] In the case of the genetically modified Methylobacteriaceae cell according to the invention, about 0.6 g L.sup.1 of the mixture of glycolic acid and lactic acid could be detected in the HPLC measurement (Y.sub.P/S77 mg.sub.glycolic acid+lactic acid g methanol.sup.1).

[0279] In most cultivations, the initial methanol concentration of 8 g L.sup.1 was depleted after approximately 22 to 24 h after induction. Only the strain M. extorquens GA1 showed a clearly measurable methanol concentration at the time of sample collection (FIG. 1A, lane 7 and FIG. 1B, lane 5). This may indicate an imbalance in the metabolism caused by the gene expression of the glyoxylate reductase. On the one hand, the enzyme expression itself can reduce the growth of the strains. But increased enzyme activity of a glyoxylate reductase can also cause a reduction of glyoxylate from the serine cycle towards glycolic acid. As a result, the microorganism lacks glyoxylate to build biomass. This deficiency can slow growth and lead to that the carbon source is not completely used up.

[0280] To confirm the presence of glycolic acid and lactic acid in the ghrA.sub.eco sample according to the invention in comparison to external standards (each 100 mg L.sup.1 lactic acid and glycolic acid), this sample was examined as described in example 1, with a GC-MS measurement (FIG. 3 to FIG. 5).

[0281] FIG. 3 shows a GC-MS chromatogram and mass spectra of a 100 mg L.sup.1 glycolic acid standard, a sample of the medium at t=0 h, a sample of M. extorquens TK 0001+pTE1887 empty vector cultivation 22-24 h after induction and a sample according to the invention of M. extorquens TK 0001+pTE1887-ghrA.sub.eco (referred to in FIG. 3 as M. extorquens GA1, containing the codon-optimised form of the ghrA.sub.eco gene) cultivated 22 to 24 h after induction. FIG. 4 shows the same samples, except for the standard, which was replaced by a 100 mg L.sup.1 lactic acid standard. The measurements verify that glycolic acid was clearly formed in the cultivation sample of M. extorquens TK 0001+pTE1887-ghrA.sub.eco (M. extorquens GA1) according to the invention compared to the glycolic acid standard (retention time=7.22 min). The mass spectrum of the obtained peak in the M. extorquens TK 0001+pTE1887-ghrA.sub.eco sample according to the invention clearly matches the mass spectrum of the glycolic acid standard (FIG. 5A). This proves the existence of glycolic acid in the M. extorquens TK 0001+pTE1887-ghrA.sub.eco sample according to the invention and thus the production of glycolic acid by this strain. In comparison, no glycolic acid can be detected in the sample of the M. extorquens TK 0001+pTE1887 empty vector strain. Surprisingly, the formation of lactic acid (retention time=6.88 min) was also detected exclusively in the sample of the M. extorquens TK 0001+pTE1887-ghrA.sub.eco cultivation according to the invention. Here, too, the mass spectrum matches that of the lactic acid standard (FIG. 5B).

[0282] This approach clearly showed that glycolic acid was produced by the genetically modified Methylorubrum cell M. extorquens TK 0001+pTE1887-ghrA.sub.eco according to the invention. Surprisingly, it was also shown that this strain produces a mixture of glycolic acid and lactic acid (see FIG. 5). FIG. 5 shows detailed mass spectra of an identical sample of the M. extorquens TK 0001+pTE1887-ghrA.sub.eco according to the invention (referred to in FIG. 5 as M. extorquens GA1, containing the codon-optimised form of the ghrA.sub.eco gene) Cultivation 22 to 24 h after induction with database evidence of the glycolic acid identity in the M. extorquens GA1 sample (A) and the lactic acid identity in the M. extorquens GA1 sample (B).

[0283] Surprisingly, in the cultivation of M. extorquens TK 0001+pTE1887-ghrA.sub.eco it could be verified by GC-MS that both glycolic acid (retention time=7.22 min) and lactic acid (retention time=6.88 min) were produced (FIGS. 3 to 5). The peak with a retention time of 6.88 min in this sample was identified as lactic acid 2TBDMS (derivative of lactic acid with MBDSTFA) with an 89-91% probability by comparison with an external standard and by database matching of the mass spectrum (FIG. 5B).

[0284] The control strain M. extorquens TK 0001+pTE1887 did not show this phenotype: neither glycolic acid nor lactic acid could be detected as products by GC-MS.

[0285] Without wanting to be bound by theory, the changes in the redox balance alter the metabolism of the genetically modified Methylobacteriaceae cell M. extorquens TK 0001+pTE1887-ghrA.sub.eco such that lactic acid is synthesised as a possible by-product of glycolic acid production. An NADH-dependent lactate dehydrogenase (KEGG database: Mex_1p4794), which uses pyruvate as a substrate, could be responsible for this lactic acid formation. An alternative possibility is that the glyoxylate reductase has a non-specific substrate utilisation that enables the enzyme to use pyruvate as an acceptor. The course of the methylglyoxal metabolic pathway is also conceivable.

[0286] It can therefore be shown that the M. extorquens TK 0001+pTE1887-ghrA.sub.eco cells according to the invention containing the codon-optimised form of the ghrA.sub.eco gene, produce a mixture of glycolic acid and lactic acid, which can serve as a starting point for polymerisation to polyglycolic acid, polylactic acid or polylactide-co-glycolide.

Example 3: Growth Experiments

[0287] Furthermore, growth experiments were carried out with M. extorquens TK 0001+pTE1887 and, according to the invention, with the strain M. extorquens TK 0001+pTE1887-ghrA.sub.eco (M. extorquens GA1) in minimal medium (reaction medium) with 10 g L.sup.1 methanol as one reactant and a mixture of 10 g L.sup.1 methanol+1.5 g L.sup.1 glyoxylate as a further reactant (FIG. 6).

[0288] FIGS. 6 A to D show diagrams in which the y-axes represent the growth curve (OD.sub.600, circles, black filled), the pH value (triangles, tip down) and the methanol (squares, unfilled), glyoxylate (diamonds, unfilled) and glycolic acid concentrations (diamonds, dark grey filled) as well as lactic acid (triangles, grey filled, tip up) of M. extorquens TK 0001+pTE1887 (A+C) and M. extorquens TK 0001+pTE1887-ghrA.sub.eco according to the invention (codon-optimised) (B+D) in the minimal medium and the time is given on the x-axis. As carbon source (reactant), thus Cx compound, 10 g L.sup.1 methanol (A+B) or 10 g L.sup.1 methanol+1.5 g L.sup.1 glyoxylate (C+D) was added. The measurement of the methanol, glyoxylate and glycolic acid concentrations was carried out by HPLC, refractive index detection and external standards. All concentrations are given in g L.sup.1. The data represent three independent biological replicates.

[0289] The glyoxylate was added at the time of induction of gene expression and serves as a test to see if an in vivo increase in glyoxylate supply leads to an increase in glycolic acid production.

[0290] In FIG. 6A it can be recognized that the reference strain M. extorquens TK 0001+pTE1887 with 10 g L.sup.1 methanol as reactant did not produce any glycolic acid and had a uniform biomass formation up to a maximum OD.sub.600 of approx. 9 after 40 h of cultivation. The significant decrease in the pH value to below 6.5 over the course of the fermentation is striking. In comparison, in a cultivation with M. extorquens TK 0001+pTE1887, the addition of glyoxylate led to slightly delayed growth and a slightly higher maximum OD.sub.600 of approx. 10 after about 42 h. In this case, too, no glycolic acid was produced (FIG. 6C). However, the pH value in this cultivation could be maintained at the initial pH value of around 7.0, which is probably due to the glyoxylate feeding.

[0291] It was shown that the recombinant strain M. extorquens TK 0001+pTE1887-ghrA.sub.eco containing the codon-optimised form of the ghrA.sub.eco according to the invention, from 10 g L.sup.1 methanol, the products glycolic acid and lactic acid were formed in increased concentrations (0.35 g L.sup.1 and 0.25 g L.sup.1 respectively in 40 h). After the methanol has been degraded, the products are completely degraded in the further course of cultivation. The formation of glycolic acid and lactic acid is accompanied by a significant slowdown in biomass growth to a maximum OD.sub.600 of 6.7 in 44 h. Furthermore, the pH value of the culture broth presumably drops in this case due to the additionally formed glycolic acid to 6.2 and rises due to the degradation of the glycolic acid to a value comparable to that of the reference strain, just under 6.5 (FIG. 6B).

[0292] In the experiment with the M. extorquens TK 0001+pTE1887-ghrA.sub.eco containing the codon-optimised form of the ghrA.sub.eco gene according to the invention, and with the additional feeding of glyoxylate, a significant increase in glycolic acid production of up to 1.0 g L.sup.1 in 44 h was achieved. The amount of lactic acid formed was comparable to that obtained in cultivation without glyoxylate supplementation (6B). This showed that glyoxylate plays an important role as a precursor in the formation of glycolic acid, and that increasing the in vivo concentration of glyoxylate results in improved glycolic acid production. In this experiment, too, the formed glycolic acid and lactic acid were metabolised after the methanol was consumed. The increased product formation in this experiment again led to a further reduction in biomass growth, wherein a maximum OD.sub.600 of around 4.5 was achieved. In contrast to the reference strain, a significant decrease in pH value can be observed in this case despite the addition of glyoxylate, as glycolic acid and lactic acid were produced. However, an increase in pH value can also be observed during the degradation of the formed glycolic acid and lactic acid after the methanol has been consumed (FIG. 6D).

[0293] In summary, the strain-specific cultivation parameters derived from the data, such as (specific growth rate), Y.sub.X/S (dry-biomass-substrate-yield), q.sub.S (specific substrate uptake rate), Y.sub.p is (product-substrate-yield) and q.sub.P (specific product formation rate) were summarized in Table 3 for the strains M. extorquens TK 0001+pTE1887 and the M. extorquens TK 0001+pTE1887-ghrA.sub.eco containing the codon-optimised form of the ghrA.sub.eco gene according to the invention. These data suggest that glycolic acid and lactic acid production is associated with a significant reduction in the biomass-substrate-yield (70% of the reference strain and 70% of the reference strain with glyoxylate feeding) and more carbon is converted into the product or has to be used to maintain the redox balance. It can also be seen that glyoxylate reduces the growth rate, indicating a potential toxic effect of the precursor. This toxicity of glyoxylate can be avoided by an optimal balancing of the in vivo glyoxylate pool.

TABLE-US-00003 TABLE 3 Summary of cultivation parameters of M. extorquens TK 0001 + pTE1887 and M. extorquens TK 0001 + pTE1887-ghr A.sub.eco (M. extorquens GA1) containing the codon-optimised form of the ghrA.sub.eco gene according to the invention, in a minimal medium with 10 g L.sup.1 methanol or additionally + 1.5 g L.sup.1 glyoxylate. Y.sub.X/S q.sub.S Y.sub.P/S q.sub.P [g.sub.DBM [g.sub.MeOH [g.sub.GA [g.sub.GA strain and condition [1 h.sup.1] g.sub.MeOH.sup.1] g.sub.DBM.sup.1 h.sup.1] g.sub.MeOH.sup.1] g.sub.DBM.sup.1 h.sup.1] M. extorquens TK 0001 + pTE1887 0.17 0.37 0.46 0.00 0.00 M. extorquens TK 0001 + pTE1887- 0.10 0.26 0.38 0.07 0.06 ghrA.sub.eco (according to the invention) M. extorquens TK 0001 + pTE1887 + 0.16 0.23 0.70 0.00 0.00 2 g L.sup.1 glyoxylate.sup.1 M. extorquens TK 0001 + pTE1887- 0.09 0.16 0.56 0.17 0.22 ghrA.sub.eco + 2 g L.sup.1 glyoxylate.sup.1 (according to the invention) .sup.1Yields are estimated from the cumulative substrate utilisation of methanol and glyoxylate. Abbreviations: , specific growth rate MeOH, methanol; GA, glycolic acid; DBM, dry biomass.

[0294] Examples 1 to 3 show that according to the invention glycolic acid and lactic acid can be produced by M. extorquens GA1 from Cx compounds in a methylotrophic fermentation process according to the invention.

[0295] It should in particular be emphasised that glycolic acid production according to the invention in M. extorquens GA1 can be significantly increased by increasing the intracellular concentration of glyoxylate, as shown in Example 3. In this case, 185% more glycolic acid was produced compared to cultivation without glyoxylate feeding.

Example 4: Experimental Data from the Fermentative Production of Glycolic Acid and Lactic Acid from Methanol

[0296] The experimental proceedings were carried out according to example 1. The strain used is the wild-type strain Methylorubrum extorquens TK 0001 DSM 1337.

[0297] The following expression vectors (1 to 4) were used: [0298] 1.) pTE1887 (expression vector, also named as empty vector; plasmid map: FIG. 8) [0299] 2) pTE1887-ghrA.sub.eco (expression vector which codon-optimised encodes the glyoxylate reductase from Escherichia coli K-12 MG1655; with SEQ ID NO: 3, plasmid map: FIG. 9) (according to the invention) [0300] 3) pTE1887-ghrA.sub.eco-ecm.sub.mea (expression vector which encodes the glyoxylate reductase from Escherichia coli K-12 MG1655 (codon-optimised) and the ethylmalonyl-CoA mutase from M. extorquens TK 0001 DSM 1337 natively; plasmid map: FIG. 10) (according to the invention) [0301] 4) pTE1887-ghrA.sub.eco-ecm.sub.rsh (expression vector encoding the glyoxylate reductase from Escherichia coli K-12 MG1655 (codon-optimised) and the ethylmalonyl-CoA mutase from Rhodobacter sphaeroides ATCC 17029 codon-optimised; plasmid map: FIG. 11) (according to the invention).

[0302] Genetically modified Methylobacteriaceae cells were produced by means of the processes described in example 1.

[0303] FIG. 10 shows the map of the vector used to generate the Methylobacteriaceae cells expressing ghrA.sub.eco-ecm.sub.mea, with the following elements: lacI gene, lacI promoter, PL/O4/A1 promoter.fwdarw.33 region.fwdarw.10 region.fwdarw.transcription start, PL/O4/A1 promoter ribosomal binding site (RBS), ghrA.sub.eco (codon-optimised), ecm.sub.mea (native), lambda T0 terminator, kanamycin resistance, mobilisation genes mobS and mob, regulatory protein RepA. Origin of replication colE1.

[0304] FIG. 11 shows the map of the vector used to generate these Methylobacteriaceae cells, expressing ghrA.sub.eco-ecm.sub.rsh, with the following elements: lacI gene, lacI promoter, PL/O4/A1 promoter.fwdarw.33 region.fwdarw.10 region.fwdarw.transcription start, PL/O4/A1 promoter ribosomal binding site (RBS), ghrA.sub.eco (codon-optimised), rsh-ecm (codon-optimised), lambda T0 terminator, kanamycin resistance, mobilisation genes mobS and mob, regulatory protein RepA. Origin of replication colE1.

[0305] Genetically modified Methylorubrum extorquens TK 0001-cells comprising an exogenous, codon-optimised nucleic acid (SEQ ID NO: 3) encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MG1655 and an exogenous, native nucleic acid sequence (SEQ ID NO: 4) encoding an ethylmalonyl-CoA mutase from the bacterium Methylorubrum extorquens TK 0001 DSM 1337, of the strain Methylorubrum extorquens Mea-GA2 were deposited on 10 Jun. 2022 at the DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany under the deposit number DSM 34287.

[0306] Genetically modified Methylorubrum extorquens TK 0001-cells comprising an exogenous, codon-optimised nucleic acid sequence (SEQ ID NO: 3) encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MG1655 and an exogenous, codon-optimised nucleic acid sequence (SEQ ID NO: 8) encoding an ethylmalonyl-CoA mutase from the bacterium Rhodobacter sphaeroides ATCC 17029, of the strain Methylorubrum extorquens Mea-GA3 were deposited on 10 Jun. 2022 at the DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany under the deposit number DSM 34288.

[0307] Fermentation experiments were carried out using culture medium as the reaction medium and methanol (reactant) as the sole carbon source.

[0308] FIG. 7 shows the time course of the biomass concentration (OD.sub.600) and the medium pH value over the course of the cultivation. At the same time, culture supernatant samples were measured using high-performance chromatography to show the substrate and product concentrations and their changes over time.

[0309] FIG. 7 shows the time in hours on the x-axis and on the y-axes the growth curve (OD.sub.600, circles, black filled), pH value (triangles, tip down), methanol (squares, unfilled) and glycolic (diamonds, dark grey filled) and lactic acid concentrations (triangles, grey filled, tip up) of M. extorquens TK 0001+pTE1887 (A), M. extorquens TK 0001+pTE1887-ghrA.sub.eco (codon-optimised) according to the invention (M. extorquens GA1) (B), M. extorquens TK 0001+pTE1887-ghrA.sub.eco-ecm.sub.mea (ghrA.sub.eco: codon-optimised; ecm.sub.mea: native) according to the invention (M. extorquens GA2) (C) and M. extorquens TK 0001+pTE1887-ghrA.sub.eco-ecm.sub.rsh (both genes codon-optimised) according to the invention (M. extorquens GA3) (D) in a culture medium with 10 g L.sup.1 methanol as the sole reactant, thus as a Cx compound.

[0310] It was again shown that the enzyme ghrA.sub.eco, allows a production of glycolic acid in the background strain M. extorquens TK 0001. Lactic acid is also produced here. In comparison, the wild-type strain, which contains the empty vector pTE1887, does not show production of glycolic acid or lactic acid. In these experiments, approx. 250 mg L.sup.1 glycolic acid and approx. 200 mg L.sup.1 lactic acid were produced with a product substrate yield of around 50 mg g.sub.methanol.sup.1 (glycolic acid+lactic acid). It can be observed that the initiated glycolic acid synthesis lowers the dry-biomass-substrate-yield (Y.sub.X/S) of the strain M. extorquens TK 0001+pTE1887-ghrA.sub.eco according to the invention (M. extorquens GA1) to 70% compared to the empty vector strain. The product yield based on the dry biomass (Y.sub.P/X) is 0.27 g g.sub.dry biomass.sup.1 (Table 4).

[0311] Surprisingly, the additionally implementation of the exogenous ethylmalonyl-CoA mutase leads to a significant improvement in glycolic acid production performance compared to the strain M. extorquens TK 0001+pTE1887-ghrA.sub.eco according to the invention. The growth of the M. extorquens TK 0001+pTE1887-ghrA.sub.eco-ecm.sub.mea strain according to the invention, with a measured growth rate () of 0.10 h.sup.1 was delayed compared to the empty vector strain (0.17 h.sup.1). The use of the ecm gene from M. extorquens TK 0001 DSM 1337 leads to an increase of the glycolic acid titer by 18% after 40 h of cultivation compared to the strain according to the invention M. extorquens TK 0001+pTE1887-ghrA.sub.eco (0.26 g L.sup.1 versus 0.22 g L.sup.1, Table 4). Likewise, the dry-biomass-substrate-yield is reduced by 49% compared to the empty vector strain and by 27% compared to the strain according to the invention M. extorquens TK 0001+pTE1887-ghrA.sub.eco.

[0312] A slight change can be observed in the product yield based on the dry biomass (Y.sub.P/X). Compared to the strain according to the invention M. extorquens TK 0001+pTE1887-ghrA.sub.eco, an 11% increase in this yield was achieved (Table 4).

[0313] The use of the exogenous codon-optimised ecm.sub.rsh gene leads to the surprising deviations of the cultivation parameters in comparison to the strain according to the invention comprising the exogenous ecm.sub.mea gene, as can be seen in Table 4. The highest measured lactic acid titer of 0.37 g L.sup.1 was observed here. A striking change concerns the dry-biomass-substrate-yield (Y.sub.X/S), which is increased by 26% (0.24 g g.sub.dry biomass.sup.1) compared to the strain according to the invention M. extorquens TK 0001+pTE1887-ghrA.sub.eco-ecm.sub.mea.

[0314] It was demonstrated that glycolic acid can be produced from methanol. The use of two exogenous ethylmalonyl-CoA mutase enzymes from two different prokaryotic strains increased the production performance of the production strains according to the invention compared to the strain according to the invention comprising ghrA.sub.eco without an exogenous ethylmalonyl-CoA mutase. In particular, the use of the ethylmalonyl-Coa mutase ecm.sub.rsh Surprisingly leads to a significantly increased and more selective lactic acid production.

TABLE-US-00004 TABLE 4 Summary of the cultivation parameters of M. extorquens TK 0001 + pTE1887 and M. extorquens TK 0001 + pTE1887-ghrA.sub.eco (codon-optimised) according to the invention (M. extorquens GA1), M. extorquens TK 0001 + pTE1887-ghrA.sub.eco-ecm.sub.mea (ghrA.sub.eco: codon- optimised; ecm.sub.mea: native) according to the invention (M. extorquens GA2) and M. extorquens TK 0001 + pTE1887-ghrA.sub.eco-ecm.sub.rsh (both genes codon-optimised) according to the invention (M. extorquens GA3) in culture medium with 10 g L.sup.1 methanol. Titer GA at Y.sub.X/S Y.sub.P/S Y.sub.P/X approx. 40 [g.sub.DBM [g.sub.GA [g.sub.GA strain and condition [1 h.sup.1] h [g L.sup.1] g.sub.MeOH.sup.1] g.sub.MeOH.sup.1] g.sub.DBM.sup.1] M. extorquens AM1 + pTE1887 0.17 0.00 0.37 0.00 0.00 +pTE1887- ghrA.sub.eco (according to the invention) 0.10 0.22 0.26 0.07 0.27 +pTE1887-ghrA.sub.eco-ecm.sub.mea (according to the invention 0.10 0.26 0.19 0.07 0.30 +pTE1887-ghrA.sub.eco-ecm.sub.rsh (according to the invention) 0.07 0.08 0.24 0.06 0.23 Abbreviations: , specific growth rate; MeOH, methanol; GA, glycolic acid; DBM, dry biomass.

Example 5: Experimental Data of the Demonstration of the Enzyme Activity of the Glyoxylate Reductase (ghrA.SUB.eco.) Expressed According to the Invention and of a Comparison Enzyme, Namely an E. coli Hydroxypyruvate Reductase (ghrB.SUB.eco.)

[0315] To provide experimental evidence for the presence of the enzyme activity of the expressed glyoxylate reductase ghrA.sub.eco and the hydroxypyruvate reductase ghrB.sub.eco (Nuez, M. F., M. T. Pellicer, J. Badia, J. Aguilar, and L. Baldoma, Biochem J, 2001. 354 (Pt 3): p. 707-15, database entry for ghrA: https://biocyc.org/gene?orgid=ECOLI&id=G6539, database entry for ghrB: https://biocyc.org/gene?orgid=ECOLI&id=EG12272) in the strain background Methylorubrum extorquens TK 0001 enzyme assays were carried out. The native form of the DNA sequences (as found in Escherichia coli K-12 MG1655) and the synthetic DNA sequences (c-optimised) that are codon-optimised for expression in Methylobacteriaceae were tested to evaluate the influence of codon optimisation on gene expression and the resulting enzyme activity.

[0316] The procedure for carrying this out and the results are summarised below.

[0317] In order to obtain sufficient biomass of the genetically modified M. extorquens TK 0001 strains containing pTE1887-ghrA.sub.eco-c-optimised (SEQ ID NO: 3), pTE1887-ghrB.sub.eco-c-optimised, pTE1887-ghrA.sub.eco-native (SEQ ID NO:1) and pTE1887-ghrB.sub.eco-native for cell disruption, the following cultivation protocol was used. The strain M. extorquens TK 0001+pTE1887 containing the empty vector, was used as a negative control. All strains were cultivated, harvested and disrupted as three independent biological replicates.

[0318] The strains were cultivated for an initial three-day preculture (in minimal medium with methanol (see example 1) in baffled shake flasks (250 mL flask volume, 50 mL culture volume) at 30 C., 150 RPM and water vapour-saturated atmosphere (New Brunswick Innova 44, Eppendorf AG, Hamburg, Germany). Subsequently, a second preculture was inoculated from the first preculture in minimal medium with methanol in baffled shake flasks (250 mL flask volume, 50 mL culture volume). The initial biomass concentration used for the inoculation corresponded to an optical density at 600 nm (OD.sub.600) of 0.1. The subsequent cultivation was carried out at 30 C., 150 rpm and in a water vapour-saturated atmosphere. On the next day, Tuesday, the main cultures were inoculated with the overgrown second precultures (50 mL minimal medium with methanol in 250 mL baffled shake flasks, initial OD.sub.600=0.05) and incubated at 30 C., 150 rpm and in a water vapour-saturated atmosphere.

[0319] After the cultures had reached an OD.sub.600 of 0.9-1.0, the gene expression of the glyoxylate reductases was induced with 1 mM IPTG (final concentration in the culture volume). Subsequently, the biomass was grown to a final OD600 of approx. 4-7.

[0320] For the actual biomass harvest, 50 mL conical centrifugation tubes were weighed empty, then each was filled with the 50 mL main culture and then centrifuged at 4,200 rpm for 15 min at 4 C. After centrifugation, the supernatant was discarded and the biomass pellets obtained were each washed with 20 mL of 50 mM Tris-HCl (pH 7.5) buffer. After that, centrifugation was repeated under the previous conditions, followed by careful removal of the supernatant with a pipette. The obtained biomass pellets were weighed and each was resuspended in 50 mM MOPS buffer (pH 6.6). For this, a buffer volume of 7 mL was used per 1 g wet pellet.

[0321] Cell disruption to obtain crude protein extracts containing the expressed glyoxylate reductases or hydroxypyruvate reductases was carried out in 2.0 mL reaction vessels. For this purpose, 1.5 mL of the cell suspension was transferred to each of these reaction vessels and subsequently disrupted six times for 30 seconds each at an amplitude of 60 by ultrasonication in an ice water bath. Between each of the six disruption cycles, the samples were cooled on ice for 1 minute. Finally, to obtain the raw protein extract, a centrifugation step at 21,500 rpm for 15 minutes at 4 C. was carried out. The protein-containing supernatant obtained was transferred to 1.5 mL reaction vessels. To ensure comparability of the enzyme assay results, the protein concentration of each raw protein extract was determined using a NanoDrop. The raw extract with the lowest measured concentration was used as the target concentration for diluting the other raw extracts with 50 mM MOPS buffer (pH 6.6). This ensured that all crude protein extracts contained the same total protein concentration in the enzyme assay. Furthermore, these prediluted crude protein extracts were diluted a further time (1:5) with 50 mM MOPS buffer (pH 6.6) and subsequently used in the enzyme assay.

[0322] The enzyme assay was carried out in 96-well microtiter plates. For this, 160 L of the diluted crude protein extracts were mixed with 20 L of 50 mM glyoxylate as a substrate and 20 L of a 2 mM cofactor stock solution (NADH or NADPH, final concentration in the assay 0.2 mM). The experimental approaches were carried out in three technical replicates. The enzyme activity was measured as the change in absorbance of NADH at 340 nm at 37 C. for up to 30 min. For the evaluation, the maximum change in absorbance over time was determined in the linear region of the reaction and multiplied by the dilution factor of five before calculating the enzyme activity in U mL.sup.1.

[0323] The enzyme activity was calculated using equation 6 and the given coefficients.

[00006]$\begin{array}{cc}\frac{U}{V_{P\mathrm{roteinrohextrakt}-Assay}}=S*\frac{V_{Assay}}{{}_{}*d*V_{Pr\mathrm{oteinrohextrakt}}}\left[\frac{U}{\mathrm{mL}}\right]& \left(\mathrm{Equation}6\right)\end{array}$

[0324] With enzyme activity: Measured in mol.sub.substrate min.sup.1, V.sub.protein crude extract assay: volume of protein crude extract used in the assay (0.00016 L), S: change in absorbance at 340 nm over time in the linear region of the reaction, corrected for the dilution factor of five (Abs..sub.340 min..sup.1), V.sub.assay: total volume of the assay (0.0002 L), : extinction coefficient of NADH/NADPH at 340 nm (6220 L mol.sup.1 cm.sup.1), d: layer thickness of the absorbing reaction mixture (0.53 cm).

[0325] To convert the enzyme activity from mol.sub.substrate min.sup.1 into the conventional unit for enzyme activity mU mL.sup.1 (1 U=1 mol.sub.substrate min.sup.1), the calculated result is multiplied by a factor of 10.sup.6.

[0326] The obtained enzyme activities were assigned to the respective expression strains and the cofactors NADH or NADPH used for a graphical comparison.

[0327] The data collected for the enzyme activities of ghrA.sub.eco-c-optimised, ghrB.sub.eco-c-optimised, ghrA.sub.eco-native, ghrB.sub.eco-native and the negative control (pTE1887 empty vector) are summarised in FIG. 12. The measurements and the standard deviation displayed are based on three biological replicates, each with three technical replicates of the assay.

[0328] As expected, the empty vector shows only a slight background activity. This was subtracted from all further measurements in order to correct for the background reaction that took place.

[0329] The enzyme activities with regard to the conversion of glyoxylate into glycolic acid shown in FIGS. 12A and 12B indicate that only the two enzymes used according to the invention, ghrA.sub.eco-c-optimised and ghrA.sub.eco-native (thus ghrA.sub.eco), exhibit sufficient activity, particularly for large-scale production. Furthermore, significant differences in enzyme activity depending on the cofactor used can be demonstrated. The assay shows that there is a clear cofactor dependency of ghrA.sub.eco and ghrB.sub.eco. Using NADH as a cofactor (FIG. 12 A), the highest enzyme activity is achieved with ghrB.sub.eco-c-optimised (10.531.50 mU mL.sup.1). The enzyme activity caused by the ghrA.sub.eco-c-optimised gene with 0.492.34 mU mL.sup.1 is significantly reduced compared to ghrB.sub.eco-c-optimised. A reduction in enzyme activity of around 95% was measured here. The enzyme activity of the NADH assays with the native genes is in a similar region: 4.611.61 mU mL.sup.1 versus 2.450.67 mU mL.sup.1 for ghrA.sub.eco-native and ghrB.sub.eco-native. Codon optimisation of ghrB.sub.eco leaded to an increase in activity of 329%. In summary, a clear dependence of the ghrB.sub.eco enzyme on NADH as a cofactor can be recognized.

[0330] In contrast, a different picture emerged when using NADPH as a cofactor (FIG. 12 B).

[0331] Here, ghrA.sub.eco-c-optimised and ghrA.sub.eco-native (33.861.29 mU mL.sup.1 and 21.761.49 mU mL.sup.1) achieve by far the highest enzyme activities measured in the tests that were present. The increase due to codon optimisation is 55% (21.761.49 versus 33.861.29 mU mL.sup.1). It can also be clearly verified that the ghrA.sub.eco enzyme has an NADPH dependency. This is underlined by the low measured activity of ghrB.sub.eco. In this case, for both the use of the condon-optimised variant of the gene (ghrB.sub.eco-c-optimised) and the native variant of the gene (ghrB.sub.eco-native) only a low enzyme activity of 3.170.29 and 0.810. 0.30 mU mL.sup.1 (FIG. 12 B) was measured, which shows that ghrB.sub.eco can be clearly distinguished from ghrA not only with regard to the observed very low NADPH dependence, but primarily also with regard to the low enzyme activity in the conversion of glyoxylate into glycolic acid.

[0332] The increased enzyme activity with NADPH as cofactor triggered by the expression of ghrA.sub.eco-c-optimised shows that the production of glycolic acid by M. extorquens is possible through the expression of this enzyme. The significantly reduced enzyme activity measured in the context of ghrB.sub.eco-c-optimised with both NADPH and NADH is not sufficient to enable glycolic acid production in vivo in M. extorquens.

[0333] The glycolic acid production observed with M. extorquens TK 0001+pTE1887-ghrA.sub.eco-c-optimised appears to be dependent on the availability of the cofactor NADPH. These results confirm the results from example 2.

[0334] Surprisingly, introducing the DNA sequence of the ghrA.sub.eco enzyme, in particular the codon-optimised DNA sequence, leads to a glycolic acid production as well as the surprising production of lactic acid.

Example 6

[0335] Expression of exogenous nucleic acid sequence encoding a glyoxylate reductase from the bacterium Escherichia in cells of further Methylobacteriaceae (invention) and other microorganisms (comparison)

[0336] Further genera of the family Methylobacteriaceae (Alphaproteobacteria) were genetically modified according to Example 1. FIG. 13 shows the microorganisms examined as examples. In particular, the following were examined as representatives of the Methylobacteriaceae: Methylorubrum, in particular M. zatmanii DSM 5688, in particular M. extorquens TK 0001 DSM 1337 (examples 2 to 5), in particular M. extorquens PA1 DSM 23939, in particular M. rhodesianum DSM 5687, a derivative of M. extorquens AM1 DSM 1338 with a deletion of a cellulase gene (M. extorquens AM1cel: https://doi.org/10.1371/journal.pone.0062957), and Methylobacterium cells, in particular M. organophilum DSM 18172, in particular M. radiotolerans DSM 760.

[0337] In addition, Methylomonas methanica DSM 25384 (Gammaproteobacteria), Methylophilus methylotrophus DSM 6330 (Betaproteobacteria) and Bacillus methanolicus DSM 16454 (Firmicutes) were examined as negative examples not belonging to the family Methylobacteriaceae. The aforementioned microorganisms are also able to metabolise methanol and were tested for the production of glycolic acid and/or lactic acid according to the invention.

[0338] The following expression vectors (1 to 4) were used: [0339] 1.) pTE1887 (expression vector, also named as empty vector; plasmid map: FIG. 8) [0340] 2) pTE1887-ghrA.sub.eco (expression vector which codon-optimised encodes the glyoxylate reductase from Escherichia coli K-12 MG1655; with SEQ ID NO: 3, plasmid map: FIG. 9) (according to the invention) [0341] 3) pTE1887-ghrA.sub.eco-ecm.sub.mea (expression vector which encodes the glyoxylate reductase from Escherichia coli K-12 MG1655 (codon-optimised) and the ethylmalonyl-CoA mutase from M. extorquens TK 0001 DSM 1337 natively; plasmid map: FIG. 10) (according to the invention) [0342] 4) pTE1887-ghrA.sub.eco-ecm.sub.rsh (expression vector encoding the glyoxylate reductase from Escherichia coli K-12 MG1655 (codon-optimised) and the ethylmalonyl-CoA mutase from Rhodobacter sphaeroides ATCC 17029 codon-optimised; plasmid map: FIG. 11) (according to the invention).

[0343] Genetically modified Methylobacteriaceae cells comprising an exogenous, codon-optimised nucleic acid sequence (SEQ ID NO: 3) encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MG1655 of the strain Methylorubrum zatmanii Mza-GA14 (M. zatmanii DSM 5688+pTE1887-ghrA.sub.eco) were deposited on 19 Jul. 2023 at the DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany under the deposit number DSM 34701.

[0344] Genetically modified Methylobacteriaceae cells comprising an exogenous, a glyoxylate reductase from the bacterium Escherichia coli K-12 MG1655 encoding codon-optimised nucleic acid sequence (SEQ ID NO: 3) of the strain Methylorubrum extorquens Mea-GA17 (M. extorquens PA1 DSM 23939+pTE1887-ghrA.sub.eco) were deposited on 19 Jul. 2023 at the DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany under the deposit number DSM 34702.

[0345] Genetically modified Methylobacteriaceae cells comprising an exogenous, codon-optimised nucleic acid sequence (SEQ ID NO: 3) encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MG1655 of the strain Methylorubrum rhodesianum Mrh-GA4 (M. rhodesianum DSM 5687+pTE1887-ghrA.sub.eco) were deposited on 19 Jul. 2023 at DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany under deposit number DSM 34697.

[0346] Genetically modified Methylobacteriaceae cells comprising an exogenous, codon-optimised nucleic acid sequence (SEQ ID NO: 3) encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MG1655 and an exogenous, native nucleic acid sequence (SEQ ID NO: 4) encoding an ethylmalonyl-CoA mutase from the bacterium Methylorubrum extorquens TK 0001 DSM 1337 of the strain Methylorubrum rhodesianum Mrh-GA5 (M. rhodesianum hodesianum DSM 5687+pTE1887-ghrA.sub.eco-ecm.sub.mea) were deposited on 19 Jul. 2023 at the DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany under the deposit number DSM 34698.

[0347] Genetically modified Methylobacteriaceae cells comprising an exogenous, codon-optimised nucleic acid sequence (SEQ ID NO: 3) encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MG1655 of the strain Methylobacterium organophilum Mor-GA 8 (M. organophilum DSM 18172+pTE1887-ghrA.sub.eco-ecm.sub.mea) were deposited on 19 Jul. 2023 at the DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany under the deposit number DSM 34699.

[0348] Genetically modified Methylobacteriaceae cells comprising an exogenous, codon-optimised nucleic acid sequence (SEQ ID NO: 3) encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MG1655 and an exogenous, native nucleic acid sequence (SEQ ID NO: 4) encoding a ethylmalonyl-CoA mutase from the bacterium Methylorubrum extorquens TK 0001 DSM 1337 of the strain Methylobacterium radiotolerans Mra-GA12 (M. radiotolerans DSM 760+pTE1887-ghrA.sub.eco-ecm.sub.mea) were deposited on 19 Jul. 2023 at the DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany under the deposit number DSM 34700.

[0349] To study the invention with the aforementioned strains, the process was carried out according to example 2. In contrast to example 2, the cultivations with the Methylobacteriaceae cells M. rhodesianum (FIG. 14) DSM 5687, M. zatmanii DSM 5688 (FIG. 15), M. radiotolerans DSM 760 (FIG. 16), M. organophilum DSM 18172 (FIG. 17), M. extorquens PA1 DSM 23939 (FIG. 18) were started with a reduced amount of reactant (Cx compound, 4 g L.sup.1 methanol) and additionally reactant was fed between ten and twelve hours after induction (fed-batch, cumulatively up to 15 g L.sup.1). Furthermore, the samples were withdrawn to determine the concentrations of glycolic acid, lactic acid and methanol after 22-28 h after induction of gene expression with 1 mM IPTG.

[0350] FIGS. 14 to 19 show the genetically modified Methylobacteriaceae cells on the x-axis and the concentration of methanol (white, unfilled bar) or the concentration of the mixture of formed glycolic acid and lactic acid (black, filled bar) in g L.sup.1 in the reaction medium on the y-axis. All sample taking times are 22 to 28 hours after induction of gene expression with 1 mM IPTG. All concentrations are given in g L.sup.1, determined by HPLC, refractive index detection and external standards.

[0351] FIG. 14 shows the screening result of glycolic acid and lactic acid production with recombinant M. rhodesianum DSM 5687 strains expressing glyoxylate reductases based on the corresponding codon-optimised genes. The first entry from the left shows the methanol concentration in the minimal medium at the beginning of cultivation. pTE1887 was used as the expression vector, which also serves as a negative control in the form of the empty vector in the reference strain M. rhodesianum DSM 5687+pTE1887 (second entry from the left on the x-axis).

[0352] Surprisingly, both the reference strain M. rhodesianum DSM 5687+pTE1887 (second entry from the left) and the genetically modified Methylobacteriaceae cells showed no glycolic acid or lactic acid production (entries from the left: 3 to 10 and 12 to 16), with the exception (black, filled bars in FIG. 14) of the genetically modified cells of M. rhodesianum DSM 5687+pTE1887-ghrA.sub.eco (in codon-optimised nucleic acid form according to SEQ ID NO: 3) according to the invention, thus a genetically modified Methylobacteriaceae cell according to the invention comprising at least one exogenous nucleic acid sequence encoding a glyoxylate reductase from the bacterium Escherichia (entry from the left: 11) and the genetically modified cells M. rhodesianum DSM 5687+pTE1887-ghrA.sub.eco-ecm.sub.mea comprising an exogenous, codon-optimised nucleic acid sequence (SEQ ID NO: 3) encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MG1655 and an exogenous native nucleic acid sequence (SEQ ID NO: 4) encoding an ethylmalonyl-CoA mutase from the bacterium Methylorubrum extorquens TK 0001 DSM 1337, thus a genetically modified Methylobacteriaceae cell according to the invention comprising at least one exogenous nucleic acid sequence encoding a glyoxylate reductase from the bacterium Escherichia (entry from the left: 17) and the genetically modified cells M. rhodesianum DSM 5687+pTE1887-ghrA.sub.eco-ecm.sub.rsh, comprising an exogenous codon-optimised nucleic acid sequence (SEQ ID NO: 3) encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MG1655 and an exogenous codon-optimised nucleic acid sequence (SEQ ID NO: 8) encoding an ethylmalonyl-CoA mutase from the bacterium Rhodobacter sphaeroides ATCC 17029, thus a genetically modified Methylobacteriaceae cell according to the invention comprising at least one exogenous nucleic acid sequence encoding a glyoxylate reductase from the bacterium Escherichia (entry from the left: 18).

[0353] FIG. 9 shows the map of the vector used to generate these Methylobacteriaceae cells, with the following elements: lacI gene, lacI promoter, PL/O4/A1 promoter.fwdarw.33 region.fwdarw.10 region.fwdarw.transcription start, PL/O4/A1 promoter ribosomal binding site (RBS), ghrA.sub.eco-c optimised, Lambda T0 terminator, kanamycin resistance, mobilisation genes mobS and mob, regulatory protein RepA. Origin of replication colE1.

[0354] FIG. 10 shows the map of the vector used to generate these Methylobacteriaceae cells expressing ghrA.sub.eco-ecm.sub.mea with the following elements: lacI gene, lacI promoter, PL/O4/A1 promoter.fwdarw.33 region.fwdarw.10 region.fwdarw.transcription start, PL/O4/A1 promoter ribosomal binding site (RBS), ghrA.sub.eco (codon-optimised), ecm.sub.mea (native), lambda T0 terminator, kanamycin resistance, mobilisation genes mobS and mob, regulatory protein RepA. Origin of replication colE1.

[0355] FIG. 11 shows the map of the vector used to generate these Methylobacteriaceae cells, expressing ghrA.sub.eco-ecm.sub.rsh, with the following elements: lacI gene, lacI promoter, PL/O4/A1 promoter.fwdarw.33 region.fwdarw.10 region.fwdarw.transcription start, PL/O4/A1 promoter ribosomal binding site (RBS), ghrA.sub.eco (codon-optimised), rsh-ecm (codon-optimised), lambda T0 terminator, kanamycin resistance, mobilisation genes mobS and mob, regulatory protein RepA. Origin of replication colE1.

[0356] The cells of M. rhodesianum DSM 5687 genetically modified according to the invention were able to produce mixtures of glycolic acid and lactic acid containing a total concentration of glycolic acid plus lactic acid of up to 0.85 g L.sup.1 (M. rhodesianum DSM5687+pTE1887-ghrA.sub.eco-ecm.sub.mea), at least 0.82 g L.sup.1 (M. rhodesianum DSM5687+pTE1887-ghrA.sub.eco), at least 0.09 g L.sup.1 (M. rhodesianum DSM5687+pTE1887-ghrA.sub.eco-ecm.sub.rsh).

[0357] These experimental data demonstrate that glycolic acid and lactic acid production according to the invention is possible within the family Methylobacteriaceae.

[0358] FIG. 15 shows the screening result of glycolic acid and lactic acid production with recombinant M. zatmanii DSM 5688 strains which, according to the invention, express the gene for glyoxylate reductase from Escherichia and, in one case, additionally the gene of an ethylmalonyl-CoA mutase from Rhodobacter sphaeroides ATCC 17029, based on the corresponding codon-optimised genes. The first entry from the left shows the methanol concentration in the minimal medium at the beginning of cultivation. pTE1887 was used as the expression vector, which also serves as a negative control in the form of the empty vector in the reference strain M. zatmanii DSM 5688+pTE1887 (second entry from the left on the x-axis).

[0359] Surprisingly, the reference strain M. zatmanii DSM 5688+pTE1887 (second entry from the left) showed no glycolic acid and lactic acid production, in contrast to (black, filled bars in FIG. 15) the genetically modified cells of M. zatmanii DSM 5688+pTE1887-ghrA.sub.eco according to the invention (in codon-optimised nucleic acid form according to SEQ ID NO: 3), thus a genetically modified Methylobacteriaceae cell according to the invention comprising at least one exogenous nucleic acid sequence encoding a glyoxylate reductase from the bacterium Escherichia (entry from the left: 3) and the genetically modified cells M. zatmanii DSM 5688+pTE1887-ghrA.sub.eco-ecm.sub.rsh comprising an exogenous, codon-optimised nucleic acid sequence (SEQ ID NO: 3) encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MG1655 and an exogenous, codon-optimised nucleic acid sequence (SEQ ID NO: 8) encoding an ethylmalonyl-CoA mutase from the bacterium Rhodobacter sphaeroides ATCC 17029, thus a genetically modified Methylobacteriaceae cell according to the invention comprising at least one exogenous nucleic acid sequence encoding a glyoxylate reductase from the bacterium Escherichia (entry from the left: 4) FIG. 9 shows the map of the vector used to generate these Methylobacteriaceae cells, with the following elements: lacI gene, lacI promoter, PL/O4/A1 promoter.fwdarw.33 region.fwdarw.10 region 4 transcription start, PL/O4/A1 promoter ribosomal binding site (RBS), ghrA.sub.eco-c optimised, Lambda T0 terminator, kanamycin resistance, mobilisation genes mobS and mob, regulatory protein RepA. Origin of replication colE1.

[0360] FIG. 11 shows the map of the vector used to generate these Methylobacteriaceae cells, expressing ghrA.sub.eco-ecm.sub.rsh, with the following elements: lacI gene, lacI promoter, PL/O4/A1 promoter.fwdarw.33 region.fwdarw.10 region.fwdarw.transcription start, PL/O4/A1 promoter ribosomal binding site (RBS), ghrA.sub.eco (codon-optimised), rsh-ecm (codon-optimised), lambda T0 terminator, kanamycin resistance, mobilisation genes mobS and mob, regulatory protein RepA. Origin of replication colE1.

[0361] Mixtures of glycolic acid and lactic acid could be produced from the genetically modified cells of M. zatmanii DSM 5688 according to the invention, containing a total concentration of glycolic acid plus lactic acid of up to 0.56 g L.sup.1 (M. zatmanii DSM 5688+pTE1887-ghrA.sub.eco), at least 0.48 g L.sup.1 (M. zatmanii DSM 5688+pTE1887-ghrA.sub.eco-ecm.sub.rsh).

[0362] These experimental data demonstrate that glycolic acid and lactic acid production according to the invention is possible within the family of Methylobacteriaceae.

[0363] FIG. 16 shows the screening result of glycolic acid and lactic acid production with a recombinant M. radiotolerans DSM 760 strain. The first entry from the left shows the methanol concentration in the minimal medium at the beginning of cultivation. pTE1887 was used as expression vector, which also serves as a negative control in the form of the empty vector in the reference strain M. radiotolerans DSM 760+pTE1887 (second entry from the left on the x-axis).

[0364] Surprisingly, the reference strain M. radiotolerans DSM 760+pTE1887 (second entry from the left) showed no glycolic acid or lactic acid production, in contrast to the genetically modified cells of M. radiotolerans DSM 760+pTE1887-ghrA.sub.eco-ecm.sub.mea according to the invention comprising an exogenous, codon-optimised nucleic acid sequence (SEQ ID NO: 3) encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MG1655 and an exogenous, native nucleic acid sequence (SEQ ID NO: 4) encoding an ethylmalonyl-CoA mutase from the bacterium Methylorubrum extorquens TK 0001 DSM 1337, thus a genetically modified Methylobacteriaceae cell according to the invention comprising at least one exogenous nucleic acid sequence encoding a glyoxylate reductase from the bacterium Escherichia (entry from the left: 3) (black filled bar in FIG. 16).

[0365] The combination of ghrA.sub.eco and ecm.sub.mea (an ethylmalonyl-CoA mutase from M. extorquens TK 0001 DSM 1337 according to the invention) leaded to the production of glycolic acid and lactic acid according to the invention.

[0366] FIG. 10 shows the map of the vector used to generate these Methylobacteriaceae cells expressing ghrA.sub.eco-ecm.sub.mea with the following elements: lacI gene, lacI promoter, PL/O4/A1 promoter.fwdarw.33 region.fwdarw.10 region.fwdarw.transcription start, PL/O4/A1 promoter ribosomal binding site (RBS), ghrA.sub.eco (codon-optimised), ecm.sub.mea (native), lambda T0 terminator, kanamycin resistance, mobilisation genes mobS and mob, regulatory protein RepA. Origin of replication colE1. Mixtures of glycolic acid and lactic acid could be produced with the genetically modified cells of M. radiotolerans DSM 760 according to the invention containing a total concentration of glycolic acid plus lactic acid of up to 0.39 g L.sup.1 (M. radiotolerans DSM 760+pTE1887-ghrA.sub.eco-ecm.sub.mea).

[0367] These experimental data demonstrate that glycolic acid and lactic acid production according to the invention is possible within the family of Methylobacteriaceae.

[0368] FIG. 17 shows the screening result of glycolic acid and lactic acid production with recombinant M. organophilum DSM 18172 strains expressing glyoxylate reductases based on the corresponding codon-optimised genes. The first entry from the left shows the methanol concentration in the minimal medium at the beginning of cultivation. pTE1887 was used as expression vector, which also serves as negative control in form of the empty vector form in the reference strain M. organophilum DSM 18172+pTE1887 (second entry from the left on the x-axis).

[0369] Surprisingly, neither the reference strain M. organophilum DSM 18172+pTE1887 (second entry from the left) nor the genetically modified Methylobacteriaceae cells showed any glycolic acid or lactic acid production (entries from the left: 3 to 10 and 12 to 18), with the exception (black, filled bars in FIG. 17) of the genetically modified cells of M. organophilum DSM 18172+pTE1887-ghrA.sub.eco (in codon-optimised nucleic acid form according to SEQ ID NO: 3) according to the invention, thus a genetically modified Methylobacteriaceae cell according to the invention comprising at least one exogenous nucleic acid sequence encoding a glyoxylate reductase from the bacterium Escherichia (entry from the left: 11) and the genetically modified cells M. organophilum DSM 18172+pTE1887-ghrA.sub.eco-ecm.sub.mea comprising an exogenous codon-optimised nucleic acid sequence (SEQ ID NO: 3) encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MG1655 and an exogenous native nucleic acid sequence (SEQ ID NO: 4) encoding an ethylmalonyl-CoA mutase from the bacterium Methylorubrum extorquens TK 0001 DSM 1337, thus a genetically modified Methylobacteriaceae cell according to the invention comprising at least one exogenous nucleic acid sequence encoding a glyoxylate reductase from the bacterium Escherichia (entry from left: 17) and the genetically modified cells M. organophilum DSM 18172+pTE1887-ghrA.sub.eco-ecm.sub.rsh comprising an exogenous codon-optimised nucleic acid sequence (SEQ ID NO: 3) encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MG1655 and an exogenous codon-optimised nucleic acid sequence (SEQ ID NO: 8) encoding an ethylmalonyl-CoA mutase from the bacterium Rhodobacter sphaeroides ATCC 17029, thus a genetically modified Methylobacteriaceae cell according to the invention comprising at least one exogenous nucleic acid sequence encoding a glyoxylate reductase from the bacterium Escherichia (entry from the left: 18).

[0370] FIG. 9 shows the map of the vector used to generate these Methylobacteriaceae cells, with the following elements: lacI gene, lacI promoter, PL/O4/A1 promoter.fwdarw.33 region.fwdarw.10 region 4 transcription start, PL/O4/A1 promoter ribosomal binding site (RBS), ghrA.sub.eco-c optimised, Lambda T0 terminator, kanamycin resistance, mobilisation genes mobS and mob, regulatory protein RepA. Origin of replication colE1.

[0371] FIG. 10 shows the map of the vector used to generate these Methylobacteriaceae cells expressing ghrA.sub.eco-ecm.sub.mea with the following elements: lacI gene, lacI promoter, PL/O4/A1 promoter.fwdarw.33 region.fwdarw.10 region.fwdarw.transcription start, PL/O4/A1 promoter ribosomal binding site (RBS), ghrA.sub.eco (codon-optimised), ecm.sub.mea (native), lambda T0 terminator, kanamycin resistance, mobilisation genes mobS and mob, regulatory protein RepA. Origin of replication colE1.

[0372] FIG. 11 shows the map of the vector used to generate these Methylobacteriaceae cells, expressing ghrA.sub.eco-ecm.sub.rsh, with the following elements: lacI gene, lacI promoter, PL/O4/A1 promoter.fwdarw.33 region.fwdarw.10 region.fwdarw.transcription start, PL/O4/A1 promoter ribosomal binding site (RBS), ghrA.sub.eco (codon-optimised), rsh-ecm (codon-optimised), lambda T0 terminator, kanamycin resistance, mobilisation genes mobS and mob, regulatory protein RepA. Origin of replication colE1.

[0373] Mixtures of glycolic acid and lactic acid could be produced from the genetically modified cells of M. organophilum DSM 18172 according to the invention, containing a total concentration of glycolic acid plus lactic acid of up to 0.13 g L.sup.1 (M. organophilum DSM 18172+pTE1887-ghrA.sub.eco), at least 0.10 g L.sup.1 (M. organophilum DSM 18172+pTE1887-ghrA.sub.eco-ecm.sub.mea), at least 0.04 g L.sup.1 (M. organophilum DSM 18172+pTE1887-ghrA.sub.eco-ecm.sub.rsh).

[0374] These experimental data demonstrate that glycolic acid and lactic acid production according to the invention is possible within the family of Methylobacteriaceae.

[0375] FIG. 18 shows the screening result of glycolic acid and lactic acid production with recombinant M. extorquens PA1 DSM 23939 strains. The first entry from the left shows the methanol concentration in the minimal medium at the beginning of cultivation. pTE1887 was used as expression vector, which also serves as a negative control in the form of the empty vector in the reference strain M. extorquens PA1 DSM 23939+pTE1887 (second entry from the left on the x-axis).

[0376] Surprisingly, the reference strain M. extorquens PA1 DSM 23939+pTE1887 (second entry from the left) showed no glycolic acid or lactic acid production, in contrast to the genetically modified cells of M. extorquens PA1 DSM 23939+pTE1887-ghrA.sub.eco (in codon-optimised nucleic acid form according to SEQ ID NO: 3) according to the invention, thus a genetically modified Methylobacteriaceae cell according to the invention comprising at least one exogenous nucleic acid sequence encoding a glyoxylate reductase from the bacterium Escherichia (black filled bar in FIG. 18) (entry from the left: 3).

[0377] FIG. 9 shows the map of the vector used to generate these Methylobacteriaceae cells, with the following elements: lacI gene, lacI promoter, PL/O4/A1 promoter.fwdarw.33 region.fwdarw.10 region 4 transcription start, PL/O4/A1 promoter ribosomal binding site (RBS), ghrA.sub.eco-c optimised, Lambda T0 terminator, kanamycin resistance, mobilisation genes mobS and mob, regulatory protein RepA. Origin of replication colE1.

[0378] Mixtures of glycolic acid and lactic acid could be produced from the genetically modified cells M. extorquens PA1 DSM 23939 according to the invention, containing a total concentration of glycolic acid plus lactic acid up to 1.50 g L.sup.1 (M. extorquens PA1 DSM 23939+pTE1887-ghrA.sub.eco) (M. extorquens GA17).

[0379] These experimental data demonstrate that the production of glycolic acid and lactic acid according to the invention is possible within the family Methylobacteriaceae.

[0380] FIG. 19 shows the screening results of glycolic acid and lactic acid production with recombinant M. extorquens AM1cel strains which express glyoxylate reductases based on the corresponding codon-optimised genes. The first entry from the left shows the methanol concentration in the minimal medium at the beginning of cultivation. pTE1887 was used as expression vector, which also serves as a negative control in the form of the empty vector in the reference strain M. extorquens AM1cel+pTE1887 (second entry from the left on the x-axis).

[0381] Surprisingly, the reference strain M. extorquens AM1cel+pTE1887 (second entry from the left) showed no glycolic acid or lactic acid production, with the exception (black filled bar in FIG. 19) of the genetically modified M. extorquens AM1 cel+pTE1887-ghrA.sub.eco (in codon-optimised nucleic acid form according to SEQ ID NO: 3) according to the invention, thus a genetically modified Methylobacteriaceae cell according to the invention comprising at least one exogenous nucleic acid sequence encoding a glyoxylate reductase from the bacterium Escherichia (entry from the left: 11).

[0382] FIG. 9 shows the map of the vector used to generate these Methylobacteriaceae cells, with the following elements: lacI gene, lacI promoter, PL/O4/A1 promoter.fwdarw.33 region.fwdarw.10 region 4 transcription start, PL/O4/A1 promoter ribosomal binding site (RBS), ghrA.sub.eco-c optimised, Lambda T0 terminator, kanamycin resistance, mobilisation genes mobS and mob, regulatory protein RepA. Origin of replication colE1.

[0383] Mixtures of glycolic acid and lactic acid could be produced from the genetically modified M. extorquens AM1cel cells according to the invention, containing a total concentration of glycolic acid plus lactic acid of up to 0.60 g L.sup.1 (M. extorquens AM1cel+pTE1887-ghrA.sub.eco).

[0384] These experimental data demonstrate that glycolic acid and lactic acid production according to the invention is possible within the family of Methylobacteriaceae. Deletion of the cellulase gene (cel) does not affect glyoxylate-glycolic acid-lactic acid-metabolism.

[0385] The results of these studies are summarised in Table 5 (below).

[0386] Further studies were carried out in methylotrophic microorganisms not belonging to the family Methylobacteriaceae. For this purpose, the strain construction procedures according to example 1 were carried out to generate genetically modified strains of Methylomonas methanica DSM 25384 (Gammaproteobacteria), Methylophilus methylotrophus DSM 6330 (Betaproteobacteria) and Bacillus methanolicus DSM 16454 (Firmicutes). This was not possible in any of the cases with the strains used. The strains studied showed no growth during the strain construction procedure described in example 1 for all vectors pTE1887, pTE1887-ghrA.sub.eco, pTE1887-ghrA.sub.eco-ecm.sub.mea and pTE1887-ghrA.sub.eco-ecm.sub.rsh (Table 5).

TABLE-US-00005 TABLE 5 Summary of the achieved glycolic acid and lactic acid titers of tested strains of the family Methylobacteriaceae and comparative examples (microorganisms not belonging to the family of the Methylobacteriaceae). Strain growth on MO Titer Ga + medium with 0.5% Growth after LA after methanol, Kanamycin introducing 22 h to 28 strain 30 (g/mL), 30 C..sup.[1] plasmids h [g L.sup.1] Deposited at DSMZ Methylorubrum rhodesianum yes yes 0.82 M. rhodesianum DSM 5687 + pTE1887- Mrh-GA4 (DSM ghrA.sub.eco 34697) Methylorubrum rhodesianum yes yes 0.85 M. rhodesianum DSM 5687 + pTE1887- Mrh-GA5 (DSM ghrAeco-ecm.sub.mea 34698) Methylorubrum rhodesianum yes yes 0.09 no DSM 5687 + pTE1887- ghrA.sub.eco-ecm.sub.rsh Methylorubrum zatmanii yes yes 0.56 M. zatmanii Mza- DSM 5688 + pTE1887- GA14 (DSM 34701) ghrA.sub.eco Methylorubrum zatmanii yes yes 0.48 no DSM 5688 + pTE1887- ghrA.sub.eco-ecm.sub.rsh Methylobacterium yes yes 0.39 M. radiotolerans radiotolerans DSM 760 + Mra-GA12 (DSM pTE1887-ghrA.sub.eco-ecm.sub.mea 34700) Methylobacterium yes yes 0.13 M. organophilum organophilum DSM 18172 + Mor-GA8 (DSM pTE1887-ghrA.sub.eco 34699) Methylobacterium yes yes 0.04 no organophilum DSM 18172 + pTE1887-ghrA.sub.eco-ecm.sub.mea Methylobacterium yes yes 0.10 no organophilum DSM 18172 + pTE1887-ghrA.sub.eco-ecm.sub.rsh Methylorubrum extorquens yes yes 1.50 M. extorquens Mea- PA1 DSM 23939 + GA17 (DSM 34702) pTE1887-ghrA.sub.eco Methylorubrum extorquens yes yes 0.60 no AM1cel + pTE1887- ghrA.sub.eco Methylomonas methanica no DSM 25384 Methylophilus yes.sup.[1] no methylotrophus DSM 6330 Bacillus methanolicus no.sup.[1] DSM 16454 .sup.[1]Temperatures used in growth tests: Methylophilus methylotrophus DSM 6330 (37 C.), Bacillus methanolicus DSM 16454 (45 C.). Abbreviations: GA, glycolic acid; LA, lactic acid.