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.

Description

[0001] DESCRIPTION

[0002] Genetically modified cells of Methylobacteriaceae for the fermentative production of glycolic acid and lactic acid from Cx compounds

[0003] The present invention relates to a genetically modified cell from the family Methylobacteriaceae comprising at least one exogenous nucleic acid sequence encoding a glyoxylate reductase from the bacterium Escherichia, a process for producing the genetically modified Methylobacteriaceae cell, a biocatalyst comprising the genetically modified Methylobacteriaceae cell, a bioreactor comprising the biocatalyst comprising the genetically modified Methylobacteriaceae cell, a process for producing a product containing glycolic acid and a process for producing polyglycolic acid, polylactic acid or polylactide-co-glycolide.

[0004] Glycolic acid, also known as hydroxyacetic acid or hydroxyethanoic acid, is an organic carboxylic acid with two carbon atoms, containing a carboxyl group and a hydroxy group at the C2 atom as functional groups. Glycolic acid has a wide range of uses in the textile industry, for example, as a dye and tanning agent; in the food industry, for example, as a flavoring and preservative or packaging material; and in the pharmaceutical industry, for example, as a skin care product (Salusjärvi, L. et al., Applied Microbiology and Biotechnology, 2019, 103(6): pp. 2525-2535; hereinafter Salusjäryi et al.). In the polymer industry, glycolic acid can be processed together with lactic acid to form a co-polymer (polylactide-co-glycolide) or in medical technology as polyglycolic acid to form a suture material that is absorbable by the body (Salusjäryi et al.; Jem, KJ and B. Tan, Advanced Industrial and Engineering Polymer Research, 2020. 3(2): p. 60-70; hereinafter Jem et al.).Currently, glycolic acid is produced industrially almost exclusively petrochemically from fossil raw materials using formaldehyde, carbon monoxide, and water. Intensive research is underway to find sustainable ways to produce the economically relevant compound glycolic acid from renewable raw materials, independent of fossil resources.

[0005] The production of glycolic acid from renewable substrates, such as D-glucose, D-xylose, D-arabinose, L-lyxose, L-arabinose, acetates, or ethanol, via microbial fermentation is known but not yet industrially established (Salusjäryi et al., Jem et al., Gädda, TM et al., Appita Journal, 2014. 67(1): p. 12). These substrates are derived from biogenic raw materials. Therefore, the use of such substrates poses a sustainability risk, as raw materials are used for chemical production that can also be used for food and feed production, such as bioethanol.

[0006] The synthesis of glycolic acid is readily accessible biotechnologically from substrates such as hexoses, pentoses, or, for example, glycolonitrile (disclosed in US Pat. No. 7,198,927 B2), formaldehyde and hydrogen cyanide (disclosed in EP 1 828 393 B1), or ethylene glycol (disclosed in EP 2 025 760 B1). In contrast, the direct biotechnological synthesis of glycolic acid from CO2 is difficult to access. This is due, among other things, to the inherent limitations of the efficiency of photosynthetic metabolism and the gas-liquid mass transfer of gas fermentation. The latter two approaches continue to be limited by low yields and conversion rates and the number of available and genetically accessible microorganisms (Frazäo, CJR and T. Walther, Chemie Ingenieur Technik, 2020. 92(11): p. 1680-1699, and Kang, NK, M. Kim, K. Baek, YK Chang, DR Ort, and Y.-S. Jin, Chemical Engineering Journal, 2022. 433: p. 133636).

[0007] Biotechnologically useful intermediates, such as methanol or formic acid, can be produced from CO2 in various ways (Bohlen, et al., Electrochemistry Communications, 2020. 110: p. 106597; Bowker, M., ChemCatChem, 2019, 11(17): p. 4238- 4246; Lenärd-Istvan Csepei, FS et al., F.-GzFdaF eV, Editor, 2016: Germany) and are also referred to as Cx compounds in the following.

[0008] Cx compounds, such as methanol or formic acid, or mixtures of these two substrates, can be used by methylotrophic microorganisms as an energy source for the production of biomass or valuable products, particularly chemicals. In the central carbon metabolism of methylotrophic microorganisms, Cx compounds such as methanol or formic acid are taken up as substrates. In the first reaction steps, for example, methanol is oxidized to formic acid. This produces the redox equivalents required for the metabolism, cytochrome c in its reduced form and NAD(P)H. Formic acid can then either be oxidized to CO2 or (like formaldehyde) be incorporated into the serine cycle. The serine cycle serves as a carbon distribution circuit for the methylotrophic microorganism and provides the main precursors required for biomass synthesis.Furthermore, the serine cycle is a starting point for other metabolic pathways that are essential for growth on Cx compounds. For example, in the serine cycle, the intermediate glyoxylate is regenerated by the linked ethylmalonyl-CoA pathway. The serine cycle intermediate glyoxylate can be converted into glycolic acid by reduction with NADH or NADPH coupled to a glyoxylate reductase (ghrA). Glyoxylate reductases (ghrA), along with hydroxypyruvate reductases (ghrB), belong to the glyoxylate / hydroxypyruvate reductases (ghr).

[0009] It is known that the M. extorquens TK 0001 genome contains a DNA sequence encoding an endogenous glyoxylate reductase (EC: 1.1.1.26, https: / / www.ncbi.nlm.nih.gov / nuccore / LT962688). However, despite the presence of the endogenous glyoxylate reductase DNA sequence and glyoxylate as the starting material in metabolism, the wild-type strain of M. extorquens TK 0001 does not produce measurable amounts of glycolic acid by HPLC or GC-MS.

[0010] It is desirable to provide a fermentative production of glycolic acid from Cx compounds such as methanol or formic acid, and to provide means for this, in particular methylotrophic microorganisms capable of converting such Cx compounds, for example, methanol, formic acid, or a mixture thereof, into glycolic acid. It is also desirable to provide a process by which glycolic acid can be obtained via an integrated process cascade in a fully renewable manner using CO2 as the sole raw material, i.e., without the consumption of fossil or biogenic resources.

[0011] The technical problem underlying the present invention is therefore to overcome the aforementioned disadvantages. In particular, the technical problem underlying the present invention is to provide a biological cell that makes it possible to convert a starting material, hereinafter also referred to as a reactant, containing at least one Cx compound, in particular methanol, formic acid, or a mixture thereof, into a product containing glycolic acid. In particular, the technical problem underlying the present invention is to provide means and methods that make it possible to obtain such a cell, in particular means and methods that are cost-effective and easy to handle.In particular, the technical problem underlying the present invention is to provide means and processes, in particular a cost-effective and easy-to-handle process, for obtaining a product containing glycolic acid. In particular, the technical problem underlying the present invention is to provide means and processes that enable a sustainable synthesis of glycolic acid that requires almost entirely, in particular without, the use of fossil resources and / or almost entirely, in particular without, biogenic raw materials and preferably starts from CO2 as the sole raw material. In particular, it is a technical problem of the present invention to provide such means and processes that are cost-effective, environmentally friendly, and easy to handle.Furthermore, the present invention is based in particular on the technical problem of providing means and methods which enable polyglycolic acid, polylactic acid or polylactide-co-glycolide to be obtained.

[0012] The technical problem is solved by the teachings of the independent claims, the dependent claims and the teaching of the description, in particular by a genetically modified Methylobacteriaceae cell comprising at least one exogenous nucleic acid sequence encoding a glyoxylate reductase from the bacterium Escherichia.

[0013] According to the invention, it is accordingly provided to provide a genetically modified Methylobacteriaceae cell, i.e. a cell that differs from the wild-type Methylobacteriaceae strain by at least one genetic modification. Furthermore, the invention provides that the genetically modified Methylobacteriaceae cell comprises at least one exogenous nucleic acid sequence, wherein the nucleic acid sequence encodes a glyoxylate reductase from the bacterium Escherichia. The genetic modification of the wild-type strain of the Methylobacteriaceae cell is accordingly at least the genetic integration of at least one exogenous nucleic acid sequence into the Methylobacteriaceae cell, wherein the exogenous nucleic acid sequence encodes a glyoxylate reductase from the bacterium Escherichia. The exogenous nucleic acid sequence can be of synthetic origin or occur naturally, in particular in Escherichia.In a preferred embodiment of the present invention, the genetically modified Methylobacteriaceae cell according to the invention comprises at least one exogenous nucleic acid sequence encoding a glyoxylate reductase, which occurs naturally or is a codon-optimized, in particular Methylobacteriaceae codon-optimized, in particular Methylorubrum codeon-optimized or Methylobacterium codon-optimized, in particular Methylorubrum extorquens codon-optimized, in particular Methylorubrum extorquens TK 0001, Methylorubrum extorquens PA1 or Methylorubrum AMI codon-optimized nucleic acid sequence.

[0014] Surprisingly, such a genetically modified Methylobacteriaceae cell according to the invention makes it possible to convert a Cx compound to glycolic acid, in particular to convert a starting material, namely a reactant containing at least one Cx compound, in particular methanol, formic acid, or a mixture thereof, to a product containing glycolic acid, in particular in amounts measurable by HPLC or GC-MS. The wild-type strain of the Methylobacteriaceae cell, which has only an endogenous nucleic acid sequence encoding a glyoxylate reductase, is, in contrast, unable to convert a Cx compound to glycolic acid, in particular to convert the starting material, namely a reactant containing at least one Cx compound, to a product containing glycolic acid, in particular in amounts measurable by HPLC or GC-MS.

[0015] The present invention thus provides a genetically modified Methylobacteriaceae cell capable of converting a reactant containing at least one Cx compound, in particular methanol, formic acid, or a mixture thereof, into a product containing glycolic acid. Cx compounds advantageously represent renewable but non-biogenic substrates for biotechnological processes. Furthermore, due to their liquid state, they are easy to handle and, unlike gases, are not limited in mass transfer in liquid reaction mixtures. The teaching of the invention makes them particularly readily accessible for glycolic acid production. According to the invention, glycolic acid can thus be produced completely renewably from CO2, provided that the CO2 conversion to a Cx compound, in particular methanol, is powered by renewable energy.In this way, PtX processes (Power-to-X) can be advantageously combined with biotechnology to create an exemplary PtXtY process (Power-to-X-to-Y).

[0016] The genetically modified Methylobacteriaceae cell according to the invention can accordingly be advantageously used in a process for producing glycolic acid from at least one Cx compound, in particular for producing a product containing glycolic acid by reacting a reactant containing at least one Cx compound, in particular methanol, formic acid, or a mixture thereof. Preferably, the product containing glycolic acid obtained by the genetically modified Methylobacteriaceae cell according to the invention additionally contains lactic acid in addition to glycolic acid. The invention provides a particularly simple, easy-to-handle, and cost-effective production process for a product containing glycolic acid, in particular glycolic acid and lactic acid, thus avoiding high equipment and cost expenditure.The present invention is also advantageous in that it enables the polymerization of glycolic acid, in particular glycolic acid and lactic acid, to produce polyglycolic acid, in particular polyglycolic acid, polylactic acid or polylactide-co-glycolide, which frequently follows a glycolic acid supply, in particular glycolic acid and lactic acid supply, and accordingly enables the production of polyglycolic acid, in particular polyglycolic acid, polylactic acid or polylactide-co-glycolide, without having to carry out cost-intensive and extensive process steps.

[0017] Without wishing to be bound by theory, the exogenous glyoxylate reductase present in the genetically modified Methylobacteriaceae cell according to the invention, encoded by the at least one exogenous nucleic acid sequence encoding a glyoxylate reductase from the bacterium Escherichia, can convert a Cx compound into glycolic acid in the serine cycle of the genetically modified Methylobacteriaceae cell according to the invention, in particular a starting material containing at least one Cx compound, in particular methanol, formic acid or a mixture thereof, can be converted into a product containing glycolic acid, in particular in amounts measurable by HPLC or GC-MS.Preferably, the glyoxylate reductase endogenously encoded in the wild-type strain of the genetically modified Methylobacteriaceae cell according to the invention does not convert any amounts measurable by HPLC or GC-MS, in particular no starting material containing at least one Cx compound, in particular methanol, formic acid or a mixture thereof, into a product containing glycolic acid in the metabolism of the wild-type strain, so that the glyoxylate reductase activity can be controlled solely by the integration of the exogenous glyoxylate reductase-encoding nucleic acid sequence in genomic or episomal form and the expression thereof, as provided for in the invention.

[0018] The genetically modified Methylobacteriaceae cell according to the invention is thus characterized by the enzymatic activity of the exogenous glyoxylate reductase, in particular its ability, brought about by the presence of the exogenous glyoxylate reductase, to convert a Cx compound to glycolic acid, in particular to be able to convert a reactant containing at least one Cx compound, in particular methanol, formic acid or a mixture thereof, in particular in a reaction medium, to a product containing glycolic acid, in particular to be able to convert it in a liquid reaction medium, in particular to catalyze this reaction enzymatically.

[0019] The genetically modified Methylobacteriaceae cell is preferably characterized accordingly in that it, in particular its genome, resembles the wild-type strain of the Methylobacteriaceae cell, in particular is identical to it, except for the presence of at least one exogenous nucleic acid sequence encoding a glyoxylate reductase from the bacterium Escherichia, which imparts to the Methylobacteriaceae cell according to the invention the advantageous enzymatic activity according to the invention, and optionally associated exogenous nucleic acid sequences of an expression vector or an expression cassette. In a preferred embodiment of the present invention, in a Methylobacteriaceae cell according to the invention, in particular its genome, in addition to the at least one nucleic acid sequence encoding glyoxylate reductase, further, in particular genetically engineered, genetic modifications compared to the wild-type strain can be present.

[0020] In a preferred embodiment of the present invention, the bacterium is Escherichia coli, in particular E. coli K-12 MGI 655.

[0021] In a preferred embodiment of the present invention, the Methylobacteriaceae cell is a Methylorubrum cell, in particular a cell of Methylorubrum extorquens, in particular Methylorubrum extorquens TK 0001, in particular Methylorubrum extorquens PA1, Methylorubrum extorquens AMI, Methylorubrum rhodesianum or Methylorubrum zatmanii.

[0022] In a preferred embodiment of the present invention, the genetically modified Methylobacteriaceae cell according to the invention is a genetically modified Methylorubrum extorquens AM1 cell, genetically modified Methylorubrum extorquens TK 0001 cell, or a genetically modified Methylorubrum extorquens PA1 cell comprising at least one exogenous nucleic acid sequence encoding a glyoxylate reductase from a bacterium Escherichia coli, in particular E. coli K-12 MGI 655.

[0023] In a preferred embodiment of the present invention, the Methylobacteriaceae cell is a Methylobacterium cell, in particular a cell of Methylobacterium organophilum or Methylobacterium radiotolerans.

[0024] In a preferred embodiment of the present invention, the Methylobacteriaceae cell is a Methylorubrum cell, in particular a cell of Methylorubrum extorquens, in particular Methylorubrum extorquens AMI, Methylorubrum extorquens TK 0001, Methylorubrum extorquens PA1, Methylorubrum rhodesianum or Methylorubrum zatmanii, or a Methylobacterium cell, in particular a cell of Methylorubrum organophilum or Methylorubrum radiotolerans.

[0025] In a preferred embodiment of the present invention, the exogenous glyoxylate reductase is encoded by a nucleic acid sequence according to SEQ ID No. 3 or a functional nucleic acid sequence equivalent thereof, wherein the functional nucleic acid sequence equivalent has a nucleic acid sequence identity of at least 30.0%, preferably 30.0 to 99.9%, preferably 40.0 to 99.9%, preferably 50.0 to 99.9%, preferably 60.0 to 99.9%, preferably 70.0 to 99.9%, preferably from 76.0 to 99.9%, preferably from 80.0 to 99.9%, preferably 90.0 to 99.9%, preferably 95.0 to 99.9%, preferably 98.0 to 99.9%, preferably 90.0 to 99.0% to the nucleic acid sequence according to SEQ ID No. 3 and wherein the encoded glyoxylate reductase is capable of converting a reactant containing at least one Cx compound, in particular methanol, formic acid or a mixture thereof, into a product containing glycolic acid.Preferably, the nucleic acid sequence identity is at least 76.0 to the nucleic acid sequence according to SEQ ID No. 3.

[0026] In a preferred embodiment of the present invention, the present invention thus relates to a genetically modified Methylobacteriaceae cell, in particular a Methylorubrum cell or Methylobacterium cell, comprising a nucleic acid sequence encoding an exogenous glyoxylate reductase, in particular a nucleic acid sequence according to SEQ ID No. 3.

[0027] In a preferred embodiment of the present invention, the present invention also relates to a genetically modified Methylobacteriaceae cell, in particular Methylorubrum cell or Methylobacterium cell, comprising a functional nucleic acid sequence equivalent of the at least one exogenous nucleic acid sequence encoding a glyoxylate reductase according to SEQ ID No. 3, wherein the functional nucleic acid sequence equivalent has a nucleic acid sequence identity of at least 30.0%, preferably 30.0 to 99.9%, preferably 40.0 to 99.9%, preferably 50.0 to 99.9%, preferably 60.0 to 99.9%, preferably 70.0 to 99.9%, preferably 76.0 to 99.9%, preferably 80.0 to 99.9%, preferably 90.0 to 99.9%, preferably 95.0 to 99.9%, preferably 98.0 to 99.9 %, preferably 90.0 to 99.0% to the nucleic acid sequence according to SEQ ID No.3 and wherein the glyoxylate reductase encoded thereby is capable of converting a reactant containing at least one Cx compound, in particular methanol, formic acid or a mixture thereof, into a product containing glycolic acid.

[0028] In a particularly preferred embodiment of the present invention, the functional nucleic acid sequence equivalent of the nucleic acid sequence according to SEQ ID No. 3 has a nucleic acid sequence with a length of at least 800, preferably at least 850, preferably at least 900, preferably at least 950, preferably at least 970 nucleic acids. According to the invention, the sequence identity of the nucleic acid sequence of the nucleic acid sequence equivalent of the nucleic acid sequence according to SEQ ID No. 3 to the nucleic acid sequence according to SEQ ID No. 3 is preferably stated over the entire length of the nucleic acid sequence of the nucleic acid sequence equivalent of the nucleic acid sequence according to SEQ ID No. 3.

[0029] In a particularly preferred embodiment of the present invention, the nucleic acid sequence according to SEQ ID No. 3 is a codon-optimized, in particular a Methylorubrum, in particular Methylorubrum extorquens, in particular Methylorubrum extorquens AMI, Methylorubrum extorquens TK 0001, in particular a Methylorubrum extorquens PA1 codon-optimized nucleic acid sequence of the native, i.e. naturally occurring, nucleic acid sequence from Escherichia, in particular E. coli, which encodes the glyoxylate reductase from Escherichia, in particular E. coli. The native nucleic acid sequence from Escherichia, which encodes the glyoxylate reductase from Escherichia, has the nucleic acid sequence according to SEQ ID No. 1 and represents a functional nucleic acid sequence equivalent of the nucleic acid sequence according to SEQ ID No. 3.

[0030] In a particularly preferred embodiment of the present invention, the functional nucleic acid sequence equivalent of the nucleic acid sequence according to SEQ ID No. 3 comprises the nucleic acid sequence according to SEQ ID No. 1.

[0031] In a preferred embodiment of the present invention, the exogenous nucleic acid sequence encoding a glyoxylate reductase from the bacterium Escherichia, in particular according to SEQ ID No. 3 or a functional nucleic acid sequence equivalent thereof, for example according to SEQ ID No. 1, encodes a glyoxylate reductase comprising, in particular consisting of, an amino acid sequence according to SEQ ID No. 2 or a functional amino acid sequence equivalent thereof.

[0032] In a preferred embodiment of the present invention, the exogenous 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 at least 30.0%, in particular 30.0 to 99.9%, preferably 40.0 to 99.9%, preferably 50.0 to 99.9%, preferably 60.0 to 99.9%, preferably 70.0 to 99.9%, preferably from 76.0 to 99.9%, preferably from 80.0 to 99.9%, preferably 85.0 to 99.9%, preferably 90.0 to 99.9%, preferably 95.0 to 99.9%, preferably 98.0 to 99.9%, to the amino acid sequence according to SEQ ID No. 2. Preferably, the amino acid sequence identity is at least 90.0% to the amino acid sequence according to SEQ ID No. 2.In a preferred embodiment of the present invention, the present invention relates to a genetically modified Methylobacteriaceae cell, in particular Methylorubrum cell or Methylobacterium cell, comprising a functional amino acid sequence equivalent of the amino acid sequence of SEQ ID No. 2, wherein the functional amino acid sequence equivalent has an amino acid sequence identity of at least 30.0%, in particular 30.0 to 99.9%, preferably 40.0 to 99.9%, preferably 50.0 to 99.9%, preferably 60.0 to 99.9%, preferably 70.0 to 99.9%, preferably 76.0 to 99.9%, preferably 80.0 to 99.9%, preferably 85.0 to 99.9%, preferably 90.0 to 99.9%, preferably 95.0 to 99.9%, preferably 98.0 to 99.9%, to the amino acid sequence according to SEQ ID No. 2 and the is capable of converting a reactant containing at least one Cx compound, in particular methanol, formic acid or a mixture thereof, into a product containing glycolic acid.

[0033] In a particularly preferred embodiment of the present invention, the functional amino acid sequence equivalent of the amino acid sequence according to SEQ ID No. 2 has an amino acid sequence with a length of at least 300, preferably at least 310, preferably at least 320, preferably at least 325 amino acids. According to the invention, the sequence identity of the amino acid sequence of the amino acid sequence equivalent of the amino acid sequence according to SEQ ID No. 2 to the amino acid sequence according to SEQ ID No. 2 is preferably stated over the entire length of the amino acid sequence of the amino acid sequence equivalent of the amino acid sequence according to SEQ ID No. 2.

[0034] In a preferred embodiment of the present invention, the Cx compound is a Cx compound with x = 1, 2 or 4, in particular x=1.

[0035] In a preferred embodiment of the present invention, the Cx compound is formic acid, methanol, methane, methylamine, acetic acid or succinic acid or a mixture thereof.

[0036] In a preferred embodiment of the present invention, the Cx compound is methanol.

[0037] In a preferred embodiment of the present invention, the Cx compound is formic acid.

[0038] In a preferred embodiment of the present invention, the reactant contains at least one Cx compound, in particular formic acid, methanol, methane, methylamine, acetic acid, or succinic acid, or a mixture thereof; in particular, the reactant consists of at least one compound thereof. In a preferred embodiment of the present invention, the product obtained by reacting a reactant containing at least one Cx compound contains glycolic acid, in particular, consists of this.

[0039] In a preferred embodiment of the present invention, the product obtained by the reaction of a reactant containing at least one Cx compound contains glycolic acid and lactic acid, in particular consists of these.

[0040] In a preferred embodiment of the present invention, the glycolic acid-containing product contains glycolic acid and lactic acid, in particular 1 to 99 wt.%, in particular 2 to 98 wt.%, in particular 10 to 90 wt.%, in particular 30 to 80 wt.%, in particular 40 to 70 wt.%, in particular 50 wt.%, in particular 60 wt.% glycolic acid and in particular 1 to 99 wt.%, in particular 2 to 98 wt.%, in particular 10 to 90 wt.%, in particular 20 to 70 wt.%, in particular 30 to 60 wt.%, in particular 50 wt.%, in particular 40 wt.% lactic acid (in each case based on the total dry weight of the product obtained) or consists of these proportions.

[0041] In a preferred embodiment of the present invention, the growth rate gmax of a genetically modified Methylobacteriaceae cell according to the invention, in particular in a reaction medium having an initial concentration of up to 10 g L' 1 of a reactant containing at least one Cx compound, in particular consisting of methanol, at least 0.05 h' 1 , at least 0.10 h' 1 , in particular at least 0.15 h' 1 , in particular at least 0.18 h' 1 , in particular at least 0.20 h' 1 , in particular at least 0.21 h' 1 , especially 0.10 to 0.30 h' 1 , especially 0.15 to 0.25 h' 1 , especially 0.20 to 0.22 h' 1 , in particular 0.21 h' 1 .

[0042] In a preferred embodiment of the present invention, the titer of a reaction medium containing the product containing glycolic acid, in particular glycolic acid and lactic acid, which after the reaction of a reactant containing at least one Cx compound, in particular consisting of methanol, by a genetically modified Methylobacteriaceae cell according to the invention, in a reaction medium having an initial concentration of up to 10 g L' 1 Educt, especially after 40 h reaction time, at least 0.01 g L' is obtained 1 , at least 0.10 g L' 1 , in particular at least 0.15 g L' 1 , in particular at least 0.20 g L' 1 , in particular at least 0.25 g L' 1 , in particular at least 0.50 g L- 1 , in particular at least 0.75 g L' 1 , in particular at least 1.00 g L' 1 and in particular 1.50 g L' 1(each based on the weight of the product per liter of reaction medium). In a preferred embodiment of the present invention, a genetically modified Methylobacteriaceae cell according to the invention converts a starting material containing at least one Cx compound, in particular consisting of methanol, into a product containing glycolic acid, in particular glycolic acid and lactic acid, in particular in a reaction medium having an initial concentration of up to 10 g L' 1 of the reactant with a dry biomass substrate yield (Yx / s) of at least 10 mg g' 1 , in particular at least 50 mg g' 1 , in particular at least 100 mg g' 1 , in particular at least 150 mg g' 1 , in particular at least 200 mg g' 1 , in particular 10 to 350 mg g' 1 , especially 50 to 320 mg g' 1 , especially 100 to 300 mg g' 1 , especially 200 to 300 mg g' 1 , in particular 280 mg g' 1(each based on dry biomass of genetically modified Methylobacteriaceae cell according to the invention per gram of the reactant).

[0043] In a preferred embodiment of the present invention, the biodry mass substrate yield (Yx / s) of a genetically modified Methylobacteriaceae cell according to the invention decreases in relation to the biodry mass substrate yield (Yx / s) of the wild-type strain in the conversion of a reactant containing at least one Cx compound, in particular consisting of methanol, to a product containing glycolic acid, in particular glycolic acid and lactic acid, in particular in a reaction medium having an initial concentration of up to 10 g L' 1 of the reactant to less than 95%, in particular less than 90%, in particular less than 80%, in particular less than 70%, in particular to 68%.

[0044] In a preferred embodiment of the present invention, a genetically modified Methylobacteriaceae cell according to the invention converts a starting material containing at least one Cx compound, in particular consisting of methanol, into a product containing glycolic acid, in particular glycolic acid and lactic acid, in particular in a reaction medium having an initial concentration of up to 10 g L' 1 of the reactant, with a product-substrate yield (Yp / s) of at least 10 mg g' 1 , in particular at least 50 mg g' 1 , in particular at least 80 mg g' 1 , in particular at least 100 mg g' 1 , in particular at least 110 mg g' 1 , especially 10 to 200 mg g' 1 , especially 50 to 180 mg g' 1 , especially 80 to 150 mg g' 1 , especially 100 to 130 mg g' 1 , especially 120 mg g' 1 (each based on the weight of the product per gram of reactant).

[0045] In a preferred embodiment of the present invention, a genetically modified Methylobacteriaceae cell according to the invention converts a starting material containing at least one Cx compound, in particular consisting of methanol, into a product containing glycolic acid, in particular glycolic acid and lactic acid, in particular in a reaction medium having an initial concentration of up to 10 g L' 1 of the reactant with a product biodry mass yield (Yp / x) of at least 0.10 g g' 1 , in particular at least 0.20 g g' 1 , in particular at least 0.30 g g' 1 , in particular at least 0.40 g g' 1 , in particular at least 0.50 g g' 1 , in particular 0.10 to 0.80 g g' 1 , in particular 0.20 to 0.70 g g' 1 , in particular 0.30 to 0.60 g g' 1 , in particular 0.40 to 0.50 g g' 1 , in particular 0.50 g g' 1(each based on the weight of the product per gram of dry biomass of the genetically modified Methylobacteriaceae cell according to the invention).

[0046] In a preferred embodiment of the present invention, the genetically modified Methylobacteriaceae cell according to the invention comprises at least one exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase.

[0047] In a preferred embodiment of the present invention, the genetically modified Methylobacteriaceae cell according to the invention comprises at least one exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase, which occurs naturally or a codon-optimized, in particular Methylobacteriaceae codon-optimized nucleic acid sequence, in particular a Methylobacterium, in particular Methylorubrum, in particular Methylorubrum extorquens, in particular Methylorubrum extorquens AMI, Methylorubrum extorquens TK 0001 or Methylorubrum extorquens PAI codon-optimized nucleic acid sequence.

[0048] In a preferred embodiment of the present invention, the exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase originates 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.

[0049] In a preferred embodiment of the present invention, the genetically modified Methylobacteriaceae cell according to the invention is a genetically modified Methylobacteriaea cell, in particular a Methylorubrum extorquens AMI, Methylorubrum extorquens PA1, Methylorubrum extorquens TK 0001 cell, comprising at least one exogenous nucleic acid sequence encoding a glyoxylate reductase from a bacterium Escherichia coli, in particular E. coli K-12 MG1655, and at least one exogenous nucleic acid sequence encoding an ethylmalonyl-CoA.In a preferred embodiment of the present invention, the genetically modified Methylobacteriaceae cell according to the invention is a genetically modified Methylobacteriaceae cell, in particular a Methylorubrum extorquens AMI, Methylorubrum extorquens PA1, Methylorubrum extorquens TK 0001 cell, comprising at least one exogenous nucleic acid sequence encoding a glyoxylate reductase from a bacterium Escherichia coli K-12 MGI 655 and at least one exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase from a bacterium Methylorubrum extorquens TK 0001 DSM 1337.

[0050] In a preferred embodiment of the present invention, the genetically modified Methylobacteriaceae cell according to the invention is a genetically modified Methylobacteriaceae cell, in particular a Methylorubrum extorquens AMI, Methylorubrum extorquens PA1, Methylorubrum extorquens TK 0001 cell, comprising at least one exogenous nucleic acid sequence encoding a glyoxylate reductase from a bacterium Escherichia coli K-12 MGI 655 and at least one exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase from a bacterium Rhodobacter sphaeroides ATCC 17029.

[0051] In a preferred embodiment of the present invention, the present invention also relates to a genetically modified Methylobacteriaceae cell which comprises at least two different exogenous nucleic acid sequences, i.e. a genetically modified Methylobacteriaceae cell which, in addition to the at least one exogenous nucleic acid sequence encoding a glyoxylate reductase from the bacterium Escherichia, comprises at least one further exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase.

[0052] Surprisingly, such a genetically modified Methylobacteriaceae cell according to the invention enables an increased glycolic acid yield, in particular glycolic acid and lactic acid yield, compared to the glycolic acid yield, in particular glycolic acid and lactic acid yield, obtained by the reaction of a reactant containing at least one Cx compound, in particular methanol, formic acid or a mixture thereof, by a Methylobacteriaceae cell according to the invention, comprising at least one exogenous nucleic acid sequence encoding a glyoxylate reductase from the bacterium Escherichia, which does not have an exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase, in particular none 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, coding nucleic acid sequence.The invention therefore surprisingly increases not only the glycolic acid yield but also the lactic acid yield.

[0053] Preferably, the conversion of Cx compounds to glycolic acid, in particular glycolic acid and lactic acid, by a Methylobacteriaceae cell according to the invention, additionally comprising the 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, is higher than the conversion by a Methylorubrum cell according to the invention without this at least one additional exogenous nucleic acid sequence.

[0054] Without wishing to be bound by theory, the exogenous ethylmalonyl-CoA mutase present in the genetically modified Methylobacteriaceae cell according to the invention increases the amount of glyoxylate in the serine cycle of the genetically modified Methylobacteriaceae cell according to the invention, which is converted by the exogenous glyoxylate reductase present in the genetically modified Methylobacteriaceae cell according to the invention. This preferably leads to an improved glycolic acid yield compared to the glycolic acid yield obtained by a Methylobacteriaceae cell according to the invention comprising at least one exogenous nucleic acid sequence encoding a glyoxylate reductase from the bacterium Escherichia, which does not have an exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase.The increased lactic acid yield also observed may be due, without being bound to theory, to a complex interplay with the metabolism of a reducing equivalent supply and an increased availability of the metabolite pyruvate, the precursor molecule of lactic acid.

[0055] In a particularly preferred embodiment, the present invention relates to a genetically modified Methylobacteriaceae cell according to the invention comprising a codon-optimized nucleic acid sequence of a nucleic acid sequence from Rhodobacter sphaeroides encoding an ethylmalonyl-CoA mutase, in particular a Methylobacteriaceaea, in particular Methylobacterium, in particular a Methylorubrum, in particular Methylorubrum extorquens, in particular Methylorubrum extorquens TK 0001, in particular Methylorubrum extorquens AMI, in particular Methylorubrum extorquens PAl codon-optimized nucleic acid sequence, in particular it has SEQ ID No. 8.In a particularly preferred embodiment, the present invention relates to a genetically modified Methylobacteriaceae cell according to the invention comprising a codon-optimized nucleic acid sequence of a nucleic acid sequence from Methylorubrum extorquens encoding an ethylmalonyl -Co A mutase, in particular a Methylobacteriaceae, in particular Methylobacterium, in particular Methylorubrum, in particular Methylorubrum extorquens, in particular Methylorubrum extorquens TK 0001, in particular Methylorubrum extorquens AMI, in particular Methylorubrum extorquens PAl-codon-optimized nucleic acid sequence, in particular it has SEQ ID No. 13.

[0056] In a preferred embodiment, the present invention relates to a genetically modified Methylobacteriaceae cell according to the invention comprising a functional nucleic acid sequence equivalent of a nucleic acid sequence encoding an ethylmalonyl-CoA mutase according to SEQ ID No. 8 or 13.

[0057] The native nucleic acid sequences of the ethylmalonyl-CoA mutase from Methylorubrum or Rhodobacter according to SEQ ID Nos. 4 and 6 are also understood in the context of the present invention as functional equivalents of the codon-optimized nucleic acid sequences derived therefrom, in particular the native nucleic acid sequence according to SEQ ID No. 6 represents a functional nucleic acid sequence equivalent of the codon-optimized nucleic acid sequence according to SEQ ID No. 8 and the native nucleic acid sequence according to SEQ ID No. 4 represents a functional nucleic acid sequence equivalent of the codon-optimized nucleic acid sequence according to SEQ ID No. 13.

[0058] In a preferred embodiment of the present invention, the ethylmalonyl-CoA mutase is encoded by a codon-optimized nucleic acid sequence (SEQ ID No. 13 or 8) of a native nucleic acid sequence according to SEQ ID No. 4 or 6 or a functional equivalent thereof, in particular of the native nucleic acid sequence itself, i.e. a nucleic acid sequence according to SEQ ID No. 4 or 6, wherein the functional nucleic acid sequence equivalent has a nucleic acid sequence identity of at least 30.0%, preferably 30.0 to 99.9%, preferably 40.0 to 99.9%, preferably 50.0 to 99.9%, preferably 60.0 to 99.9%, preferably 70.0 to 99.9%, preferably 76.0 to 99.9%, preferably 80.0 to 99.9%, preferably 90.0 to 99.9%, preferably 95.0 to 99.9%, preferably 98.0 to 99.9%, preferably 90.0 to 99.0%, to the codon-optimized nucleic acid sequence according to SEQ ID No. 13 or 8, wherein the functional equivalent has the enzymatic activity of an ethylmalonyl-CoA mutase.In a preferred embodiment of the present invention, the present invention also relates to a genetically modified Methylobacteriaceae cell according to the invention comprising a functional nucleic acid sequence equivalent of the at least one exogenous codon-optimized nucleic acid sequence encoding an ethylmalonyl-CoA mutase according to SEQ ID No. 13 or 8, i.e. for example a native nucleic acid sequence according to SEQ ID No. 4 or 6, wherein the functional nucleic acid sequence equivalent has a nucleic acid sequence identity of at least 30.0%, preferably 30.0 to 99.9%, preferably 40.0 to 99.9%, preferably 50.0 to 99.9%, preferably 60.0 to 99.9%, preferably 70.0 to 99.9%, preferably 76.0 to 99.9%, preferably 80.0 to 99.9%, preferably 90.0 to 99.9%, preferably 95.0 to 99.9%, preferably 98.0 to 99.9%, preferably 90.0 to 99.0% to the codon-optimized nucleic acid sequence according to SEQ ID No.13 or 8 and wherein the modified Methylobacteriaceae cell is capable of converting a reactant containing at least one Cx compound, in particular methanol, formic acid or a mixture thereof, into a product containing glycolic acid.

[0059] In a particularly preferred embodiment of the present invention, the functional nucleic acid sequence equivalent of the codon-optimized nucleic acid sequence according to SEQ ID No. 8 comprises the native nucleic acid sequence according to SEQ ID No. 6.

[0060] In a particularly preferred embodiment of the present invention, the functional nucleic acid sequence equivalent of the codon-optimized nucleic acid sequence according to SEQ ID No. 13 comprises the native nucleic acid sequence according to SEQ ID No. 4.

[0061] In a preferred embodiment of the present invention, 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 at least 30.0%, in particular 30.0 to 99.9%, preferably 40.0 to 99.9%, preferably 50.0 to 99.9%, preferably 60.0 to 99.9%, preferably 70.0 to 99.9%, preferably 76.0 to 99.9%, preferably 80.0 to 99.9%, preferably 85.0 to 99.9%, preferably 90.0 to 99.9%, preferably 95.0 to 99.9%, preferably 98.0 to 99.9%, to the amino acid sequence according to SEQ ID No. 5 or 7 and the enzymatic activity of a ethylmalonyl-CoA mutase.

[0062] In a preferred embodiment of the present invention, the present invention relates to a genetically modified Methylobacteriaceae cell according to the invention comprising a functional amino acid sequence equivalent of the amino acid sequence of SEQ ID No. 5 or 7, wherein the functional amino acid sequence equivalent has an amino acid sequence identity of at least 30.0%, in particular 30.0 to 99.9%, preferably 40.0 to 99.9%, preferably 50.0 to 99.9%, preferably 60.0 to 99.9%, preferably 70.0 to 99.9%, preferably 76.0 to 99.9%, preferably 80.0 to 99.9%, preferably 85.0 to 99.9%, preferably 90.0 to 99.9%, preferably 95.0 to 99.9%, preferably 98.0 to 99.9%, to the amino acid sequence according to SEQ ID No.5 or 7 and wherein the modified Methylobacteriaceae cell is capable of converting a reactant containing at least one Cx compound, in particular methanol, formic acid or a mixture thereof, into a product containing glycolic acid, in particular glycolic acid and lactic acid.

[0063] In a preferred embodiment of the present invention, the growth rate pimax of a genetically modified Methylobacteriaceae cell according to the invention additionally comprising at least one exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase in a reaction medium having an initial concentration of up to 10 g L' 1 , especially 10 g L' 1 , of a reactant containing at least one Cx compound, in particular consisting of methanol, at least 0.05 h' 1 , at least 0.10 h' 1 , in particular at least 0.12 h' 1 , in particular at least 0.14 h' 1, in particular at least 0.16 h' 1 , especially 0.10 to 0.25 h' 1 , especially 0.12 to 0.22 h' 1 , especially 0.15 to 0.20 h' 1 , especially 0.16 h' 1 , especially 0.19 h' 1 .

[0064] In a preferred embodiment of the present invention, the titer of a reaction medium containing the product containing glycolic acid, in particular glycolic acid and lactic acid, which after the reaction of a starting material containing at least one Cx compound, in particular consisting of methanol, by a genetically modified Methylobacteriaceae cell according to the invention additionally comprising at least one exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase, in particular in a reaction medium having an initial concentration of up to 10 g L' 1 , especially 10 g L' 1 ' of the reactant, especially after 40 h reaction time, at least 0.10 g L' is obtained1 , in particular at least 0.20 g L' 1 , in particular at least 0.30 g L' 1 , in particular at least 0.40 g L' 1 , in particular 0.10 to 80 g L' 1 , in particular 0.20 to 70 g L' 1 , in particular 0.30 to 60 g L' 1 , in particular 0.40 to 55 g L' 1 , in particular 0.49 g L' 1 , in particular 0.52 g L' 1 (each based on the weight of the product per liter of reaction medium).

[0065] In a preferred embodiment of the present invention, the titer obtained by the reaction of a reactant containing at least one Cx compound, in particular consisting of methanol, by the genetically modified Methylobacteriaceae cell according to the invention, additionally comprising at least one exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase, in a reaction medium having an initial concentration of up to 10 g L' 1of the starting material, in particular after a reaction time of 40 hours, is increased by at least 10%, in particular at least 30%, in particular at least 50%, in particular at least 60%, in particular 69%, in particular 79%, compared to the titer obtained by the reaction using the genetically modified Methylobacteriaceae cell according to the invention without the at least one exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase.

[0066] In a preferred embodiment of the present invention, a genetically modified Methylobacteriaceae cell according to the invention additionally comprising at least one exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase converts a reactant containing at least one Cx compound, in particular consisting of methanol, to a product containing glycolic acid, in particular glycolic acid and lactic acid, in particular in a reaction medium having an initial concentration of up to 10 g L' 1 , especially 10 g L'1 , of the reactant with a dry biomass substrate yield (Yx / s) of at least 10 mg g' 1 , in particular at least 50 mg g' 1 , in particular at least 100 mg g' 1 , in particular at least 150 mg g' 1 , in particular at least 200 mg g' 1 , in particular 10 to 350 mg g' 1 , especially 50 to 320 mg g' 1 , especially 100 to 300 mg g' 1 , especially 200 to 300 mg g' 1 , especially 210 mg g' 1 , especially 270 mg g' 1 (each based on dry biomass of genetically modified Methylobacteriaceae cell according to the invention per gram of the reactant).

[0067] In a preferred embodiment of the present invention, the biomass substrate yield (Yx / s) of a genetically modified Methylobacteriaceae cell according to the invention additionally comprising at least one exogenous nucleic acid sequence encoding an ethylmalonyl-CoA decreases in relation to the biomass substrate yield (Yx / s) of the wild-type strain in the conversion of a starting material containing at least one Cx compound, in particular consisting of methanol, to a product containing glycolic acid, in particular glycolic acid and lactic acid, in particular in a reaction medium having an initial concentration of up to 10 g L' 1 , especially 10 g L' 1 , of the reactant, to less than 95%, in particular less than 90%, in particular less than 80%, in particular less than 70%, in particular 68%, in particular 51%.

[0068] In a preferred embodiment of the present invention, the biomass substrate yield (Yx / s) of a genetically modified Methylobacteriaceae cell according to the invention additionally comprising at least one exogenous nucleic acid sequence encoding an ethylmalonyl-CoA decreases in relation to the biomass substrate yield (Yx / s) of the genetically modified Methylobacteriaceae cell according to the invention without the at least one exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase in the conversion of a reactant containing at least one Cx compound, in particular consisting of methanol, to a product containing glycolic acid, in particular glycolic acid and lactic acid, in particular in a reaction medium having an initial concentration of up to 10 g L' 1 , especially 10 g L' 1 , of the reactant to less than 99%, in particular less than 97%, in particular 96%, in particular 75%.

[0069] In a preferred embodiment of the present invention, the biomass substrate yield (Yx / s) of a genetically modified Methylobacteriaceae cell according to the invention additionally comprising at least one exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase from the bacterium Rhodobacter sphaeroides, in particular Rhodobacter sphaeroides ATCC 17029, in relation to the biomass substrate yield (Yx / s) of a genetically modified Methylobacteriaceae cell according to the invention comprising at least one exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase from the bacterium Methylorubrum extorquens, in particular Methylorubrum extorquens TK 0001 DSM 1337, in the conversion of a starting material containing at least one Cx compound, in particular consisting of methanol, to a product containing glycolic acid, in particular glycolic acid and lactic acid, in particular in a Reaction medium having an initial concentration of up to 10 g L' 1, especially 10 g L' 1 , of the reactant by at least 5%, in particular at least 10%, in particular at least 20%, in particular at least 25%, in particular 28%.

[0070] In a preferred embodiment of the present invention, the genetically modified Methylobacteriaceae cell according to the invention additionally comprising at least one exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase uses a reactant containing at least one Cx compound, in particular consisting of methanol, to produce a product containing glycolic acid, in particular glycolic acid and lactic acid, in particular in a reaction medium having an initial concentration of up to 10 g L' 1 , especially 10 g L' 1 , of the reactant with a product-substrate yield (Yp / s) of at least 10 mg g' 1 , in particular at least 50 mg g' 1 , in particular at least 80 mg g' 1 , in particular at least 100 mg g' 1, in particular at least 140 mg g' 1 , in particular 10 to 250 mg g' 1 , especially 50 to 200 mg g' 1 , especially 80 to 180 mg g' 1 , especially 100 to 160 mg g' 1 , in particular 150 mg g' 1 (based on the weight of the product per gram of reactant).

[0071] In a preferred embodiment of the present invention, the product-substrate yield (Yp / s) of a genetically modified Methylobacteriaceae cell according to the invention additionally comprising at least one exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase increases in relation to the product-substrate yield (Yp / s) of the genetically modified Methylobacteriaceae cell according to the invention without the at least one exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase in the conversion of a reactant containing at least one Cx compound, in particular consisting of methanol, to a product containing glycolic acid, in particular glycolic acid and lactic acid, in particular in a reaction medium comprising up to 10 g L' 1 , especially 10 g L' 1 , of the reactant by at least 10%, in particular at least 15%, in particular at least 20%, in particular 25%.

[0072] In a preferred embodiment of the present invention, the genetically modified Methylobacteriaceae cell according to the invention additionally comprising at least one exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase converts a reactant containing at least one Cx compound, in particular consisting of methanol, to a product containing glycolic acid, in particular glycolic acid and lactic acid, in particular in a reaction medium having an initial concentration of up to 10 g L' 1 , especially 10 g L' 1 , of the reactant with a product biodry mass yield (Yp / x) of at least 0.10 g g' 1 , in particular at least 0.30 g g' 1 , in particular at least 0.40 g g' 1 , in particular at least 0.50 g g' 1 , in particular at least 0.60 g g' 1 , in particular 0.10 to 0.99 g g' 1 , in particular 0.30 to 0.90 g g' 1 , in particular 0.40 to 0.80 g g' 1, in particular 0.50 to 0.75 g g' 1 , in particular 0.70 g g' 1 , in particular 0.71 gg' 1 (each based on the weight of the product per gram of dry biomass of the genetically modified Methylobacteriaceae cell according to the invention).

[0073] In a preferred embodiment of the present invention, the product biodry mass yield (Yp / x) of a genetically modified Methylobacteriaceae cell according to the invention additionally comprising at least one exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase increases in relation to the product biodry mass yield (Yp / x) of the genetically modified Methylobacteriaceae cell according to the invention without the at least one exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase in the conversion of a reactant containing at least one Cx compound, in particular consisting of methanol, to a product containing glycolic acid, in particular glycolic acid and lactic acid, in particular in a reaction medium having an initial concentration of up to 10 g L' 1 , especially 10 g L' 1, of the reactant by at least 10%, in particular at least 20%, in particular at least 30%, in particular at least 35%, in particular 40%, in particular 42%.

[0074] In a preferred embodiment of the present invention, the Methylobacteriaceae cell is a cell of Methylorubrum extorquens, in particular Methylorubrum extorquens TK 0001 and in particular Methylorubrum extorquens PA1.

[0075] In a preferred embodiment of the present invention, the at least one exogenous nucleic acid sequence encoding a glyoxylate reductase is integrated in the chromosome of the Methylobacteriaceae cell or is present extrachromosomally, in particular is integrated in the cell in an episomal expression vector or minichromosome.

[0076] In a preferred embodiment of the present invention, the at least one exogenous nucleic acid sequence encoding a glyoxylate reductase is stably integrated in the chromosome of the Methylobacteriaceae cell or is stably present extrachromosomally.

[0077] In a preferred embodiment of the present invention, more than one copy, in particular 2, 3, 4, 5, 6 or more copies of the exogenous nucleic acid sequence encoding a glyoxylate reductase are present in the genome of the Methylobacteriaceae cell, preferably stably integrated in the chromosome, or, preferably stably, extrachromosomally.

[0078] In a preferred embodiment of the present invention, the at least one exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase is integrated in the chromosome of the Methylobacteriaceae cell or is present extrachromosomally, in particular is integrated in the cell in an episomal expression vector or minichromosome.

[0079] In a preferred embodiment of the present invention, the at least one exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase is stably integrated in the chromosome of the Methylobacteriaceae cell or is stably present extrachromosomally.

[0080] In a preferred embodiment of the present invention, more than one copy, in particular 2, 3, 4, 5, 6 or more copies of the exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase, are present in the genome of the Methylobacteriaceae cell, preferably stably integrated in the chromosome, or, preferably stably, extrachromosomally. In a preferred embodiment of the present invention, the genetically modified Methylobacteriaceae cell is the Methylorubrum cell Methylorubrum extorquens Mea-GA1, Methylorubrum extorquens Mea-GA2 or Methylorubrum extorquens Mea-GA3, each deposited on June 10, 2022 at the DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany under the deposit numbers DSM 34286, DSM 34287 and DSM 34288. All deposits were made under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

[0081] In a preferred embodiment of the present invention, the present invention relates to a genetically modified Methylorubrum extorquens TK 0001 cell comprising at least one exogenous codon-optimized nucleic acid sequence encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MG1655, in particular cells of the strain Methylorubrum extorquens Mea-GAI deposited on June 10, 2022 at the DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany under the deposit number DSM 34286.

[0082] In a preferred embodiment of the present invention, the present invention relates to a genetically modified Methylorubrum extorquens TK 0001 cell comprising an exogenous codon-optimized nucleic acid sequence encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MGI 655 and an exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase from the bacterium Methylorubrum extorquens TK 0001 DSM 1337, in particular cells of the strain Methylorubrum extorquens Mea-GA2 deposited on June 10, 2022 at the DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany under the deposit number DSM 34287.

[0083] In a preferred embodiment of the present invention, the present invention relates to a genetically modified Methylorubrum extorquens TK 0001 cell comprising an exogenous codon-optimized nucleic acid sequence encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MGI 655 and an exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase from the bacterium Rhodobacter sphaeroides ATCC 17029, in particular cells of the strain Mea-GA3 deposited on June 10, 2022 at the DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany under the deposit number DSM 34288.

[0084] In a preferred embodiment of the present invention, the genetically modified Methylobacteriaceae cell is a cell of the 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), Methyl obacterium radiotolerans Mra-GA12 (DSM 34700), or Methyl obacterium organophilum Mor-GA8 (DSM 34699), each deposited on July 19, 2023, with the DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany. All deposits were made in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

[0085] In a preferred embodiment of the present invention, the present invention relates to a genetically modified Methylobacteriaceae cell comprising an exogenous codon-optimized nucleic acid sequence (SEQ ID No. 3) encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MGI 655, in particular cells of the strain Methylorubrum zatmanii Mza-GA14 (M. zatmanii DSM 5688 + pTE1887-ghrA eC o) deposited on 19 July 2023 with the DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany under the deposit number DSM 34701.

[0086] In a preferred embodiment of the present invention, the present invention relates to a genetically modified Methylobacteriaceae cell comprising an exogenous codon-optimized nucleic acid sequence (SEQ ID No. 3) encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MGI 655 of the strain Methylorubrum extorquens Mea-GA17 (M. extorquens PA1 DSM 23939 + pTE1887-ghrA eC o) deposited on 19 July 2023 with the DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany under the deposit number DSM 34702.

[0087] In a preferred embodiment of the present invention, the present invention relates to a genetically modified Methylobacteriaceae cell comprising an exogenous codon-optimized nucleic acid sequence (SEQ ID No. 3) encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MGI 655 of the strain Methylorubrum rhodesianum Mrh-GA4 (M. rhodesianum DSM 5687 + pTE1887-ghrA eC o) deposited on 19 July 2023 with the DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany under the deposit number DSM 34697.

[0088] In a preferred embodiment of the present invention, the present invention relates to a genetically modified Methylobacteriaceae cell comprising an exogenous codon-optimized nucleic acid sequence (SEQ ID No. 3) encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MGI 655 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 DSM 5687 + pTE1887-ghrA e co-ecm m ea) deposited on 19 July 2023 with the DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany under the deposit number DSM 34698.

[0089] In a preferred embodiment of the present invention, the present invention relates to a genetically modified Methylobacteriaceae cell comprising an exogenous codon-optimized nucleic acid sequence (SEQ ID No. 3) encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MGI 655 of the strain Methylobacterium organophilum Mor-GA8 (M. organophilum DSM 18172 + pTE1887-ghrA e co-ecm m ea) deposited on 19 July 2023 with the DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany under the deposit number DSM 34699.

[0090] In a preferred embodiment of the present invention, the present invention relates to a genetically modified Methylobacteriaceae cell comprising an exogenous codon-optimized nucleic acid sequence (SEQ ID No. 3) encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MGI 655 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 Methylobacterium radiotolerans Mra-GA12 (M. radiotolerans DSM 760 + pTE1887-ghrA e co-ecm m ea) deposited on 19 July 2023 with the DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany under the deposit number DSM 34700.

[0091] In a particularly preferred embodiment of the present invention, this relates to the specifically deposited Methylobacteriaceae cells, in particular the specifically deposited Methylorubrum strains and respective derivatives thereof.

[0092] In a preferred embodiment of the present invention, the at least one exogenous nucleic acid sequence encoding a glyoxylate reductase from the bacterium Escherichia is functionally linked to at least one regulatory unit to form 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.

[0093] In a preferred embodiment of the present invention, the at least one exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase, in particular an ethylmalonyl-CoA mutase 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, is functionally linked to at least one regulatory unit to form 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.

[0094] In a preferred embodiment, the expression cassette is present in a vector, in particular an expression vector, in particular an episomal expression vector, in particular pTE1887.

[0095] In a particularly preferred embodiment, the at least one exogenous nucleic acid sequence encoding a glyoxylate reductase and the at least one exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase can be present on the same expression vector or on different expression vectors.

[0096] In a preferred embodiment of the present invention, the promoter is an inducible promoter, in particular an IPT G-inducible promoter, in particular the PL / O4 / AI promoter.

[0097] A further aspect of the present invention is a method for producing a genetically modified Methylobacteriaceae cell according to the invention, comprising the method steps: 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 which enable the uptake and, preferably stable, subsequent integration of the at least one exogenous nucleic acid sequence into the Methylobacteriaceae cell, and c) obtaining the at least one exogenous,a genetically modified Methylobacteriaceae cell containing a nucleic acid sequence encoding glyoxylate reductase from the bacterium Escherichia.

[0098] In a particularly preferred embodiment, the present invention relates to an aforementioned method, wherein in method step a) additionally 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 or genome editing system comprising this nucleic acid sequence is provided, in method step b) the Methylobacteriaceae cell is transformed with the exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase, in particular the expression cassette comprising this, and in method step c) at least one exogenous genetically modified Methylobacteriaceae cell comprising a glyoxylate reductase, which additionally contains at least one exogenous,a nucleic acid sequence encoding ethylmalonyl-CoA mutase.

[0099] In a preferred embodiment of the present invention, the transformation according to process step b) is carried out by means of chemical, physical and / or electrical transformation methods, in particular electroporation.

[0100] The present invention also relates to a genetically modified Methylobacteriaceae cell which can be produced, in particular has been produced, by means of a method according to the invention.

[0101] A further aspect of the present invention is a genetically modified Methylobacteriaceae cell according to the invention, wherein the cell is present in a living, dead, lyophilized form, in the form of a cell lysate or a cell extract, and wherein the cell lysate or cell extract, in particular a protein extract, was obtained from a genetically modified Methylobacteriaceae cell according to the invention. According to the invention, the genetically modified Methylobacteriaceae cell according to the invention, optionally dead, lyophilized, or in the form of a cell lysate or cell extract, has the property provided by the invention of converting at least one Cx compound in a reaction medium to glycolic acid and optionally lactic acid.

[0102] In a preferred embodiment of the present invention, the cell, which is present in living, dead, lyophilized form or in the form of a cell lysate or a cell extract, catalyzes at least the conversion of at least one Cx compound to glycolic acid, in particular the conversion of a reactant containing at least one Cx compound to a product containing glycolic acid, in particular glycolic acid and lactic acid.

[0103] A further aspect of the present invention is a biocatalyst comprising a genetically modified Methylobacteriaceae cell according to the invention or a genetically modified Methylobacteriaceae cell according to the invention that is dead, lyophilized or in the form of a cell lysate or a cell extract, wherein said cell is arranged, in particular immobilized, on a carrier.

[0104] In a preferred embodiment of the present invention, the carrier is an organic carrier or an inorganic carrier. In a preferred embodiment of the present invention, the carrier comprises a naturally occurring organic carrier, in particular consists of this, in particular wherein the carrier is selected from the group consisting of chitin, agar, agarose, alginate, carrageenan, and a combination thereof.

[0105] In a preferred embodiment of the present invention, the carrier comprises a synthetic organic carrier, in particular consists of this, in particular wherein the carrier is selected from the group consisting of polyvinyl alcohol (PVA), polyurethane, acrylamide, polypropyleneammonium and a combination thereof.

[0106] In a preferred embodiment of the present invention, the carrier comprises an inorganic carrier, in particular consists of this, in particular wherein the carrier is selected from the group consisting of activated carbon, zeolite, ceramic, clay, anthracite, porous glass and a combination thereof.

[0107] In a preferred embodiment of the present invention, the carrier is a composite mixture of an organic carrier and an inorganic carrier, in particular comprising or consisting of polyvinyl alcohol sodium alginate (PVA-NA), polyvinyl alcohol guar gum (PVA-GG) or both.

[0108] In a preferred embodiment of the present invention, the biocatalyst according to the invention catalyzes at least the conversion of at least one Cx compound to glycolic acid, in particular the conversion of a reactant containing at least one Cx compound, in particular consisting thereof, to a product containing glycolic acid, in particular glycolic acid and lactic acid, in particular consisting thereof. A further aspect of the present invention is a bioreactor comprising a genetically modified Methylobacteriaceae cell according to the invention or a biocatalyst according to the invention, wherein the genetically modified Methylobacteriaceae cell or the biocatalyst according to the invention is present in particular in a reaction medium in the bioreactor.

[0109] A further aspect of the present invention is a process for producing glycolic acid from at least one Cx compound, in particular a product containing glycolic acid from a starting material containing at least one Cx compound, where x is preferably 1, 2 or 4, comprising the process steps: x) providing a genetically modified Methylobacteriaceae cell according to the invention or a biocatalyst according to the invention, a reaction medium and at least one Cx compound, in particular a starting material containing at least one Cx compound, y) reacting the at least one Cx compound, in particular the starting material, in the reaction medium under conditions which enable the formation of glycolic acid from the Cx compound, and z) obtaining glycolic acid, in particular the product containing glycolic acid, from the reaction medium.

[0110] In a preferred embodiment of the present invention, the Methylobacteriaceae cell according to the invention provided in process step x) or the biocatalyst according to the invention provided in process step x) is present in a form suspended in the reaction medium or in an immobilized form.

[0111] In a preferred embodiment of the present invention, the reaction medium provided in process step x) and / or used in process step y) is an aqueous salt-containing solution, in particular a culture medium, in particular a minimal medium, in particular a minimal medium consisting of up to 10 g of a Cx compound, in particular methanol, methane, formic acid, methylamine, acetic acid or succinic acid or a mixture thereof, per liter of reaction medium, 1 g of ammonium sulfate, 450 mg of magnesium sulfate heptahydrate, 3.2 mg of calcium chloride dihydrate, 7.4 mg of trisodium citrate dihydrate, 190 pg of zinc sulfate heptahydrate, 110 pg of manganese chloride tetrahydrate, 2.75 mg of iron sulfate heptahydrate, 1.36 mg of ammonium heptamolybdate tetrahydrate, 140 pg of copper sulfate pentahydrate, 260 pg of cobalt chloride hexahydrate, 390 pg of sodium tungstate, 30 pg boric acid, 2.02 g potassium dihydrogen phosphate and 4.14 g disodium hydrogen phosphate dihydrate.In a preferred embodiment of the present invention, the reaction medium provided in process step x) comprises the reactant containing at least one Cx compound.

[0112] In a preferred embodiment of the present invention, the reaction medium provided in process step x) at the beginning of process step y) comprises the reactant containing at least one Cx compound in a concentration of 1 to 100 g, in particular 5 to 90 g, in particular 6 to 80 g, in particular 7 to 70 g, in particular 8 to 40 g, in particular 9 to 30 g, in particular 10 to 20 g of Cx compound per liter of reaction medium.

[0113] In a preferred embodiment of the present invention, the reaction medium provided in process step x) comprises coenzyme B 12.

[0114] In a preferred embodiment of the present invention, the reactant used according to the invention, containing at least one Cx compound, is the sole carbon source in the reaction medium. Accordingly, in a preferred embodiment, a reaction medium is used that has the reactant used, containing at least one Cx compound, as the sole carbon source for the Methylobacteriaceae cells.

[0115] According to the invention, it is preferably provided that the reaction in process step y) takes place with continuous or batchwise addition of glyoxylate.

[0116] In a preferred embodiment of the present invention, the Cx compound of the reactant provided in process step x) and reacted in process step y) is formic acid, methanol, methane, methylamine, acetic acid, succinic acid or a mixture thereof.

[0117] In a preferred embodiment of the present invention, the reactant provided in process step x) and reacted in process step y) consists of formic acid, methanol, methane, methylamine, acetic acid, succinic acid or a mixture thereof.

[0118] In a preferred embodiment of the present invention, x = 1 for the Cx compound of the reactant provided in process step x) and reacted in process step y) containing at least one Cx compound. In a preferred embodiment of the present invention, the Cx compound of the reactant provided in process step x) and reacted in process step y) is methanol, formic acid, or a mixture thereof.

[0119] In a preferred embodiment of the present invention, the reactant provided in process step x) and reacted in process step y) consists of methanol, formic acid or a mixture thereof.

[0120] In a preferred embodiment of the present invention, the

[0121] Educt from methanol provided in process step x) and converted in process step y).

[0122] In a preferred embodiment of the present invention, the

[0123] Educt from formic acid provided in process step x) and converted in process step y).

[0124] In a preferred embodiment of the present invention, the reactant provided in process step x) and reacted in process step y) contains methanol and formic acid, in particular 1 to 99% by weight, in particular 2 to 98% by weight, in particular 10 to 90% by weight, in particular 30 to 70% by weight, in particular 40 to 60% by weight, in particular 50% by weight, of methanol and in particular 1 to 99% by weight, in particular 2 to 98% by weight, in particular 10 to 90% by weight, in particular 30 to 70% by weight, in particular 40 to 60% by weight, in particular 50% by weight of formic acid (in each case based on the total weight of the reactant provided in process step x)) or consists of these proportions.

[0125] In a preferred embodiment of the present invention, the reactant provided in process step x) containing at least one Cx compound, in particular methanol, formic acid or a mixture thereof, in particular methanol, is present at the beginning of process step y) in an initial concentration of 1 to 20 g L' 1 , especially 3 to 17 g L' 1 , especially 5 to 15 g L' 1 , especially 7 to 13 g L' 1 , especially 9 to 11 g L' 1 , especially 10 g L' 1 , in the reaction medium.

[0126] In a preferred embodiment of the present invention, the reactant provided in process step x) is methanol and is present at the beginning of process step y) in an initial concentration of 1 to 20 g L' 1 , especially 3 to 17 g L' 1 , especially 5 to 15 g L' 1 , especially 7 to 13 g L' 1 , especially 9 to 11 g L' 1, especially 10 g L' 1 , in the reaction medium.

[0127] In a preferred embodiment of the present invention, the Cx compound, in particular CI compound, in particular methanol, formic acid or a mixture thereof, provided in process step x) and reacted in process step y) is prepared from CO2, CO or a mixture in a process step w), in particular a process step w) which is operated with renewable energy, in particular electrical current from solar, wind, geothermal or hydropower energy.

[0128] In a preferred embodiment of the present invention, the Cx compound provided in process step x) and reacted in process step y), in particular methanol, formic acid or mixtures thereof, is produced from CO2, in particular synthesis gas comprising a mixture of CO2, CO and H2, in a process step w), in particular by means of a heterogeneous catalytic chemical process, in particular an electrochemical process.

[0129] In a preferred embodiment of the present invention, the Cx compound, in particular acetic acid, provided in process step x) and reacted in process step y) is produced from CO2, in particular synthesis gas comprising a mixture of CO2, CO and H2, in a process step w) by means of gas fermentation.

[0130] In a preferred embodiment of the present invention, the Cx compound, in particular methanol, provided in process step x) and converted in process step y) is produced from CO2, in particular synthesis gas comprising a mixture of CO2, CO and H2, or CO2, H2O and electric current, or CO2 and H2, in a process step w) by means of an electrochemical process, biochemical process, bioelectrochemical process or gas fermentation.

[0131] In a preferred embodiment of the present invention, the CO2 used in process step w), in particular synthesis gas, is produced by chemical conversion, in particular thermo-catalytic conversion, of organic substances or materials, in particular sewage sludge and other biogenic residues and waste materials.

[0132] In a preferred embodiment of the present invention, the

[0133] In process step w) the synthesis gas used is produced from sewage sludge.

[0134] In a preferred embodiment of the present invention, the

[0135] In process step w) the CO2 used is obtained from the atmosphere or from industrial exhaust gases.

[0136] In a preferred embodiment, the present invention thus makes it possible to enable a sustainable synthesis of glycolic acid and lactic acid that is cost-effective, environmentally friendly, and easy to handle, and that requires almost no, in particular no, use of fossil resources and / or almost no, in particular no, use of biogenic raw materials. According to the invention, glycolic acid and lactic acid are therefore advantageously obtained via an integrated process cascade in a completely renewable manner from CO2 as the sole raw material, i.e., without the consumption of fossil or biogenic resources. According to the invention, glycolic acid is therefore advantageously produced completely renewably from CO2 by the present invention.

[0137] In a preferred embodiment of the present invention, the reaction medium in process step y) has a temperature of 20 to 40 °C, in particular 22 to 38 °C, in particular 24 to 36 °C, in particular 28 to 32 °C, in particular 30 °C.

[0138] In a preferred embodiment of the present invention, process step y) is carried out in a water vapor-saturated atmosphere.

[0139] In a preferred embodiment of the present invention, the reaction medium at the beginning of process step y) has a pH of pH 4 to 8, in particular 5 to 7, in particular 6, in particular 6.8.

[0140] In a preferred embodiment of the present invention, the reaction medium has a pH of pH 0 to 6, in particular 0 to 4, in particular 0 to 3, in particular 1 to 2, after 40 h reaction time of process step y).

[0141] In a preferred embodiment of the present invention, the reaction according to process step y) is carried out under mechanical agitation, in particular shaking or stirring.

[0142] In a preferred embodiment of the present invention, in process step y) stirring is carried out at 50 to 1000 rpm, in particular 50 to 500 rpm, in particular 50 to 250 rpm, in particular 100 to 200 rpm, in particular 150 rpm (rpm: revolutions per minute).

[0143] In a preferred embodiment of the present invention, the reaction medium obtained in process step z) comprises the product containing at least glycolic acid.

[0144] In a preferred embodiment of the present invention, the product obtained in process step z) in the reaction medium containing glycolic acid is glycolic acid or a product containing glycolic acid and lactic acid. In a preferred embodiment of the present invention, the product obtained in process step z) contains glycolic acid and lactic acid, in particular 1 to 99 wt.%, in particular 2 to 98 wt.%, in particular 10 to 90 wt.%, in particular 30 to 80 wt.%, in particular 40 to 70 wt.%, in particular 50%, in particular 60 wt.% of glycolic acid and in particular 1 to 99 wt.%, in particular 2 to 98 wt.%, in particular 10 to 90 wt.%, in particular 20 to 70 wt.%, in particular 30 to 60 wt.%, in particular 50%, in particular 40 wt.% of lactic acid (in each case based on the total weight of the product obtained in process step z)) or consists of these proportions.

[0145] In a preferred embodiment of the present invention, the

[0146] Product obtained from glycolic acid in process step z).

[0147] In a preferred embodiment of the present invention, the

[0148] Product obtained from glycolic acid and lactic acid in process step z).

[0149] In a preferred embodiment of the present invention, after process step z), in a process step zl), the product containing glycolic acid and optionally lactic acid is isolated from the reaction medium, in particular separated from the reaction medium and the genetically modified Methylobacteriaceae cell according to the invention or the biocatalyst according to the invention, in particular by decanting, salting out with a base, in particular NaOH or KOH, filtration, in particular membrane filtration or column filtration, or ion exchange chromatography in combination with HPLC, extraction and / or distillation.

[0150] In a preferred embodiment of the present invention, the process for producing a product containing glycolic acid is a continuous process.

[0151] A further aspect of the present invention is a process for producing polyglycolic acid, polylactic acid or polylactide-co-glycolide, comprising carrying out a process according to the invention for producing glycolic acid, in particular a product containing glycolic acid and optionally lactic acid, and subsequently polymerizing the glycolic acid, lactic acid or glycolic acid and lactic acid obtained from these processes.

[0152] In the context of the present invention, “genetically modified Methylobacteriaceae cell according to the invention” is understood to mean a genetically modified Methylobacteriaceae cell which preferably resembles, in particular is identical to, the wild-type strain of the Methylobacteriaceae cell, with the exception of the presence of at least one exogenous nucleic acid sequence encoding a glyoxylate reductase from the bacterium Escherichia, which imparts to the Methylobacteriaceae cell according to the invention the enzymatic glyoxylate reductase activity advantageous according to the invention, and optionally exogenous nucleic acid sequences of an expression vector or an expression cassette associated therewith, and optionally the at least one exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase and optionally exogenous nucleic acid sequences of an expression vector or an expression cassette associated therewith.

[0153] In the context of the present invention, "one" genetically modified Methylobacteriaceae cell is understood to mean one, two, several, many, or an indefinable number of Methylobacteriaceae cells. In a preferred embodiment of the present invention, a Methylobacteriaceae cell is also understood to be a Methylobacteriaceae strain, in particular a Methylorubrum extorquens strain, in particular a Methylorubrum rhodesianum strain, in particular a Methylorubrum zatmanii strain, in particular a Methylorubrum extorquens TK 0001 strain, in particular a Methylorubrum extorquens AMI strain, in particular a Methylorubrum extorquens PA1 strain, in particular a Methylobacterium organophilum strain, or in particular a Methylobacterium radiotolerans strain.

[0154] In the context of the present invention, a “derivative” of a deposited Methylobacteriaceae cell or a deposited Methylobacteriaceae strain, in particular a deposited Methylorubrum strain or cell or a deposited Methylobacterium strain or cell is understood to mean a Methylobacteriaceae cell, in particular a Methylobacteriaceae strain, in particular a Methylorubrum cell, in particular a Methylorubrum strain, or a Methylobacterium cell, in particular a Methylobacterium strain, which is distinguished by the presence of the features provided according to the invention, in particular the integration of the nucleic acid sequence encoding exogenous glyoxylate reductase, and which is obtained from a deposited Methylobacteriaceae cell and whose genome has been modified while retaining the features according to the invention.

[0155] In the context of the present invention, an “exogenous nucleic acid sequence” of an organism, in particular a microorganism, in particular a bacterium, is understood to mean a nucleic acid sequence introduced into a recipient organism by means of recombinant, i.e. genetic engineering, process steps.

[0156] In connection with the present invention, in a preferred embodiment, an “exogenous nucleic acid sequence” of an organism, in particular a microorganism, in particular a bacterium, is understood to mean a nucleic acid sequence originating from another microorganism strain, in particular another organism species, in particular another bacterium species, i.e. not endogenous, and thus not native or not occurring in the wild-type strain or wild-type species.

[0157] In the context of the present invention, a “glyoxylate reductase” is understood to mean an enzyme which is capable of enzymatically catalyzing the conversion of glyoxylate, in particular to glycolic acid, in particular using a cofactor, in particular NADH or NADPH.

[0158] In connection with the present invention, a “glyoxylate reductase” (ghrA) of the present invention in a preferred embodiment has a KM value of at most 2.0, in particular at most 1.5, in particular at most 1.0, in particular at most 0.6 mM, in particular 0.6 mM, for glyoxylate.

[0159] In the context of the present invention, a “glyoxylate reductase” (ghrA) of the present invention in a preferred embodiment has a KM value of at least 0.9, in particular at least 1.0 mM, in particular 1.0 mM, for hydroxypyruvate.

[0160] In connection with the present invention, a "glyoxylate reductase" (ghrA) of the present invention in a preferred embodiment has a KM value of at most 2.0, in particular at most 1.5, in particular at most 1.0, in particular at most 0.6 mM, in particular 0.6 mM for glyoxylate and a KM value of at least 0.9, in particular at least 1.0 mM, in particular 1.0 mM, for hydroxypyruvate.

[0161] In the context of the present invention, a “glyoxylate reductase” (ghrA) of the present invention, in a preferred embodiment, has a KM value of at most 2.0 for glyoxylate and a KM value of at least 0.9 for hydroxypyruvate.

[0162] Glyoxylate reductase is preferably NADPH dependent.

[0163] In contrast, a "hydroxypyruvate reductase" (ghrB) has a KM value of at least 3.0, in particular at least 4.0, in particular at least 5.0, in particular at least 6.0 mM, in particular at least 6.6 mM, in particular 6.6 mM for glyoxylate. In particular, a hydroxypyruvate reductase (ghrB) has a KM value of at most 0.6, in particular at most 0.7 mM, in particular 0.7 mM, for hydroxypyruvate.

[0164] In particular, a “hydroxypyruvate reductase” (ghrB) has a KM value of at least 3.0, in particular at least 4.0, in particular at least 5.0, in particular at least 6.0 mM, in particular at least 6.6 mM, in particular 6.6 mM for glyoxylate and a KM value of at most 0.6, in particular at most 0.7 mM, in particular 0.7 mM, for hydroxypyruvate.

[0165] Hydroxypyruvate reductase is preferably NADH-dependent.

[0166] For the definition of the Michaelis-Menten constant, KM, which is defined as the substrate concentration at which the half-maximal turnover rate of a specific enzyme is reached under fixed reaction conditions, the Lineweaver-Burk evaluation method is preferred as the calculation method, described in Lineweaver, H. and Burk, D. (1934) Determination of the enzyme dissociation constants. J. Am. Chem. Soc. 56, 658-666.

[0167] In connection with the present invention, a "glyoxylate reductase" of the present invention, in a preferred embodiment, has a higher enzyme activity in an NADPH-dependent conversion of glyoxylate to glycolate than in an NADH-dependent conversion of glyoxylate to glycolate, in particular an at least 3-fold higher enzyme activity, in particular under conditions as specified in the enzyme assay according to Example 5.

[0168] In the context of the present invention, a "cell of a methylotrophic bacterium" is understood to mean, in particular, a cell belonging to the Methylobacteriaceae family. In particular, these cells are capable of carrying out the serine cycle (https: / / doi.org / 10.1002 / 9781118960608. gbm02024, https: / / doi.org / 10.llll / 1462-2920.12736, https: / / doi.org / 10.3389 / fmicb.2021.740610).

[0169] The serine cycle is a methylotrophic metabolic pathway that facilitates the assimilation of Cl substrates, such as methanol, formate / formic acid, and methylamines, in microbial metabolism for the formation of biomass or chemical products / intermediates of this metabolism. It is a defined sequence of enzymatically catalyzed reactions.

[0170] The cycle starts with glycine. In a first step, the eponymous amino acid L-serine is formed from the carbon assimilated from the CI (i.e., methanol, formic acid, etc.) in the form of 5,10-methylenetetrahydrofolate and a molecule of water and glycine by a glycine hydroxymethyltransferase (EC 2.1.2.1). Tetrahydrofolate is cleaved off, which prepares it for further carbon assimilation. In subsequent steps in the serine cycle, the L-serine is deaminated by a transaminase to hydroxypyruvate. The cleaved NH3 equivalent is used for the transamination of glyoxylate to glycine to keep the cycle going. The aforementioned hydroxypyruvate is reduced by a hydroxypyruvate reductase with NAD(P)H to glycerate, which is then phosphorylated by a kinase to form 3-phosphoglycerate.In two consecutive reaction steps, 3-phosphoglycerate is converted into phosphoenolpyruvate by a phosphoglyceromutase (EC 5.4.2.11) and a water-releasing enolase (EC 4.2.1.11). The phosphoenolpyruvate is carboxylated to oxaloacetate by phosphoenolpyruvate carboxylase (EC 4.1.1.31) using bicarbonate / dissolved CO2. The phosphoenolpyruvate is finally converted via L-malate to L-malyl-CoA with the use of NADH and ATP, as well as a cofactor A (CoA) molecule. Acetyl-CoA is then cleaved off, forming glyoxylate. With this reaction, carried out by a malonyl-CoA lyase (EC 4.1.3.24), the cycle closes, and further assimilation of a single carbon can begin. (Anthony, CW (2011). "How half a century of research was required to understand bacterial growth on Cl and C2 compounds; the story of the serine cycle and the ethylmalonyl-CoA pathway." Science progress 94 Pt 2: 109-137).

[0171] The serine cycle can be detected by the presence of the metabolite hydroxypyruvate. In addition, the characteristic labeling of glycine, serine, and glyoxylate can be measured in labeling studies using 13C-labeled Cl substrate and unlabeled CO2 (https: / / doi.org / 10.1186 / 1752-0509-5-189).

[0172] In the context of the present invention, “M. extorquens” means Methylorubrum extorquens, “M. rhodesianum” means Methylorubrum rhodesianum, “M. zatmanii” means Methylorubrum zatmanii, “M. organophilum” means Methyl obacterium organophilum, and “M. radiotolerans” means Methylobacterium radiotolerans.

[0173] In the context of the present invention, “pTE1887” is understood to mean a specific expression vector.

[0174] In the context of the present invention, “ghrA eCo" is understood to mean a nucleic acid sequence encoding the glyoxylate reductase from Escherichia coli K-12 MG1655. This nucleic acid sequence can be the native ("ghrA eC o-native”) or a codon-optimized (“ghrA eC oc-optimized”) nucleic acid sequence.

[0175] In the context of the present invention, “pTE1887-ghrA eC o" is understood to mean an expression vector containing the nucleic acid sequence encoding the glyoxylate reductase from Escherichia coli K-12 MG1655. In the context of the present invention, "ecm me "a" means the nucleic acid sequence encoding the ethylmalonyl-CoA mutase from M. extorquens TK 0001 DSM 1337. This nucleic acid sequence can be the native or a codon-optimized nucleic acid sequence.

[0176] In the context of the present invention, “pTE1887- ghrA e co-ecm mea" is understood to mean an expression vector which contains the nucleic acid sequence encoding the glyoxylate reductase from Escherichia coli K-12 MG1655 and the nucleic acid sequence encoding the ethylmalonyl-CoA mutase from M. extorquens TK 0001 DSM 1337.

[0177] In the context of the present invention, “ecm rs h" means the nucleic acid sequence encoding the ethylmalonyl-CoA mutase from Rhodobacter sphaeroides ATCC 17029. This nucleic acid sequence can be the native or a codon-optimized nucleic acid sequence.

[0178] In the context of the present invention, “pTE1887- ghrAeco-ecm rs h" is understood to mean an expression vector which contains the nucleic acid sequence encoding the glyoxylate reductase from Escherichia coli K-12 MG1655 and the nucleic acid sequence encoding the ethylmalonyl-CoA mutase from Rhodobacter sphaeroides ATCC 17029.

[0179] In the context of the present invention, “functional nucleic acid sequence equivalent” is understood to mean a nucleic acid sequence equivalent of a nucleic acid sequence encoding a glyoxylate reductase or an ethylmalonyl-CoA mutase, wherein the nucleic acid equivalent has at least one difference at at least one nucleotide position to the nucleic acid sequence, that is to say has at least one additional nucleotide, i.e. an inserted nucleotide or at least one missing nucleotide, i.e. a deleted nucleotide, or has at least one exchanged nucleotide, and wherein the nucleic acid equivalent encodes an amino acid sequence with the enzymatic activity of a glyoxylate reductase or an ethylmalonyl-CoA mutase.In the context of the present invention, “codon-optimized” is understood to mean that the nucleic acid sequence of a wild-type gene, which is to be integrated as an exogenous nucleic acid sequence into a Methylobacteriaceae host cell, in particular from E. coli, is optimized for expression, i.e. transcription and translation in the host cell, prior to integration by genetically engineered exchange of codons, and in particular of those codons which in the exogenous nucleic acid sequence are usually not used or not used optimally by the translation system of the host cell, i.e. the Methylobacteriaceae cell, in particular Methylorubrum extorquens, in particular Methylorubrum extorquens AMI, Methylorubrum extorquens TK 0001, in particular Methylorubrum extorquens PAI cell.For example, through in vitro mutagenesis, the corresponding Methylobacteriaceae-preferred codons are incorporated instead, without altering the amino acid sequence encoded by the nucleic acid sequence. In the context of the present invention, a codon-optimized nucleic acid sequence is therefore a nucleic acid sequence optimized for expression in a Methylobacteriaceae cell. Codon optimization can also be performed if the exogenous nucleic acid sequence originates from the same bacterial species as the host cell, but an improvement in expression is nevertheless desired. Codon optimization can preferably be carried out according to the following overview (Table 1):

[0180] In the context of the present invention, “functional nucleic acid sequence equivalent of a codon-optimized nucleic acid” is understood to include, but is not limited to, the native, naturally occurring nucleic acid.

[0181] Table 1 : Codon optimization In the context of the present invention, “functional amino acid sequence equivalent” is understood to mean an amino acid sequence equivalent of an amino acid sequence of a glyoxylate reductase or an ethylmalonyl-CoA mutase, wherein the amino acid equivalent has at least one difference at at least one amino acid position to the amino acid sequence, i.e. has at least one additional amino acid, i.e. an inserted amino acid or at least one missing amino acid, i.e. a deleted amino acid, or has at least one exchanged amino acid, and wherein the amino acid equivalent has the enzymatic activity of a glyoxylate reductase or an ethylmalonyl-CoA mutase.

[0182] In the context of the present invention, the "identity of nucleic acid or amino acid sequences" is understood to mean a degree of identity in % determined by a sequence comparison. This sequence comparison is fundamentally based on the BLAST algorithm, which is established and commonly used in the prior art (cf., for example, Altschul et al. (1990) "Basic local alignment search tool", J. Mol. Biol. 215:403-410, and Altschul et al. (1997): "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402) and is essentially performed by matching similar sequences of nucleotides or amino acids in the nucleic acid or amino acid sequences to one another. A tabular assignment of the relevant positions is referred to as alignment. Another algorithm available in the prior art is the FASTA algorithm.Sequence alignments, especially multiple sequence alignments, are created using computer programs. Commonly used programs include the Clustal series (see, for example, Chenna et al. (2003) "Multiple sequence alignment with the Clustal series of programs," Nucleic Acids Res. 31:3497-3500), T-Coffee (see, for example, Notredame et al. (2000) "T-Coffee: A novel method for multiple sequence alignments," J. Mol. Biol. 302:205-217), or programs based on these programs or algorithms. Sequence alignments can also be performed using the computer program Vector NTI® Suite 10.3 (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California, USA) with the specified standard parameters. Its AlignX module for sequence comparisons is based on ClustalW. Unless otherwise stated, the sequence identity reported herein is determined using the NCBI Constraint-based Multiple Alignment Tool (COBALT) (https: / / www.ncbi.nlm.nih.gov / , as of January 26, 2019).2022), whereby SEQ ID Nos. 1 to 8 were used as a reference for determining the percentage sequence differences. Such a comparison also allows a statement to be made about the similarity of the compared sequences to one another. It is given here as percent "identity", i.e. the proportion of identical nucleotides or amino acid residues at the same positions or at corresponding positions in an alignment. Identity statements can be made for entire polypeptides or genes or only for individual regions. Identical regions of different nucleic acid or amino acid sequences are therefore defined by similarities in the sequences. Such regions often have identical functions. They can be small and comprise only a few nucleotides or amino acids. Unless otherwise stated, identity statements in the present teaching refer to the total length of the respective nucleic acid or amino acid sequence specified.

[0183] In the context of the present invention, an “amino acid sequence” is understood to mean a sequence of linearly linked amino acids, in particular a protein, in particular a polypeptide.

[0184] In the context of the present invention, a "nucleic acid sequence" is understood to mean a sequence of linearly linked nucleotides, in particular a nucleic acid molecule, in particular a gene, in particular a protein-coding region of a gene. In a particularly preferred embodiment, the nucleic acid sequence is a DNA sequence.

[0185] In the context of the present invention, “ethylmalonyl-CoA mutase” is understood to mean a coenzyme B1 2-dependent enzyme with intramolecular isomerase activity which is responsible for the conversion of ethylmalonyl-CoA to methylsuccinyl-CoA in the ethylmalonyl-CoA metabolism and which preferably has the EC classification EC 5.4.99.63.

[0186] In the context of the present invention, a "Cx compound" is understood to mean a chemical compound containing carbon (C), hydrogen (H), and oxygen (O) that contains x carbon atoms, where x is preferably a natural number. According to the invention, x is preferably 1, 2, or 4, in particular 1. The Cx compound preferably contains only C, H, and O atoms and therefore no other atoms.

[0187] In the context of the present invention, formic acid also means formate, acetic acid also means acetate and succinic acid also means succinate.

[0188] In the context of the present invention, “integration of an exogenous nucleic acid sequence into a Methylobacteriaceae cell” or “presence of an exogenous nucleic acid sequence in a Methylobacteriaceae cell” is understood to mean that the respective nucleic acid sequence referred to is present chromosomally or extrachromosomally, preferably chromosomally, in the genome of the cell.

[0189] In a preferred embodiment, the exogenous nucleic acid sequence is present in a stably integrated form, wherein a stable integration of a nucleic acid is such an integration that is detectable and capable of expression in the microorganism for at least 2, 3, 5, 10, 20 or 50 generations of the microorganism.

[0190] In the context of the present invention, "maximum growth rate" (gmax) is understood to mean the rate of cell division, i.e., microbial growth, of the Methylobacteriaceae cell according to the invention in the reaction medium, in particular liquid culture medium. The calculation of p max is based on the measured values ​​of the optical density of the culture medium at 600 nm wavelength, measured in the photometer (ODeoo) over the time course of the process step. The calculation of p max can be performed using Equation 1, taking into account the measured values ​​of the ODeoo in the growth interval of the fastest observed growth. (Equation 1)

[0191] Equation 1 with t y - t x as the time interval of the growth interval of the fastest observed growth and t y > t xThe time interval is typically given in hours (h). In the context of the present invention, "dry biomass substrate yield" (Yx / s) is understood to mean the mass of microbial dry biomass (biomass completely dried to constant weight) in the reaction medium, in particular liquid culture medium, expressed in a weight unit such as grams (X, gx), which can be produced by the specific microbial strain from one gram of the Cx compound (S, gcx). The calculation is carried out graphically using linear regression of the changes in the measured values ​​of the dry biomass (AX(t)) as a function of the mass of the Cx compound (ACx(t)) over time in the process step according to Equation 2. According to Equation 2, Yx / s is thus the gradient of the time-linearly correlated change in dry biomass X as a function of the change in the mass of the Cx compound. The unit of Yx / s is typically given in gx per gcx. (Equation 2) In the context of the present invention, "product-substrate yield" (Yp / s) refers to the mass of product, expressed in a weight unit such as grams (P, gp), that can be produced by the specific microbial strain from one gram of the Cx compound (S, gcx). The calculation is performed graphically using linear regression of the changes in the measured values ​​of the product mass (AP(t)) as a function of the mass of the Cx compound (ACx(t)) over time in the process step according to Equation 3. According to Equation 3, Yp / s is thus the slope of the time-linearly correlated change in the product mass P as a function of the change in the mass of the Cx compound. The unit of Yp / s is typically given in gp per gcx. (Equation 3)

[0192] In the context of the present invention, "product dry biomass yield" (Yp / x) is understood to mean the mass of product, expressed in a weight unit such as grams (P, gp), produced by the specific microbial strain per gram of dry biomass (X, gx) formed during microbial growth. The calculation is carried out graphically using linear regression of the changes in the measured values ​​of the product mass (AP(t)) as a function of the dry biomass (AX(t)) over time in the process step according to equation 4. According to equation 4, Yp / x is thus the gradient of the time-linearly correlated change in the product mass P as a function of the change in the dry biomass formed. The unit of Yp / x is typically given in gp per gx.

[0193] Y P / x = i™ [g] (Equation 4)

[0194] In the context of the present invention, "dry biomass" is understood to mean the mass X(t) of microbial dry biomass (biomass completely dried to constant weight) in the reaction medium, in particular liquid culture medium with the volume v(t) at time t, expressed in a weight unit such as grams (X, gx). The dry biomass X can be determined from the measured values ​​of the ODeoo using the correlation factor z according to equation 5, where z = 0.305 gx per 1 ODeoo.

[0195] X(t) = 0.305 * ODeoo(t) * v [gx] (Equation 5) In the context of the present invention, the abbreviation "NAD" refers to nicotinamide adenine dinucleotide. In the context of the present invention, "NADH" refers to the reduced form of NAD. In the context of the present invention, the abbreviation "NADP" refers to nicotinamide adenine dinucleotide phosphate. In the context of the present invention, "NADPH" refers to the reduced form of NADP. In the context of the present invention, “NADH / NADPH analogue” is understood to mean a chemical compound, for example thionicotinamide adenine dinucleotide (S-NAD), nicotinic acid adenine dinucleotide (O-NAD), nicotinamide hypoxanthine dinucleotide (NHD), nicotinamide guanine dinucleotide, or other compounds which have a similar, preferably the same activity as NADH and / or NADPH.

[0196] In the context of the present invention, a “reactant” is understood to mean a starting material, in particular at least one Cx compound, in particular one Cx compound or two or more or many Cx compounds, in particular a composition of Cx compounds.

[0197] In the context of the present invention, a “product” is understood to mean at least glycolic acid, in particular glycolic acid alone, preferably glycolic acid and lactic acid, in particular a composition of compounds containing glycolic acid, in particular consisting of the compounds glycolic acid and lactic acid.

[0198] In the context of the present invention, a “reaction” is understood to mean a chemical reaction, in particular a catalyzed chemical reaction, in particular an enzymatically catalyzed reaction.

[0199] In connection with the present invention, a “reaction medium” is understood to mean a liquid medium, in particular a liquid aqueous medium, in which a reaction, in particular an enzymatically catalysed reaction, can take place, in particular a reaction brought about by microorganisms or components of microorganisms, in particular a culture medium, in particular a minimal medium.

[0200] In the context of the present invention, the term "obtaining a product" means that the product obtained in a previous process step by reacting the reactant, i.e., a starting material, is made available from the respective reaction medium, in particular culture medium or solvent, and in particular is isolated from it. Obtaining a product is therefore to be understood in particular as concentrating, in particular isolating, the product. The processes used for this purpose can be physical, chemical, and / or biological processes.

[0201] In the context of the present invention, “compound” is understood to mean one molecule or several identical molecules.

[0202] In the context of the present invention, a composition containing glycolic acid is the product of a reaction according to the invention in process step b).

[0203] If quantitative information, in particular percentage information, of components of a product or composition is given in connection with the present invention, these add up to 100% of the composition and / or product together with the other explicitly stated or expertly apparent further components of the composition or product, unless explicitly stated otherwise or apparent to a person skilled in the art.

[0204] In the context of the present invention, the term "at least one" is understood to mean a quantity that expresses a number of 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10, etc. In a particularly preferred embodiment, the term "at least one" can represent exactly 1. In a further preferred embodiment, the term "at least one" can also mean 2 or 3 or 4 or 5 or 6 or 7.

[0205] If in connection with the present invention a “presence”, a “containing”, a “having” or a “content” of a component is expressly mentioned or implied, this means that the respective component is present, in particular is present in a measurable amount.

[0206] If in connection with the present invention a "presence", a "containing" or a "having" of a component in an amount of 0 [unit], in particular mg / kg, pg / kg or wt.%, is expressly mentioned or implied, this means that the respective components are not present in a measurable amount, in particular are not present.

[0207] The number of decimal places specified corresponds to the precision of the measurement method used. If, in the context of the present invention, the first and second decimal places or the second decimal place are not specified for a number, they should be set to zero.

[0208] In the context of the present invention, the term "and / or" means that all members of a group linked by the term "and / or" are disclosed both alternatively to one another and cumulatively among one another in any combination. For the expression "A, B, and / or C," this means that the following disclosure content is to be understood: a) A or B or C or b) (A and B), or c) (A and C), or d) (B and C), or e) (A and B and C).

[0209] In the context of the present invention, the terms "comprising" and "having" are understood to mean that, in addition to the elements explicitly covered by these terms, further, not explicitly mentioned elements can be added. In the context of the present invention, these terms are also understood to mean that only the explicitly mentioned elements are covered and no further elements are present. In this particular embodiment, the meaning of the terms "comprising" and "having" is synonymous with the term "consisting of". Furthermore, the terms "comprising" and "having" also encompass compositions which, in addition to the explicitly mentioned elements, also contain further elements not mentioned, which, however, are of a functional and qualitatively subordinate nature. In this embodiment, the terms "comprising" and "having" are synonymous with the term "consisting essentially of".

[0210] The designation “DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany” stands for “Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstraße 7B, 38124 Braunschweig”.

[0211] Further preferred embodiments of the present invention emerge from the subclaims.

[0212] The invention is described in more detail below, without limiting the general inventive concept, using examples and associated figures.

[0213] The sequence listing shows: SEQ ID No. 1 represents the native nucleic acid sequence encoding a glyoxylate reductase from the bacterium Escherichia coli (K-12 MG1655), in particular also referred to as ghrAeco-native, i.e. a functional nucleic acid sequence equivalent of the nucleic acid sequence according to SEQ ID No. 3.

[0214] SEQ ID NO: 2 the amino acid sequence encoded by SEQ ID NO: 1 and 3.

[0215] SEQ ID No. 3 represents a Methylobacteriaceae codon-optimized nucleic acid sequence (ghrA eco - c-optimized) of the native nucleic acid sequence according to SEQ ID No. 1 encoding a glyoxylate reductase from the bacterium Escherichia coli (K-12 MG1655).

[0216] SEQ ID No. 4 represents the native nucleic acid sequence encoding an ethylmalonyl-CoA mutase from the bacterium Methylorubrum extorquens (TK 0001 DSM 1337), also referred to as ecm me a, i.e. a functional nucleic acid sequence equivalent of the codon-optimized nucleic acid sequence according to SEQ ID No. 13.

[0217] SEQ ID NO: 5 comprises the amino acid sequence encoded by SEQ ID NO: 4 and 13.

[0218] SEQ ID No. 6 represents the native nucleic acid sequence encoding an ethylmalonyl-CoA mutase from the bacterium Rhodobacter sphaeroides (ATCC 17029), in particular also referred to as ecmrsh, i.e. a functional nucleic acid sequence equivalent of the codon-optimized nucleic acid sequence according to SEQ ID No. 8.

[0219] SEQ ID No. 7 the amino acid sequence encoded by SEQ ID Nos. 6 and 8.

[0220] SEQ ID No. 8 represents a Methylobacteriaceae codon-optimized nucleic acid sequence of the native nucleic acid sequence according to SEQ ID No. 6 encoding an ethylmalonyl-CoA mutase from the bacterium Rhodobacter sphaeroides (ATCC 17029).

[0221] SEQ ID No. 9 represents the nucleic acid sequence of the expression vector pTE1887, with the corresponding plasmid map shown in Figure 8.

[0222] SEQ ID No. 10 represents the nucleic acid sequence of the expression vector pTE1887-ghrA eCo, with the corresponding plasmid map shown in Figure 9.

[0223] SEQ ID No. 11 represents the nucleic acid sequence of the expression vector pTE1 887-ghrA eco-CCnimea, with the corresponding plasmid map shown in Figure 10. SEQ ID No. 12 represents the nucleic acid sequence of the expression vector pTE1887-EcoGoxRed l-ecmrsh, with the corresponding plasmid map shown in Figure 11.

[0224] SEQ ID No. 13 represents a Methylobacteriaceae codon-optimized nucleic acid sequence of the native nucleic acid sequence according to SEQ ID No. 4 encoding an ethylmalonyl-CoA mutase from the bacterium Methylorubrum extorquens (TK 0001 DSM 1337).

[0225] The figures show:

[0226] Figure 1 shows the screening results for glycolic acid production in recombinant, i.e. genetically modified, M. extorquens TK 0001 strains that have and express codon-optimized genes of the glyoxylate reductases (A), screening results according to 1A in (B and C), whereby enzyme activities of the glyoxylate reductases from the biomass used according to 1(A) expressed with the expression vector pTE1887 in the strain background M. extorquens TK 0001 with NADH (B) and NADPH (C) as cofactors are shown,

[0227] Figure 2 shows an HPLC chromatogram comparison of the culture samples (22 to 24 h after induction) of the genetically modified Methylobacteriaceae cells M. extorquens TK 0001 glyoxylate reductase strains that have and express codon-optimized genes of the glyoxylate reductases,

[0228] Figure 3 a GC-MS chromatogram and mass spectra of the glycolic acid peak (retention time: 7.22 min) of a 100 mg L' 1Glycolic acid standards, a sample of the reaction medium at time t = 0 h, a sample of the M. extorquens TK 0001 + pTE1887 empty vector culture after induction, and a sample of the + pTE1887-ghrA eC oc-optimized (M. extorquens GAI) cultivation after induction,

[0229] Figure 4 a GC-MS chromatogram and mass spectra of the lactic acid peak (retention time: 6.88 min) of a 100 mg L' 1Lactic acid standards, a sample of the reaction medium at time t = 0 h, a sample of the M. extorquens TK 0001 + pTE1887 empty vector culture 22 - 24 h after induction, and an inventive sample of the M. extorquens TK 0001 + pTE1887-ghrAeco-c-optimized (M. extorquens GAI) culture 22 to 24 h after induction, Figure 5 shows a detailed view of the mass spectra of the glycolic acid peak (A) and the lactic acid peak (B) of a sample of the M. extorquens GAI culture 22 to 24 h after induction and database detection of the glycolic acid identity (A) and the lactic acid identity (B) in the M. extorquens GAI sample,

[0230] Figure 6 shows 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- ghrAeco-c-optimised (M. extorquens GAI) (B+D) according to the invention in reaction medium, namely minimal medium, using 8 g L' as carbon source. 1 Methanol (A+B) or 9 g L' 1 Methanol + 1.5 g L' 1 Glyoxylate (C+D) was added,

[0231] Figure 7 shows the growth curve (OD600), pH, methanol, glycolic acid and lactic acid concentrations of M. extorquens TK 0001 + pTE1887 (A), inventive M. extorquens TK 0001 + pTE1887-ghrA eC oc-optimized (M. extorquens GAI) (B), inventive M. extorquens TK 0001 + pTE1887- ghrAeco-c-optimized-eemmea (M. extorquens GA2) (C) and inventive M. extorquens TK 0001 + pTE1887-ghrA e co-c-optimized-ecm rsh (M. extorquens GA3) (D) in reaction medium with 9 g L' 1 Methanol as the sole substrate,

[0232] Figure 8 shows the plasmid map of the expression vector pTE1887,

[0233] Figure 9 the plasmid map of the expression vector pTE1887-ghrA eC oc-optimized,

[0234] Figure 10 the plasmid map of the expression vector pTE1887-ghrA e co-c-optimized-ecm m ea,

[0235] Figure 11 the plasmid map of the expression vector pTE1887-ghrA eC oc-optimized-ecm r sh,

[0236] Figure 12 shows the results of the glyoxylate reductase enzyme activity tests of ghrA eco and ghrBeco in native and codon-optimized DNA sequence expressed with the expression vector pTEl 887 in the strain background M. extorquens TK 0001,

[0237] Figure 13 shows the taxonomic classification of the methylotrophic microorganisms tested with the expression vectors according to the invention,

[0238] Figure 14 shows the screening result for glycolic acid and lactic acid production 22 h to 28 h after induction of gene expression in recombinant, i.e. genetically modified, M. rhodesianum DSM 5687 strains that contain and express codon-optimized genes of glyoxylate reductases and, in some strains, additionally ethylmalonyl-CoA mutases.

[0239] Figure 15 shows the screening result for glycolic acid and lactic acid production 22 h to 28 h after induction of gene expression in recombinant, i.e. genetically modified, M. zatmanii DSM 5688 strains which have and express the codon-optimized gene of glyoxylate reductase from Escherichia according to the invention and, in one strain, additionally a codon-optimized gene of ethylmalonyl-CoA mutase from Rhodobacter sphaeroides ATCC 17029,

[0240] Figure 16 shows the screening result for glycolic acid and lactic acid production 22 h to 28 h after induction of gene expression in a recombinant, i.e. genetically modified, M. radiotolerans DSM 760 strain, which has and expresses the inventive combination of the codon-optimized gene of glyoxylate reductase from Escherichia and additionally a native gene of ethylmalonyl-CoA mutase from M. extorquens TK 0001 DSM 1337,

[0241] Figure 17 shows the screening result for glycolic acid and lactic acid production 22 h to 28 h after induction of gene expression in recombinant, i.e. genetically modified, M. organophilum DSM 18172 strains that contain and express codon-optimized genes of glyoxylate reductases and, in some strains, additionally ethylmalonyl-CoA mutases.

[0242] Figure 18 shows the screening result for glycolic acid and lactic acid production 22 h to 28 h after induction of gene expression of a recombinant, i.e. genetically modified, M. extorquens PA1 DSM 23939 strain, which according to the invention has and expresses the codon-optimized gene of glyoxylate reductase from Escherichia coli K12 1655,

[0243] Figure 19 shows the screening results for glycolic acid and lactic acid production 22 to 28 hours after induction of gene expression in recombinant, i.e., genetically modified, M. extorquens AM1Acel (based on strain DSM 1338) strains that contain and express codon-optimized glyoxylate reductase genes. Examples

[0244] Example 1: Production of genetically modified Methylobacteriaceae cells

[0245] Using bioinformatics methods, 12 different exogenous glyoxylate reductases were identified using the KEGG database (www.genome.jp / kegg / ) and the Brenda Enzymes database (https: / / www.brenda-enzymes.org / ), and the corresponding DNA and amino acid sequences were extracted. Only glyoxylate reductases found 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 summarized 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): pp. 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).

[0246] Table 2: Summary of the enzymes tested

[0247]

[0248] The heterologous enzymes from Pseudomonas fluorescens PfO-l, 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-optimized form for Methylobacteriaceae (BioCat GmbH, Heidelberg, Germany, Table 1) to support optimal 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" via PCR (Kozak, M., Gene, 1999, 234(2): pp. 187-208). Subsequently, both gene variants of SEQ ID Nos. 1 and 3 were tested. This results in 14 variants of the tested glyoxylate reductases.

[0249] The synthetic genes were cloned in codon-optimized form using Gibson assembly onto the episomal expression vector pTE1887 (Carrillo, M. et al., ACS Synthetic Biology, 2019, 8(11): pp. 2451-2456) under the control of the PL / O4 / AI promoter (IPTG-inducible) (Figure 8). Figure 8 shows the vector with the following elements: lacl gene, lacl promoter, PL / O4 / A1 promoter -33 region -10 region transcription start^PL / O4 / AI promoter ribosomal binding site (RBS), lambda TO terminator, kanamycin resistance, mobilization genes mobS and mobL, regulatory protein RepA, origin of replication colEl.

[0250] For this cloning, the expression vector was cleaved with the restriction enzyme NcoI. The sequence identity and correctness of the constructs were ensured by sequencing. Subsequently, the prepared constructs and a wild-type strain of Methylobacteriaceae cells, in particular M. extorquens TK 0001 cells and in particular M. extorquens PA1, were provided according to process step a). Following this, each of them was transformed into the Methylobacteriaceae cells using electroporation according to process step b), and a genetically modified Methylobacteriaceae cell was obtained according to process step c). Clones of the Methylobacteriaceae cells, i.e., genetically modified Methylobacteriaceae cells carrying the individually prepared constructs containing the synthetic genes in codon-optimized form, were selected on minimal medium agar plates using kanamycin as a selection marker.Colony PCR was used to verify the presence of the expression vectors and the expected sequence size of the PCR product representing the cloned gene in the resulting individual clones. The verified strains were preserved as cryocultures at -80°C.

[0251] In order to test the ability of the genetically modified Methylobacteriaceae strains to produce glycolic acid, the strains were provided with a minimal medium as the reaction medium, in particular also referred to as the culture medium, and a Cx compound with x=1, namely methanol (Cui, L.-Y. et al., Biochemical Engineering Journal, 2017, 119: pp. 67-73) as the starting material (inventive process step x)), cultivated in baffled shake flasks (250 mL flask volume, 50 mL culture volume) at 30 °C, 150 RPM (revolutions per minute) and a water vapor-saturated atmosphere (inventive process step y)) (New Brunswick™ Innova 44, Eppendorf AG, Hamburg, Germany) and a product containing glycolic acid was obtained in the reaction medium (process step z)). Analogously, cultivation is also possible with formic acid, which can also be used as a starting material for glycolic acid production.

[0252] The main cultures were inoculated from precultures grown under identical conditions (final ODeoo between 3 and 5) to a starting ODeoo of 0.05. Once the cultures had reached an ODeoo of 1.0, gene expression of the codon-optimized glyoxylate reductase genes was induced with 1 mM IPTG (final concentration in the culture volume). To detect glycolic acid production, a 1 mL sample volume of the minimal medium was taken from the culture volume before inoculation, and a 1 mL sample volume each from all cultures was taken from the culture volume before induction, immediately after induction, and approximately 20 hours after induction. After separating the biomass from the reaction medium by centrifugation, the samples were analyzed using high-performance liquid chromatography (HPLC) and refractory index detection (RID) for the concentrations of methanol, formic acid, glyoxylate, glycolic acid, and lactic acid.The HPLC measurement was carried out to separate the analytes using a Synergi™ 4 pm Hydro-RP 80A, LC column 250 x 4.6 mm (Phenomenex Inc., Torrance, CA, USA) and 20 mM K2HPO4 (pH 1.5) as eluent at 30 °C and 0.5 mL min'. 1 Flow rate for 20 minutes per sample. Identification and quantification of the analytes were performed using external standards of known concentration. The unequivocal detection of glycolic acid in the culture samples was achieved by gas chromatography coupled with mass spectrometry (GC-MS) using a glycolic acid standard (100 mg L' 1). For this purpose, the -OH or -NH groups contained in the culture samples and the standard were converted into the corresponding tert-butyldimethylsilyl ethers (TBDMS) by derivatization. For this purpose, a volume of 50 pL of standard or 50 pL of sample was freeze-dried by lyophilization and then resuspended in 50 pL DMF + 0.1% (v / v) pyridine. Derivatization was carried out with 50 pL of N-methyl-N-tert-butyldimethylsilyltrifluoroacetamide (MBDSTFA, Macherey-Nagel) and incubation at 80 °C for 30 minutes. Precipitations formed were removed by centrifugation, and the samples were subsequently analyzed by GC-MS. The GC method was specified with a carrier gas flow of 1.7 mL min' 1 , an inlet temperature of 250 °C, an interface temperature of 230 °C, and a quadrupole temperature of 150 °C. Separation of the analytes was achieved by a temperature gradient: 120 °C (2 min), ramp 8 °C min' 1 up to 200 °C and 10 °C min' 1up to 325 °C. The analytes were qualified using the MS in scan mode (m / z 50 to 750).

[0253] Genetically modified Methylobacteriaceae cells comprising an exogenous codon-optimized nucleic acid sequence (SEQ ID No. 3) of the strain Methylorubrum extorquens Mea-GAl encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MGI 655 were deposited on June 10, 2022, with the DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany, under the accession number DSM 34286.

[0254] Example 2: Screening of functional glyoxylate reductases in M. extorquens TK 0001

[0255] The glyoxylate reductase-encoding nucleic acid sequences listed in Table 2 in codon-optimized form were cloned into the pTE1887 expression vector as described in Example 1, and the corresponding genetically modified Methylobacteriaceae strains were constructed. The reference strain used was M. extorquens TK 0001 strain containing the pTE1887 vector, which does not carry a recombinant plasmid but carries the pTE1887 empty vector.

[0256] In a first experimental step, these initially constructed strains were tested for their ability to produce glycolic acid, as described in Example 1. The results are summarized in Figure 1.

[0257] Figure 1A shows a bar chart where the x-axis represents the genetically modified Methylobacteriaceae cells and the y-axis represents the concentration of glycolic acid (black filled bar) in g L' 1in the reaction medium. All sampling times were 22 to 24 hours after induction of gene expression with 1 mM IPTG. To determine the amount of methanol absorbed, the reaction medium was measured at time t = 0 h. All concentrations are in g L' 1 determined by HPLC, refractory index detection and external standards.

[0258] Figure 1A shows the screening result for glycolic acid production in recombinant M. extorquens TK 0001 strains expressing glyoxylate reductases, starting from the corresponding codon-optimized genes. 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. extorquens TK 0001 + pTE1887 (first entry from the left on the x-axis).

[0259] Surprisingly, both the reference strain M. extorquens TK 0001 + pTE1887 (first entry from the left) and the genetically modified Methylobacteriaceae cells showed no glycolic acid production (entries from 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 eC o (in codon-optimized nucleic acid form according to SEQ ID No. 3), i.e., 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: 4, the only entry showing a black bar). Figure 9 shows the map of the vector used to generate these Methylorubrum cells with the following elements: lacl gene, lacl promoter, PL / O4 / A1 promoter -33 Region -10 Region transcription start, PL / O4 / A1 promoter Ribosomal binding site (RBS), ghrA eC oc-optimized, lambda T0 terminator, kanamycin resistance, mobilization genes mobS and mob, regulatory protein RepA. Origin of replication colEl.

[0260] Figure IB shows a bar chart where the x-axis represents the genetically modified Methylobacteriaceae cells and the y-axis represents the enzyme activity in mU mg' 1 (white, open bar: NADH as cofactor).

[0261] Figure IC shows a bar chart where the x-axis represents the genetically modified Methylobacteriaceae cells and the y-axis represents the enzyme activity in mU mg' 1 (grey filled bar: NADPH as cofactor).

[0262] Figures 1B and 1C show the screening result of an enzyme assay using recombinant M. extorquens TK 0001 strains expressing glyoxylate reductases, starting from the corresponding codon-optimized genes. The enzyme assay was performed analogously to Example 5. The biomass used in 1A was used. In the case of 1B, the enzyme assay was performed using NADH as the redox cofactor. In the case of 1C, the enzyme assay was performed using NADPH as the redox cofactor.

[0263] 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 non-inventive Methylobacteriaceae cell containing the glyoxylate reductase ghrB eco (Entry from left: 5).

[0264] In Figure 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) show no measurable glyoxylate reductase enzyme activity with NADPH as a cofactor. Only the genetically modified Methylobacteriaceae cells (entries from the left: 3 to 7 and 10) showed increased glyoxylate reductase enzyme activity, with the genetically modified Methylobacteriaceae cells of M. extorquens TK 0001 + pTE1887-ghrA according to the invention. eC o (in codon-optimized nucleic acid form according to SEQ ID No. 3), i.e. 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, exhibited a high glyoxylate reductase enzyme activity (entry from left: 4).

[0265] Enzyme activity of glyoxylate reductase Tiit was not detectable and was not associated with glycolic acid production. Only the enzyme activity of ghrA eC o, i.e. the glyoxylate reductase from / :. coli according to the invention, is associated with glycolic acid production.

[0266] The genetically modified Methylobacteriaceae cell according to the invention comprising M. extorquens TK 0001 + pTE1887-ghrA eC oc-optimized therefore shows NADPH but not NADH dependence.

[0267] Figure 2 shows HPLC chromatograms of the culture samples according to Figure 1 (22 to 24 hours after induction) of the genetically modified Methylobacteriaceae cells M. extorquens TK 0001 glyoxylate reductase strains. It is clearly evident that only the genetically modified Methylobacteriaceae cell according to the invention, M. extorquens TK 0001 + pTE1887-ghrA eC o (referred to in Figure 2 as M. extorquens GAI, containing the codon-optimized form of ghrAeco Gens) is the only strain that produces a mixture of glycolic acid and lactic acid (glycolic acid retention time = 6.20 min and lactic acid retention time = 9.1 min) (comparison standards - lane 1 and lane 2 - with M. extorquens TK 0001 + pTE1887- ghrAeco, M. extorquens GAI - lane 7 in Figure 1A and lane 5 in Figure 1B). The lack of glycolic acid and lactic acid production when using the non-inventive glyoxylate reductases indicates a lack of functionality. These enzymes could be hydroxypyruvate reductases that reduce the hydroxypyruvate also produced in the serine cycle to D-glycerate in a NAD(P)H-dependent manner. In this case, as shown in Figure 1 and Figure 2, no accumulation of glycolic acid would be observed.

[0268] Compared to external standards, this sample was analyzed using GC-MS to confirm the presence of glycolic acid and lactic acid in the sample, thus verifying glycolic acid production and the surprising lactic acid production by expression of the ghrAeco enzyme. Additional peaks were observed: glyoxylate (retention time = 5.40 min), methanol (retention time = 7.6 min), and peaks not described here (retention time = 6.50 min and 8.00 min).

[0269] In the genetically modified Methylobacteriaceae cell according to the invention, about 0.6 g L' 1 the mixture of glycolic acid and lactic acid (

[0270] In most cultures, after approximately 22 to 24 h after induction, the initial methanol concentration of 8 g L' 1depleted. Only the strain M. extorquens GAI showed a clearly measurable methanol concentration at the time of sampling (Figure 1A lane 7 and Figure 1B lane 5). This may indicate a metabolic imbalance caused by the gene expression of glyoxylate reductase. On the one hand, enzyme expression itself can reduce the growth of the strains. But increased enzyme activity of a glyoxylate reductase can also fundamentally cause a withdrawal of glyoxylate from the serine cycle into glycolic acid. As a result, the microorganism lacks glyoxylate to build biomass. This deficiency can slow growth and lead to the carbon source not being completely used up.

[0271] To determine the presence of glycolic acid and lactic acid in the ghrA according to the invention eco Sample compared to external standards (100 mg L' each 1To confirm the presence of lactic acid and glycolic acid, this sample was analyzed by GC-MS measurement as described in Example 1 (Figure 3 to Figure 5).

[0272] Figure 3 shows a GC-MS chromatogram and mass spectra of a 100 mg L' 1 Glycolic acid standards, a sample of the medium at time t = 0 h, a sample of the M. extorquens TK 0001 + pTE1887 empty vector culture 22 - 24 h after induction and a sample according to the invention of the M. extorquens TK 0001 + pTE1887-ghrA eC o (referred to in Figure 3 as M. extorquens GAI, containing the codon-optimized form of ghrA eco Gens) cultivation 22 to 24 hours after induction. Figure 4 shows the same samples, except for the standard, which was cultured against a 100 mg L' 1 Lactic acid standard was exchanged. The measurements show that, compared with the glycolic acid standard (retention time = 7.22 min), in the culture sample according to the invention of M. extorquens TK 0001 + pTE1887- ghrAeC o (M. extorquens GAI) clearly produced glycolic acid. The mass spectrum of the peak obtained in the inventive M. extorquens TK 0001 + pTE1887-ghrA eC The o-sample clearly agrees with the mass spectrum of the glycolic acid standard (Figure 5A). This confirms the presence of glycolic acid in the M. extorquens TK 0001 + pTE1887-ghrA strain according to the invention. eC o sample, thus proving the production of glycolic acid by this strain. In comparison, no glycolic acid could 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 observed exclusively in the sample of the inventive M. extorquens TK 0001 + pTE1887-ghrA. eC o Cultivation. Here, too, the mass spectrum agrees with that of the lactic acid standard (Figure 5B)

[0273] This procedure clearly demonstrated that glycolic acid was produced by the genetically modified Methylorubrum cell M. extorquens TK 0001 + pTE1887-ghrA eC o was produced. Surprisingly, it was also shown that this strain produces a mixture of glycolic acid and lactic acid (see Figure 5). Figure 5 shows detailed images of the mass spectra in comparison from an identical sample of the inventive M. extorquens TK 0001 + pTE1887-ghrA. eC o (referred to in Figure 5 as M. extorquens GAI, containing the codon-optimized form of ghrA eco Gens) Cultivation 22 to 24 h after induction with database detection of glycolic acid identity in the M. extorquens GAI sample (A) and lactic acid identity in the M. extorquens GAI sample (B).

[0274] Surprisingly, GC-MS demonstrated the production of both glycolic acid (retention time = 7.22 min) and lactic acid (retention time = 6.88 min) in the cultivation of M. extorquens TK 0001 + pTE1887-ghrAeco (Figure 3 to Figure 5). The peak with a retention time of 6.88 min in this sample was identified with a probability of 89-91% as lactic acid 2xTBDMS (lactic acid derivative with MBDSTFA) by comparison with an external standard and database comparison of the mass spectrum (Figure 5B).

[0275] The control strain M. extorquens TK 0001 + pTE1887 did not exhibit this phenotype: Neither glycolic acid nor lactic acid could be detected as products by GC-MS. Without wishing 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 according to the invention. eCo so that lactic acid is synthesized as a possible byproduct of glycolic acid production. An NADH-dependent lactate dehydrogenase (KEGG database: Mex_lp4794), which uses pyruvate as a substrate, could be responsible for this lactic acid formation. An alternative possibility is that glyoxylate reductase exhibits nonspecific substrate utilization, allowing the enzyme to use pyruvate as an acceptor. The methylglyoxal pathway is also conceivable.

[0276] It can therefore be shown that the M. extorquens TK 0001 + pTE1887- ghrAeco cells according to the invention, containing the codon-optimized form of ghrA eco Genes, produce a mixture of glycolic acid and lactic acid, which can serve as a starting point for polymerization to polyglycolic acid, polylactic acid or polylactide-co-glycolide.

[0277] Example 3: Growth experiments

[0278] 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 eC o (M. extorquens GAI) in minimal medium (reaction medium) with 10 g L' 1 Methanol as starting material and a mixture of 10 g L' 1 Methanol + 1.5 g L' 1 Glyoxylate was used as a further reactant (Figure 6).

[0279] Figure 6 A to D show diagrams in which the y-axes show the growth curve (ODeoo, circles, filled in black), the pH value (triangles, vertex at the bottom) and the methanol (squares, unfilled), glyoxylate (diamonds, unfilled) and glycolic acid concentrations (diamonds, filled in dark grey) as well as lactic acid concentrations (triangles, filled in grey, vertex at the top) of M. extorquens TK 0001 + pTE1887 (A+C) and inventive M. extorquens TK 0001 + pTE1887-ghrA eCo (codon-optimized) (B+D) in minimal medium and time on the x-axis. As carbon source (educt), i.e. Cx compound, 10 g L' 1 Methanol (A+B) or 10 g L' 1 Methanol + 1.5 g L' 1 Glyoxylate (C+D) was added. The methanol, glyoxylate, and glycolic acid concentrations were measured using HPLC, refractory index detection, and external standards. All concentrations are expressed in g L' 1 The data represent three independent biological replicates.

[0280] The glyoxylate was added at the time of induction of gene expression and serves as a test to determine whether an in vivo increase in glyoxylate supply leads to an increase in glycolic acid production. Figure 6A shows that the reference strain M. extorquens TK 0001 + pTE1887 with 10 g L' 1Using methanol as a starting material, no glycolic acid was produced and consistent biomass formation occurred, reaching a maximum ODeoo of approximately 9 after 40 h of cultivation. The significant reduction in pH to below 6.5 during the course of fermentation was 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 ODeoo of approximately 10 after approximately 42 h. In this case, too, no glycolic acid was produced (Figure 6C). However, the pH in this cultivation could be maintained at the initial pH of approximately 7.0, which is likely due to the glyoxylate feeding.

[0281] It was shown that the recombinant strain M. extorquens TK 0001 + pTE1887-ghrA according to the invention eC o, containing the codon-optimized form of ghrA eco Gens, from 10 g L' 1Methanol the products glycolic acid and lactic acid in increased concentrations (-0.35 g L' 1 respectively 0.25 g L' 1 in 40 h). After the methanol has been degraded, the products are completely degraded again during the further course of cultivation. The formation of glycolic acid and lactic acid is accompanied by a significant slowing of biomass growth to a maximum ODeoo of 6.7 in 44 h. Furthermore, the pH of the culture broth presumably decreases in this case to as low as 6.2 due to the additional glycolic acid formation and increases due to the degradation of glycolic acid to a value comparable to that of the reference strain, at just under 6.5 (Figure 6B).

[0282] In the experiment with the inventive M. extorquens TK 0001 + pTE1887-ghrA eC o, containing the codon-optimized form of ghrA eco Gens, and feeding glyoxylate resulted in a significant increase in glycolic acid production up to 1.0 g L' 1in 44 h. The amount of lactic acid produced was comparable to the culture without glyoxylate supplementation (Figure 6B). This demonstrated that glyoxylate plays an important role as a precursor to glycolic acid formation, and that increasing the in vivo concentration of glyoxylate results in improved glycolic acid production. In this experiment, the glycolic and lactic acid produced were also metabolized after the methanol had been consumed. The increased product formation in this experiment again led to a further reduction in biomass growth, with a maximum ODeoo of around 4.5 being reached. In contrast to the reference strain, a significant reduction in pH was observed in this case despite the addition of glyoxylate, as glycolic and lactic acid were produced.Analogously, the increase in pH during the degradation of the formed glycolic acid and lactic acid can also be observed after the methanol has been consumed (Figure 6D). In summary, the strain-specific cultivation parameters derived from the data, such as p (specific growth rate), Yx / s (dry biomass-substrate yield), qs (specific substrate uptake rate), Yp / s (product-substrate yield), and qp (specific product formation rate), are summarized in Table 3 for the strains M. extorquens TK 0001 + pTE1887 and the inventive M. extorquens TK 0001 + pTE1887-ghrA. eco , containing the codon-optimized form of ghrA ecoThese data suggest that glycolic and lactic acid production is associated with a significant reduction in dry biomass substrate yield (70% of the reference strain and 70% of the glyoxylate-fed reference strain), and that more carbon is converted into the product or must be used to maintain the redox balance. It is also evident that glyoxylate reduces the growth rate, suggesting a potential toxic effect of the precursor. This glyoxylate toxicity can be avoided by optimally balancing the in vivo glyoxylate pool.

[0283] Table 1. Summary of cultivation parameters of M. extorquens TK 0001 + pTE1887 and the inventive AT. extorquens TK 0001 + pTE1887-ghrA eC o (M. extorquens GAI), containing the codon-optimized form of the ghrAeco gene, in minimal medium with 10 g L' 1 Methanol or additionally + 1.5 g L -1Glyoxylates. Abbreviations: p, specific growth rate; MeOH, methanol; GS, glycolic acid; BTM, dry biomass.

[0284]

[0285] 1 Yields are estimated by the cumulative substrate utilization of methanol and glyoxylate.

[0286] Examples 1 to 3 show that glycolic acid and lactic acid can be produced according to the invention with M. extorquens GAI from Cx compounds in a methylotrophic fermentation process.

[0287] It is particularly noteworthy that the glycolic acid production according to the invention in M. extorquens GAI can be significantly enhanced by increasing the intracellular concentration of glyoxylate, as shown in Example 3. In this case, 185% more glycolic acid was produced compared to the culture without glyoxylate feeding.

[0288] Example 4: Experimental data of the fermentative glycolic acid-lactic acid production from methanol

[0289] The experimental procedure was carried out according to Example 1. The strain used was the wild-type strain Methylorubrum extorquens TK 0001 DSM 1337.

[0290] The following expression vectors (1 to 4) were used:

[0291] 1.) pTE1887 (expression vector, also called empty vector; plasmid map: Figure 8)

[0292] 2.) pTE1887-ghrA eC o (Expression vector encoding the glyoxylate reductase from Escherichia coli K-12 MG1655 in a codon-optimized manner; with SEQ ID No. 3, plasmid map: Figure 9) (according to the invention) 3.) pTE1887-ghrAeco-ecm m ea (expression vector natively encoding glyoxylate reductase from Escherichia coli K-12 MGI 655 (codon-optimized) and ethylmalonyl-CoA mutase from M. extorquens TK 0001 DSM 1337; plasmid map: Figure 10) (according to the invention)

[0293] 4.) pTE1887- ghrAeco-eemrsh (expression vector expressing the glyoxylate reductase from Escherichia coli K-12 MGI 655 (codon-optimized) and the ethylmalonyl-CoA mutase from Rhodobacter sphaeroides ATCC 17029 (plasmid map: Figure 11) (according to the invention).

[0294] Genetically modified Methylobacteriaceae cells were produced using the methods described in Example 1.

[0295] Figure 10 shows the map of the vector used to generate Methylobacteriaceae cells expressing the ghrA e co-ecm m ea was used with the following elements: lacl gene, lacl promoter, PL / O4 / A1 promoter -33 Region -10 Region transcription start, PL / O4 / A1 promoter Ribosomal binding site (RBS), ghrA eco (codon-optimized), ecm me a (native), Lambda T0 terminator, kanamycin resistance, mobilization genes mobS and mob, regulatory protein RepA. Origin of replication colEl.

[0296] Figure 11 shows the map of the vector used to generate these Methylobacteriaceae cells expressing the ghrA eC o-ecm r sh was used with the following elements: lacl gene, lacl promoter, PL / O4 / A1 promoter -33 Region -10 Region Transcription start, PL / O4 / A1 promoter Ribosomal binding site (RBS), ghrA eco (codon-optimized), rsh-ecm (codon-optimized), lambda T0 terminator, kanamycin resistance, mobilization genes mobS and mob, regulatory protein RepA. Origin of replication colEl.

[0297] Genetically modified Methylorubrum extorquens TK 0001 cells comprising an exogenous codon-optimized nucleic acid sequence (SEQ ID No. 3) encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MGI 655 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 June 10, 2022, at the DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany under the accession number DSM 34287.

[0298] Genetically modified Methylorubrum extorquens TK 0001 cells comprising an exogenous codon-optimized nucleic acid sequence (SEQ ID No. 3) encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MGI 655 and an exogenous codon-optimized 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 June 10, 2022, at the DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany under the accession number DSM 34288.

[0299] Fermentation experiments were carried out using culture medium as reaction medium and methanol (reactant) as the sole carbon source.

[0300] Figure 7 shows the temporal progression of biomass concentration (ODeoo) and medium pH over the course of cultivation. At the same time, culture supernatant samples were analyzed using high-performance chromatography to visualize the substrate and product concentrations and their changes over time.

[0301] Figure 7 shows on the x-axis the time in hours and on the y-axes the growth curve (ODeoo, circles, filled in black), pH value (triangles, apex bottom), methanol (squares, unfilled) and glycolic acid (diamonds, filled in dark grey) as well as lactic acid concentrations (triangles, filled in grey, apex top) of M. extorquens TK 0001 + pTE1887 (A), inventive M. extorquens TK 0001 + pTE1887- ghrAeco (codon-optimised) (M. extorquens GAI) (B), inventive M. extorquens TK 0001 + pTE1887-ghrAeco-ecm me a (ghrAeco: codon-optimized; ecm mea: native) (M. extorquens GA2) (C) and inventive M. extorquens TK 0001 + pTE1887-ghrA eC o-ecm r sh (both genes codon-optimized) (M. extorquens GA3) (D) in culture medium with 10 g L' 1 Methanol as the sole reactant, i.e. as a Cx compound.

[0302] It was again demonstrated that the enzyme ghrAeco allows the production of glycolic acid in the M. extorquens TK 0001 strain background. Lactic acid is also produced in this strain. In comparison, the wild-type strain, which contains the empty vector pTE1887, shows no production of glycolic acid or lactic acid. In these experiments, approximately 250 mg L' 1 Glycolic acid and approx. 200 mg L' 1 Lactic acid produced with a product substrate yield of around 50 mg gMethanoi' 1(Glycolic acid + lactic acid). It can be observed that the initiated glycolic acid synthesis reduces the dry biomass substrate yield (Yx / s) of the inventive strain M. extorquens TK 0001 + pTE1887-ghrAeco (M. extorquens GAI) to 70% compared to the empty vector strain. The product yield based on dry biomass (Yp / x) is 0.27 g g dry biomass (Table 4). Surprisingly, the additional implementation of the exogenous ethylmalonyl-CoA mutases leads to a significant improvement in glycolic acid production performance compared to the inventive strain M. extorquens TK 0001 + pTE1887-ghrA. eC o. The growth of the inventive strain M. extorquens TK 0001 + pTE1887-ghrA eC o- emmea was with a measured growth rate (p) of 0.10 h' 1 delayed compared to the empty vector strain (0.17 h' 1The use of the ecm gene from M. extorquens TK 0001 DSM 1337 led to an increase in 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 eC o (0.26 g L' 1 versus 0.22 g L' 1 , Table 4). The dry biomass substrate yield is also 49% higher compared to the empty vector strain and compared to the strain according to the invention M. extorquens TK 0001 + pTE1887-ghrA eC reduced by 27%.

[0303] A slight change in the product yield relative to the dry biomass (Yp / x) is observed. Compared to the inventive strain M. extorquens TK 0001 + pTE1887-ghrA eC o an increase of this yield by 11% was achieved (Table 4).

[0304] The use of exogenous codon-optimized ecm rs h gene resulted in comparison to the strain according to the invention comprising the exogenous ecm mea-gene, to the surprising deviations in the cultivation parameters shown in Table 4. Here, the highest measured lactic acid titer of 0.37 g L' 1 A striking change concerns the dry biomass substrate yield (Yx / s), which, compared to the strain according to the invention M. extorquens TK 0001 + pTE1887-ghrA e co-ecm m ea is increased by 26% (0.24 g dry biomass)•

[0305] It was demonstrated that glycolic acid production from methanol is possible. The respective 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. eco without an exogenous ethylmalonyl-CoA mutase. In particular, the use of the ethylmalonyl-CoA mutase ecm rsh surprisingly leads to a significantly increased and more selective lactic acid production.

[0306] Table 4: Summary of cultivation parameters of M. extorquens TK 0001 + pTE1887 and inventive M. extorquens TK 0001 + pTE1887-ghrA eC o (codon-optimized) (M. extorquens GAI), inventive M. extorquens TK 0001 + pTE1887- ghrAeco-ecnimea (ghrA eco : codon-optimized; ecm me a: native) (M. extorquens GA2) and inventive M. extorquens TK 0001 + pTE1887-ghrA eC o-ecm r sh (both genes codon-optimized) (M. extorquens GA3) in culture medium with 10 g L' 1 Methanol. Abbreviations: p, specific growth rate; MeOH, methanol; GS, glycolic acid; BTM, dry biomass.

[0307] Example 5: Experimental data for the detection of the enzyme activity of the glyoxylate reductase (ghrA) expressed according to the invention ec0) and a reference enzyme, namely an E. coli hydroxypyruvate reductase (ghrB ec0 ):

[0308] To provide experimental evidence of the presence of the enzyme activity of the expressed glyoxylate reductase ghrA eco and the hydroxypyruvate reductase ghrB eco (Nunez, MF, MT 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) Enzyme assays were performed in the Methylorubrum extorquens TK 0001 strain background. The native form of the DNA sequences (as found in Escherichia coli K-12 MG1655) as well as the synthetic DNA sequences codon-optimized for expression in Methylobacteriaceae (c-optimized) were tested to evaluate the influence of codon optimization on gene expression and the resulting enzyme activity.

[0309] The implementation procedure and the results are summarized below.

[0310] To obtain sufficient biomass of the genetically modified M. extorquens TK 0001 strains containing pTE1887-ghrA eC oc-optimized (SEQ ID No. 3), pTE1887-ghrB eC oc-optimized, pTE1887-ghrA eC o-native (SEQ ID No. 1) and pTE1887-ghrB eC To obtain native cells for cell disruption, the following culture protocol was used. The strain M. extorquens TK 0001 + pTE1887, containing the empty vector, was included as a negative control. All strains were cultured, harvested, and disrupted as three independent biological replicates.

[0311] The strains were cultured 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 a water vapor-saturated atmosphere (New Brunswick™ Innova 44, Eppendorf AG, Hamburg, Germany). Subsequently, a second preculture was inoculated from the overgrown 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 inoculation corresponded to an optical density at 600 nm (ODeoo) of 0.1. The subsequent cultivation was carried out at 30 °C, 150 rpm, and a water vapor-saturated atmosphere.The next day, Tuesday, the main cultures were inoculated with the second precultures (50 mL minimal medium with methanol in a 250 mL baffled shake flask, initial OD = 0.05) and incubated at 30 °C, 150 rpm, and under a water vapor-saturated atmosphere. After the cultures had reached an OD of 0.9–1.0, glyoxylate reductase gene expression was induced with 1 mM IPTG (final concentration in the culture volume). The biomass was then grown to a final OD of approximately 4–7.

[0312] For the actual biomass harvest, 50 mL conical centrifuge tubes were empty, weighed, filled with the 50 mL of grown main culture, and finally centrifuged at 4,200 rpm for 15 min at 4 °C. After the centrifugation step, the supernatant was discarded, and the resulting biomass pellets were washed with 20 mL of 50 mM Tris-HCl (pH 7.5) buffer each. This was followed by further centrifugation under the previous conditions, followed by careful removal of the supernatant with a pipette. The resulting biomass pellets were weighed and resuspended in 50 mM MOPS buffer (pH 6.6). For this purpose, a buffer volume of 7 mL was used per 1 g of wet pellet.

[0313] Cell disruption to obtain crude protein extracts containing the expressed glyoxylate or hydroxypyruvate reductases was performed in 2.0 mL reaction vessels. 1.5 mL of the cell suspension was transferred to each reaction vessel and then disrupted six times in an ice-water bath for 30 seconds each at an amplitude of 60 using ultrasound. Between each of the six disruption cycles, the samples were chilled on ice for 1 minute. Finally, to obtain the crude protein extract, a centrifugation step at 21,500 rpm for 15 minutes at 4 °C followed. The resulting protein-containing supernatant was transferred to 1.5 mL reaction vessels. To ensure comparability of the enzyme assay results, the protein concentration of the respective crude protein extracts was determined using a NanoDrop™.The crude extract with the lowest measured concentration was used as the target concentration for diluting the other crude 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 pre-diluted crude protein extracts were diluted once more (1:5) with 50 mM MOPS buffer (pH 6.6) and then used in the enzyme assay.

[0314] The enzyme assay was performed in 96-well microtiter plates. For this purpose, 160 μL of the diluted crude protein extracts were spiked with 20 μL of 50 mM glyoxylate as substrate and 20 μL of 2 mM cofactor stock solution (NADH or NADPH, final concentration in the assay 0.2 mM). The experimental setups were carried out in three technical replicates. Enzyme activity was measured as the change in NADH absorbance at 340 nm at 37 °C for up to 30 min. For evaluation, the maximum change in absorbance over time in the linear range of the reaction was determined and multiplied by a dilution factor of five before calculating the enzyme activity in U mL. 1 .

[0315] The enzyme activity was calculated using Equation 6 and the given coefficients. (Equation 6) With enzyme activity: Measured in mol substrate min.' 1, Vprotein crude extract assay: Volume of crude protein extract used in the assay (0.00016 L), S: Change in absorbance at 340 nm over time in the linear range of the reaction corrected for the dilution factor of five (Abs.34o Min.' 1 ), VAssa y : total volume of the assay (0.0002 L), s : extinction coefficient of NADH / NADPH at 340 nm (6220 L mol' 1 cm' 1 ), d: layer thickness of the absorbing reaction mixture (0.53 cm).

[0316] For a conversion of enzyme activity from mol substrate min' 1 into the conventional unit for enzyme activity mU mL' 1 (1 U = 1 pmol substrate min' 1 ) the calculated result is multiplied by a factor of 10 6 multiplied.

[0317] The obtained enzyme activities were assigned to the respective expression strains and the used cofactors NADH or NADPH for a graphical comparison.

[0318] The collected data for the enzyme activities of ghrA eC oc-optimized, ghrB eC oc-optimized, ghrAeco-native, ghrB eC o-native and the negative control (pTE1887-empty vector) are summarized in Figure 12. The measurements and the standard deviation shown are based on three biological replicates, each with three technical replicates of the assay.

[0319] As expected, the empty vector shows only a slight background activity. This was subtracted from all other measured values ​​to correct for the background reaction.

[0320] The enzyme activities shown in Figures 12A and 12B with regard to the conversion of glyoxylate to glycolic acid show that only the two enzymes used according to the invention, ghrA eC oc-optimized and ghrA eC o-native (also ghrA eco) show sufficient activity, especially for large-scale production. Furthermore, significant differences in enzyme activity can be detected depending on the cofactor used. The assay shows a clear cofactor dependence of ghrA eco and ghrBeco. Using NADH as a cofactor (Figure 2 A), the highest enzyme activity is achieved with ghrB eC oc-optimized achieved (10.53 ± 1.50 mU mL' 1 ). The enzyme activity caused by the gene ghrA eC oc-optimized, is 0.49 ± 2.34 mU mL' 1 significantly reduced compared to ghrBeco-c-optimized. A reduction in enzyme activity of approximately 95% was measured. The enzyme activity of the NADH assays with the native genes is in a similar range: 4.61 ± 1.61 mU mL' 1 versus 2.45 ± 0.67 mU mL' 1 for ghrA eC o-native and ghrBeco-native. Codon optimization of ghrB ecoled to an increase in activity of 329%. In summary, a clear dependence of ghrB eco Enzyme recognizes NADH as a cofactor.

[0321] In contrast, a different picture emerged when NADPH was used as a cofactor (Figure 12 B).

[0322] Here you can use ghrA eC oc-optimized and ghrAe C0 -native (33.86 ± 1.29 mU mL' 1 and 21.76 ± 1.49 mU mL' 1 ) the highest enzyme activities measured in the present tests were achieved. The increase through codon optimization is 55% (21.76 ± 1.49 versus 33.86 ± 1.29 mU mL' 1 ). It can also be clearly demonstrated that the ghrA eco Enzyme is NADPH dependent. This is underlined by the low measured activity of ghrB eco In this case, both the use of the codon-optimized variant of the gene (ghrB eCoc-optimized) and the native variant of the gene (ghrBeco-native) only showed an enzyme activity of 3.17 ± 0.29 and 0.81 ± 0.30 mU mL', respectively. 1 measured (Figure 12 B), which shows that ghrBeco is clearly distinguishable 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 to glycolic acid.

[0323] The increased enzyme activity with NADPH as a cofactor triggered by the expression of ghrAeco-c-optimized shows that glycolic acid production by M. extorquens is enabled by the expression of this enzyme. The significantly reduced enzyme activity with both NADPH and NADH, which was associated with ghrB eC oc-optimized, is not sufficient to enable glycolic acid production in M. extorquens in vivo.

[0324] Those with M. extorquens TK 0001 + pTE1887-ghrA eCThe observed glycolic acid production in oc-optimized cultures appears to be dependent on the availability of NADPH as a cofactor. These results confirm the results from Example 2.

[0325] Surprisingly, the introduction of the DNA sequence of ghrA eC o enzyme, especially the codon-optimized DNA sequence, leads to glycolic acid production as well as to a surprising production of lactic acid.

[0326] Example 6:

[0327] Expression of an exogenous nucleic acid sequence encoding a glyoxylate reductase from the bacterium Escherichia in cells of other Methylobacteriaceae (invention) and other microorganisms (comparison). Further genera of the Methylobacteriaceae (Alphaproteobacteria) family were genetically modified according to Example 1. Figure 13 shows examples of the microorganisms investigated. In particular, 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 AMI DSM 1338 with a deletion of a cellulase gene (M. extorquens AM1Acel: https: / / doi.org / 10.1371 / journal.pone.0062957), and Methylobacterium cells, in particular M. organophilum DSM 18172, in particular M. radiotolerans DSM 760, were investigated as representative of the Methylobacterium species.

[0328] Additionally, Methylomonas methanica DSM 25384 (Gammaproteobacteria), Methylophilus methylotrophus DSM 6330 (Betaproteobacteria), and Bacillus methanolicus DSM 16454 (Firmicutes) were investigated as negative examples not belonging to the Methylophilus acteriaceae family. These microorganisms are also capable of metabolizing methanol and were tested for glycolic acid and / or lactic acid production according to the invention.

[0329] The following expression vectors (1 to 4) were used:

[0330] 1.) pTE1887 (expression vector, also called empty vector; plasmid map: Figure 8)

[0331] 2.) pTE1887-ghrA eC o (Expression vector encoding the glyoxylate reductase from Escherichia coli K-12 MG1655 in a codon-optimized manner; with SEQ ID No. 3, plasmid map: Figure 9) (according to the invention)

[0332] 3.) pTE1887-ghrAeco-ecm mea (expression vector natively encoding the glyoxylate reductase from Escherichia coli K-12 MGI 655 (codon-optimized) and the ethylmalonyl-CoA mutase from M. extorquens TK 0001 DSM 1337; plasmid map: Figure 10) (according to the invention)

[0333] 4.) pTE1887-ghrAeco-ecm r sh (expression vector which codon-optimizes the glyoxylate reductase from Escherichia coli K-12 MGI 655 and the ethylmalonyl-CoA mutase from Rhodobacter sphaeroides ATCC 17029; plasmid map: Figure 11) (according to the invention).

[0334] Genetically modified Methylobacteriaceae cells comprising an exogenous codon-optimized nucleic acid sequence (SEQ ID No. 3) encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MGI 655 of the strain Methylorubrum zatmanii Mza-GA14 (M. zatmanii DSM 5688 + pTE1887-ghrA eCo) were deposited on 19 July 2023 with the DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany under the deposit number DSM 34701.

[0335] Genetically modified Methylobacteriaceae cells comprising an exogenous codon-optimized nucleic acid sequence (SEQ ID No. 3) encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MGI 655 of the strain Methylorubrum extorquens Mea-GA17 (M. extorquens PA1 DSM 23939 + pTE1887-ghrA eC o) were deposited on 19 July 2023 with the DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany under the deposit number DSM 34702.

[0336] Genetically modified Methylobacteriaceae cells comprising an exogenous codon-optimized nucleic acid sequence (SEQ ID No. 3) encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MGI 655 of the strain Methylorubrum rhodesianum Mrh-GA4 (M. rhodesianum DSM 5687 + pTE1887-ghrA eC o) were deposited on 19 July 2023 with the DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany under the deposit number DSM 34697.

[0337] Genetically modified Methylobacteriaceae cells comprising an exogenous codon-optimized nucleic acid sequence (SEQ ID No. 3) encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MGI 655 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 DSM 5687 + pTE1887-ghrA e co-ecm mea) were deposited on 19 July 2023 with the DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany under the deposit number DSM 34698.

[0338] Genetically modified Methylobacteriaceae cells comprising an exogenous codon-optimized nucleic acid sequence (SEQ ID No. 3) encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MGI 655 of the strain Methylobacterium organophilum Mor-GA8 (M. organophilum DSM 18172 + pTE1887-ghrA e co-ecm m ea) were deposited on 19 July 2023 at the DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany under the deposit number DSM 34699.

[0339] Genetically modified Methylobacteriaceae cells comprising an exogenous codon-optimized nucleic acid sequence (SEQ ID No. 3) encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MGI 655 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 radiotolerans Mra-GA12 (M. radiotolerans DSM 760 + pTE1887-ghrA e co-ecm m ea) were deposited on 19 July 2023 with the DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany under the deposit number DSM 34700.

[0340] In order to investigate the invention with the aforementioned strains, the procedure was as in Example 2. In contrast to Example 2, the cultivations with the Methylobacteriaceae cells used were M. rhodesianum (Figure 14) DSM 5687, M. zatmanii DSM 5688 (Figure 15), M. radiotolerans DSM 760 (Figure 16), M. organophilum DSM 18172 (Figure 17), M. extorquens PA1 DSM 23939 (Figure 18) with a reduced amount of starting material (Cx compound, 4 g L' 1 methanol) and between ten and twelve hours after induction, additional reactant was added (fed-batch, cumulative up to 15 g L' 1 ). In addition, samples for determination of glycolic acid, lactic acid, and methanol concentrations were taken 22–28 h after induction of gene expression with 1 mM IPTG.

[0341] Figures 14 to 19 show the genetically modified Methylobacteriaceae cells on the x-axis and the concentration of methanol (white, open bar) or the concentration of the mixture of glycolic acid and lactic acid formed (black, filled bar) in g L' on the y-axis. 1 in the reaction medium. All sampling times were 22 to 28 hours after induction of gene expression with 1 mM IPTG. All concentrations are in g L' 1 determined by HPLC, refractory index detection and external standards.

[0342] Figure 14 shows the screening results for glycolic acid and lactic acid production using recombinant M. rhodesianum DSM 5687 strains expressing glyoxylate reductases, starting from the corresponding codon-optimized genes. The first panel from the left shows the methanol concentration in the minimal medium at the start 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 panel from the left on the x-axis).

[0343] 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 and lactic acid production (entries from the left: 3 to 10 and 12 to 16), with the exception (black, filled bars in Figure 14) of the genetically modified cells of M. rhodesianum DSM 5687 + pTE1887-ghrA according to the invention. eCo (in codon-optimized nucleic acid form according to SEQ ID No. 3), i.e. 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: 11) and the genetically modified cells M. rhodesianum DSM 5687 + pTE1887-ghrA eC o- ecmmea, comprising an exogenous codon-optimized 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, i.e. 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. rhodesianum DSM 5687 + pTE1887-ghrAeC o-ecm r sh, comprising an exogenous codon-optimized nucleic acid sequence (SEQ ID No. 3) encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MGI 655 and an exogenous codon-optimized nucleic acid sequence (SEQ ID No. 8) encoding an ethylmalonyl-CoA mutase from the bacterium Rhodobacter sphaeroides ATCC 17029, i.e. 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).

[0344] Figure 9 shows the map of the vector used to generate these Methylobacteriaceae cells with the following elements: lacl gene, lacl promoter, PL / O4 / A1 promoter - 33 region -10 region transcription start, PL / O4 / A1 promoter ribosomal binding site (RBS), ghrAeco-c optimized, lambda T0 terminator, kanamycin resistance, mobilization genes mobS and mob, regulatory protein RepA. Origin of replication colEl.

[0345] Figure 10 shows the map of the vector used to generate Methylobacteriaceae cells expressing the ghrA e co-ecm m ea was used with the following elements: lacl gene, lacl promoter, PL / O4 / A1 promoter -33 Region -10 Region transcription start, PL / O4 / A1 promoter Ribosomal binding site (RBS), ghrA eco (codon-optimized), ecm me a (native), Lambda T0 terminator, kanamycin resistance, mobilization genes mobS and mob, regulatory protein RepA. Origin of replication colEl.

[0346] Figure 11 shows the map of the vector used to generate these Methylobacteriaceae cells expressing the ghrA eC o-ecm rsh was used with the following elements: lacl gene, lacl promoter, PL / O4 / A1 promoter -33 Region -10 Region Transcription start, PL / O4 / A1 promoter Ribosomal binding site (RBS), ghrA eco (codon-optimized), rsh-ecm (codon-optimized), lambda T0 terminator, kanamycin resistance, mobilization genes mobS and mob, regulatory protein RepA. Origin of replication colEl.

[0347] Mixtures of glycolic acid and lactic acid containing a total concentration of glycolic acid plus lactic acid up to 0.85 g L' were obtained with the cells of M. rhodesianum DSM 5687 genetically modified according to the invention. 1 (M. rhodesianum DSM5687 + pTE1887- ghrAeco-eemmea), at least 0.82 g L' 1 (M. rhodesianum DSM5687 + pTE1887-ghrA eC o), at least 0.09 g L' 1 (M. rhodesianum DSM5687 + pTE1887-ghrA eC o-ecm r sh) are produced.

[0348] These experimental data demonstrate that glycolic acid and lactic acid production according to the invention is possible within the family Methyl obacteriaceae.

[0349] Figure 15 shows the screening result for glycolic acid and lactic acid production with recombinant M. zatmanii DSM 5688 strains that express the glyoxylate reductase gene from Escherichia and, in one case, additionally the ethylmalonyl-CoA mutase gene from Rhodobacter sphaeroides ATCC 17029, based on the corresponding codon-optimized genes. The first entry from the left shows the methanol concentration in the minimal medium at the start of cultivation. pTE1887 was used as the expression vector, which, in the form of the empty vector in the reference strain M. zatmanii DSM 5688 + pTE1887, also serves as a negative control (second entry from the left on the x-axis).

[0350] 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 Figure 15) the genetically modified cells of M. zatmanii DSM 5688 + pTE1887-ghrA according to the invention. eC o (in codon-optimized nucleic acid form according to SEQ ID No. 3), i.e. 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: 3) and the genetically modified cells M. zatmanii DSM 5688 + pTE1887-ghrA eC o-ecm rsh, comprising an exogenous codon-optimized nucleic acid sequence (SEQ ID No. 3) encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MGI 655 and an exogenous codon-optimized nucleic acid sequence (SEQ ID No. 8) encoding an ethylmalonyl-CoA mutase from the bacterium Rhodobacter sphaeroides ATCC 17029, i.e. 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).

[0351] Figure 9 shows the map of the vector used to generate these Methylobacteriaceae cells with the following elements: lacl gene, lacl promoter, PL / O4 / A1 promoter - 33 region -10 region transcription start, PL / O4 / A1 promoter ribosomal binding site (RBS), ghrAeco-c optimized, lambda TO terminator, kanamycin resistance, mobilization genes mobS and mob, regulatory protein RepA, origin of replication colEl.

[0352] Figure 11 shows the map of the vector used to generate these Methylobacteriaceae cells expressing the ghrA eC o-ecm r sh was used with the following elements: lacl gene, lacl promoter, PL / O4 / A1 promoter -33 Region -10 Region Transcription start, PL / O4 / A1 promoter Ribosomal binding site (RBS), ghrA eco (codon-optimized), rsh-ecm (codon-optimized), lambda TO terminator, kanamycin resistance, mobilization genes mobS and mob, regulatory protein RepA. Origin of replication colEl.

[0353] Mixtures of glycolic acid and lactic acid containing a total concentration of glycolic acid plus lactic acid up to 0.56 g L' could be obtained with the cells of M. zatmanii DSM 5688 genetically modified according to the invention. 1 (M. zatmanii DSM 5688 + pTE1887-ghrA eC o), at least 0.48 g L' 1 (M. zatmanii DSM 5688 + pTE1887-ghrA eC o-ecm rsh). These experimental data demonstrate that the glycolic acid and lactic acid production according to the invention is possible within the Methylobacteriaceae family.

[0354] Figure 16 shows the screening results for glycolic acid and lactic acid production of a recombinant M. radiotolerans DSM 760 strain. The first panel from the left shows the methanol concentration in the minimal medium at the start of cultivation. pTE1887 was used as the expression vector, which, in the form of the empty vector in the reference strain M. radiotolerans DSM 760 + pTE1887, also serves as a negative control (second panel from the left on the x-axis).

[0355] Surprisingly, the reference strain M. radiotolerans DSM 760 + pTE1887 (second entry from the left) showed no glycolic acid and lactic acid production in contrast to the genetically modified cells of M. radiotolerans DSM 760 + pTE1887-ghrAeco-ecm according to the invention. mea, comprising an exogenous codon-optimized nucleic acid sequence (SEQ ID No. 3) encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MGI 655 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, i.e. 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: 3) (black solid bar in Figure 16). The combination of ghrA eco and ecm me a (an ethylmalonyl-CoA mutase from M. extorquens TK 0001 DSM 1337 according to the invention) led to glycolic acid and lactic acid production according to the invention.

[0356] Figure 10 shows the map of the vector used to generate Methylobacteriaceae cells expressing the ghrA e co-ecm mea was used with the following elements: lacl gene, lacl promoter, PL / O4 / A1 promoter -33 Region -10 Region transcription start, PL / O4 / A1 promoter Ribosomal binding site (RBS), ghrA eco (codon-optimized), ecm me a (native), Lambda T0 terminator, kanamycin resistance, mobilization genes mobS and mob, regulatory protein RepA. Origin of replication colEl.

[0357] Mixtures of glycolic acid and lactic acid containing a total concentration of glycolic acid plus lactic acid up to 0.39 g L' could be obtained with the cells of M. radiotolerans DSM 760 genetically modified according to the invention. 1 (M. radiotolerans DSM 760 + pTE1887- ghrAeco-eemmea).

[0358] These experimental data demonstrate that the inventive glycolic acid and lactic acid production is possible within the family Methylobacteriaceae.

[0359] Figure 17 shows the screening results for glycolic acid and lactic acid production using recombinant M. organophilum DSM 18172 strains expressing glyoxylate reductases, starting from the corresponding codon-optimized genes. The first entry from the left shows the methanol concentration in the minimal medium at the start 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. organophilum DSM 18172 + pTE1887 (second entry from the left on the x-axis).

[0360] Surprisingly, both the reference strain M. organophilum DSM 18172 + pTE1887 (second entry from the left) and the genetically modified Methylobacteriaceae cells showed no glycolic acid and lactic acid production (entries from the left: 3 to 10 and 12 to 18), with the exception (black, filled bars in Figure 17) of the genetically modified cells of M. organophilum DSM 18172 + pTE1887-ghrA according to the invention. eCo (in codon-optimized nucleic acid form according to SEQ ID No. 3), i.e. 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: 11) and the genetically modified cells M. organophilum DSM 18172 + pTE1887-ghrA eC o- ecmmea, comprising an exogenous codon-optimized 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, i.e. 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-ghrAeC o-ecm r sh, comprising an exogenous codon-optimized nucleic acid sequence (SEQ ID No. 3) encoding a glyoxylate reductase from the bacterium Escherichia coli K-12 MGI 655 and an exogenous codon-optimized nucleic acid sequence (SEQ ID No. 8) encoding an ethylmalonyl-CoA mutase from the bacterium Rhodobacter sphaeroides ATCC 17029, i.e. 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).

[0361] Figure 9 shows the map of the vector used to generate these Methylobacteriaceae cells with the following elements: lacl gene, lacl promoter, PL / O4 / A1 promoter - 33 region -10 region transcription start, PL / O4 / A1 promoter ribosomal binding site (RBS), ghrAeco-c optimized, lambda T0 terminator, kanamycin resistance, mobilization genes mobS and mob, regulatory protein RepA. Origin of replication colEl.

[0362] Figure 10 shows the map of the vector used to generate Methylobacteriaceae cells expressing the ghrA e co-ecm m ea was used with the following elements: lacl gene, lacl promoter, PL / O4 / A1 promoter -33 Region -10 Region transcription start, PL / O4 / A1 promoter Ribosomal binding site (RBS), ghrA eco (codon-optimized), ecm me a (native), Lambda T0 terminator, kanamycin resistance, mobilization genes mobS and mob, regulatory protein RepA. Origin of replication colEl.

[0363] Figure 11 shows the map of the vector used to generate these Methylobacteriaceae cells expressing the ghrA eC o-ecm rsh was used with the following elements: lacl gene, lacl promoter, PL / O4 / A1 promoter -33 Region -10 Region Transcription start, PL / O4 / A1 promoter Ribosomal binding site (RBS), ghrA eco (codon-optimized), rsh-ecm (codon-optimized), lambda T0 terminator, kanamycin resistance, mobilization genes mobS and mob, regulatory protein RepA. Origin of replication colEl.

[0364] Mixtures of glycolic acid and lactic acid containing a total concentration of glycolic acid plus lactic acid up to 0.13 g L' could be obtained using the cells of M. organophilum DSM 18172 genetically modified according to the invention. 1 (M. organophilum DSM 18172 + pTE1887-ghrA eC o), at least 0.10 g L' 1 (M. organophilum DSM 18172 + pTE1887-ghrAeco-ecirimea), at least 0.04 g L' 1 (M. organophilum DSM 18172 + pTE1887-ghrA eC o- ecmrsh) are produced.

[0365] These experimental data demonstrate that the inventive glycolic acid and lactic acid production is possible within the family Methylobacteriaceae.

[0366] Figure 18 shows the screening results for glycolic acid and lactic acid production using recombinant M. extorquens PA1 DSM 23939 strains. The first panel from the left shows the methanol concentration in the minimal medium at the start 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. extorquens PA1 DSM 23939 + pTE1887 (second panel from the left on the x-axis).

[0367] Surprisingly, the reference strain M. extorquens PA1 DSM 23939 + pTE1887 (second entry from the left) showed no glycolic acid and lactic acid production, in contrast to the genetically modified cells of M. extorquens PA1 DSM 23939 + pTE1887-ghrA according to the invention. eCo (in codon-optimized nucleic acid form according to SEQ ID No. 3), i.e. 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 Figure 18) (entry from the left: 3).

[0368] Figure 9 shows the map of the vector used to generate these Methylobacteriaceae cells with the following elements: lacl gene, lacl promoter, PL / O4 / A1 promoter - 33 region -10 region transcription start, PL / O4 / A1 promoter ribosomal binding site (RBS), ghrAeco-c-optimized, lambda TO terminator, kanamycin resistance, mobilization genes mobS and mob, regulatory protein RepA. Origin of replication colEl.

[0369] It was possible to produce mixtures of glycolic acid and lactic acid containing a total concentration of glycolic acid plus lactic acid up to 1.50 g L' with the cells of M. extorquens PA1 DSM 23939 genetically modified according to the invention. 1 (M. extorquens PA1 DSM 23939 + pTE1887-ghrA eC o) (M. extorquens GA17) are produced.

[0370] These experimental data demonstrate that the inventive glycolic acid and lactic acid production is possible within the family Methylobacteriaceae.

[0371] Figure 19 shows the screening results for glycolic acid and lactic acid production using recombinant M. extorquens AMlAcel strains expressing glyoxylate reductases, starting from the corresponding codon-optimized genes. The first panel from the left shows the methanol concentration in the minimal medium at the start 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. extorquens AMlAcel + pTE1887 (second panel from the left on the x-axis).

[0372] Surprisingly, the reference strain M. extorquens AMlAcel + pTE1887 (second entry from the left) showed no glycolic acid and lactic acid production, with the exception (black solid bar in Figure 19) of the genetically modified cells of M. extorquens AMlAcel + pTE1887-ghrA according to the invention. eCo (in codon-optimized nucleic acid form according to SEQ ID No. 3), i.e. 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: 11).

[0373] Figure 9 shows the map of the vector used to generate these Methylobacteriaceae cells with the following elements: lacl gene, lacl promoter, PL / O4 / A1 promoter - 33 region -10 region transcription start, PL / O4 / A1 promoter ribosomal binding site (RBS), ghrAeco-c-optimized, lambda TO terminator, kanamycin resistance, mobilization genes mobS and mob, regulatory protein RepA. Origin of replication colEl.

[0374] It was possible to use the cells of M. extorquens AM1Acel genetically modified according to the invention to produce mixtures of glycolic acid and lactic acid containing a total concentration of glycolic acid plus lactic acid up to 0.60 g L' 1(M. extorquens AMlAcel + pTE1887-ghrA eC o) are produced.

[0375] These experimental data demonstrate that the glycolic acid and lactic acid production according to the invention is possible within the Methylobacteriaceae family. Deletion of the cellulase gene (Acel) does not affect glyoxylate-glycolic acid-lactic acid metabolism.

[0376] The results of these studies are summarized in Table 5 (below).

[0377] Further studies were conducted on methylotrophic microorganisms that do not belong to the Methylobacteriaceae family. For this purpose, the strain construction procedures according to Example 1 were used 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 case with the strains used. The strains tested showed pTE1887, pTE1887-ghrA for all vectors used. eC o, pTE1887-ghrA e co-ecm m ea and pTE1887-ghrA eC o-ecm r sh no growth during the strain construction procedure described in Example 1 (Table 5).

[0378] Table 5: Summary of the achieved glycolic acid and lactic acid titers of tested strains of the Methyl obacteriaceae family and control samples (microorganisms not belonging to the Methyl obacteriaceae family). Abbreviations: GS, glycolic acid; MS, lactic acid.

[0379] 1 Temperatures used in growth test: Methylophilus methylotrophus DSM 6330 (37

[0380] °C), Bacillus methanolicus DSM 16454 (45 °C).

Claims

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

2. Genetically modified cell according to claim 1, wherein the Methylobacteriaceae cell is a Methylorubrum cell, in particular a cell of Methylorubrum extorquens, in particular Methylorubrum extorquens AMI, Methylorubrum extorquens TK 0001, Methylorubrum extorquens PA1, Methylorubrum rhodesianum or Methylorubrum zatmanii, or a Methylobacterium cell, in particular a cell of Methylorubrum organophilum or Methylorubrum radiotolerans.

3. Genetically modified Methylobacteriaceae cell according to claim 1 or 2, wherein the bacterium is Escherichia coli, in particular E. coli K-12 MG1655.

4. A genetically modified Methylobacteriaceae cell according to any one of the preceding claims, 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 according to SEQ ID No.

2.

5. Genetically modified Methylobacteriaceae cell according to one of the preceding claims, comprising 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. Genetically modified Methylobacteriaceae cell according to 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.A genetically modified Methylobacteriaceae cell according to any one of the preceding claims, wherein the at least one exogenous nucleic acid sequence encoding glyoxylate reductase and / or encoding ethylmalonyl-CoA mutase is integrated into the chromosome of the Methylobacteriaceae cell or is present extrachromosomally, in particular integrated into the cell in an episomal expression vector. A genetically modified Methylobacteriaceae cell according to any one of the preceding claims, 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 10June 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 Methylorubrum radiotolerans Mra-GA12 (DSM 34700) or Methylorubrum organophilum Mor-GA8 (DSM 34699), each deposited on 19 July 2023 at the DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany, or a derivative thereof. A genetically modified Methylobacteriaceae cell according to any one of the preceding claims, wherein the at least one exogenous nucleic acid sequence encoding glyoxylate reductase and / or encoding ethylmalonyl-CoA mutase is functionally linked. additionally linked to at least one regulatory unit to form 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.A method for producing a genetically modified Methylobacteriaceae cell according to any one of claims 1 to 9, comprising the method steps: 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 which enable the uptake and, optionally stable, integration of the at least one exogenous nucleic acid sequence into the Methylobacteriaceae cell, and c) obtaining the at least one exogenous genetically modified Methylobacteriaceae cell comprising a glyoxylate reductase.Method according to claim 10, wherein in method 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 method step b) the Methylobacteriaceae cell is transformed with the exogenous nucleic acid sequence encoding an ethylmalonyl-CoA mutase, in particular the expression cassette comprising this, and in method step c) at least one exogenous genetically modified Methylobacteriaceae cell comprising a glyoxylate reductase, which additionally comprises at least one exogenous ethylmalonyl-CoA mutase encoding nucleic acid sequence is obtained. A genetically modified Methylobacteriaceae cell according to any one of claims 1 to 9 or produced by the method of claim 10 or 11, wherein the cell is present in a living or dead form or lyophilized or in the form of a cell lysate or cell extract obtained from a genetically modified Methylobacteriaceae cell according to any one of claims 1 to 9 or produced by the method of claim 10 or 11. A biocatalyst comprising a genetically modified Methylobacteriaceae cell according to any one of claims 1 to 9 or 12 or produced by the method of claim 10 or 11, wherein the cell is arranged on a support. A bioreactor comprising a genetically modified Methylobacteriaceae cell according to any one of claims 1 to 9 or 12 or produced by the method of claim 10 or 11, or a biocatalyst according to claim 12.A process for producing a product containing glycolic acid from a starting material containing at least one Cx compound, comprising the process steps: x) providing a genetically modified Methylobacteriaceae cell according to any one of the preceding claims 1 to 9 or 12 or a biocatalyst according to claim 13, a reaction medium, and the starting material containing at least one Cx compound, y) reacting the starting material 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. The process according to 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. The process according to claim 15 or 16, wherein the product containing glycolic acid is a product containing glycolic acid and lactic acid. Process according to one of claims 15 to 17, wherein the Cx compound is produced from CO2, in particular synthesis gas comprising a mixture of CO2, CO, and H2, in particular by means of a heterogeneous catalytic chemical process. Process according to claim 18, wherein the CO2, in particular synthesis gas, is produced by chemical conversion of organic substances or materials, in particular sewage sludge and other biogenic residues and waste materials. Process for producing polyglycolic acid, polylactic acid, or polylactide-co-glycolide, comprising carrying out a process according to one of claims 15 to 19 and subsequently polymerizing the glycolic acid, lactic acid, or mixture of glycolic acid and lactic acid obtained from these processes.

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