Transgenic methylobacteriaceae cells for fermentative production of glycolic acid and lactic acid from Cx compounds

Abstract

The present invention relates to a transgenic methylobacteriaceae cell comprising at least one exogenous nucleic acid sequence encoding a glyoxylate reductase derived from Escherichia bacteria; the invention relates to a preparation method of transgenic methylobacteriaceae cells. A biocatalyst comprising a transgenic cell of the family Methylobacteriaceae; a bioreactor comprising a transgenic methylobacteriaceae cell; the invention discloses a preparation method of a product containing glycollic acid and lactic acid. The invention also provides a preparation method of polyglycolic acid, polylactic acid or a polylactic acid-glycolic acid copolymer.

Description

Technical Field

[0001] The present invention relates to a transgenic Methylobacteriaceae cell comprising at least one exogenous nucleic acid sequence encoding a glyoxylate reductase derived from a bacterium of the genus Escherichia; a method for preparing a transgenic Methylobacteriaceae cell; a biocatalyst comprising a transgenic Methylobacteriaceae cell; a bioreactor comprising a biocatalyst of a transgenic Methylobacteriaceae cell; a method for preparing a product containing glycolic acid and lactic acid; a method for preparing polyglycolic acid, polylactic acid or a polylactic acid-glycolic acid copolymer. Background Art

[0002] Glycolic acid, also known as hydroxyacetic acid or hydroxyethanoic acid, is an organic carboxylic acid with two carbon atoms, and the C 2 atom contains a carboxyl group and a hydroxyl group as functional groups. Glycolic acid is widely used in: the textile industry, for example, as a dye and a tanning agent; the food industry, for example, as a flavoring agent and a preservative or a packaging material; the pharmaceutical industry, for example, as a skin care product ( et al., Applied Microbiology and Biotechnology, Vol. 103(6), 2019, pp. 2525-2535; hereinafter referred to as et al.). In the polymer industry, glycolic acid can be processed together with lactic acid into a copolymer (polylactic acid-glycolic acid copolymer), or in medical technology, as polyglycolic acid to form absorbable sutures for the human body ( et al.; K.J. Jem and B. Tan, Advanced Industrial and Engineering Polymer Research, Vol. 3(2), 2020, pp. 60-70; hereinafter referred to as Jem et al.). Currently, glycolic acid is almost entirely industrially produced from fossil raw materials through a petrochemical process using formaldehyde, carbon monoxide and water. Efforts are being made to find sustainable ways to produce economically valuable glycolic acid compounds using renewable raw materials without relying on fossil resources.

[0003] It is well known that glycolic acid can be produced by microbial fermentation using renewable substrates such as D-glucose, D-xylose, D-arabinose, L-lyxose, L-arabinose, acetate or ethanol, etc., but industrial production has not been achieved ( et al., Jem et al., et al., Appita Journal, Vol. 67(1), 2014, p. 12). Such substrates are all from biological raw materials. Therefore, using such substrates poses risks in terms of sustainability because the raw materials can be used for chemical production, as well as for food and feed production, such as bioethanol.

[0004] Glycolic acid can be easily synthesized from substrates by biotechnology, such as hexoses, pentoses, or glycolonitrile disclosed in patent document US7,198,927B2, formaldehyde and hydrogen cyanide disclosed in patent document EP1828393B1, or ethylene glycol disclosed in patent document EP2025760B1. In contrast, the direct synthesis of glycolic acid from CO 2 by biotechnology is difficult to achieve biotechnologically. The main reasons are the limitations of photosynthetic metabolic efficiency or the inherent limitations of gas-liquid mass transfer in gas fermentation. The latter two methods are also limited by low yields and low conversion rates, as well as the number of available genetically modifiable microorganisms ( and T. Walther, Chemie Ingenieur Technik, 2020, 92(11), pp. 1680-1699; N.K. Kang, M. Kim, K. Baek, Y.K. Chang, D.R. Ort, and Y.-S. Jin, Chemical Engineering Journal, 2022, 433, p. 133636).

[0005] Biotechnologically available intermediates (such as methanol or formic acid) can be produced from CO 2 in various ways (Bohlen et al., Electrochemistry Communications, 2020, 110, p. 106597; M. Bowker, ChemCatChem, 2019, 11(17), pp. 4238-4246; F.S. Lénárd-Istvan Csepei et al., F.-G.z.F.d.a.F.e.V., Editor, 2016, Germany), hereinafter referred to as Cx compounds.

[0006] Cx compounds (such as methanol or formic acid or a mixture of these two substrates) can be used by methylotrophic microorganisms as an energy source for the construction of biomass or economic products, especially chemical products. In the central carbon metabolism of methylotrophic microorganisms, Cx compounds (such as methanol or formic acid) are used as substrates. For example, in the first reaction step, methanol is oxidized to formic acid. This generates the redox equivalents of reduced cytochrome C and NAD(P)H required for metabolism. Subsequently, formic acid can be oxidized to CO 2or enter the serine cycle (like formaldehyde). The serine cycle acts as a carbon distribution circuit in methylotrophic microorganisms, providing the main precursors required for biomass synthesis. In addition, the serine cycle is also the starting point for other metabolic pathways necessary for Cx compound growth. For example, in the serine cycle, the intermediate glyoxylate can be regenerated through the connected ethylmalonyl-CoA metabolic pathway. The serine cycle intermediate glyoxylate can be reduced by NADH or NADPH coupled with glyoxylate reductase (ghrA) to form glycolate. Glyoxylate reductase (ghrA) and hydroxypyruvate reductase (ghrB) belong to the glyoxylate / hydroxypyruvate reductase (ghr).

[0007] It is well known that there is a DNA sequence encoding endogenous glyoxylate reductase (EC: 1.1.1.26, https: / / www.ncbi.nlm.nih.gov / nuccore / LT962688) in the genome of Methylobacterium extorquens TK 0001. However, although there is an endogenous glyoxylate reductase DNA sequence and glyoxylate is the starting compound of metabolism, the wild-type strain Methylobacterium extorquens does not produce any glycolate measurable by HPLC or GC-MS.

[0008] There is a need to provide a method for fermenting glycolate from Cx compounds (such as methanol or formic acid), and a means for converting such Cx compounds (such as methanol or formic acid or a mixture thereof) into glycolate, particularly methylotrophic microorganisms. There is also a need to provide a method for obtaining glycolate in a completely renewable manner through an integrated cascade process using CO 2 as the sole raw material, that is, without consuming any fossil or biological resources. Summary of the Invention

[0009] In view of the above, the technical problem to be solved by the present invention is to overcome the above defects. Specifically, the technical problem of the present invention is to provide a biological cell that can convert a starting material (hereinafter also referred to as a reactant) containing at least one Cx compound (particularly methanol, formic acid or a mixture thereof) into a glycolate-containing product. Specifically, the technical problem of the present invention is to provide means and methods for obtaining such cells, particularly means and methods that are low-cost and easy to operate. Specifically, the technical problem of the present invention is to provide means and methods for obtaining a glycolate-containing product, particularly means and methods that are low-cost and easy to operate. Specifically, the technical problem of the present invention is to provide means and methods for sustainable synthesis of glycolate that hardly use, particularly do not use, fossil resources and / or hardly use, particularly do not use, biological raw materials, preferably using CO 2As the sole raw material. Specifically, the technical problem of the present invention is to provide means and methods that are low-cost, environmentally friendly, and easy to operate. Additionally, the technical problem of the present invention is especially to provide means and methods capable of obtaining polyglycolic acid, polylactic acid, or a polylactic acid-glycolic acid copolymer.

[0010] To solve the above technical problems, the present invention proposes the technical solutions of the independent claims, dependent claims, and the specification, especially a transgenic Methylobacteriaceae cell, which contains at least one exogenous nucleic acid sequence encoding glyoxylate reductase derived from bacteria of the genus Escherichia.

[0011] According to the present invention, there is provided a transgenic Methylobacteriaceae cell, that is, a cell that has deviated from the wild-type strain of Methylobacteriaceae after at least one genetic transformation. In addition, according to the present invention, the transgenic Methylobacteriaceae cell contains at least one exogenous nucleic acid sequence that encodes glyoxylate reductase derived from bacteria of the genus Escherichia. Correspondingly, the genetic transformation of the wild-type strain of Methylobacteriaceae cells is at least the integration of at least one exogenous nucleic acid sequence gene into the Methylobacteriaceae cells, and this exogenous nucleic acid sequence encodes glyoxylate reductase derived from bacteria of the genus Escherichia. The exogenous nucleic acid sequence can be synthetic or naturally occurring, especially present in Escherichia. In a preferred embodiment of the present invention, the transgenic Methylobacteriaceae cell of the present invention contains at least one exogenous nucleic acid sequence encoding glyoxylate reductase, which is naturally occurring or codon-optimized, especially codon-optimized for Methylobacteriaceae, especially codon-optimized for Methylobacterium, especially codon-optimized for the genus Methylobacterium, especially codon-optimized for Methylobacterium extorquens, especially codon-optimized for Methylobacterium extorquens TK 0001, Methylobacterium extorquens PA1, or Methylobacterium extorquens AM1.

[0012] Surprisingly, the transgenic Methylobacteriaceae cell of the present invention can convert Cx compounds into glycolic acid, especially convert the starting material (i.e., the reactant containing at least one Cx compound, especially methanol, formic acid, or a mixture thereof) into a glycolic acid-containing product, and the specific amount can be determined by HPLC or GC-MS. In contrast, the wild-type strain of Methylobacteriaceae cells that only contains the endogenous nucleic acid sequence encoding glyoxylate reductase cannot convert Cx compounds into glycolic acid, especially cannot convert the starting material (i.e., the reactant containing at least one Cx compound) into a glycolic acid-containing product, especially the specific amount can be determined by HPLC or GC-MS.

[0013] The present invention provides a transgenic Methylobacteriaceae cell that can convert a reactant containing at least one Cx compound (especially methanol, formic acid or a mixture thereof) into a glycolic acid-containing product. Cx compounds represent favorable substrates that are renewable but not of biological origin in biotechnological processes. Because they are in liquid form, they are easy to handle, unlike gases, and mass transfer in the liquid reaction mixture is not restricted. The preparation of glycolic acid can be easily achieved using the technical solution of the present invention. According to the present invention, as long as CO 2 is converted into a Cx compound (especially methanol) using renewable energy, glycolic acid can be produced completely in a renewable manner from CO 2 . In this way, the combination of the PtX process (Power-to-X) and biotechnology can advantageously form a demonstration PtXtY process (Power-to-X-to-Y).

[0014] Accordingly, the transgenic Methylobacteriaceae cell of the present invention can be advantageously used in a method for producing glycolic acid from at least one Cx compound, especially for producing a glycolic acid-containing product by reacting with a reactant containing at least one Cx compound (especially methanol, formic acid or a mixture thereof). Preferably, the glycolic acid-containing product obtained by the transgenic Methylobacteriaceae cell of the present invention contains lactic acid in addition to glycolic acid. According to the present invention, a very simple, easy-to-operate and low-cost method for preparing a product containing glycolic acid, especially a product containing glycolic acid and lactic acid, is provided, thereby avoiding high expenditures related to equipment and costs. Another advantage of the present invention is that usually after providing glycolic acid, especially glycolic acid and lactic acid, polymerization of glycolic acid, especially glycolic acid, lactic acid or a glycolic acid-lactic acid copolymer is carried out to produce polyglycolic acid, especially polyglycolic acid, polylactic acid or a glycolic acid-lactic acid copolymer, so that polyglycolic acid, especially polyglycolic acid, polylactic acid or a glycolic acid-lactic acid copolymer can be prepared without performing high-cost and time-consuming process steps.

[0015] Without being bound by theory, through the exogenous glyoxylate reductase present in the transgenic Methylobacteriaceae cells of the present invention (encoded by at least one exogenous nucleic acid sequence encoding a glyoxylate reductase derived from a bacterium of the genus Escherichia), Cx compounds can be converted to glycolic acid in the serine cycle of the transgenic Methylobacteriaceae cells of the present invention, in particular, a reactant containing at least one Cx compound (especially methanol, formic acid or a mixture thereof) can be converted to a glycolic acid-containing product, and in particular, the specific amount can be determined by HPLC or GC-MS. Preferably, the endogenous glyoxylate reductase encoded in the wild-type strain of the transgenic Methylobacteriaceae cells of the present invention does not convert any product with an amount measurable by HPLC or GC-MS in the metabolism of the wild-type strain, and in particular, does not convert a reactant containing at least one Cx compound (especially methanol, formic acid or a mixture thereof) to a glycolic acid-containing product. Therefore, the activity of glyoxylate reductase is completely controlled by integrating the exogenous nucleic acid sequence encoding glyoxylate reductase in genomic or free form and its expression according to the present invention.

[0016] Therefore, the transgenic Methylobacteriaceae cells of the present invention are characterized by the enzymatic activity of the exogenous glyoxylate reductase, in particular, being capable of converting Cx compounds to glycolic acid due to the presence of the exogenous glyoxylate reductase, especially converting a reactant containing at least one Cx compound (especially methanol, formic acid or a mixture thereof) to a glycolic acid-containing product, especially in the reaction matrix, especially being able to carry out the conversion in a liquid reaction matrix, especially being able to enzymatically catalyze this reaction.

[0017] A preferred feature of the transgenic Methylobacteriaceae cells is that the transgenic Methylobacteriaceae cells (especially their genomes) are similar to, especially the same as, the wild-type strain of Methylobacteriaceae cells, except for the presence of at least one exogenous nucleic acid sequence encoding a glyoxylate reductase derived from a bacterium of the genus Escherichia, which confers favorable enzymatic activity on the Methylobacteriaceae cells of the present invention, possibly accompanied by the exogenous nucleic acid sequence of an expression vector or expression cassette. In a preferred embodiment of the present invention, in the Methylobacterium of the present invention (especially in its genome), in addition to at least one nucleic acid sequence encoding glyoxylate reductase, there may be further genetic modifications, especially genetic engineering modifications, compared to the wild-type strain.

[0018] In a preferred embodiment of the present invention, the bacterium is Escherichia coli, especially Escherichia coli K-12 MG1655.

[0019] In a preferred embodiment of the present invention, the Methylobacteriaceae cells are Methylobacterium cells, especially Methylobacterium extorquens cells, especially Methylobacterium extorquens TK 0001, especially Methylobacterium extorquens PA1, Methylobacterium extorquens AM1, Methylobacterium rhodesianum or Methylobacterium zatmanii.

[0020] In a preferred embodiment of the present invention, the transgenic Methylobacteriaceae cell of the present invention is a transgenic Methylobacterium extorquens AM1 cell, a transgenic Methylobacterium extorquens TK 0001 cell or a transgenic Methylobacterium extorquens PA1 cell, which contains at least one exogenous nucleic acid sequence encoding glyoxylate reductase derived from Escherichia coli (especially Escherichia coli K-12 MG1655).

[0021] In a preferred embodiment of the present invention, the Methylobacteriaceae cell is a Methylobacterium cell, especially a Methylobacterium organophilum cell or a Methylobacterium radiotolerans cell.

[0022] In a preferred embodiment of the present invention, the Methylobacteriaceae cell is a Rhodobacter cell, especially a Methylobacterium extorquens cell, especially Methylobacterium extorquens AM1, Methylobacterium extorquens TK 0001, Methylobacterium extorquens PA1, Methylobacterium rhodesianum cell or Methylobacterium zatmanii, or a Methylobacterium cell, especially a Methylobacterium organophilum cell or a Methylobacterium radiotolerans cell.

[0023] In a preferred embodiment of the present invention, the exogenous glyoxylate reductase is encoded by the nucleic acid sequence of SEQ ID No: 3 or a functionally equivalent nucleic acid sequence, and the nucleic acid sequence identity of the functionally equivalent nucleic acid sequence with the nucleic acid sequence of SEQ ID No: 3 is 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%, and the glyoxylate reductase encoded by it can convert a reactant containing at least one Cx compound (especially methanol, formic acid or a mixture thereof) into a glycolate product. Preferably, the nucleic acid sequence identity with the nucleic acid sequence of SEQ ID No: 3 is at least 76.0%.

[0024] Therefore, in a preferred embodiment of the present invention, the present invention relates to a transgenic Methylobacteriaceae cell, especially a Rhodobacter cell or a Methylobacterium cell, which contains a nucleic acid sequence encoding an exogenous glyoxylate reductase, especially the nucleic acid sequence of SEQ ID NO: 3.

[0025] In a preferred embodiment of the present invention, the present invention also relates to a genetically modified Methylobacteriaceae cell, particularly a Methylobacterium cell or a Methylorubrum cell, which comprises at least one functional equivalent nucleic acid sequence of the exogenous SEQ ID No:3 nucleic acid sequence encoding glyoxylate reductase, and the nucleic acid sequence identity of the functional equivalent nucleic acid sequence with the SEQ ID No:3 nucleic acid sequence is 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%, and the glyoxylate reductase encoded by it can convert a reactant containing at least one Cx compound (particularly methanol, formic acid or a mixture thereof) into a glycolate product.

[0026] In a particularly preferred embodiment of the present invention, the functional equivalent nucleic acid sequence of the SEQ ID No:3 nucleic acid sequence has a nucleic acid sequence length of at least 800 nucleic acids, preferably at least 850 nucleic acids, preferably at least 900 nucleic acids, preferably at least 950 nucleic acids, preferably at least 970 nucleic acids. According to the present invention, the functional equivalent nucleic acid sequence of the SEQ ID No:3 nucleic acid sequence exhibits nucleic acid sequence identity with the SEQ ID No:3 nucleic acid sequence over the entire nucleic acid sequence length of the equivalent nucleic acid sequence of the SEQ ID No:3 nucleic acid sequence.

[0027] In a particularly preferred embodiment of the present invention, the SEQ ID No:3 nucleic acid sequence is a nucleic acid sequence obtained by codon optimization of a native nucleic acid sequence (i.e., a naturally occurring nucleic acid sequence) derived from the genus Escherichia (particularly Escherichia coli), particularly by codon optimization of the genus Methylobacterium (especially Methylobacterium extorquens, particularly Methylobacterium extorquens AM1, Methylobacterium extorquens TK 0001, especially Methylobacterium extorquens PA1), and this nucleic acid sequence encodes a glyoxylate reductase derived from the genus Escherichia (particularly Escherichia coli). The native Escherichia genus nucleic acid sequence encoding the glyoxylate reductase derived from the genus Escherichia comprises the SEQ ID No:1 nucleic acid sequence, which represents the functional equivalent nucleic acid sequence of the SEQ ID No:3 nucleic acid sequence.

[0028] In a particularly preferred embodiment of the present invention, the functional equivalent nucleic acid sequence of the SEQ ID No:3 nucleic acid sequence comprises the SEQ ID No:1 nucleic acid sequence.

[0029] In a preferred embodiment of the present invention, an exogenous nucleic acid sequence encoding glyoxylate reductase derived from bacteria of the genus Escherichia (particularly the nucleic acid sequence of SEQ ID No: 3 or a functionally equivalent nucleic acid sequence thereof, such as the nucleic acid sequence of SEQ ID No: 1) encodes a glyoxylate reductase comprising the amino acid sequence of SEQ ID No: 2 or a functionally equivalent amino acid sequence thereof (particularly consisting of the same).

[0030] In a preferred embodiment of the present invention, the exogenous glyoxylate reductase comprises the amino acid sequence of SEQ ID No: 2 or a functionally equivalent amino acid sequence thereof, and the amino acid sequence identity of the functionally equivalent amino acid sequence with the amino acid sequence of SEQ ID No: 2 is at least 30.0%, particularly 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%. Preferably, the amino acid sequence identity with the amino acid sequence of SEQ ID No: 2 is at least 90.0%.

[0031] In a preferred embodiment of the present invention, the present invention relates to a transgenic Methylobacteriaceae cell, particularly a Rhodobacter cell or a Methylobacterium cell, which comprises a functionally equivalent amino acid sequence of the amino acid sequence of SEQ ID No: 2, and the amino acid sequence identity of the functionally equivalent amino acid sequence with the amino acid sequence of SEQ ID No: 2 is at least 30.0%, particularly 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%, and it is capable of converting a reactant containing at least one Cx compound (particularly methanol, formic acid or a mixture thereof) into a glycolic acid-containing product.

[0032] In a particularly preferred embodiment of the present invention, the functionally equivalent amino acid sequence of the amino acid sequence of SEQ ID No: 2 has an amino acid sequence with a length of at least 300 amino acids, preferably at least 310 amino acids, preferably at least 320 amino acids, preferably at least 325 amino acids. According to the present invention, over the entire amino acid sequence length of the equivalent amino acid sequence of the amino acid sequence of SEQ ID No: 2, the amino acid sequence of the equivalent amino acid sequence of the amino acid sequence of SEQ ID No: 2 exhibits amino acid sequence identity with the amino acid sequence of SEQ ID No: 2.

[0033] In a preferred embodiment of the present invention, the Cx compound is a Cx compound where x = 1, 2 or 4, particularly a Cx compound where x = 1.

[0034] 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.

[0035] In a preferred embodiment of the present invention, the Cx compound is methanol.

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

[0037] In a preferred embodiment of the present invention, the reactant contains at least one Cx compound, particularly formic acid, methanol, methane, methylamine, acetic acid or succinic acid or a mixture thereof, particularly the reactant consists of at least one of these compounds. In a preferred embodiment of the present invention, a glycolic acid-containing product, particularly a product consisting of glycolic acid, is obtained by reacting a reactant containing at least one Cx compound.

[0038] In a preferred embodiment of the present invention, a product containing glycolic acid and lactic acid, particularly a product consisting of glycolic acid and lactic acid, is obtained by reacting a reactant containing at least one Cx compound.

[0039] In a preferred embodiment of the present invention, the glycolic acid-containing product contains glycolic acid and lactic acid, particularly 1 wt.% to 99 wt.%, particularly 2 wt.% to 98 wt.%, particularly 10 wt.% to 90 wt.%, particularly 30 wt.% to 80 wt.%, particularly 40 wt.% to 70 wt.%, particularly 50 wt.%, particularly 60 wt.% of glycolic acid and particularly 1 wt.% to 99 wt.%, particularly 2 wt.% to 98 wt.%, particularly 10 wt.% to 90 wt.%, particularly 20 wt.% to 70 wt.%, particularly 30 wt.% to 60 wt.%, particularly 50 wt.%, particularly 40 wt.% of lactic acid (both calculated based on the total dry weight of the obtained product) or consists of the above proportions.

[0040] In a preferred embodiment of the present invention, particularly in a reaction matrix with an initial concentration of at most 10 g L of a reactant containing at least one Cx compound (particularly composed of methanol), the growth rate μ of the transgenic Methylobacteriaceae cells of the present invention -1 is at least 0.05 h max -1 , particularly at least 0.10 h -1 , particularly at least 0.15 h -1 , particularly at least 0.18 h -1 , particularly at least 0.20 h -1 , particularly at least 0.21 h -1 , particularly 0.10 h -1 to 0.30 h -1 , particularly 0.15 h -1 to 0.25 h -1 , particularly 0.20 h -1 to 0.22 h -1 , particularly 0.21 h -1 .

[0041] In a preferred embodiment of the present invention, after reacting a reactant containing at least one Cx compound (particularly composed of methanol) with the transgenic Methylobacteriaceae cells of the present invention in a reaction matrix with an initial concentration of at most 10 g L (particularly after 40 hours of reaction time), a product is obtained. The product contains glycolic acid, particularly glycolic acid and lactic acid. The titer of the reaction matrix containing the product is at least 0.01 g L -1 -1 , particularly at least 0.10 g L -1 , particularly at least 0.15 g L -1 , particularly at least 0.20 g L -1 , particularly at least 0.25 g L -1 , particularly at least 0.50 g L- -1 , particularly at least 0.75 g L -1 , particularly at least 1.00 g L -1 , particularly 1.50 g L -1 (both calculated by the weight of the product per liter of the reaction matrix).

[0042] In a preferred embodiment of the present invention, the transgenic Methylobacteriaceae cells of the present invention transform a reactant containing at least one Cx compound (particularly composed of methanol) to produce a product containing glycolic acid (particularly containing glycolic acid and lactic acid). Particularly in a reaction matrix with an initial concentration of at most 10 g L of the reactant, the dry biomass to substrate yield (Y -1 ) is at least 10 mg g X / S -1 ​​​, particularly at least 50 mg / g -1 , particularly at least 100 mg / g -1 , particularly at least 150 mg / g -1 , particularly at least 200 mg / g -1 , particularly 10 mg / g -1 to 350 mg / g -1 , particularly 50 mg / g -1 to 320 mg / g -1 , particularly 100 mg / g -1 to 300 mg / g -1 , particularly 200 mg / g -1 to 300 mg / g -1 , particularly 280 mg / g -1 (both calculated based on the dry biomass of the transgenic Methylobacteriaceae cells of the present invention per gram of reactant).

[0043] In a preferred embodiment of the present invention, particularly in a reaction medium with an initial concentration of the reactant of at most 10 g / L -1 after converting the reactant containing at least one Cx compound (particularly composed of methanol) into a product containing glycolic acid (particularly containing glycolic acid and lactic acid), the dry biomass yield of the transgenic Methylobacteriaceae cells of the present invention on the substrate (Y X / S ) is reduced to less than 95%, particularly less than 90%, particularly less than 80%, particularly less than 70%, particularly 68% compared to the dry biomass yield of the wild-type strain on the substrate (Y X / S ).

[0044] In a preferred embodiment of the present invention, the transgenic Methylobacteriaceae cells of the present invention convert a reactant containing at least one Cx compound (particularly composed of methanol) to produce a product containing glycolic acid (particularly containing glycolic acid and lactic acid), particularly in a reaction medium with an initial concentration of the reactant of at most 10 g / L -1 the product yield on the substrate (Y P / S ) is at least 10 mg / g -1 , particularly at least 50 mg / g -1 , particularly at least 80 mg / g -1 , particularly at least 100 mg / g -1 , particularly at least 110 mg / g -1 , particularly 10 mg / g -1 to 200 mg / g -1 , particularly 50 mg / g -1 to 180 mg / g -1 , particularly 80 mg / g -1 to 150 mg / g -1, particularly 100 mg / g -1 to 130 mg / g -1 , particularly 120 mg / g -1 (both calculated as the weight of the product per gram of the reactant).

[0045] In a preferred embodiment of the present invention, the transgenic Methylobacteriaceae cells of the present invention transform a reactant containing at least one Cx compound (particularly composed of methanol) to produce a product containing glycolic acid (particularly containing glycolic acid and lactic acid), particularly in a reaction medium with an initial concentration of the reactant of at most 10 g / L -1 , the product-to-dry-biomass yield (Y P / X ) is at least 0.10 g / g -1 , particularly at least 0.20 g / g -1 , particularly at least 0.30 g / g -1 , particularly at least 0.40 g / g -1 , particularly at least 0.50 g / g -1 , particularly 0.10 g / g -1 to 0.80 g / g -1 , particularly 0.20 g / g -1 to 0.70 g / g -1 , particularly 0.30 g / g -1 to 0.60 g / g -1 , particularly 0.40 g / g -1 to 0.50 g / g -1 , particularly 0.50 g / g -1 (both calculated as the weight of the product per gram of dry biomass of the transgenic Methylobacteriaceae cells of the present invention).

[0046] In a preferred embodiment of the present invention, the transgenic Methylobacteriaceae cells of the present invention contain at least one exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase.

[0047] In a preferred embodiment of the present invention, the transgenic Methylobacteriaceae cells of the present invention contain at least one exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase, which is naturally occurring or codon-optimized, particularly a nucleic acid sequence codon-optimized for Methylobacteriaceae, particularly codon-optimized for the genus Methylobacterium, particularly for the genus Methylorubrum (particularly Methylobacterium extorquens, especially Methylobacterium extorquens AM1, Methylobacterium extorquens TK 0001 or Methylobacterium extorquens PA1).

[0048] In a preferred embodiment of the present invention, the exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase is derived from at least one bacterium selected from the group consisting of Methylobacterium extorquens, in particular Methylobacterium extorquens TK 0001 DSM 1337; and Rhodobacter sphaeroides, in particular Rhodobacter sphaeroides ATCC 17029.

[0049] In a preferred embodiment of the present invention, the transgenic Methylobacteriaceae cell of the present invention is a transgenic Methylobacteriaceae cell comprising at least one exogenous nucleic acid sequence encoding glyoxylate reductase derived from Escherichia coli (in particular Escherichia coli K-12 MG1655) and at least one exogenous nucleic acid sequence encoding ethylmalonyl-CoA, in particular Methylobacterium extorquens AM1, Methylobacterium extorquens PA1, Methylobacterium extorquens TK 0001 cells.

[0050] In a preferred embodiment of the present invention, the transgenic Methylobacteriaceae cell of the present invention is a transgenic Methylobacteriaceae cell comprising at least one exogenous nucleic acid sequence encoding glyoxylate reductase derived from Escherichia coli K-12 MG 1655 and at least one exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase derived from Methylobacterium extorquens TK 0001 DSM 1337, in particular Methylobacterium extorquens AM1, Methylobacterium extorquens PA1, Methylobacterium extorquens TK 0001 cells.

[0051] In a preferred embodiment of the present invention, the transgenic Methylobacteriaceae cell of the present invention is a transgenic Methylobacteriaceae cell comprising at least one exogenous nucleic acid sequence encoding glyoxylate reductase derived from Escherichia coli K-12 MG1655 and at least one exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase derived from Rhodobacter sphaeroides ATCC 17029, in particular Methylobacterium extorquens AM1, Methylobacterium extorquens PA1, Methylobacterium extorquens TK 0001 cells.

[0052] In a preferred embodiment of the present invention, the present invention also relates to a transgenic Methylobacteriaceae cell comprising at least two different exogenous nucleic acid sequences, i.e., in addition to at least one exogenous nucleic acid sequence, the transgenic Methylobacteriaceae cell further comprises a nucleic acid sequence encoding glyoxylate reductase derived from a bacterium of the genus Escherichia and at least one additional exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase.

[0053] Surprisingly, the glyoxylate yield (especially the glyoxylate and lactate yields) of the transgenic Methylobacteriaceae cells of the present invention is increased, specifically compared to the glyoxylate yield (especially the glyoxylate and lactate yields) obtained by transforming a reactant containing at least one Cx compound (especially methanol, formic acid or a mixture thereof) with the Methylobacteriaceae cells of the present invention (comprising at least one exogenous nucleic acid sequence encoding a glyoxylate reductase derived from a bacterium of the genus Escherichia, but not containing an exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase, especially not containing an exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase derived from at least one bacterium selected from the group consisting of Methylobacterium extorquens, especially Methylobacterium extorquens TK 0001 DSM 1337; Rhodobacter sphaeroides, especially Rhodobacter sphaeroides ATCC 17029). Therefore, particularly surprisingly, the present invention not only increases the glyoxylate yield but also increases the lactate yield.

[0054] Preferably, with the Methylobacteriaceae cells of the present invention, which additionally comprise at least one exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase, especially derived from at least one bacterium selected from the group consisting of Methylobacterium extorquens, especially Methylobacterium extorquens TK 0001 DSM 1337; Rhodobacter sphaeroides, especially Rhodobacter sphaeroides ATCC 17029, the conversion rate of converting a Cx compound into glyoxylate (especially glyoxylate and lactate) is higher than that of the Methylobacteriaceae cells of the present invention that do not contain the at least one additional exogenous nucleic acid sequence.

[0055] Without being bound by theory, the exogenous ethylmalonyl-CoA mutase present in the transgenic Methylobacteriaceae cells of the present invention increases the amount of glyoxylate converted by the exogenous glyoxylate reductase present in the transgenic Methylobacteriaceae cells of the present invention in the serine cycle of the transgenic Methylobacteriaceae cells of the present invention. This preferably increases the glyoxylate yield, specifically compared to the glyoxylate yield obtained by the Methylobacteriaceae cells of the present invention comprising at least one exogenous nucleic acid sequence encoding a glyoxylate reductase derived from a bacterium of the genus Escherichia and not containing an exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase. Without being bound by theory, an increase in the lactate yield is also observed, possibly due to the complex interaction between the supply of reducing equivalents in metabolism and the increased availability of the metabolite pyruvate (the precursor molecule of lactate).

[0056] In a particularly preferred embodiment, the present invention relates to a transgenic Methylobacteriaceae cell comprising a codon-optimized nucleic acid sequence encoding ethylmalonyl-CoA mutase derived from Rhodobacter sphaeroides, especially a nucleic acid sequence optimized by Methylobacteriaceae codons, especially by Methylobacterium genus (especially Methylobacterium extorquens, especially Methylobacterium extorquens TK 0001, especially Methylobacterium extorquens AM1, especially Methylobacterium extorquens PA1) codons, especially a nucleic acid sequence containing SEQ ID No: 8.

[0057] In a particularly preferred embodiment, the present invention relates to a transgenic Methylobacteriaceae cell comprising a codon-optimized nucleic acid sequence encoding ethylmalonyl-CoA mutase derived from the nucleic acid sequence of Methylobacterium extorquens, in particular a nucleic acid sequence optimized with Methylobacteriaceae codons, in particular with Methylobacterium genus (in particular Methylobacterium extorquens, especially Methylobacterium extorquens TK 0001, especially Methylobacterium extorquens AM1, especially Methylobacterium extorquens PA1) codons, especially a nucleic acid sequence containing SEQ ID No:13.

[0058] In a preferred embodiment, the present invention relates to a transgenic Methylobacteriaceae cell comprising a functionally equivalent nucleic acid sequence of the nucleic acid sequence encoding ethylmalonyl-CoA mutase according to SEQ ID No:8 or SEQ ID No:13.

[0059] For the purposes of the present invention, the native nucleic acid sequence of ethylmalonyl-CoA mutase derived from Rhodobacter or Rhodobacter genus according to SEQ ID No:4 and SEQ ID No:6 shall refer to a functionally equivalent nucleic acid sequence of a codon-optimized nucleic acid sequence derived from said nucleic acid sequence, in particular the native nucleic acid sequence of SEQ ID No:6 represents a functionally equivalent nucleic acid sequence of the codon-optimized nucleic acid sequence of SEQ ID No:8, and the native nucleic acid sequence of SEQ ID No:4 represents a functionally equivalent nucleic acid sequence of the codon-optimized nucleic acid sequence of SEQ ID No:13.

[0060] In a preferred embodiment of the present invention, ethylmalonyl-CoA mutase is encoded by a codon-optimized nucleic acid sequence (SEQ ID No:13 or SEQ ID No:8) of the native nucleic acid sequence of SEQ ID No:4 or SEQ ID No:6 or a functionally equivalent nucleic acid sequence thereof, in particular the native nucleic acid sequence itself (i.e., the nucleic acid sequence of SEQ ID No:4 or SEQ ID No:6), wherein the nucleic acid sequence identity of the functionally equivalent nucleic acid sequence with the codon-optimized nucleic acid sequence of SEQ ID No:13 or SEQ ID No:8 is 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%, and the functionally equivalent nucleic acid sequence has the enzymatic activity of ethylmalonyl-CoA mutase.

[0061] In a preferred embodiment of the present invention, the present invention also relates to a transgenic Methylobacteriaceae cell comprising at least one functional equivalent nucleic acid sequence encoding an ethylmalonyl-CoA mutase and corresponding to the codon-optimized nucleic acid sequence of SEQ ID No: 13 or SEQ ID No: 8 (i.e., for example, the native nucleic acid sequence of SEQ ID No: 4 or SEQ ID No: 6), the nucleic acid sequence identity of the functional equivalent nucleic acid sequence with the codon-optimized nucleic acid sequence of SEQ ID No: 13 or SEQ ID No: 8 being at least 30.0%, preferably from 30.0% to 99.9%, preferably from 40.0% to 99.9%, preferably from 50.0% to 99.9%, preferably from 60.0% to 99.9%, preferably from 70.0% to 99.9%, preferably from 76.0% to 99.9%, preferably from 80.0% to 99.9%, preferably from 90.0% to 99.9%, preferably from 95.0% to 99.9%, preferably from 98.0% to 99.9%, preferably from 90.0% to 99.0%, and the transgenic Methylobacteriaceae cell being capable of converting a reactant containing at least one Cx compound (in particular methanol, formic acid or a mixture thereof) into a glycolate-containing product.

[0062] In a particularly preferred embodiment of the present invention, the functional equivalent nucleic acid sequence of the codon-optimized nucleic acid sequence of SEQ ID No: 8 comprises the native nucleic acid sequence of SEQ ID No: 6.

[0063] In a particularly preferred embodiment of the present invention, the functional equivalent nucleic acid sequence of the codon-optimized nucleic acid sequence of SEQ ID No: 13 comprises the native nucleic acid sequence of SEQ ID No: 4.

[0064] In a preferred embodiment of the present invention, the ethylmalonyl-CoA mutase comprises the amino acid sequence of SEQ ID No: 5 or SEQ ID No: 7 or a functional equivalent amino acid sequence, the amino acid sequence identity of the functional equivalent amino acid sequence with the amino acid sequence of SEQ ID No: 5 or SEQ ID No: 7 being at least 30.0%, in particular from 30.0% to 99.9%, preferably from 40.0% to 99.9%, preferably from 50.0% to 99.9%, preferably from 60.0% to 99.9%, preferably from 70.0% to 99.9%, preferably from 76.0% to 99.9%, preferably from 80.0% to 99.9%, preferably from 85.0% to 99.9%, preferably from 90.0% to 99.9%, preferably from 95.0% to 99.9%, preferably from 98.0% to 99.9%, and having the enzymatic activity of ethylmalonyl-CoA mutase.

[0065] In a preferred embodiment of the present invention, the present invention relates to a transgenic Methylobacteriaceae cell comprising a functional equivalent amino acid sequence of the amino acid sequence of SEQ ID No: 5 or SEQ ID No: 7, the amino acid sequence identity of the functional equivalent amino acid sequence with the amino acid sequence of SEQ ID No: 5 or SEQ ID No: 7 being at least 30.0%, particularly 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% SEQ, and the transgenic Methylobacteriaceae cell being capable of converting a reactant containing at least one Cx compound (particularly methanol, formic acid or a mixture thereof) into a glycolic acid-containing product, particularly a product containing glycolic acid and lactic acid.

[0066] In a preferred embodiment of the present invention, in a reaction medium having an initial concentration of at most 10 g L of a reactant containing at least one Cx compound (particularly consisting of methanol) -1 、particularly 10 g L -1 the growth rate μ of the transgenic Methylobacteriaceae cell of the present invention additionally comprising at least one exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase max is at least 0.05 h -1 、particularly at least 0.10 h -1 、particularly at least 0.12 h -1 、particularly at least 0.14 h -1 、particularly at least 0.16 h -1 、particularly 0.10 h -1 to 0.25 h -1 、particularly 0.12 h -1 to 0.22 h -1 、particularly 0.15 h -1 to 0.20 h -1 、particularly 0.16 h -1 、particularly 0.19 h -1 。

[0067] In a preferred embodiment of the present invention, particularly when the initial concentration of the reactant is at most 10 g L -1 、particularly 10 g L -1In the reaction medium, after reacting a reactant containing at least one Cx compound (especially composed of methanol) with transgenic Methylobacteriaceae cells of the present invention additionally containing at least one exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase (especially after a reaction time of 40 hours), a product is obtained which contains glycolic acid, especially glycolic acid and lactic acid, and the titer of the reaction medium containing the product is at least 0.10 g L -1 , especially at least 0.20 g L -1 , especially at least 0.30 g L -1 , especially at least 0.40 g L -1 , especially 0.10 g L -1 to 80 g L -1 , especially 0.20 g L -1 to 70 g L -1 , especially 0.30 g L -1 to 60 g L -1 , especially 0.40 g L -1 to 55 g L -1 , especially 0.49 g L -1 , especially 0.52 g L -1 (both calculated as the weight of the product per liter of the reaction medium).

[0068] In a preferred embodiment of the present invention, compared with the titer obtained by reacting with transgenic Methylobacteriaceae cells of the present invention that do not contain at least one exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase, in a reaction medium with an initial reactant concentration of at most 10 g L -1 , after reacting a reactant containing at least one Cx compound (especially composed of methanol) with transgenic Methylobacteriaceae cells of the present invention additionally containing at least one exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase, especially after a reaction time of 40 hours, the titer is increased by at least 10%, especially at least 30%, especially at least 50%, especially at least 60%, especially 69%, especially 79%.

[0069] In a preferred embodiment of the present invention, transgenic Methylobacteriaceae cells of the present invention additionally containing at least one exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase are used to transform a reactant containing at least one Cx compound (especially composed of methanol) to produce a product containing glycolic acid (especially containing glycolic acid and lactic acid), especially in a reaction medium with an initial reactant concentration of at most 10 g L -1 , especially 10 g L -1 , the dry biomass to substrate yield (Y X / S ) is at least 10 mg g -1 , especially at least 50 mg g -1, especially at least 100 mg / g -1 , especially at least 150 mg / g -1 , especially at least 200 mg / g -1 , especially 10 mg / g -1 to 350 mg / g -1 , especially 50 mg / g -1 to 320 mg / g -1 , especially 100 mg / g -1 to 300 mg / g -1 , especially 200 mg / g -1 to 300 mg / g -1 , especially 210 mg / g -1 , especially 270 mg / g -1 (per gram of dry biomass of the transgenic Methylobacteriaceae cells of the present invention in the reactants).

[0070] In a preferred embodiment of the present invention, especially in a reaction substrate with an initial concentration of the reactant of at most 10 g / L -1 , especially 10 g / L -1 , after converting a reactant containing at least one Cx compound (especially composed of methanol) into a product containing glycolic acid (especially containing glycolic acid and lactic acid), the dry biomass yield of the transgenic Methylobacter cells of the present invention additionally containing at least one exogenous nucleic acid sequence encoding ethylmalonyl-CoA for the substrate (Y X / S ) is reduced to less than 95%, especially less than 90%, especially less than 80%, especially less than 70%, especially 68%, especially 51% compared to the biomass yield of the wild-type strain for the substrate (Y X / S ).

[0071] In a preferred embodiment of the present invention, especially in a reaction substrate with an initial concentration of the reactant of at most 10 g / L -1 , especially 10 g / L -1 , after converting a reactant containing at least one Cx compound (especially composed of methanol) into a product containing glycolic acid (especially containing glycolic acid and lactic acid), the biomass yield of the transgenic Methylobacteriaceae cells of the present invention additionally containing at least one exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase for the substrate (Y X / S ) is reduced to less than 99%, especially less than 97%, especially to 96%, especially 75% compared to the biomass yield of the transgenic Methylobacteriaceae cells of the present invention without at least one exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase for the substrate (Y X / S ).

[0072] In a preferred embodiment of the present invention, especially in a reaction substrate with an initial concentration of the reactant of at most 10 g / L-1 , especially 10 g L -1 In a reaction matrix of, after converting a reactant containing at least one Cx compound (especially composed of methanol) into a product containing glycolic acid (especially containing glycolic acid and lactic acid), the present invention additionally includes biomass of at least one transgenic Methylobacteriaceae cell containing an exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase derived from Rhodobacter sphaeroides (especially Rhodobacter sphaeroides ATCC 17029), and the product yield on substrate (Y X / S ) is at least 5% higher, especially at least 10% higher, especially at least 20% higher, especially at least 25% higher, especially 28% higher, compared to the product yield on substrate (Y X / S ) of the present invention including biomass of at least one transgenic Methylobacteriaceae cell containing an exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase derived from Methylobacterium extorquens (especially Methylobacterium extorquens TK 0001 DSM 1337).

[0073] In a certain preferred embodiment of the present invention, the present invention additionally includes at least one transgenic Methylobacteriaceae cell containing an exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase to convert a reactant containing at least one Cx compound (especially composed of methanol) to produce a product containing glycolic acid (especially containing glycolic acid and lactic acid), especially in a reaction matrix where the initial concentration of the reactant is at most 10 g L -1 , especially 10 g L -1 , and the product yield on substrate (Y P / S ) is at least 10 mg g -1 , especially at least 50 mg g -1 , especially at least 80 mg g -1 , especially at least 100 mg g -1 , especially at least 140 mg g -1 , especially 10 mg g -1 to 250 mg g -1 , especially 50 mg g -1 to 200 mg g -1 , especially 80 mg g -1 to 180 mg g -1 , especially 100 mg g -1 to 160 mg g -1 , especially 150 mg g -1 (calculated as the weight of the product per gram of the reactant).

[0074] In a certain preferred embodiment of the present invention, especially in a reaction matrix where the initial concentration of the reactant is at most 10 g L -1 , especially 10 g L -1In a reaction medium, after converting a reactant containing at least one Cx compound (especially composed of methanol) into a product containing glycolic acid (especially containing glycolic acid and lactic acid), the product of the transgenic Methylobacteriaceae cells of the present invention additionally containing at least one exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase has a substrate yield (Y P / S ) that is at least 10% higher, especially at least 15% higher, especially at least 20% higher, especially 25% higher, compared to the substrate yield (Y P / S ) of the product of the transgenic Methylobacteriaceae cells of the present invention that do not contain at least one exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase.

[0075] In a preferred embodiment of the present invention, the transgenic Methylobacteriaceae cells of the present invention additionally containing at least one exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase convert a reactant containing at least one Cx compound (especially composed of methanol) to produce a product containing glycolic acid (especially containing glycolic acid and lactic acid), especially in a reaction medium where the initial concentration of the reactant is at most 10 g L -1 、especially 10 g L -1 , and the product-to-dry-biomass yield (Y P / X ) is at least 0.10 g g -1 , especially at least 0.30 g g -1 , especially at least 0.40 g g -1 , especially at least 0.50 g g -1 , especially at least 0.60 g g -1 , especially 0.10 mg g -1 to 0.99 g g -1 , especially 0.30 mg g -1 to 0.90 g g -1 , especially 0.40 mg g -1 to 0.80 mg g -1 , especially 0.40 mg g -1 to 0.75 mg g -1 , especially 0.70 g g -1 、especially 0.71 g g -1 (both calculated as the weight of the product per gram of dry biomass of the transgenic Methylobacteriaceae cells of the present invention).

[0076] In a preferred embodiment of the present invention, especially in a reaction medium where the initial concentration of the reactant is at most 10 g L -1 、especially 10 g L -1In a reaction matrix, after converting a reactant containing at least one Cx compound (especially composed of methanol) into a product containing glycolic acid (especially containing glycolic acid and lactic acid), the product of the transgenic Methylobacteriaceae cells of the present invention additionally containing at least one exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase has a dry biomass yield (Y P / S ) that is at least 10%, especially at least 20%, especially at least 30%, especially at least 35%, especially at least 40%, especially 42% higher than the dry biomass yield (Y P / S ) of the product of the transgenic Methylobacteriaceae cells of the present invention that do not contain at least one exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase.

[0077] In a preferred embodiment of the present invention, the Methylobacteriaceae cells are Methylobacterium extorquens cells, especially Methylobacterium extorquens TK 0001 cells, especially Methylobacterium extorquens PA1 cells.

[0078] In a preferred embodiment of the present invention, at least one exogenous nucleic acid sequence encoding glyoxylate reductase is integrated into the chromosome of the Methylobacteriaceae cells or exists extrachromosomally, especially integrated into a free expression vector or microchromosome within the cell.

[0079] In a preferred embodiment of the present invention, at least one exogenous nucleic acid sequence encoding glyoxylate reductase is stably integrated into the chromosome of the Methylobacteriaceae cells or stably exists extrachromosomally.

[0080] In a preferred embodiment of the present invention, the copy number of the exogenous nucleic acid sequence encoding glyoxylate reductase in the genome of the Methylobacteriaceae cells is more than one, especially 2, 3, 4, 5, 6 or more, preferably stably integrated into the chromosome or preferably stably existing extrachromosomally.

[0081] In a preferred embodiment of the present invention, at least one exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase is integrated into the chromosome of the Methylobacteriaceae cells or exists extrachromosomally, especially integrated into a free expression vector or microchromosome within the cell.

[0082] In a preferred embodiment of the present invention, at least one exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase is stably integrated into the chromosome of the Methylobacteriaceae cells or stably exists extrachromosomally.

[0083] In a preferred embodiment of the present invention, the copy number of the exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase in the genome of the Methylobacteriaceae cells is more than one, especially 2, 3, 4, 5, 6 or more, preferably stably integrated into the chromosome or preferably stably existing extrachromosomally.

[0084] In a preferred embodiment of the present invention, the transgenic Methylobacteriaceae cells are Methylobacterium extorquens Mea-GA1, Methylobacterium extorquens Mea-GA2 or Methylobacterium extorquens Mea-GA3 of the genus Methylobacterium, all of which were deposited at the DSMZ - German Collection of Microorganisms and Cell Cultures in Braunschweig, Germany on June 10, 2022, with the deposit accession numbers DSM 34286, DSM 34287, and DSM 34288. All deposits comply with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, which is widely applied in international patent procedures.

[0085] In a preferred embodiment of the present invention, the present invention relates to transgenic Methylobacterium extorquens TK 0001 cells, which contain at least one exogenous codon-optimized nucleic acid sequence encoding glyoxylate reductase derived from Escherichia coli K-12 MG1655, particularly cells of the Methylobacterium extorquens Mea-GA1 strain, which were deposited at the DSMZ - German Collection of Microorganisms and Cell Cultures in Braunschweig, Germany on June 10, 2022, with the deposit accession number DSM 34286.

[0086] In a preferred embodiment of the present invention, the present invention relates to transgenic Methylobacterium extorquens TK 0001 cells, which contain an exogenous codon-optimized nucleic acid sequence encoding glyoxylate reductase derived from Escherichia coli K-12 MG1655 and an exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase derived from Methylobacterium extorquens TK 0001 DSM 1337, particularly cells of the Methylobacterium extorquens Mea-GA2 strain, which were deposited at the DSMZ - German Collection of Microorganisms and Cell Cultures in Braunschweig, Germany on June 10, 2022, with the deposit accession number DSM 34287.

[0087] In a preferred embodiment of the present invention, the present invention relates to transgenic Methylobacterium extorquens TK 0001 cells, which contain an exogenous codon-optimized nucleic acid sequence encoding glyoxylate reductase derived from Escherichia coli K-12 MG1655 and an exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase derived from Rhodobacter sphaeroides ATCC 17029, particularly cells of the Methylobacterium extorquens Mea-GA3 strain, which were deposited at the DSMZ - German Collection of Microorganisms and Cell Cultures in Braunschweig, Germany on June 10, 2022, with the deposit accession number DSM 34288.

[0088] In a preferred embodiment of the present invention, the transgenic Methylobacteriaceae cell is a cell of Methylobacterium rhodesianum Mrh-GA4 strain (DSM 34697), a cell of Methylobacterium rhodesianum Mrh-GA5 strain (DSM 34698), a cell of Methylobacterium zatmanii Mza-GA14 strain (DSM 34701), a cell of Methylobacterium extorquens Mea-GA17 strain (DSM 34702), a cell of Methylobacterium radiotolerans Mra-GA12 strain (DSM 34700), or a cell of Methylobacterium organophilum Mor-GA8 strain (DSM 34699), which was deposited at the DSMZ - German Collection of Microorganisms and Cell Cultures in Braunschweig, Germany on July 19, 2023. All deposits comply with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, which is widely applied in international patent procedures.

[0089] In a preferred embodiment of the present invention, the present invention relates to a transgenic Methylobacteriaceae cell comprising an exogenous codon-optimized nucleic acid sequence (SEQ ID No: 3) encoding glyoxylate reductase derived from Escherichia coli K-12 MG1655, particularly a cell of Methylobacterium zatmanii Mza-GA14 strain (M. zatmanii DSM 5688 + pTE1887-ghrA eco ), which was deposited at the DSMZ - German Collection of Microorganisms and Cell Cultures in Braunschweig, Germany on July 19, 2023, with the deposit accession number DSM 34701.

[0090] In a preferred embodiment of the present invention, the present invention relates to a transgenic Methylobacteriaceae cell comprising an exogenous codon-optimized nucleic acid sequence (SEQ ID No: 3) encoding glyoxylate reductase derived from Escherichia coli K-12 MG1655, particularly a cell of Methylobacterium extorquens Mea-GA17 strain (M. extorquens PA1 DSM 23939 + pTE1887-ghrA eco ), which was deposited at the DSMZ - German Collection of Microorganisms and Cell Cultures in Braunschweig, Germany on July 19, 2023, with the deposit accession number DSM 34702.

[0091] In a preferred embodiment of the present invention, the present invention relates to a transgenic Methylobacteriaceae cell comprising an exogenous codon-optimized nucleic acid sequence (SEQ ID No: 3) encoding glyoxylate reductase derived from Escherichia coli K-12 MG1655, particularly a cell of Methylobacterium rhodesianum Mrh-GA4 strain (M. rhodesianum DSM 5687 + pTE1887-ghrA eco ), which was deposited at the DSMZ - German Collection of Microorganisms and Cell Cultures in Braunschweig, Germany on July 19, 2023, with the deposit accession number DSM 34697.

[0092] In a preferred embodiment of the present invention, the present invention relates to a transgenic Methylobacteriaceae cell, which contains an exogenous codon-optimized nucleic acid sequence (SEQ ID No: 3) encoding glyoxylate reductase derived from Escherichia coli K-12 MG1655 and an exogenous native nucleic acid sequence (SEQ ID No: 4) encoding ethylmalonyl-CoA mutase derived from Methylobacterium extorquens TK 0001 DSM 1337, particularly Methylobacterium rhodesianum strain Mrh-GA5 (M. rhodesianum DSM 5687 + pTE1887-ghrA eco -ecm mea ), which was deposited at the DSMZ - German Collection of Microorganisms and Cell Cultures in Braunschweig, Germany on July 19, 2023, with the deposit accession number DSM 34698.

[0093] In a preferred embodiment of the present invention, the present invention relates to a transgenic Methylobacteriaceae cell, which contains an exogenous codon-optimized nucleic acid sequence (SEQ ID No: 3) encoding glyoxylate reductase derived from Escherichia coli K-12 MG1655, particularly Methylobacterium organophilum strain Mor-GA8 (M. organophilum DSM 18172 + pTE1887-ghrA eco -ecm mea ), which was deposited at the DSMZ - German Collection of Microorganisms and Cell Cultures in Braunschweig, Germany on July 19, 2023, with the deposit accession number DSM 34699.

[0094] In a preferred embodiment of the present invention, the present invention relates to a transgenic Methylobacteriaceae cell, which contains an exogenous codon-optimized nucleic acid sequence (SEQ ID No: 3) encoding glyoxylate reductase derived from Escherichia coli K-12 MG1655 and an exogenous native nucleic acid sequence (SEQ ID No: 4) encoding ethylmalonyl-CoA mutase derived from Methylobacterium extorquens TK 0001 DSM 1337, particularly Methylobacterium radiotolerans strain Mra-GA12 (M. radiotolerans DSM 760 + pTE1887-ghrA eco -ecm mea ), which was deposited at the DSMZ - German Collection of Microorganisms and Cell Cultures in Braunschweig, Germany on July 19, 2023, with the deposit accession number DSM 34700.

[0095] In a particularly preferred embodiment of the present invention, the present invention relates to specifically deposited Methylobacteriaceae cells, particularly specifically deposited Rhodobacter strains and their various derivatives.

[0096] In a preferred embodiment of the present invention, at least one exogenous nucleic acid sequence encoding glyoxylate reductase derived from Escherichia bacteria is functionally linked to at least one other regulatory unit to form an expression cassette, particularly a promoter, especially an inducible promoter, a derepressible promoter or a constitutive promoter, an enhancer, a ribosome binding site and / or a terminator.

[0097] In a preferred embodiment of the present invention, at least one exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase (especially an exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase derived from at least one bacterium selected from the group consisting of Methylobacterium extorquens, especially Methylobacterium extorquens TK 0001 DSM 1337; Rhodobacter sphaeroides, especially Rhodobacter sphaeroides ATCC 17029) is functionally linked to at least one regulatory unit to form an expression cassette, particularly a promoter, especially an inducible promoter, a derepressible promoter or a constitutive promoter, an enhancer, a ribosome binding site and / or a terminator.

[0098] In a preferred embodiment, the expression cassette is present on a vector, especially an expression vector, especially an episomal expression vector, especially pTE1887.

[0099] In a particularly preferred embodiment, at least one exogenous nucleic acid sequence encoding glyoxylate reductase and at least one exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase may be present on the same expression vector or on different expression vectors.

[0100] In a preferred embodiment of the present invention, the promoter is an inducible promoter, especially an IPTG-inducible promoter, especially the P L / O4 / A1 promoter.

[0101] On the other hand, the present invention relates to a method for preparing the transgenic Methylobacteriaceae cells of the present invention, comprising the following process steps:

[0102] (a) Providing Methylobacteriaceae cells, especially wild-type cells, and an expression vector or a genome editing system comprising at least one exogenous nucleic acid sequence encoding glyoxylate reductase derived from Escherichia bacteria, especially an expression cassette comprising the nucleic acid sequence;

[0103] (b) Transforming the Methylobacteriaceae cells with the expression vector or the genome editing system under conditions allowing the uptake and subsequent stable integration of at least one exogenous nucleic acid sequence into the Methylobacteriaceae cells;

[0104] (c) Obtaining at least one exogenous transgenic Methylobacteriaceae cell containing a nucleic acid sequence encoding glyoxylate reductase derived from Escherichia bacteria.

[0105] In a particularly preferred embodiment, the present invention relates to the above method, wherein in process step (a), at least one exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase is additionally provided, in particular derived from at least one bacterium selected from the group consisting of: Methylobacterium extorquens, in particular Methylobacterium extorquens TK 0001 DSM 1337; Rhodobacter sphaeroides, in particular Rhodobacter sphaeroides ATCC 17029, and in particular an expression cassette or genome editing system containing such nucleic acid sequence is provided; in process step (b), a Methylobacteriaceae cell is transformed with an exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase, in particular an expression cassette containing such nucleic acid sequence; in process step (c), at least one exogenous transgenic Methylobacteriaceae cell containing glyoxylate reductase is obtained, which additionally contains at least one exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase.

[0106] In a preferred embodiment of the present invention, the transformation according to process step (b) is carried out by a chemical, physical and / or electrical transformation process, in particular electroporation.

[0107] The present invention also relates to transgenic Methylobacteriaceae cells that can be produced by the method of the present invention, in particular transgenic Methylobacteriaceae cells produced by the method of the present invention.

[0108] Another aspect of the present invention relates to a transgenic Methylobacteriaceae cell of the present invention, which is in the form of a live cell, dead cell, frozen and dried cell, cell lysate or cell extract, and the cell lysate or cell extract (in particular a protein extract) is obtained from the transgenic Methylobacteriaceae cell of the present invention. According to the present invention, the transgenic Methylobacteriaceae cell of the present invention may be in the form of a dead cell, frozen and dried cell or cell lysate or cell extract, and has the characteristic of converting at least one Cx compound into glycolic acid and optionally lactic acid in a reaction matrix provided by the present invention.

[0109] In a preferred embodiment of the present invention, a cell in the form of a live cell, dead cell, frozen and dried cell or cell lysate or cell extract catalyzes at least the conversion of at least one Cx compound into glycolic acid, in particular catalyzes the conversion of a reactant containing at least one Cx compound into a product containing glycolic acid, in particular a product containing glycolic acid and lactic acid.

[0110] Another aspect of the present invention is a biocatalyst comprising a transgenic Methylobacteriaceae cell of the present invention or a cell in the form of a dead cell, frozen and dried cell or cell lysate or cell extract of the transgenic Methylobacteriaceae cell of the present invention, and the cells are arranged on a carrier, in particular immobilized on a carrier.

[0111] 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, particularly consisting of, and in particular, the carrier is selected from the group consisting of: chitin, agar, agarose, alginate, carrageenan, and combinations thereof.

[0112] In a preferred embodiment of the present invention, the carrier comprises a synthetic organic carrier, particularly consisting of, and in particular, the carrier is selected from the group consisting of: polyvinyl alcohol (PVA), polyurethane, acrylamide, polyammonium acrylate, and combinations thereof.

[0113] In a preferred embodiment of the present invention, the carrier comprises an inorganic carrier, particularly consisting of, and in particular, the carrier is selected from the group consisting of: activated carbon, zeolite, ceramics, clay, anthracite, porous glass, and combinations thereof.

[0114] In a preferred embodiment of the present invention, the carrier is a composite mixture of an organic carrier and an inorganic carrier, particularly including sodium alginate polyvinyl alcohol (PVA-NA), guar gum polyvinyl alcohol (PVA-GG), or both.

[0115] In a preferred embodiment of the present invention, the biocatalyst of the present invention catalyzes at least one Cx compound to be converted into glycolic acid, particularly catalyzes a reactant containing at least one Cx compound (particularly consisting of) to be converted into a glycolic acid-containing product, particularly a product containing glycolic acid and lactic acid (particularly consisting of).

[0116] Another aspect of the present invention relates to a bioreactor comprising the transgenic Methylobacteriaceae cells of the present invention or the biocatalyst of the present invention, wherein the transgenic Methylobacteriaceae cells of the present invention or the biocatalyst of the present invention are particularly present in the reaction matrix within the bioreactor.

[0117] Another aspect of the present invention relates to a method for producing glycolic acid using at least one Cx compound, particularly for producing a glycolic acid-containing product using a reactant containing at least one Cx compound. Preferably, x = 1, 2, or 4. The method comprises the following process steps:

[0118] (x) Providing the transgenic Methylobacteriaceae cells of the present invention or the biocatalyst of the present invention, a reaction matrix, and at least one Cx compound, particularly a reactant containing at least one Cx compound;

[0119] (y) Reacting at least one Cx compound (particularly the reactant) in the reaction matrix under conditions allowing the formation of glycolic acid from the Cx compound;

[0120] (z) Obtaining glycolic acid, particularly a glycolic acid-containing product, from the reaction matrix.

[0121] In a preferred embodiment of the present invention, the methylobacteriaceae cells of the present invention provided in process step (x) or the biocatalyst of the present invention provided in process step (x) are present in the reaction matrix in a suspended or immobilized form.

[0122] In a preferred embodiment of the present invention, the reaction matrix provided in process step (x) and / or used in process step (y) is an aqueous saline solution, in particular a culture medium, in particular a basal medium, especially a basal medium containing at most 10 g of Cx compounds (in particular methanol, methane, formic acid, methylamine, acetic acid or succinic acid or a mixture thereof) per liter of reaction matrix, 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 μg of zinc sulfate heptahydrate, 110 μg of manganese chloride tetrahydrate, 2.75 mg of ferrous sulfate heptahydrate, 1.36 mg of ammonium heptamolybdate tetrahydrate, 140 μg of copper sulfate pentahydrate, 260 μg of cobalt chloride hexahydrate, 390 μg of sodium tungstate, 30 μg of boric acid, 2.02 g of potassium dihydrogen phosphate and 4.14 g of disodium hydrogen phosphate dihydrate.

[0123] In a preferred embodiment of the present invention, the reaction matrix provided in process step (x) comprises a reactant containing at least one Cx compound.

[0124] In a preferred embodiment of the present invention, the reaction matrix provided in process step (x) comprises a reactant containing at least one Cx compound at the start of process step (y), at a concentration of 1 g to 100 g, in particular 5 g to 90 g, in particular 6 g to 80 g, in particular 7 g to 70 g, in particular 8 g to 40 g, in particular 9 g to 30 g, in particular 10 g to 20 g of Cx compound per liter of reaction matrix.

[0125] In a preferred embodiment of the present invention, the reaction matrix provided in process step (x) comprises coenzyme B12.

[0126] In a preferred embodiment of the present invention, the reactant containing at least one Cx compound used in the present invention is the sole carbon source in the reaction matrix. Correspondingly, in a preferred embodiment, the reaction matrix used comprises a reactant containing at least one Cx compound as the sole carbon source for the methylobacteriaceae cells.

[0127] According to the present invention, preferably, the reaction in process step (y) is carried out with continuous or intermittent addition of glyoxylic acid.

[0128] In a preferred embodiment of the present invention, the Cx compound in 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.

[0129] In a preferred embodiment of the present invention, the reactants provided in process step (x) and reacted in process step (y) are composed of formic acid, methanol, methane, methylamine, acetic acid, succinic acid or a mixture thereof.

[0130] In a preferred embodiment of the present invention, for the Cx compound in the reactant containing at least one Cx compound provided in process step (x) and reacted in process step (y), x = 1.

[0131] In a preferred embodiment of the present invention, the Cx compound in the reactants provided in process step (x) and reacted in process step (y) is methanol, formic acid or a mixture thereof.

[0132] In a preferred embodiment of the present invention, the reactants provided in process step (x) and reacted in process step (y) are composed of methanol, formic acid or a mixture thereof.

[0133] In a preferred embodiment of the present invention, the reactants provided in process step (x) and reacted in process step (y) are composed of methanol.

[0134] In a preferred embodiment of the present invention, the reactants provided in process step (x) and reacted in process step (y) are composed of formic acid.

[0135] In a preferred embodiment of the present invention, the reactants provided in process step (x) and reacted in process step (y) contain methanol and formic acid, especially 1 wt.% to 99 wt.%, especially 2 wt.% to 98 wt.%, especially 10 wt.% to 90 wt.%, especially 30 wt.% to 70 wt.%, especially 40 wt.% to 60 wt.%, especially 50 wt.% of methanol and especially 1 wt.% to 99 wt.%, especially 2 wt.% to 98 wt.%, especially 10 wt.% to 90 wt.%, especially 30 wt.% to 70 wt.%, especially 40 wt.% to 60 wt.%, especially 50 wt.% of formic acid (both calculated based on the total weight of the reactants provided in process step (x)), or are composed of the above proportions.

[0136] In a preferred embodiment of the present invention, the reactant containing at least one Cx compound (especially methanol, formic acid or a mixture thereof, especially methanol) provided in process step (x) has an initial concentration of 1 g / L -1 to 20 g / L -1 、especially 3 g / L -1 to 17 g / L -1 、especially 5 g / L -1 to 15 g / L -1 、especially 7 g / L -1 to 13 g / L -1, especially 9 g L -1 to 11 g L-1, especially 10 g L -1 is present in the reaction medium.

[0137] In a preferred embodiment of the present invention, the reactant provided in process step (x) is methanol, at an initial concentration of 1 g L -1 to 20 g L -1 , especially 3 g L -1 to 17 g L -1 , especially 5 g L -1 to 15 g L -1 , especially 7 g L -1 to 13 g L -1 , especially 9 g L -1 to 11 g L -1 , especially 10 g L -1 is present in the reaction medium.

[0138] In a preferred embodiment of the present invention, the Cx compound (especially a C1 compound, in particular methanol, formic acid or a mixture thereof) provided in process step (x) and reacted in process step (y) is prepared from CO in process step (w) 2 , CO or a mixture, especially in process step (w) operating with renewable energy (especially electricity generated from solar, wind, geothermal or hydro energy).

[0139] In a preferred embodiment of the present invention, the Cx compound (especially methanol, formic acid or a mixture thereof) provided in process step (x) and reacted in process step (y) is prepared from CO in process step (w) especially by chemical heterogeneous catalysis (especially electrochemistry) 2 (especially comprising CO 2 , CO and H 2 mixture of synthesis gas).

[0140] In a preferred embodiment of the present invention, the Cx compound (especially acetic acid) provided in process step (x) and reacted in process step (y) is prepared from CO in process step (w) by gas fermentation 2 (especially comprising CO 2 , CO and H 2 mixture of synthesis gas).

[0141] In a preferred embodiment of the present invention, the Cx compound (especially methanol) provided in process step (x) and reacted in process step (y) is prepared from CO in process step (w) by electrochemistry, biochemistry, bioelectrochemistry or gas fermentation 2 (especially from a mixture comprising CO2 , CO and H 2 mixture or CO 2 , H 2 O and current mixture or CO 2 and H 2 mixture (syngas) is obtained.

[0142] In a preferred embodiment of the present invention, the CO used in process step (w) 2 (especially syngas) is obtained by chemical conversion (especially thermocatalytic conversion) of organic substances or materials (especially sludge and other biological residues and wastes).

[0143] In a preferred embodiment of the present invention, the syngas used in process step (w) is obtained from sludge.

[0144] In a preferred embodiment of the present invention, the CO used in process step (w) 2 is obtained from the atmosphere or industrial waste gas.

[0145] In a preferred embodiment, glycolic acid and lactic acid can be continuously synthesized using the present invention. This synthesis method has low cost, environmental friendliness, and easy operation, and hardly uses fossil resources at all, especially without using any fossil resources, and / or hardly uses biological raw materials at all, especially without using any biological raw materials. The advantage of the present invention is that glycolic acid and lactic acid are obtained in a completely renewable manner from CO 2 as the sole raw material through an integrated cascade process, that is, without consuming any fossil or biological resources. The advantage of the present invention is that glycolic acid is produced from CO in a completely renewable manner using the present invention 2 .

[0146] In a preferred embodiment of the present invention, the temperature of the reaction substrate in process step (y) is 20°C to 40°C, especially 22°C to 38°C, especially 24°C to 36°C, especially 28°C to 32°C, especially 30°C.

[0147] In a preferred embodiment of the present invention, process step (y) is carried out in a water vapor-saturated atmosphere.

[0148] In a particularly preferred embodiment of the present invention, at the start of process step (y), the pH value of the reaction substrate is 4 to 8, especially 5 to 7, especially 6, especially 6.8.

[0149] In a preferred embodiment of the present invention, after 40 hours of reaction time in process step (y), the pH of the reaction substrate is 0 to 6, especially 0 to 4, especially 0 to 3, especially 1 to 2.

[0150] In a preferred embodiment of the present invention, the reaction according to process step (y) is carried out under mechanical stirring, especially shaking or agitation.

[0151] In a preferred embodiment of the present invention, the stirring speed in process step (y) is 50 rpm to 1000 rpm, particularly 50 rpm to 500 rpm, particularly 50 rpm to 250 rpm, particularly 100 rpm to 200 rpm, particularly 150 rpm (rpm: revolutions per minute).

[0152] In a preferred embodiment of the present invention, the reaction substrate obtained in process step (z) comprises a product containing at least glycolic acid.

[0153] In a preferred embodiment of the present invention, in process step (z), a product containing glycolic acid, glycolic acid or a product containing glycolic acid and lactic acid is obtained in the reaction substrate.

[0154] In a preferred embodiment of the present invention, the product obtained in process step (z) contains glycolic acid and lactic acid, particularly 1 wt.% to 99 wt.%, particularly 2 wt.% to 98 wt.%, particularly 10 wt.% to 90 wt.%, particularly 30 wt.% to 80 wt.%, particularly 40 wt.% to 70 wt.%, particularly 50 wt.%, particularly 60 wt.% of glycolic acid and particularly 1 wt.% to 99 wt.%, particularly 2 wt.% to 98 wt.%, particularly 10 wt.% to 90 wt.%, particularly 20 wt.% to 70 wt.%, particularly 30 wt.% to 60 wt.%, particularly 50 wt.%, particularly 40 wt.% of lactic acid (both calculated based on the total weight in process step (z)) or consists of the above proportions.

[0155] In a preferred embodiment of the present invention, the product obtained in process step (z) consists of glycolic acid.

[0156] In a preferred embodiment of the present invention, the product obtained in process step (z) consists of glycolic acid and lactic acid.

[0157] In a preferred embodiment of the present invention, after process step (z), in process step (z1), the product containing glycolic acid and optionally lactic acid is separated from the reaction substrate, particularly from the reaction substrate and the transgenic Methylobacterium of the present invention or the biocatalyst of the present invention, particularly by decantation, salting out with a base (particularly NaOH or KOH), filtration (particularly membrane filtration or column filtration) or ion exchange chromatography combined with HPLC, extraction and / or distillation.

[0158] In a preferred embodiment of the present invention, the method for preparing the glycolic acid-containing product is a continuous method.

[0159] On the other hand, the present invention also relates to a method for preparing polyglycolic acid, polylactic acid or a polylactic acid-glycolic acid copolymer, which includes performing the method of the present invention to prepare glycolic acid, particularly to prepare a product containing glycolic acid and optionally lactic acid, and then polymerizing the glycolic acid, lactic acid or glycolic acid and lactic acid obtained therefrom.

[0160] For the purposes of the present invention, the "Genetically Modified Methylobacteriaceae Cell" of the present invention shall refer to such a genetically modified Methylobacteriaceae cell that is preferably similar to, particularly identical to, the wild-type strain of Methylobacteriaceae cell, except that there is at least one exogenous nucleic acid sequence encoding glyoxylate reductase derived from Escherichia coli. According to the present invention, the exogenous nucleic acid sequence of the expression vector or expression cassette that may be related is advantageously imparted to the Methylobacteriaceae cell of the present invention with the enzymatic activity of glyoxylate reductase, and optionally at least one exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase and the exogenous nucleic acid sequence of the expression vector or expression cassette that may be related.

[0161] For the purposes of the present invention, "a genetically modified Methylobacteriaceae cell" shall refer to one, two, several, multiple or an indefinite number of Methylobacteriaceae cells. In a preferred embodiment of the present invention, the Methylobacteriaceae cell is also referred to as a Methylobacteriaceae strain, particularly Methylobacterium extorquens strain, particularly Methylobacterium rhodesianum strain, particularly Methylobacterium zatmanii strain, particularly Methylobacterium extorquens TK 0001 strain, particularly Methylobacterium extorquens AM1 strain, particularly Methylobacterium extorquens PA1 strain, particularly Methylobacterium organophilum strain, particularly Methylobacterium radiotolerans strain.

[0162] For the purposes of the present invention, a "derivative" of a deposited Methylobacteriaceae cell or a deposited Methylobacteriaceae strain, particularly a deposited Methylorubrum strain or cell or a deposited Methylobacterium strain or cell, shall refer to a Methylobacteriaceae cell (particularly a Methylobacteriaceae strain), particularly a Methylorubrum cell (particularly a Methylorubrum strain) or a Methylobacterium cell (particularly a Methylobacterium strain), which has the characteristics according to the present invention, particularly integrates an exogenous nucleic acid sequence encoding glyoxylate reductase, is obtained from the deposited Methylobacteriaceae cell, and its genome is modified while retaining the characteristics of the present invention.

[0163] For the purposes of the present invention, an "Exogenous Nucleic Acid Sequence" of an organism, particularly a microorganism, especially a bacterium, shall refer to a nucleic acid sequence introduced into a recipient organism by recombination (i.e., genetic engineering).

[0164] For the present invention, in a preferred embodiment, the "foreign nucleic acid sequence" of an organism, particularly a microorganism, especially a bacterium, is derived from another microbial strain, particularly another organism, especially another bacterium, i.e., it is non-endogenous, so it refers to a non-native or non-wild-type strain or non-wild-type nucleic acid sequence.

[0165] For the present invention, "Glyoxylate Reductase" shall refer to an enzyme capable of enzymatically converting glyoxylate, particularly to glycolate, particularly using cofactors (especially NADH or NADPH).

[0166] For the present invention, in a preferred embodiment, the glyoxylate K M value of the "glyoxylate reductase (ghrA)" of the present invention is at most 2.0, particularly at most 1.5, particularly at most 1.0, particularly at most 0.6, particularly 0.6 mM.

[0167] For the present invention, in a preferred embodiment, the hydroxypyruvate K M value of the "glyoxylate reductase (ghrA)" of the present invention is at least 0.9, particularly at least 1.0, particularly 1.0 mM.

[0168] For the present invention, in a preferred embodiment, the glyoxylate K M value of the "glyoxylate reductase (ghrA)" of the present invention is at most 2.0, particularly at most 1.5, particularly at most 1.0, particularly at most 0.6, particularly 0.6 mM, and the hydroxypyruvate K M value is at least 0.9, particularly at least 1.0, particularly 1.0 mM.

[0169] For the present invention, in a preferred embodiment, the glyoxylate K M value of the "glyoxylate reductase (ghrA)" of the present invention is at most 2.0, and the hydroxypyruvate K M value is at least 0.9.

[0170] Glyoxylate reductase is preferably NADPH-dependent.

[0171] In contrast, the glyoxylate K M value of "hydroxypyruvate reductase (ghrB)" is at least 3.0, particularly at least 4.0, particularly at least 5.0, particularly at least 6.0, particularly at least 6.6, particularly 6.6 mM. In particular, the hydroxypyruvate K M value of "hydroxypyruvate reductase (ghrB)" is at most 0.6, particularly at most 0.7, particularly 0.7 mM.

[0172] In particular, the glyoxylate KM The value is at least 3.0, particularly at least 4.0, particularly at least 5.0, particularly at least 6.0, particularly at least 6.6, particularly 6.6 mM, for hydroxypyruvate K M The value is at most 0.6, particularly at most 0.7, particularly 0.7 mM.

[0173] Hydroxypyruvate reductase is preferably NADH-dependent.

[0174] Regarding the Michaelis-Menten Constant K M , which is defined as the substrate concentration at which a specific enzyme reaches half of its maximum conversion rate under fixed reaction conditions, and the calculation method is preferably the double reciprocal plot method (Lineweaver-Burk), see "Determination of the enzyme dissociation constants" by H. Lineweaver and D. Burk (1934), J. Am. Chem. Soc. Vol. 56, pp. 658–666.

[0175] For the purposes of the present invention, in one preferred embodiment, the "glyoxylate reductase" of the present invention exhibits higher enzyme activity in the NADPH-dependent conversion of glyoxylate to glycolate than in the NADH-dependent conversion of glyoxylate to glycolate, particularly at least 3-fold higher enzyme activity, especially under the conditions specified in the enzyme assay of Example 5.

[0176] For the purposes of the present invention, "Methylotrophic Bacterium cells" particularly refer to cells belonging to the family Methylobacteriaceae. In particular, such cells are capable of performing the serine cycle (https: / / doi.org / 10.1002 / 9781118960608.gbm02024; https: / / doi.org / 10.1111 / 1462-2920.12736; https: / / doi.org / 10.3389 / fmicb.2021.740610).

[0177] The serine cycle is a methylotrophic metabolic pathway that can assimilate C1 substrates, such as methanol, formate / formic acid, and methylamine, during microbial metabolism, thereby forming biomass or chemical products / intermediates of this metabolism, which is a series of defined enzymatic reactions.

[0178] This cycle starts with glycine. In the first step, glycine hydroxymethyltransferase (EC 2.1.2.1) generates the eponymous amino acid L-serine from C1 assimilated carbon (i.e., methanol, formic acid, etc.) in the form of 5,10-methylene tetrahydrofolate, water molecules, and glycine. In this process, tetrahydrofolate is decomposed for new carbon assimilation. Subsequently, L-serine is deaminated to hydroxypyruvate by transaminase in the serine cycle. The decomposed NH3 is used in equal amounts for the transamination of glyoxylate to glycine to maintain the operation of the cycle. The above hydroxypyruvate is reduced to glyceric acid by hydroxypyruvate reductase with NAD(P)H and then phosphorylated to 3-phosphoglyceric acid by kinase. In two consecutive reaction steps, 3-phosphoglyceric acid is converted to phosphoenolpyruvate by phosphoglycerate mutase (EC 5.4.2.11) and enolase (EC 4.2.1.11) with water cleavage. Phosphoenolpyruvate is carboxylated to oxaloacetate by phosphoenolpyruvate carboxylase (EC4.1.1.31) using bicarbonate / dissolved carbon dioxide. Finally, under the action of NADH, ATP, and cofactor A (CoA) molecules, phosphoenolpyruvate is converted to L-malyl-CoA through L-malate. Then, acetyl-CoA is cleaved to form glyoxylate. This reaction is completed by malyl-CoA lyase (EC 4.1.3.24). After the cycle ends, further assimilation of single carbon can begin. See C.W. Anthony (2011) "How half a century of research was required to understand bacterial growth on C1 and C2 compounds; the story of the serine cycle and the ethylmalonyl-CoA pathway", Science Progress, Volume 94, Part 2, pp. 109-137.

[0179] The serine cycle can be verified by the presence of the metabolite hydroxypyruvate. In addition, when conducting labeling studies using 13C-labeled C1 substrates and unlabeled CO 2 the labeling characteristics of glycine, serine, and glyoxylate can be determined (https: / / doi.org / 10.1186 / 1752-0509-5-189).

[0180] For the purposes of this invention, "M. extorquens" shall refer to Methylobacterium extorquens, "M. rhodesianum" shall refer to Methylobacterium rhodesianum, "M. zatmanii" shall refer to Methylobacterium zatmanii, "M. organophilum" shall refer to Methylobacterium organophilum, and "M. radiotolerans" shall refer to Methylobacterium radiotolerans.

[0181] For the purposes of the present invention, the term "pTE1887" shall refer to a specific expression vector.

[0182] For the purposes of the present invention, "ghrA eco " shall refer to a nucleic acid sequence encoding glyoxylate reductase derived from Escherichia coli K-12 MG1655. This nucleic acid sequence can be the native nucleic acid sequence (ghrA eco -nativ) or a codon-optimized nucleic acid sequence ("ghrA eco -c-optimiert").

[0183] For the purposes of the present invention, "pTE1887-ghrA eco " shall refer to an expression vector containing a nucleic acid sequence encoding glyoxylate reductase derived from Escherichia coli K-12 MG1655.

[0184] For the purposes of the present invention, "ecm mea " shall refer to a nucleic acid sequence encoding ethylmalonyl-CoA mutase derived from Methylobacterium extorquens TK 0001 DSM 1337. This nucleic acid sequence can be the native nucleic acid sequence or a codon-optimized nucleic acid sequence.

[0185] For the purposes of the present invention, "pTE1887-ghrA eco -ecm mea " shall refer to an expression vector containing a nucleic acid sequence encoding glyoxylate reductase derived from Escherichia coli K-12 MG1655 and a nucleic acid sequence encoding ethylmalonyl-CoA mutase derived from Methylobacterium extorquens TK 0001 DSM 1337.

[0186] For the purposes of the present invention, "ecm rsh " shall refer to a nucleic acid sequence encoding ethylmalonyl-CoA mutase derived from Rhodobacter sphaeroides ATCC 17029. This nucleic acid sequence can be the native nucleic acid sequence or a codon-optimized nucleic acid sequence.

[0187] For the purposes of the present invention, "pTE1887-ghrAeco-ecm rsh " shall refer to an expression vector containing a nucleic acid sequence encoding glyoxylate reductase derived from Escherichia coli K-12 MG1655 and a nucleic acid sequence encoding ethylmalonyl-CoA mutase derived from Rhodobacter sphaeroides ATCC 17029.

[0188] For the purposes of the present invention, "Functional Nucleic Acid Sequence Equivalent" shall refer to an equivalent nucleic acid sequence of a nucleic acid sequence encoding glyoxylate reductase or ethylmalonyl-CoA mutase, which equivalent nucleic acid sequence differs from said nucleic acid sequence by at least one difference at at least one nucleotide position, i.e. has at least one additional nucleotide (i.e. an inserted nucleotide) or at least one deleted nucleotide (i.e. a deleted nucleotide) or at least one substituted nucleotide, and which equivalent nucleic acid sequence encodes an amino acid sequence exhibiting the enzymatic activity of glyoxylate reductase or ethylmalonyl-CoA mutase. For the purposes of the present invention, "codon-optimised" shall mean that the nucleic acid sequence of a wild-type gene is integrated as a foreign nucleic acid sequence into a Methylobacterium host cell (especially derived from Escherichia coli), and before integration, is optimised by genetic engineering exchange of codons for expression (i.e. transcription and translation) in the host cell, especially those codons which are not normally used or are poorly used by the translation system of the host cell, i.e. Methylobacterium cells, especially Methylobacterium extorquens cells, in particular Methylobacterium extorquens AM1 cells, Methylobacterium extorquens TK 0001 cells, Methylobacterium extorquens PA1 cells. For example, by in vitro mutagenesis, the corresponding preferred codons of Methylobacterium are incorporated without changing the amino acid sequence encoded by the nucleic acid sequence. For the purposes of the present invention, a codon-optimised nucleic acid sequence is a nucleic acid sequence optimised for expression in Methylobacterium cells. If necessary, codon optimisation can also be carried out if the foreign nucleic acid sequence is derived from the same bacterial species as the host cell but still needs to be expressed. Preferably, codon optimisation can be carried out according to the following overview (Table 1):

[0189] For the purposes of the present invention, the "Functional Nucleic Acid Sequence Equivalent of a Codon-Optimised Nucleic Acid" is likewise but not limited to native nucleic acids (naturally occurring nucleic acids).

[0190] Table 1: Codon optimisation

[0191]

[0192] For the purposes of the present invention, "Functional Amino Acid Sequence Equivalent" shall refer to an equivalent amino acid sequence of an amino acid sequence encoding glyoxylate reductase or ethylmalonyl-CoA mutase, which amino acid equivalent sequence differs from said amino acid sequence by at least one difference at at least one amino acid position, i.e. has at least one additional amino acid (i.e. an inserted amino acid) or at least one deleted amino acid (i.e. a deleted amino acid) or at least one substituted amino acid, and which amino acid equivalent sequence exhibits the enzymatic activity of glyoxylate reductase or ethylmalonyl-CoA mutase.

[0193] For the purposes of the present invention, "Identity of Nucleic Acid or Amino Acid Sequences" shall mean the degree of identity (percentage) determined by sequence comparison. This sequence comparison is basically based on the BLAST algorithm established and commonly used in the prior art (see, for example, Altschul et al. (1990) "Basic local alignment search tool", Journal Of Molecular Biology, Vol. 215, pp. 403-410; Altschul et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Research, Vol. 25, pp. 3389-3402), the principle of which is to correlate similar nucleotides or amino acids in nucleic acid sequences or amino acid sequences. Associating relevant positions in tabular form is called alignment. Another algorithm available in the prior art is the FASTA algorithm. Computer programs are used to create sequence comparisons (alignments), especially multiple sequence alignments. For example, the Clustal series is commonly used (see, for example, Chenna et al. (2003) "Multiple sequence alignment with the Clustal series of programs", Nucleic Acids Research, Vol. 31, pp. 3497-3500), T-Coffee (see Z.B. Notredame et al. (2000) "T-Coffee: A novel method for multiple sequence alignments", Journal Of Molecular Biology, Vol. 302, pp. 205-217), or programs based on these programs or algorithms. Computer programs with specified standard parameters, such as Vector, can also be used. Sequence comparisons (alignments) were performed using the AlignX module in the Vector NTI Advance 10.3 (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California, USA), where the AlignX module for sequence comparison is based on ClustalW. The sequence identity referred to herein was determined using the NCBI Constraint-based Multiple Alignment Tool (COBALT) (https: / / www.ncbi.nlm.nih.gov / , as of January 26, 2022), with SEQ ID No: 1 to SEQ ID No: 8 used as the basis for determining the percentage of sequence differences, unless otherwise stated.

[0194] Such comparisons can also illustrate the similarity between the compared sequences. In this case, this similarity is given as a percentage of "identity", which is the proportion of identical nucleotides or amino acid residues at the same or corresponding positions in the alignment. Identity information can be obtained for the entire polypeptide or gene or only for individual regions. Thus, the identical regions of different nucleic acid or amino acid sequences are defined by the matching points in the sequences. These regions usually have the same function. These regions may be very small, containing only a few nucleotides or amino acids. The identity information in the present invention should refer to the total length of the given nucleic acid sequence or amino acid sequence, unless otherwise stated.

[0195] For the purposes of the present invention, "Amino Acid Sequence" shall mean a sequence of linearly linked amino acids, particularly a protein, especially a polypeptide.

[0196] For the purposes of the present invention, "Nucleic Acid Sequence" shall mean a sequence of linearly linked nucleotides, particularly a nucleic acid molecule, especially a gene, particularly the protein-coding region of a gene. In a particularly preferred embodiment, the nucleic acid sequence is a DNA sequence.

[0197] For the purposes of the present invention, "Ethylmalonyl-CoA-Mutase" shall mean an enzyme that is dependent on coenzyme B12, has intramolecular isomerase activity, and is responsible for converting ethylmalonyl-CoA to methylsuccinyl-CoA in the metabolism of ethylmalonyl-CoA, and the EC classification is preferably EC 5.4.99.63.

[0198] For the purposes of the present invention, "Cx-Compound" shall mean a carbon (C), hydrogen (H), and oxygen (O) compound containing x carbon atoms, where x is preferably a natural number. According to the present invention, preferably x = 1, 2, or 4, especially 1. The Cx-compound preferably contains only C, H, and O atoms and no other atoms.

[0199] For the purposes of the present invention, formic acid also refers to formate, acetic acid also refers to acetate, and succinic acid also refers to succinate.

[0200] For the purposes 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" shall mean that the nucleic acid sequence is present in the genome of the cell in chromosomal or extrachromosomal form (preferably chromosomal form).

[0201] In a preferred embodiment, the exogenous nucleic acid sequence is stably integrated, where stable integration of the nucleic acid shall mean integration that can still be detected and is capable of being expressed in at least 2, 3, 5, 10, 20, or 50 generations of the microorganism.

[0202] For the purposes of the present invention, "maximum growth rate (μ max )" shall mean the cell division rate of the Methylobacteriaceae cells of the present invention in a reaction substrate, particularly a liquid substrate, i.e., the microorganism growth rate. The calculation of μ max is based on the optical density measurement values of the culture medium at a wavelength of 600 nm measured with a photometer (OD 600 ) over the time course of the process step. The calculation of μ max can use Equation 1, taking into account the OD 600 measurement values within the fastest growth interval observed.

[0203]

[0204] In Equation 1, t y –t x is the time interval of the fastest growth interval observed, where t y >t x . This time interval is usually in hours (h). For the purposes of the present invention, "Biological Dry Matter - Substrate - Yield (Y X / S )" shall mean the mass of microbial dry biomass (completely dried to constant weight) that can be formed by a specific microbial strain in a reaction substrate, particularly a liquid substrate, using one gram of Cx compound (S, g Cx ), expressed in weight units such as grams (X, g X ). According to Equation 2, it is calculated through the linear regression graph of the change in dry biomass measurement values (ΔX(t)) and the mass of Cx compound (ΔCx(t)) as a function of time. According to Equation 2, Y X / S is the slope of the linear change of dry biomass X as a function of the change in the mass of Cx compound over time. Y X / S is usually in units of g X / g Cx .

[0205]

[0206] For the present invention, "Product-Substrate-Yield (Y P / S )" shall refer to the mass of the product formed by a specific microbial strain using one gram of Cx compound (S, g Cx ), expressed in equal weight units such as grams (P, g P ). According to Equation 3, it is calculated through the linear regression graph of the change in the measured value of the product mass (ΔP(t)) and the change in the mass of the Cx compound (ΔCx(t)) as a function of time. According to Equation 3, Y P / S is the slope of the linear change of the product mass P as a function of the change in the mass of the Cx compound over time. Y P / S is usually in units of g P / g Cx .

[0207]

[0208] For the present invention, "Product-Biological Dry Matter-Yield (Y P / X )" refers to the mass of the product per gram of dry biological matter (X, g X ) that can be formed by a specific microbial strain during the growth of the microorganism, expressed in equal weight units such as grams (P, g P ). According to Equation 4, it is calculated through the linear regression graph of the change in the measured value of the product mass (ΔP(t)) and the dry biological matter (ΔX(t)) as a function of time. According to Equation 4, Y P / X is the slope of the linear change of the product mass P as a function of the change in the dry biological matter formed over time. Y P / X is usually in units of g P / g X .

[0209]

[0210] For the present invention, "dry biological matter" shall refer to the mass X(t) of the dry biological matter of the microorganism (the biological matter is completely dried to a constant weight) in the reaction substrate, especially in the liquid culture medium, with a volume of v(t) at time t, expressed in equal weight units such as grams (X, g(X)). The dry biological matter X can be determined from the OD 600 measurement value and the correlation coefficient z, where z = 0.305 g X / 1 OD 600 .

[0211] X(t) = 0.305 * OD 600 (t) * v [gX (Formula 5)

[0212] For the purposes of the present invention, the abbreviation "NAD" shall refer to nicotinamide adenine dinucleotide. For the purposes of the present invention, "NADH" shall refer to the reduced form of NAD. For the purposes of the present invention, the abbreviation "NADP" shall refer to nicotinamide adenine dinucleotide phosphate. For the purposes of the present invention, "NADPH" shall refer to the reduced form of NADP. For the purposes of the present invention, "NADH / NADPH analog" shall refer to a chemical compound, such as thio-nicotinamide adenine dinucleotide (S-NAD), nicotinic acid adenine dinucleotide (O-NAD), nicotinamide hypoxanthine dinucleotide (NHD), nicotinyl guanine dinucleotide or other compounds having activity similar to, preferably identical to, NADH and / or NADPH.

[0213] For the purposes of the present invention, "reactant" shall refer to a starting material, in particular at least one Cx compound, in particular a Cx compound or two or more Cx compounds, in particular a composition of Cx compounds.

[0214] For the purposes of the present invention, "product" shall refer to at least glycolic acid, in particular solely glycolic acid, preferably glycolic acid and lactic acid, in particular a composition of compounds containing glycolic acid, in particular a compound composed of glycolic acid and lactic acid.

[0215] For the purposes of the present invention, "reaction" shall refer to a chemical reaction, in particular a chemically catalyzed reaction, in particular an enzyme-catalyzed reaction.

[0216] For the purposes of the present invention, "reaction matrix" shall refer to a liquid matrix, in particular a liquid aqueous matrix, in which a reaction can occur, in particular an enzyme-catalyzed reaction, in particular a reaction caused by a microorganism or a microbial component, in particular a culture medium, in particular a basal medium.

[0217] For the purposes of the present invention, "obtaining the product" shall mean that the product obtained by reacting the reactant (i.e., the starting material) in the previous process step can be obtained from the corresponding reaction matrix, in particular a culture medium or a solvent, in particular isolated therefrom. In particular, obtaining the product means concentrating the product, in particular isolating the product. The processes employed for this purpose can be physical, chemical and / or biological processes.

[0218] For the purposes of the present invention, "compound" shall refer to one molecule or several identical molecules.

[0219] For the purposes of the present invention, the composition containing glycolic acid is the product of the reaction in process step (b) of the present invention.

[0220] If the context of the present invention specifies quantitative information (especially percentages) of certain components of a product or composition, such information, together with other components of the composition or product that are explicitly stated or clear to those skilled in the art, totals 100% of the composition and / or product, unless otherwise explicitly stated or denied by those skilled in the art.

[0221] For the purposes of the present invention, the term "at least one" shall mean a number represented by 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" may specifically represent the number 1. In a more preferred embodiment, the term "at least one" may also represent the numbers 2 or 3 or 4 or 5 or 6 or 7.

[0222] If the context of the present invention explicitly or implicitly indicates the "presence", "comprising", "having" or "containing" of a certain component, it means the presence of the corresponding component, especially in a measurable amount.

[0223] If the context of the present invention explicitly or implicitly indicates that the "presence", "comprising" or "containing" of a certain component is 0 [unit], especially mg / kg, μg / kg or wt.%, it means that the corresponding component does not exist within the measurable range, especially does not exist at all.

[0224] The specified decimal places correspond to the precision of the measurement method used.

[0225] For the purposes of the present invention, if the first and second decimal places or the second decimal place are specified in a number, they are set to zero.

[0226] For the purposes of the present invention, the term "and / or" shall mean that all members of a group connected by the phrase "and / or" can be substituted for each other and can also be combined arbitrarily with each other. For the expression "A, B and / or C", it should be understood as: (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.

[0227] For the purposes of the present invention, the terms "include / includes / including" and "have / has / having" shall mean that there may be other elements not explicitly covered in addition to the elements explicitly covered by these terms. For the purposes of the present invention, these terms may also mean that only the explicitly covered elements are included and there are no other elements. In a specific embodiment of the present invention, the terms "include" and "have" are synonymous with the term "consist of". In addition, the terms "include" and "have" also cover compositions that include, in addition to the explicitly covered elements, other elements that are functionally and qualitatively subordinate although not mentioned. In an embodiment of the present invention, the terms "include" and "have" are synonymous with the term "consist essentially of".

[0228] The institutional name "German Collection of Microorganisms and Cell Cultures DSMZ, Braunschweig, Germany" represents "Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstraβe 7B, 38124 Braunschweig".

[0229] For more preferred embodiments of the present invention, please refer to the dependent claims.

[0230] The present invention will be described in detail below with reference to the accompanying drawings and examples, but the general concept of the present invention is not limited.

[0231] In the sequence listing:

[0232] SEQ ID No:1 represents the native nucleic acid sequence encoding glyoxylate reductase derived from Escherichia coli (K-12 MG1655), especially also called ghrA eco -nativ, that is, a functionally equivalent nucleic acid sequence of the nucleic acid sequence of SEQ ID No:3.

[0233] SEQ ID No:2 represents the amino acid sequence encoded by SEQ ID No:1 and SEQ ID No:3.

[0234] SEQ ID No:3 represents the Methylobacteriaceae codon-optimized nucleic acid sequence (ghrA eco -c-optimised) of the native nucleic acid sequence of SEQ ID No:1 encoding glyoxylate reductase derived from Escherichia coli (K-12 MG1655).

[0235] SEQ ID No:4 represents the native nucleic acid sequence encoding ethylmalonyl-CoA mutase derived from Methylobacterium extorquens (TK 0001 DSM 1337), especially also called ecm mea , that is, a functionally equivalent nucleic acid sequence of the codon-optimized nucleic acid sequence of SEQ ID No:13.

[0236] SEQ ID No:5 represents the amino acid sequence encoded by SEQ ID No:4 and SEQ ID No:13.

[0237] SEQ ID No:6 represents the native nucleic acid sequence encoding ethylmalonyl-CoA mutase derived from Rhodobacter sphaeroides (ATCC 17029), especially also called ecm rsh , that is, a functionally equivalent nucleic acid sequence of the codon-optimized nucleic acid sequence of SEQ ID No:8.

[0238] SEQ ID No:7 represents the amino acid sequence encoded by SEQ ID No:6 and SEQ ID No:8.

[0239] SEQ ID No:8 represents the Methylobacteriaceae codon-optimized nucleic acid sequence of the native nucleic acid sequence of SEQ ID No:6, which encodes ethylmalonyl-CoA mutase derived from Rhodobacter sphaeroides (ATCC 17029).

[0240] SEQ ID No:9 represents the nucleic acid sequence of the expression vector pTE1887. For the relevant plasmid map, see Figure 8 。

[0241] SEQ ID No:10 represents the nucleic acid sequence of the expression vector pTE1887-ghrA eco For the relevant plasmid map, see Figure 9 。

[0242] SEQ ID No:11 represents the nucleic acid sequence of the expression vector pTE1887-ghrA eco -ecm mea For the relevant plasmid map, see Figure 10 。

[0243] SEQ ID No:12 represents the nucleic acid sequence of the expression vector pTE1887-EcoGoxRed_1-ecm rsh For the relevant plasmid map, see Figure 11 。

[0244] SEQ ID No:13 represents the Methylobacteriaceae codon-optimized nucleic acid sequence of the native nucleic acid sequence of SEQ ID No:4, which encodes ethylmalonyl-CoA mutase derived from Methylobacterium extorquens (TK 0001 DSM 1337). BRIEF DESCRIPTION OF THE DRAWINGS

[0245] Figure 1 Shows the screening results of glycolic acid production in the recombinant (i.e., transgenic) Methylobacterium extorquens TK 0001 strain, which contains and expresses the codon-optimized glyoxylate reductase gene (A), and also shows the screening results according to 1A (B and C), where according to Figure 1 (A), in the background of the M. extorquens TK 0001 strain, shows the enzyme activity of glyoxylate reductase in the biomass expressed by the expression vector pTE1887, using NADH (B) and NADPH (C) as cofactors;

[0246] Figure 2Shows the HPLC chromatogram comparison of a culture sample (22 to 24 hours after induction) of the transgenic Methylobacteriaceae cell M. extorquens TK 0001 glyoxylate reductase strain, which contains and expresses the codon-optimized glyoxylate reductase gene;

[0247] Figure 3 Shows the gas chromatography-mass spectrometry (GC-MS) chromatogram of the glyoxylate peak (retention time 7.22 minutes) of a 100 mg L -1 glycolate standard, the reaction matrix sample at time t = 0 h, the culture sample after induction of M. extorquens TK 0001 + pTE1887 vector, and the culture sample of the present invention + pTE1887-ghrA eco -c-optimized (M. extorquens GA1) after 22 to 24 hours of induction;

[0248] Figure 4 Shows the gas chromatography-mass spectrometry (GC-MS) chromatogram of the lactate peak (retention time 6.88 minutes) of a 100 mg L -1 lactate standard, the reaction matrix sample at time t = 0 h, the culture sample after 22 - 24 hours of induction of M. extorquens TK 0001 + pTE1887 vector, and the culture sample of the present invention M. extorquens TK 0001 + pTE1887-ghrA eco -c-optimized (M. extorquens GA1) after 22 to 24 hours of induction;

[0249] Figure 5 Shows the mass spectrometry detail diagrams of the glyoxylate peak (A) and lactate peak (B) of the culture sample of M. extorquens GA1 after 22 to 24 hours of induction, as well as the database references for glyoxylate identity (A) and lactate identity (B) in the M. extorquens GA1 sample;

[0250] Figure 6 Shows the growth process (OD600), pH value, and methanol concentration, glyoxylate concentration, glycolate concentration, and lactate concentration of M. extorquens TK 0001 + pTE1887 (A + C) and the present invention M. extorquens TK 0001 + pTE1887-ghrA eco -c-optimized (M. extorquens GA1) (B + D) in the reaction matrix (i.e., the basal medium), where 8 g L -1 methanol (A + B) or 9 g L -1 methanol + 1.5 g L -1Glyoxylic acid (C+D) as a carbon source;

[0251] Figure 7 Shows the reaction medium with 9 g L -1 M. extorquens TK 0001 + pTE1887(A), M. extorquens TK 0001 + pTE1887-ghrA of the present invention eco -c-optimized (M. extorquens GA1)(B), M. extorquens TK 0001 + pTE1887-ghrA of the present invention eco -c-optimized-ecm mea (M. extorquens GA2)(C) and M. extorquens TK 0001 + pTE1887-ghrA of the present invention eco -c-optimized-ecm rsh (M. extorquens GA3)(D) in terms of growth process (OD600), pH value, methanol concentration, and glyoxylic acid and lactic acid concentrations;

[0252] Figure 8 Shows the plasmid map of the expression vector pTE1887;

[0253] Figure 9 Shows the expression vector pTE1887-ghrA eco -c-optimized plasmid map;

[0254] Figure 10 Shows the expression vector pTE1887-ghrA eco -c-optimized-ecm mea plasmid map;

[0255] Figure 11 Shows the expression vector pTE1887-ghrA eco -c-optimized-ecm rsh plasmid map;

[0256] Figure 12 Shows the ghrA in the native DNA sequence and codon-optimized DNA sequence expressed by the expression vector pTE1887 in the background of M. extorquens TK 0001 strain eco and ghrB eco glyoxylic acid reductase activity test results;

[0257] Figure 13Shows the taxonomic classification of methylotrophic microorganisms tested with the expression vectors of the present invention;

[0258] Figure 14 Shows the screening results of the production of glycolate and lactate 22 to 28 hours after induced gene expression in recombinant (i.e., transgenic) M. rhodesianum DSM 5687 strains, which contain and express the codon-optimized glyoxylate reductase gene, and some strains also contain and express the ethylmalonyl-CoA mutase gene;

[0259] Figure 15 Shows the screening results of the production of glycolate and lactate 22 to 28 hours after induced gene expression in recombinant (i.e., transgenic) M. zatmanii DSM 5688 strains, which contain and express the codon-optimized glyoxylate reductase gene of the present invention, and one strain also contains and expresses the codon-optimized ethylmalonyl-CoA mutase gene derived from Rhodobacter sphaeroides ATCC 17029;

[0260] Figure 16 Shows the screening results of the production of glycolate and lactate 22 to 28 hours after induced gene expression in recombinant (i.e., transgenic) M. radiotolerans DSM 760 strains, which contain and express the combination of the codon-optimized glyoxylate reductase gene derived from Escherichia coli and the ethylmalonyl-CoA mutase gene originally derived from M. extorquens TK 0001 DSM 1337;

[0261] Figure 17 Shows the screening results of the production of glycolate and lactate 22 to 28 hours after induced gene expression in recombinant (i.e., transgenic) M. organophilum DSM 18172 strains, which contain and express the codon-optimized glyoxylate reductase gene, and some strains also contain and express the ethylmalonyl-CoA mutase gene;

[0262] Figure 18 Shows the screening results of the production of glycolate and lactate 22 to 28 hours after induced gene expression in recombinant (i.e., transgenic) M. extorquens PA1 DSM 23939 strains, which contain and express the codon-optimized glyoxylate reductase gene derived from Escherichia coli K12 1655 of the present invention;

[0263] Figure 19 Shows the screening results of the production of glycolate and lactate 22 to 28 hours after induced gene expression in recombinant (i.e., transgenic) M. extorquens AM1Δcel strains (based on DSM 1338 strains), which contain and express the codon-optimized glyoxylate reductase gene. Detailed implementation mode

[0264] Example 1: Preparation of transgenic Methylobacteriaceae cells

[0265] By 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 relevant DNA sequences and amino acid sequences were extracted. Only glyoxylate reductases present in prokaryotes or Saccharomyces cerevisiae were considered. In addition, the native glyoxylate reductase derived from Methylobacterium extorquens TK 0001 was also selected. Table 2 summarizes the 13 selected glyoxylate reductases. In particular, the glyoxylate reductase derived from Thermococcus kodakarensis was identified as an NADH-dependent enzyme (Ohshima et al., European Journal of Biochemistry, Vol. 268(17), 2001, pp. 4740-4747). The effect of specific redox equivalents on glycolate production can be significant, depending on the availability of specific redox equivalents in the cytoplasm and the adaptability of the metabolic network to the intervention (overexpression of glyoxylate reductase).

[0266] Table 2: Overview of tested enzymes

[0267]

[0268]

[0269] Heterologous enzymes derived from Pseudomonas fluorescens Pf0-1, Thermococcus kodakarensis, Pyrococcus furiosus DSM 3638, Saccharomyces cerevisiae, Thermus thermophilus HB27, Escherichia coli K-12 MG1655, and Acetobacter were encoded by synthetic genes in the codon-optimized form of Methylobacteriaceae (BioCat GmbH, Heidelberg, Germany, Table 1) to support optimal gene expression. The homologous gene derived from Methylobacterium extorquens (SEQ ID No:1) had the start codon "TTG", so the start codon was changed to "ATG" by PCR (M. Kozak, Gene, Vol. 234(2), 1999, pp. 187-208). Subsequently, two gene variants of SEQ ID No:1 and SEQ ID No:3 were tested. In this way, a total of 14 glyoxylate reductase variants were tested.

[0270] In promoter P L / O4 / A1Under the control of an (IPTG-inducible promoter), the synthetic gene was cloned in codon-optimized form into the episomal expression vector pTE1887 (M. Carrillo et al., ACS Synthetic Biology, 2019, Vol. 8(11), pp. 2451-2456) by means of Gibson assembly technology. See Figure 8 . Figure 8 The vector shown has the following elements: the lacI gene, the lacI promoter, the PL / O4 / A1 promoter → -33 region → -10 region → transcription start point, the PL / O4 / A1 promoter ribosome binding site (RBS), the Lambda T0 terminator, kanamycin resistance, the mobilization genes mobS and mobL, the regulatory protein RepA, and the replication origin colE1.

[0271] In this cloning process, the expression vector was cut with the restriction enzyme NcoI. The sequence identity and correctness of the construct were ensured by sequencing. Subsequently, the resulting construct and wild-type strain of Methylobacteriaceae cells, especially Methylobacterium extorquens TK 0001 cells, particularly Methylobacterium extorquens PA1, were provided according to process step (a), transformed into Methylobacteriaceae cells by means of electroporation technology according to process step (b), and transgenic Methylobacteriaceae cells were obtained according to process step (c). Kanamycin was used as a selection marker, and Methylobacteriaceae cell clones, i.e., transgenic Methylobacteriaceae cells carrying the individually produced construct containing the codon-optimized synthetic gene, were selected on minimal medium agar plates. The presence of the expression vector and the expected sequence sizes of the PCR products representing the cloned genes in each of the obtained clones were verified by colony PCR. The verified strains were cryopreserved at -80 °C.

[0272] To test the ability of the transgenic Methylobacterium strains to produce glyoxylate, the strains were cultured in a shake flask (250 mL flask, 50 mL culture) at 150 RPM (revolutions per minute) in a steam-saturated atmosphere at 30 °C with minimal medium as the reaction substrate (especially also referred to as the culture medium) and a Cx compound with x = 1 (i.e., methanol) as the reactant (L.-Y. Cui et al., Biochemical Engineering Journal, 2017, Vol. 119, pp. 67-73) (process step (x) of the present invention), and glyoxylate-containing products were obtained in the reaction substrate (process step (z)). Similarly, cultivation can also be carried out with formic acid, which can also be used as a reactant for producing glyoxylate. TM Innova44, Eppendorf AG, Hamburg, Germany), and glyoxylate-containing products were obtained in the reaction substrate (process step (z)). Similarly, cultivation can also be carried out with formic acid, which can also be used as a reactant for producing glyoxylate.

[0273] When inoculating the main culture, start with a preculture grown under the same conditions (final OD600 (between 3 and 5), starting OD 600 is 0.05. After the culture OD 600 reaches 1.0, gene expression of the codon-optimized glyoxylate reductase gene is induced with 1 mM IPTG (final concentration in the culture). To detect the production of glycolic acid, 1 mL of sample is extracted from the culture broth before inoculation, and 1 mL of sample is extracted from the cultures at approximately 20 hours before induction, at the start of induction, and after induction. After centrifuging the biomass from the reaction matrix, the concentrations of methanol, formic acid, glyoxylate, glycolic acid, and lactic acid in the samples are analyzed using high-performance liquid chromatography (HPLC) and refractive index detection (RID). Using Synergi TM 4 μm Hydro-RP80A, 250 x 4.6 mm LC column (Phenomenex Inc., Torrance, CA, USA) and 20 mM K 2 HPO 4 (pH 1.5) as the eluent, HPLC measurements are performed at a temperature of 30 °C and a flow rate of 0.5 mL min -1 for 20 minutes for each sample to separate the analytes. External standard method is used to identify and quantify the analytes at known concentrations.

[0274] Using a glycolic acid standard (100 mg L -1 ), glycolic acid in the culture samples is clearly detected by gas chromatography-mass spectrometry (GC-MS). For this purpose, the -OH or -NH groups contained in the culture samples and the standard are converted into the corresponding tert-butyldimethylsilyl ethers (TBDMS) by derivatization. For this, 50 μL of the standard or 50 μL of the sample is lyophilized and then resuspended in 50 μL of DMF + 0.1% (v / v) pyridine. Derivatization is carried out using 50 μL of N-methyl-N-tert-butyldimethylsilyltrifluoroacetamide (MBDSTFA, Macherey-Nagel) and incubated at 80 °C for 30 minutes. The precipitate is removed by centrifugation, and then the samples are analyzed by gas chromatography-mass spectrometry. The carrier gas flow rate for gas chromatography is 1.7 mL min -1 , the inlet temperature is 250 °C, the interface temperature is 230 °C, and the quadrupole temperature is 150 °C. Temperature gradient is used to separate the analytes: 120 °C (2 minutes), rising to 200 °C at 8 °C min -1 and then rising to 325 °C at 10 °C min -1 . The analytes are identified by MS scan mode (m / z 50 - 750).

[0275] The genetically modified Methylobacteriaceae cells contain an exogenous codon-optimized nucleic acid sequence (SEQ ID No: 3) encoding glyoxylate reductase from Escherichia coli K-12 MG1655 of Methylobacterium extorquens Mea-GA1 strain, which was deposited at the DSMZ - German Collection of Microorganisms and Cell Cultures in Braunschweig, Germany on June 10, 2022, with the deposit accession number DSM 34286.

[0276] Example 2: Screening for functional glyoxylate reductase in Methylobacterium extorquens TK 0001

[0277] As described in Example 1, the codon-optimized nucleic acid sequences encoding glyoxylate reductase listed in Table 2 were cloned into the pTE1887 expression vector, and the corresponding genetically modified Methylobacteriaceae strains were constructed. The Methylobacterium extorquens TK0001 strain containing pTE1887-Vektor was used as a reference strain, which does not carry a recombinant plasmid but carries pTE1887-Leervektor.

[0278] In the first experiment, as described in Example 1, the ability of these preliminarily constructed strains to produce glycolic acid was examined, and the results are shown in Figure 1 .

[0279] Figure 1 In the bar graph shown in A, the x-axis represents the genetically modified Methylobacteriaceae cells, and the y-axis represents the concentration of glycolic acid in the reaction substrate (black solid bars), in g L -1 units. All sampling times were 22 to 24 hours after induction of gene expression with 1 mM IPTG. To determine the methanol uptake, the reaction substrate was measured at t = 0 h. All concentrations were in g L -1 units and were determined by HPLC, refractive index detection, and the external standard method.

[0280] Figure 1 A shows the screening results of glycolic acid production in the recombinant Methylobacterium extorquens TK 0001 strain expressing glyoxylate reductase, based on the corresponding codon-optimized gene. Using pTE1887 as the expression vector, it can also be used as a negative control in the form of an empty vector of the reference strain M.extorquens TK 0001 + pTE1887 (the first bar from the left on the x-axis).

[0281] Surprisingly, neither the reference strain M.extorquens TK 0001 + pTE1887 (the first bar from the left) nor the genetically modified Methylobacteriaceae cells showed glycolic acid production (the second, third, and fifth to fifteenth bars from the left), with the exception of the genetically modified Methylobacteriaceae cells of the present invention, M.extorquens TK 0001 + pTE1887-ghrA eco(Codon-optimized nucleic acid form of SEQ ID No: 3), that is, the transgenic Methylobacteriaceae cells of the present invention contain at least one exogenous nucleic acid sequence encoding glyoxylate reductase derived from bacteria of the genus Escherichia (the fourth from the left, the only entry showing a black bar). Figure 9 Shows the vector map for generating these Methylobacterium cells, with the following elements: lacI gene, lacI promoter, PL / O4 / A1 promoter → -33 region → -10 region → transcription start point, PL / O4 / A1 promoter ribosome binding site (RBS), ghrA eco -c-optimized, LambdaT0 terminator, kanamycin resistance, mobilization genes mobS and mob, regulatory protein RepA, replication origin colE1.

[0282] Figure 1 In the bar graph shown in B, the x-axis represents transgenic Methylobacteriaceae cells, and the y-axis represents enzyme activity (white hollow bars: NADH as cofactor), in mUmg -1 per unit.

[0283] Figure 1 In the bar graph shown in C, the x-axis represents transgenic Methylobacteriaceae cells, and the y-axis represents enzyme activity (gray solid bars: NADPH as cofactor), in mU mg -1 per unit.

[0284] Figure 1 B and Figure 1 C show the screening results of enzyme assays of recombinant Methylobacterium organophilum TK 0001 strains expressing glyoxylate reductase, based on the corresponding codon-optimized genes. The enzyme assay is similar to Example 5. The biomass used is the same as in 1A. Figure 1 In the case of B, the enzyme assay was performed with NADH as the redox cofactor. Figure 1 In the case of C, the enzyme assay was performed with NADPH as the redox cofactor.

[0285] Figure 1 In the case of B, all tested Methylobacteriaceae cells containing recombinant glyoxylate reductase did not show measurable glyoxylate reductase activity with NADH as cofactor, except for Methylobacteriaceae cells containing glyoxylate reductase ghrB eco of the present invention (the fifth from the left).

[0286] Figure 1In C, the reference strain M. extorquens TK 0001 + pTE1887 (the first from the left) and several tested Methylobacteriaceae cells containing recombinant glyoxylate reductase (the second, eighth, ninth, eleventh to fifteenth from the left) did not show measurable glyoxylate reductase activity when using NADPH as a cofactor. Only the transgenic Methylobacteriaceae cells (the third to seventh and tenth from the left) showed enhanced glyoxylate reductase activity, and the transgenic Methylobacteriaceae cells of the present invention were derived from M. extorquens TK 0001 + pTE1887-ghrA eco (In the codon-optimized nucleic acid form according to SEQ ID No: 3, that is, the transgenic Methylobacteriaceae cells of the present invention contain at least one exogenous nucleic acid sequence encoding glyoxylate reductase derived from Escherichia bacteria and show high glyoxylate reductase activity (the fourth from the left).

[0287] The enzyme activity of glyoxylate reductase Tlit could not be measured and was not related to the production of glycolic acid. Only ghrA eco (that is, the glyoxylate reductase of the present invention derived from Escherichia coli) has an enzyme activity related to the production of glycolic acid.

[0288] Therefore, the transgenic Methylobacteriaceae cells of the present invention contain M. extorquens TK 0001 + pTE1887-ghrA eco -c-optimized, so it shows NADPH-dependence and does not show NADH-dependence.

[0289] Figure 2 Shows according to Figure 1 (22 to 24 hours after induction) the HPLC chromatogram of the culture of the transgenic Methylobacteriaceae cell M. extorquens TK 0001 glyoxylate reductase strain. It can be clearly seen that only the transgenic Methylobacteriaceae cell M. extorquens TK 0001 + pTE1887-ghrA of the present invention eco ( Figure 2 referred to as M. extorquens GA1 in eco and containing the codon-optimized form of the ghrA eco gene) is the only strain of the glycolic acid and lactic acid mixture (glycolic acid retention time = 6.20 minutes, lactic acid retention time = 9.1 minutes) (control standard - the first and second lanes - M. extorquens TK 0001 + pTE1887-ghrA eco ), M. extorquens GA1- Figure 1 the seventh lane in A and Figure 1The fifth in B). When using glyoxylate reductase other than the present invention, the production of glycolate (and lactate) is insufficient, indicating a lack of functionality. These enzymes may be hydroxypyruvate reductases that reduce hydroxypyruvate produced in the serine cycle to D-glycerate according to NAD(P)H. In this case, as Figure 1 and Figure 2 shown, no accumulation of glycolate is observed.

[0290] Compared with the external standard method, samples were measured by gas chromatography-mass spectrometry (GC-MS) to confirm the presence of glycolate and lactate in the samples, thereby verifying the production of glycolate and prominent lactate due to the expression of the ghrA eco enzyme. Other peaks: glyoxylate (retention time = 5.40 minutes), methanol (retention time = 7.6 minutes), peaks not described herein (retention time = 6.50 minutes and 8.00 minutes).

[0291] In the transgenic Methylobacteriaceae cells of the present invention, HPLC measurement can detect a mixture of about 0.6 g L -1 of glycolate and lactate (Y P / S ~77 mg 乙醇酸+乳酸 g 甲醇 -1 ).

[0292] During most cultivation processes, the initial methanol concentration of 8 g L -1 is depleted about 22 to 24 hours after induction. Only the Methylobacterium extorquens strain GA1 showed a significantly measurable methanol concentration at the time of sampling ( Figure 1 lane 7 in A and Figure 1 lane 5 in B). This may indicate that the gene expression of glyoxylate reductase leads to metabolic imbalance. On the one hand, the enzyme expression itself slows down the strain growth. However, the enhanced enzyme activity of glyoxylate reductase also fundamentally causes glyoxylate to withdraw from the serine cycle and form glycolate. Therefore, the microorganism lacks glyoxylate to build biomass. This lack slows down growth and results in insufficient utilization of the carbon source.

[0293] To compare with the external standard method (100 mg L -1 each of lactate and glycolate), to confirm the presence of glycolate and lactate in the ghrA eco samples of the present invention, as described in Example 1, samples were examined by gas chromatography-mass spectrometry ( Figures 3 to 5 ).

[0294] Figure 3 shows 100 mg L -1Glycolic acid standard, matrix sample at time t = 0 h, sample after culturing for 22 to 24 hours after induction of M. extorquens TK0001 + pTE1887 - vector, and M. extorquens TK 0001 + pTE1887 - ghrA of the present invention eco ( Figure 3 referred to as M. extorquens GA1 in Figure 3 , containing the gene codon - optimized form of ghrA eco ) Gas chromatography - mass spectrometry (GC - MS) chromatogram of the sample after culturing for 22 to 24 hours after induction Figure 4 Shows the same samples, except for the standard, which is replaced by 100 mg L -1 Lactic acid standard. The measurement results prove that, compared with the glycolic acid standard (retention time = 7.22 minutes), in the culture sample of M. extorquens TK 0001 + pTE1887 - ghrA eco (M. extorquens GA1) of the present invention, glycolic acid was significantly formed. The peak mass spectrum obtained from the sample of M. extorquens TK 0001 + pTE1887 - ghrA eco of the present invention is significantly consistent with the mass spectrum of the glycolic acid standard( Figure 5 A). This proves the presence of glycolic acid in the sample of M. extorquens TK 0001 + pTE1887 - ghrA eco of the present invention, so this strain produces glycolic acid. In contrast, no glycolic acid was detected in the sample of the M. extorquens TK 0001 + pTE1887 - vector strain. Unexpectedly, it was also found that in the culture sample of M. extorquens TK 0001 + pTE1887 - ghrA eco of the present invention, lactic acid (retention time = 6.88 minutes) was formed. The mass spectrum here is also consistent with the mass spectrum of the lactic acid standard( Figure 5 B)

[0295] This process can clearly show that the transgenic Methylobacterium cell M. extorquens TK 0001 + pTE1887 - ghrA eco of the present invention produces glycolic acid. Unexpectedly, this strain can also produce a mixture of glycolic acid and lactic acid (see Figure 5 ). Figure 5 Shows a comparison of M. extorquens TK 0001 + pTE1887 - ghrA eco ( Figure 5The detailed mass spectrometry image of the same sample after induction by M. extorquens GA1 (containing the codon-optimized form) for 22 to 24 hours. Database records indicate the presence of glycollate identity (A) in the M. extorquens GA1 sample and the presence of lactate identity (B) in the M. extorquens GA1 sample.

[0296] Surprisingly, during the cultivation of M. extorquens TK 0001 + pTE1887-ghrA eco glycolic acid (retention time = 7.22 minutes) and lactic acid (retention time = 6.88 minutes) can be detected according to gas chromatography-mass spectrometry (GC-MS) ( Figures 3 to 5 ). By external standard comparison and database mass spectrometry matching, the chromatographic peak with a retention time of 6.88 minutes in this sample is lactic acid 2xTBDMS (a lactic acid derivative containing MBDSTFA), with a probability of 89% to 91% ( Figure 5 B).

[0297] This phenotype was not observed in the control strain M. extorquens TK 0001 + pTE1887: no products of glycolic acid or lactic acid were detected by gas chromatography-mass spectrometry (GC-MS).

[0298] Without being bound by theory, changes in the redox balance can alter the metabolism of the transgenic Methylobacterium cell M. extorquens TK 0001 + pTE1887-ghrA of the present invention eco so as to synthesize lactic acid as a possible by-product of glycolic acid production. The NADH-dependent lactate dehydrogenase (KEGG database: Mex_1p4794) using pyruvate as a substrate may be the cause of this lactic acid formation. Another possibility is that glyoxylate reductase has a non-specific substrate effect, allowing the enzyme to use pyruvate as an acceptor. In principle, the methylglyoxal metabolic pathway can also be envisaged.

[0299] This indicates that the M. extorquens TK 0001 + pTE1887-ghrA eco cells of the present invention contain the codon-optimized form of the ghrA eco gene and are capable of producing a mixture of glycolic acid and lactic acid, which can be used as polyglycolic acid, polylactic acid, or poly(lactic acid-glycolic acid) copolymer.

[0300] Example 3: Growth experiment

[0301] In addition, using 10 g L -1 methanol as a reactant and 10 g L -1 methanol + 1.5 g L -1In a minimal medium (reaction matrix) with a mixture of glyoxylates as an additional reactant, growth experiments were carried out using M. extorquens TK 0001 + pTE1887 and M. extorquens TK 0001 + pTE1887-ghrA of the present invention eco (M. extorquens GA1) strain Figure 6 ).

[0302] Figure 6 From A to Figure 6 D, the y-axis represents the growth curves (OD eco ), pH values (inverted triangles), methanol concentrations (open squares), glyoxylate concentrations (open diamonds), glycolate concentrations (dark gray diamonds), and lactate concentrations (gray right triangles) of M. extorquens TK 0001 + pTE1887 (A + C) and M. extorquens TK 0001 + pTE1887-ghrA of the present invention 600 (codon-optimized) (B + D) in the minimal medium. The x-axis represents time. 10 g L -1 methanol (A + B) or 10 g L -1 methanol + 1.5 g L -1 glyoxylate (C + D) was added as a carbon source (reactant), i.e., Cx compound. The methanol concentration, glyoxylate concentration, and glycolate concentration were measured by HPLC, refractometry, and the external standard method, and the concentrations were all in g L -1 . These data represent three independent biological replication processes.

[0303] Adding glyoxylate during the induction of gene expression can be used to test whether an increase in the supply of glyoxylates in vivo will lead to an increase in the production of glycolate.

[0304] From Figure 6 A, it can be seen that the reference strain M. extorquens TK 0001 + pTE1887 with 10 g L -1 methanol as a reactant did not produce glycolate after 40 hours of cultivation, formed a uniform biomass, and the maximum OD 600 was approximately 9. Notably, the pH value decreased significantly to below 6.5 during the fermentation process. In contrast, during the cultivation of M. extorquens TK 0001 + pTE1887, the addition of glyoxylate led to a slightly slower growth, and the maximum OD 600 increased slightly after approximately 42 hours. In this case, glycolate was not produced either( Figure 6 C). However, the pH value of such cultivation could be maintained at the initial pH value of around 7.0, probably due to the addition of glyoxylate.

[0305] Research shows that the recombinant strain M. extorquens TK 0001 + pTE1887 - ghrA of the present invention eco contains the codon - optimized form of the ghrA eco gene and forms the products glyoxylate and lactate with increased concentrations from 10 g / L -1 methanol (about 0.35 g / L and 0.25 g / L respectively within 40 hours). -1 After methanol decomposition, these products are completely degraded during further cultivation. The formation of glyoxylate and lactate is accompanied by a significant slowdown in biomass growth, and the maximum OD -1 reaches 6.7 within 44 hours. In addition, in this case, due to the formation of additional glyoxylate, the pH value of the culture medium may drop to 6.2, and due to the degradation of glyoxylate, the pH value will rise to slightly below 6.5, reaching a value comparable to that of the reference strain ( 600 ) Figure 6 B).

[0306] In the experiment using M. extorquens TK 0001 + pTE1887 - ghrA of the present invention containing the codon - optimized form of the ghrA eco gene, when glyoxylate is fed, the glyoxylate production rate is significantly increased within 44 hours, up to 1.0 g / L eco . The amount of lactate formed is comparable to the cultivation result without feeding glyoxylate (6B). This indicates that glyoxylate is crucial as a precursor for glyoxylate formation, and increasing the in - vivo concentration of glyoxylate will lead to an increase in glyoxylate production. In this experiment, the formed glyoxylate and lactate are also metabolized after methanol consumption. In this experiment, the increase in product formation further leads to a decrease in biomass growth, and the maximum OD -1 is about 4.5. Different from the reference strain, in this case, although glyoxylate is added, the pH value still decreases significantly due to the production of glyoxylate and lactate. Similarly, when methanol is depleted and the formed glyoxylate and lactate decompose, an increase in the pH value may also be observed ( 600 ) Figure 6 D).

[0307] Table 3 summarizes the strain - specific culture parameters obtained from the data, such as the μ (specific growth rate), Y eco (biomass yield on substrate), q eco (specific substrate uptake rate), Y X / S (product yield on substrate), and q S (specific product formation rate) of strains such as M. extorquens TK 0001 + pTE1887 and M. extorquens TK 0001 + pTE1887 - ghrA of the present invention containing the codon - optimized form of the ghrA P / S gene P(Specific product formation rate). These data indicate that the production of glyoxylate and lactate is associated with a significant decrease in the yield of dry biomass on the substrate (70% of the reference strain and 70% of the reference strain fed with glyoxylate), and more carbon is converted into products or must be used to maintain the redox balance. It can also be seen that glyoxylate reduces the growth rate, so the precursor may have a potential toxic effect. By optimizing the balance of the intracellular glyoxylate pool, this toxicity of glyoxylate can be avoided.

[0308] Table 1: Summary of cultures containing 10 g L -1 methanol or additional +1.5 g L -1 glyoxylate in minimal medium of M. extorquens TK 0001 + pTE1887 and M. extorquens TK 0001 + pTE1887-ghrA eco containing the codon-optimized form of the ghrA eco gene (M. extorquens GA1). Abbreviations: μ: specific growth rate; MeOH: methanol; GS: glyoxylate; BTM: dry biomass.

[0309]

[0310] 1 Yields were estimated based on the cumulative substrate utilization of methanol and glyoxylate.

[0311] Examples 1 to 3 show that during the methylotrophic fermentation process according to the present invention, glyoxylate and lactate can be produced from Cx compounds using M. extorquens GA1.

[0312] It should be particularly emphasized that, as shown in Example 3, according to the present invention, the glyoxylate yield in M. extorquens GA1 can be significantly increased by increasing the intracellular glyoxylate concentration. In this case, the yield of glyoxylate is increased by 185% compared to the culture without adding glyoxylate.

[0313] Example 4: Experimental data on the production of glyoxylate and lactate using methanol fermentation

[0314] The experimental procedure was referred to Example 1. The strain used was the wild-type strain M. extorquens TK 0001 DSM 1337.

[0315] The following expression vectors (1 to 4) were used:

[0316] 1. pTE1887 (expression vector, also known as empty vector; plasmid map: Figure 8 )

[0317] 2. pTE1887-ghrA eco(Expression vector encoding glyoxylate reductase derived from Escherichia coli K-12 MG1655 in a codon-optimized manner; SEQ ID No: 3, plasmid map: Figure 9 )(According to the present invention)

[0318] 3. pTE1887-ghrA eco -ecm mea (Expression vector encoding glyoxylate reductase (codon-optimized) derived from Escherichia coli K-12 MG1655 and ethylmalonyl-CoA mutase derived from Methylobacterium extorquens TK 0001 DSM 1337; plasmid map: Figure 10 )(According to the present invention)

[0319] 4. pTE1887-ghrA eco -ecm rsh (Expression vector with codon optimization for glyoxylate reductase (codon-optimized) derived from Escherichia coli K-12 MG1655 and ethylmalonyl-CoA mutase derived from Rhodobacter sphaeroides ATCC 17029; plasmid map: Figure 11 )(According to the present invention).

[0320] Transgenic Methylobacteriaceae cells were prepared by the method described in Example 1.

[0321] Figure 10 Shows the vector map for generating Methylobacteriaceae cells expressing ghrA eco -ecm mea with the following elements: lacI gene, lacI promoter, PL / O4 / A1 promoter → -33 region → -10 region → transcription start point, PL / O4 / A1 promoter ribosome binding site (RBS), ghrA eco (codon-optimized), ecm mea (native), lambdaT0 terminator, kanamycin resistance, mobilization genes mobS and mob, regulatory protein RepA, replication origin colE1.

[0322] Figure 11 Shows the vector map for generating Methylobacteriaceae cells expressing ghrA eco -ecm rsh with the following elements: lacI gene, lacI promoter, PL / O4 / A1 promoter → -33 region → -10 region → transcription start point, PL / O4 / A1 promoter ribosome binding site (RBS), ghrA eco (codon-optimized), rsh-ecm (codon-optimized), lambdaT0 terminator, kanamycin resistance, mobilization genes mobS and mob, regulatory protein RepA, replication origin colE1.

[0323] The genetically modified Methylobacterium extorquens TK 0001 cell contains an exogenous codon-optimized nucleic acid sequence encoding glyoxylate reductase derived from Escherichia coli K-12 MG1655 (SEQ ID No: 3) and an exogenous native nucleic acid sequence encoding ethylmalonyl-CoA mutase derived from Methylobacterium extorquens TK 0001 DSM 1337 (SEQ ID No: 4), namely, the Methylobacterium extorquens Mea-GA2 strain cell, which was deposited at the DSMZ - German Collection of Microorganisms and Cell Cultures in Braunschweig, Germany on June 10, 2022, with the deposit accession number DSM 34287.

[0324] The genetically modified Methylobacterium extorquens TK 0001 cell contains an exogenous codon-optimized nucleic acid sequence encoding glyoxylate reductase derived from Escherichia coli K-12 MG1655 (SEQ ID No: 3) and an exogenous codon-optimized nucleic acid sequence encoding ethylmalonyl-CoA mutase derived from Rhodobacter sphaeroides ATCC 17029 (SEQ ID No: 8), namely, the Methylobacterium extorquens Mea-GA3 strain cell, which was deposited at the DSMZ - German Collection of Microorganisms and Cell Cultures in Braunschweig, Germany on June 10, 2022, with the deposit accession number DSM 34288.

[0325] In the fermentation experiment, the culture medium was used as the reaction substrate, and methanol (the reactant) was used as the sole carbon source.

[0326] Figure 7 The time course of the biomass concentration (OD 600 ) and the pH value of the culture medium during cultivation is shown. Meanwhile, the culture supernatant samples were measured by high-performance chromatography to observe the substrate concentration, the product concentration, and their changes over time.

[0327] Figure 7 In, the x-axis represents time (in hours), and the y-axis represents M. extorquens TK 0001 + pTE1887 (A), M. extorquens GA1 (B) of the present invention, M. extorquens TK0001 + pTE1887-ghrA -1 in the culture medium with methanol as the sole reactant (i.e., as a Cx compound), M. extorquens TK0001 + pTE1887-ghrA eco (codon-optimized) (M. extorquens GA1) (B), M. extorquens TK0001 + pTE1887-ghrA eco -ecm mea (ghrA eco : codon-optimized; ecm mea: Wild type (M. extorquens GA2) (C) and M. extorquens TK 0001 + pTE1887-ghrA of the present invention eco -ecm rsh (Both genes were codon-optimized) Growth process of (M. extorquens GA3) (D) (OD 600 , black solid circles), pH value (inverted triangles), methanol concentration (hollow squares), glyoxylate concentration (dark gray diamonds), and lactate concentration (gray right triangles).

[0328] This again indicates that in the background of M. extorquens TK 0001 strain, the ghrA eco enzyme allows the production of glyoxylate and also produces lactate. In contrast, the wild-type strain containing the empty vector pTE1887 does not produce glyoxylate or lactate. In these experiments, approximately 250 mg L -1 of glyoxylate and approximately 200 mg L -1 of lactate were produced, and the product yield on the substrate was approximately 50 mg g 甲醇 -1 (glyoxylate + lactate). It can be observed that compared with the empty vector strain, the glyoxylate synthesis initiated by the present invention in the exogenous strain M. extorquens TK 0001 + pTE1887-ghrA eco (M. extorquens GA1) reduced the product yield on the substrate (Y X / S ) by 70%. The product yield based on dry biomass (Y P / X ) was 0.27 g g 干生物质 -1 (Table 4).

[0329] Surprisingly, compared with the M. extorquens TK 0001 + pTE1887-ghrA eco strain of the present invention, additional implementation of exogenous ethylmalonyl-CoA mutase significantly improved the performance of glyoxylate production. Compared with the empty vector strain (0.17 h -1 ), the growth of the M. extorquens TK 0001 + pTE1887-ghrA eco -ecm mea strain was retarded, and the measured growth rate (μ) was 0.10 h -1 . Compared with the M. extorquens TK 0001 + pTE1887-ghrA eco strain (0.26 g L -1 versus 0.22 g L -1, compared with that in Table 4), the ecm gene of M. extorquens TK 0001DSM 1337 led to an 18% increase in the glyoxylate titer after 40 h of cultivation. Compared with the empty vector strain, the dry biomass to substrate yield decreased by 49%, and compared with the strain of the present invention M. extorquens TK 0001 + pTE1887-ghrA eco strain, the dry biomass to substrate yield also decreased by 27%.

[0330] A slight change in the product yield based on dry biomass (Y P / X ) was observed. Compared with the strain of the present invention M. extorquens TK0001 + pTE1887-ghrA eco strain, an 11% increase in yield could be achieved (Table 4).

[0331] Compared with the strain of the present invention containing the exogenous ecm mea gene, the use of the exogenous codon-optimized ecm rsh gene led to significant deviations in the culture parameters shown in Table 4. The highest lactate titer measured at this time was 0.37 g L -1 . An obvious change was the dry biomass to substrate yield (Y X / S ), which increased by 26% (0.24 g g eco -ecm mea ) compared with the strain of the present invention M. extorquens TK 0001 + pTE1887-ghrA Biotrockenmasse -1 .

[0332] It has been proven that it is possible to produce glyoxylate using methanol. Compared with the strain of the present invention containing ghrA eco but without exogenous ethylmalonyl-CoA mutase, the use of two exogenous ethylmalonyl-CoA mutases from two different prokaryotic strains respectively improved the production performance of the strain prepared by the present invention. In particular, the use of the ethylmalonyl-CoA mutase ecm rsh could unexpectedly and significantly increase the lactate yield with stronger selectivity.

[0333] Table 4: Summary of M. extorquens TK 0001 + pTE1887 and the strain of the present invention M. extorquens TK 0001 + pTE1887-ghrA -1 in a methanol medium containing 10 g L eco (codon-optimized) (M. extorquens GA1), the strain of the present invention M. extorquens TK 0001 + pTE1887-ghrA eco -ecm mea (ghrAeco : Codon optimization; ecm mea : Wild type (M. extorquens GA2) and M. extorquens TK 0001 + pTE1887-ghrA of the present invention eco- ecm rsh (Both genes were codon-optimized) Culture parameters of (M. extorquens GA3). Abbreviations: μ: Specific growth rate; MeOH: Methanol; GS: Glycolate; BTM: Dry biomass.

[0334]

[0335] Example 5: Detection of glyoxylate reductase (ghrA eco ) expressed by the present invention and the experimental data of the enzyme activity of the reference enzyme, i.e., Escherichia coli hydroxypyruvate reductase (ghrB eco )

[0336] To provide experimental evidence to prove the enzyme activity of the expressed glyoxylate reductase ghrA eco and hydroxypyruvate reductase ghrB eco ( M. T. Pellicer, J. Badia, J. Aguilar and L. Baldoma, Biochemical Journal, 2001, Vol. 354 (Part 3), pp. 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 background of M. extorquens TK 0001 strain. To evaluate the effect of codon optimization on gene expression and the resulting enzyme activity, the native forms of the DNA sequences of these two genes (e.g., in Escherichia coli K-12 MG1655) and the synthetic DNA sequences (c-optimized) codon-optimized for expression in Methylobacterium were tested.

[0337] The implementation process and results are summarized as follows.

[0338] To ensure the presence of pTE1887-ghrA eco -c-optimized (SEQ ID No:3), pTE1887-ghrB eco -c-optimized, pTE1887-ghrA ecoThe transgenic M. extorquens TK 0001 strain with - nativ (SEQ ID No: 1) obtains sufficient biomass for cell disruption and the following cultivation protocol is applied. The M. extorquens TK 0001 + pTE1887 strain containing the empty vector is used as a negative control. All strains are cultivated, harvested, and disrupted as three independent biological replicates.

[0339] In shake flasks (250 mL flasks, 50 mL culture medium), the strains are pre - cultivated for three days at 30 °C in a steam - saturated atmosphere at 150 RPM (in a minimal medium containing methanol, see Example 1) (New Brunswick Innova 44, Eppendorf AG, Hamburg, Germany). TM Then, in shake flasks (250 mL flasks, 50 mL culture medium), the overgrown first pre - culture is inoculated with the minimal medium containing methanol to form a second pre - culture. The initial biomass concentration for inoculation corresponds to an optical density (OD 600 ) of 0.1 at a wavelength of 600 nm. Subsequently, the cultivation is carried out at 150 rpm in a steam - saturated atmosphere at 30 °C. On the second day (i.e., Tuesday), the overgrown second pre - culture (250 mL shake flask, 50 mL minimal medium containing methanol, initial OD 600 = 0.05) is inoculated with the main culture and cultivated at 150 rpm in a steam - saturated atmosphere at 30 °C.

[0340] After the culture OD 600 reaches 0.9 - 1.0, the gene expression of the glyoxylate reductase gene is induced with 1 mM IPTG (final concentration in the culture). Then, the biomass is cultivated to a final OD 600 of approximately 4 - 7.

[0341] For actual biomass harvest, 50 - mL conical centrifuge tubes are emptied, weighed, filled with 50 mL of the overgrown main culture, and finally centrifuged at 4200 rpm for 15 minutes at 4 °C. After the centrifugation step, the supernatant is discarded, and the obtained biomass pellets are rinsed with 20 mL each of 50 mM Tris - HCl (pH 7.5) buffer. Then, centrifugation is repeated under the previous conditions, and the supernatant is carefully removed with a pipette. The obtained biomass pellets are weighed and resuspended in 50 mM MOPS buffer (pH 6.6). For this, 7 mL of buffer is used per 1 g of wet pellets.

[0342] Cell disruption was carried out in a 2.0 mL reaction vessel to obtain a crude protein extract containing glyoxylate or hydroxypyruvate reductase. For this purpose, 1.5 mL of the cell suspension was transferred to each reaction vessel, and then sonicated six times in an ice-water bath for 30 seconds each time with an amplitude of 60. Between the six disruption cycles, the samples were cooled on ice for 1 minute. Finally, centrifugation was carried out at 21500 rpm for 15 minutes at 4 °C to obtain the crude protein extract. The resulting protein-containing supernatant was transferred to a 1.5 mL reaction vessel. To ensure the comparability of the enzyme assay results, the protein concentration of each crude protein extract was determined using NanoDrop TM The protein concentration of each crude protein extract was measured. Using the lowest measured concentration of the crude extract as the target concentration, the other crude extracts were diluted with 50 mM MOPS buffer (pH 6.6). This ensured that all crude protein extracts in the enzyme assay contained the same total protein concentration. In addition, these pre-diluted crude protein extracts were further diluted (1:5) with 50 mM MOPS buffer (pH 6.6) and then used for the enzyme assay.

[0343] The enzyme assay was carried out in a 96-well microtiter plate. For this purpose, 160 μL of the diluted crude protein extract was mixed with 20 μL of 50 mM glyoxylate (as the substrate) and 20 μL of a 2 mM cofactor stock solution (NADH or NADPH, final concentration 0.2 mM in the assay). The experimental method was repeated three times technically. At a temperature of 37 °C, over a period of up to 30 minutes, the enzyme activity was measured by the change in NADH absorbance at a wavelength of 340 nm. For the evaluation, the maximum value of the absorbance change over time within the linear range of the reaction was measured, multiplied by the dilution factor, and then the enzyme activity was calculated in U mL -1 units.

[0344] The enzyme activity was calculated using Equation 6 and the specified coefficients.

[0345]

[0346] Enzyme activity: in mol Substrat Min -1 units; V Proteinrohextrakt-Assay : the volume of the crude protein extract used in the assay (0.00016 L); S: within the linear range of the reaction, the change in absorbance over time at a wavelength of 340 nm corrected with a dilution factor of 5 (Abs. 340 Min. -1 ); V Assay : the total assay volume (0.0002 L); ε λ : the extinction coefficient of NADH / NADPH at a wavelength of 340 nm (6220 L mol -1 cm -1 ); d: the layer thickness of the absorption reaction mixture (0.53 cm).

[0347] To convert enzyme activity from mol Substrat min -1 to the conventional unit of enzyme activity mU mL -1 (1 U = 1 μmol Substrat min -1 ), the calculated result should be multiplied by the factor 10 6 .

[0348] Assign the obtained enzyme activities to the individual expression strains and perform graphical comparison with the cofactors NADH or NADPH.

[0349] Figure 12 Summarizes the enzyme activity data of ghrA eco -c-optimized, ghrB eco -c-optimized, ghrA eco -native, ghrB eco -native and the negative control (pTE1887-Leervector). The measured values and standard deviations shown in the figure are based on three biological replicates, each with three technical replicates.

[0350] As expected, the empty vector only shows slight background activity. Subtract this value from all other measured values to correct for the background reaction that occurs.

[0351] Figure 12 A and Figure 12 B for the enzyme activity of glyoxylate conversion to glycolate indicate that only the two enzymes ghrA eco -c-optimized and ghrA eco -native (i.e., ghrA eco ) used in the present invention show sufficient activity and are particularly suitable for large-scale production. Depending on the cofactor used, significant differences in enzyme activity can also be detected. The determination results show that ghrA eco and ghrB eco are significantly dependent on the cofactor. Using NADH as the cofactor( Figure 12 A), the enzyme activity of ghrB eco -c-optimized is the highest (10.53 ± 1.50 mU mL -1 ). Compared with the ghrB eco -c-optimized gene, the enzyme activity of the ghrA eco -c-optimized gene is significantly decreased, only 0.49 ± 2.34 mU mL -1 . The enzyme activity measured at this time is reduced by about 95%. The enzyme activity range for NADH determination with the native gene is similar: ghrAeco -nativ and ghrB eco -nativ was 4.61 ± 1.61 mU mL -1 compared with 2.45 ± 0.67 mU mL -1 . ghrB eco codon optimization increased the activity by 329%. In summary, it can be seen that ghrB eco enzyme significantly depends on NADH as a cofactor.

[0352] In contrast, when using NADPH as a cofactor, a different situation occurs ( Figure 12 B).

[0353] Here, in the current test, using ghrA eco -c-optimized and ghrA eco -nativ (33.86 ± 1.29 mU mL -1 and 21.76 ± 1.49 mU mL -1 ) showed the highest enzyme activity. The increase of 55% due to codon optimization (21.76 ± 1.49 and 33.86 ± 1.29 mU mL -1 ). It can also be clearly shown that ghrA eco enzyme has NADPH dependence. The lower activity measurement of ghrB eco also emphasizes this point. In this case, whether using the codon-optimized gene variant (ghrB eco -c-optimized) or the native gene variant (ghrB eco -nativ), the measured enzyme activities were only 3.17 ± 0.29 mU mL -1 or 0.81 ± 0.30 mU mL -1 ([[]] Figure 12 B), which indicates that the difference between ghrB eco and ghrA lies not only in the observed extremely low NADPH dependence, but mainly in the lower enzyme activity for the conversion of glyoxylate to glycolate.

[0354] After expressing ghrA eco -c-optimized, the enzyme activity with NADPH as a cofactor was enhanced, indicating that M. extorquens can produce glycolate by expressing this enzyme. The measurement results of ghrB eco -c-optimized showed that the enzyme activity was significantly reduced under the action of NADPH and NADH, which was not sufficient to produce glycolate in M. extorquens.

[0355] Using M. extorquens TK 0001 + pTE1887-ghrAeco When -c-optimized, it was observed that the production of glycolic acid seems to depend on the availability of NADPH as a cofactor. These results confirm the results of Example 2.

[0356] Surprisingly, the introduction of ghrA eco The DNA sequence of the enzyme, especially the codon-optimized DNA sequence, can not only produce glycolic acid, but also prominently produce lactic acid.

[0357] Example 6: Expression of an exogenous nucleic acid sequence encoding glyoxylate reductase derived from Escherichia bacteria in other Methylobacteriaceae cells (the present invention) and other microbial cells (comparative examples)

[0358] According to Example 1, other classes of Methylobacteriaceae (Alphaproteobacteria) were genetically modified. Figure 13 Examples of the microorganisms examined are shown. In particular, the following representative strains of Methylobacteriaceae were examined: Methylorubrum, especially M. zatmanii DSM 5688, especially M. extorquens TK 0001 DSM 1337 (Examples 2 to 5), especially M. extorquens PA1 DSM 23939, especially M. rhodesianum DSM 5687, i.e., a derivative of M. extorquens AM1Δcel that has the cellulase gene deleted (M. extorquens AM1Δcel: https: / / doi.org / 10.1371 / journal.pone.0062957); Methylobacterium cells, especially M. organophilum DSM18172, especially M. radiotolerans DSM 760.

[0359] In addition, Methylomonas methanica DSM 25384 (Gammaproteobacteria), Methylophilus methylotrophus DSM 6330 (Betaproteobacteria), and Bacillus methanolicus DSM 16454 (Firmicutes) were examined as negative examples that do not belong to Methylobacter. The above microorganisms can also metabolize methanol, and the production of glycolic acid and / or lactic acid was tested according to the present invention.

[0360] The following expression vectors (1 to 4) were used:

[0361] 1. pTE1887 (expression vector, also known as empty vector; plasmid map: Figure 8 )

[0362] 2. pTE1887-ghrA eco (expression vector encoding glyoxylate reductase derived from Escherichia coli K-12 MG1655 in a codon-optimized manner; SEQ ID No: 3, plasmid map: Figure 9 )(according to the present invention)

[0363] 3. pTE1887-ghrA eco -ecm mea (expression vector encoding glyoxylate reductase (codon-optimized) derived from Escherichia coli K-12 MG1655 and ethylmalonyl-CoA mutase derived from Methylobacterium extorquens TK 0001 DSM 1337; plasmid map: Figure 10 )(according to the present invention)

[0364] 4. pTE1887-ghrA eco -ecm rsh (expression vector with codon optimization for glyoxylate reductase (codon-optimized) derived from Escherichia coli K-12 MG1655 and ethylmalonyl-CoA mutase derived from Rhodobacter sphaeroides ATCC 17029; plasmid map: Figure 11 )(according to the present invention).

[0365] The transgenic Methylobacteriaceae cell contains an exogenous codon-optimized nucleic acid sequence (SEQ ID No: 3) encoding glyoxylate reductase derived from Escherichia coli K-12 MG1655 of the Methylobacterium zatmanii Mza-GA14 strain (M. zatmanii DSM 5688 + pTE1887-ghrA eco ), which was deposited at the DSMZ - German Collection of Microorganisms and Cell Cultures in Braunschweig, Germany on July 19, 2023, with the deposit accession number DSM 34701.

[0366] The transgenic Methylobacteriaceae cell contains an exogenous codon-optimized nucleic acid sequence (SEQ ID No: 3) encoding glyoxylate reductase derived from Escherichia coli K-12 MG1655 of the Methylobacterium extorquens Mea-GA17 strain (M. extorquens PA1 DSM 23939 + pTE1887-ghrA eco ), which was deposited at the DSMZ - German Collection of Microorganisms and Cell Cultures in Braunschweig, Germany on July 19, 2023, with the deposit accession number DSM 34702.

[0367] The genetically modified Methylobacteriaceae cell contains an exogenous codon-optimized nucleic acid sequence (SEQ ID No: 3) encoding glyoxylate reductase derived from Escherichia coli K-12 MG1655, strain Mrh-GA4 of Methylobacterium rhodesianum (M. rhodesianum DSM 5687 + pTE1887-ghrA eco ), which was deposited on July 19, 2023 at the DSMZ - German Collection of Microorganisms and Cell Cultures in Braunschweig, Germany, with the deposit accession number DSM 34697.

[0368] The genetically modified Methylobacteriaceae cell contains an exogenous codon-optimized nucleic acid sequence (SEQ ID No: 3) encoding glyoxylate reductase derived from Escherichia coli K-12 MG1655 and an exogenous native nucleic acid sequence (SEQ ID No: 4) encoding ethylmalonyl-CoA mutase derived from Methylobacterium extorquens TK 0001 DSM 1337, especially strain Mrh-GA5 of Methylobacterium rhodesianum (M. rhodesianum DSM 5687 + pTE1887-ghrA eco -ecm mea ), which was deposited on July 19, 2023 at the DSMZ - German Collection of Microorganisms and Cell Cultures in Braunschweig, Germany, with the deposit accession number DSM 34698.

[0369] The genetically modified Methylobacteriaceae cell contains an exogenous codon-optimized nucleic acid sequence (SEQ ID No: 3) encoding glyoxylate reductase derived from Escherichia coli K-12 MG1655, strain Mor-GA8 of Methylobacterium organophilum (M. organophilum DSM 18172 + pTE1887-ghrA eco -ecm mea ), which was deposited on July 19, 2023 at the DSMZ - German Collection of Microorganisms and Cell Cultures in Braunschweig, Germany, with the deposit accession number DSM 34699.

[0370] The genetically modified Methylobacteriaceae cell contains an exogenous codon-optimized nucleic acid sequence (SEQ ID No: 3) encoding glyoxylate reductase derived from Escherichia coli K-12 MG1655 and an exogenous native nucleic acid sequence (SEQ ID No: 4) encoding ethylmalonyl-CoA mutase derived from Methylobacterium extorquens TK 0001 DSM 1337, especially strain Mra-GA12 of Methylobacterium radiotolerans (M. radiotolerans DSM 760 + pTE1887-ghrA eco -ecm mea ), which was deposited on July 19, 2023 at the DSMZ - German Collection of Microorganisms and Cell Cultures in Braunschweig, Germany, with the deposit accession number DSM 34700.

[0371] The present invention was tested with the above-mentioned strains using the method of Example 2. The difference from Example 2 is that the amount of reactants (Cx compound, containing 4 g L Figure 14 ) at the start of cultivation with Methylobacterium rhodesianum DSM 5687( Figure 15 ), Methylobacterium zatmanii DSM 5688( Figure 16 ), Methylobacterium radiotolerans DSM760( Figure 17 ), Methylobacterium organophilum DSM 18172( Figure 18 ), and Methylobacterium extorquens PA1 DSM 23939( -1 methanol) was reduced, and additional reactants were fed in batches within 10 to 12 hours after induction (cumulative up to 15 g L -1 ). In addition, samples for measuring the concentrations of glycolic acid, lactic acid, and methanol were collected 22 - 28 hours after inducing gene expression with 1 mM IPTG.

[0372] Figures 14 to 19 In , the x-axis represents transgenic Methylobacterium cells, and the y-axis represents the concentration of methanol in the reaction substrate (white hollow bars) or the concentration of the mixture of glycolic acid and lactic acid formed (black solid bars), in g L -1 . All sampling times were 22 to 28 hours after inducing gene expression with 1 mM IPTG. All concentrations were in g L -1 and were determined by HPLC, refractive index detection, and external standard method.

[0373] Figure 14 shows the screening results for the production of glycolic acid and lactic acid in the recombinant Methylobacterium rhodesianum DSM 5687 strain expressing glyoxylate reductase, based on the corresponding codon-optimized gene. The first bar from the left represents the methanol concentration in the basal medium at the start of cultivation. pTE1887 was used as the expression vector and also as a negative control in the form of an empty vector in the reference strain M. rhodesianum DSM 5687 + pTE1887 (the second bar from the left on the x-axis).

[0374] Surprisingly, neither the reference strain M. rhodesianum DSM 5687 + pTE1887 (the second bar from the left) nor the transgenic Methylobacterium cells showed the production of glycolic acid and lactic acid (the third to tenth and twelfth to sixteenth bars from the left), with the exception of: the transgenic cells of the present invention ( Figure 14 the black solid bars in ) M. rhodesianum DSM 5687 + pTE1887-ghrA eco(Codon-optimized nucleic acid form of SEQ ID No: 3), that is, the transgenic Methylobacteriaceae cells of the present invention contain at least one exogenous nucleic acid sequence encoding glyoxylate reductase derived from Escherichia bacteria (the 11th from the left); transgenic cell M. rhodesianum DSM 5687 + pTE1887-ghrA eco -ecm mea , containing an exogenous codon-optimized nucleic acid sequence encoding glyoxylate reductase derived from Escherichia coli K-12 MG1655 (SEQ ID No: 3) and an exogenous native nucleic acid sequence encoding ethylmalonyl-CoA mutase derived from Methylobacterium extorquens TK 0001 DSM 1337 (SEQ ID No: 4), that is, the transgenic Methylobacteriaceae cells of the present invention contain at least one exogenous nucleic acid sequence encoding glyoxylate reductase derived from Escherichia bacteria (the 17th from the left); transgenic cell M. rhodesianum DSM 5687 + pTE1887-ghrA eco -ecm rsh , containing an exogenous codon-optimized nucleic acid sequence encoding glyoxylate reductase derived from Escherichia coli K-12 MG1655 (SEQ ID No: 3) and an exogenous codon-optimized nucleic acid sequence encoding ethylmalonyl-CoA mutase derived from Rhodobacter sphaeroides ATCC 17029 (SEQ ID No: 8), that is, the transgenic Methylobacteriaceae cells of the present invention contain at least one exogenous nucleic acid sequence encoding glyoxylate reductase derived from Escherichia bacteria (the 18th from the left).

[0375] Figure 9 Shows the vector map for generating these Methylobacteriaceae cells, having the following elements: lacI gene, lacI promoter, PL / O4 / A1 promoter → -33 region → -10 region → transcription start point, PL / O4 / A1 promoter ribosome binding site (RBS), ghrA eco -c-optimized, LambdaT0 terminator, kanamycin resistance, mobilization genes mobS and mob, regulatory protein RepA, replication origin colE1.

[0376] Figure 10 Shows the vector map for generating Methylobacteriaceae cells expressing ghrA eco -ecm mea , having the following elements: lacI gene, lacI promoter, PL / O4 / A1 promoter → -33 region → -10 region → transcription start point, PL / O4 / A1 promoter ribosome binding site (RBS), ghrA eco (Codon-optimized), ecm mea(Native), lambdaT0 terminator, kanamycin resistance, mobilization genes mobS and mob, regulatory protein RepA, replication origin colE1.

[0377] Figure 11 Shows a vector map for generating Methylobacteriaceae cells expressing ghrA eco -ecm rsh with the following elements: lacI gene, lacI promoter, PL / O4 / A1 promoter → -33 region → -10 region → transcription start point, PL / O4 / A1 promoter ribosome binding site (RBS), ghrA eco (Codon-optimized), rsh-ecm (codon-optimized), lambdaT0 terminator, kanamycin resistance, mobilization genes mobS and mob, regulatory protein RepA, replication origin colE1.

[0378] Using the transgenic cell M. rhodesianum DSM 5687 of the present invention, a mixture of glycolic acid and lactic acid can be produced, and the total concentration of glycolic acid plus lactic acid can be as high as 0.85 g L -1 of (M. rhodesianum DSM5687 + pTE1887-ghrA eco -ecm mea ), at least 0.82 g L -1 (M. rhodesianum DSM5687 + pTE1887-ghrA eco ), at least 0.09 g L -1 (M. rhodesianum DSM5687 + pTE1887-ghrA eco -ecm rsh ).

[0379] These experimental data indicate that it is possible for Methylobacteriaceae cells to produce glycolic acid and lactic acid according to the present invention.

[0380] Figure 15 Shows the screening results of the recombinant Methylobacterium zatmanii DSM 5688 strain for producing glycolic acid and lactic acid. According to the present invention, this strain contains a glyoxylate reductase gene derived from the genus Escherichia, and in some cases, depending on the corresponding codon-optimized gene, it also expresses an ethylmalonyl-CoA mutase derived from Rhodobacter sphaeroides ATCC 17029. The first from the left represents the methanol concentration in the basal medium at the start of the culture. pTE1887 is used as an expression vector and also as a negative control in the form of an empty vector of the reference strain M. zatmanii DSM 5688 + pTE1887 (the second from the left on the x-axis).

[0381] Surprisingly, the reference strain M. zatmanii DSM 5688 + pTE1887 (second from the left) did not show production of glyoxylate and lactate, in comparison with: the transgenic cells of the present invention ( Figure 15 the black solid bars in) M. zatmanii DSM 5688 + pTE1887 - ghrA eco (codon - optimized nucleic acid form of SEQ ID No: 3), that is, the transgenic Methylobacteriaceae cells of the present invention contain at least one exogenous nucleic acid sequence encoding glyoxylate reductase derived from Escherichia bacteria (third from the left); the transgenic cell M. zatmanii DSM 5688 + pTE1887 - ghrA eco -ecm rsh , which contains an exogenous codon - optimized nucleic acid sequence encoding glyoxylate reductase derived from Escherichia coli K - 12 MG1655 (SEQ ID No: 3) and an exogenous codon - optimized nucleic acid sequence encoding ethylmalonyl - CoA mutase derived from Rhodobacter sphaeroides ATCC 17029 (SEQ ID No: 8), that is, the transgenic Methylobacteriaceae cells of the present invention contain at least one exogenous nucleic acid sequence encoding glyoxylate reductase derived from Escherichia bacteria (fourth from the left).

[0382] Figure 9 The vector maps for generating these Methylobacteriaceae cells are shown, having the following elements: lacI gene, lacI promoter, PL / O4 / A1 promoter → - 33 region → - 10 region → transcription start point, PL / O4 / A1 promoter ribosome - binding site (RBS), ghrA eco -c - optimiert, LambdaT0 terminator, kanamycin resistance, mobilization genes mobS and mob, regulatory protein RepA, replication origin colE1.

[0383] Figure 11 Shows the vector maps for generating Methylobacteriaceae cells expressing ghrA eco -ecm rsh having the following elements: lacI gene, lacI promoter, PL / O4 / A1 promoter → - 33 region → - 10 region → transcription start point, PL / O4 / A1 promoter ribosome - binding site (RBS), ghrA eco (codon - optimized), rsh - ecm (codon - optimized), lambdaT0 terminator, kanamycin resistance, mobilization genes mobS and mob, regulatory protein RepA, replication origin colE1.

[0384] Using the transgenic cell M. zatmanii DSM 5688 of the present invention, a mixture of glycolic acid and lactic acid can be produced, and the total concentration of glycolic acid plus lactic acid can be as high as 0.56 g / L -1 (M. zatmanii DSM 5688 + pTE1887-ghrA eco ), at least 0.48 g / L -1 (M. zatmanii DSM 5688 + pTE1887-ghrA eco -ecm rsh ).

[0385] These experimental data indicate that it is possible for Methylobacteriaceae cells according to the present invention to produce glycolic acid and lactic acid.

[0386] Figure 16 The screening results of the recombinant radiation-resistant Methylobacterium DSM 760 strain producing glycolic acid and lactic acid are shown. The first bar from the left represents the methanol concentration in the basal medium at the start of cultivation. pTE1887 was used as the expression vector and also as a negative control in the form of an empty vector of the reference strain M. radiotolerans DSM 760 + pTE1887 (the second bar from the left on the x-axis).

[0387] Surprisingly, the reference strain M. radiotolerans DSM 760 + pTE1887 (the second bar from the left) did not show the production of glycolic acid and lactic acid. In contrast, the transgenic cell of the present invention M. radiotolerans DSM 760 + pTE1887-ghrA eco -ecm mea , which contains an exogenous codon-optimized nucleic acid sequence encoding glyoxylate reductase derived from Escherichia coli K-12 MG1655 (SEQ ID No: 3) and an exogenous native nucleic acid sequence encoding ethylmalonyl-CoA mutase derived from Methylobacterium extorquens TK 0001 DSM 1337 (SEQ ID No: 4), that is, the transgenic Methylobacteriaceae cell of the present invention contains at least one exogenous nucleic acid sequence encoding glyoxylate reductase derived from Escherichia coli bacteria (the solid black bar in Figure 16 ).

[0388] ghrA eco and ecm mea (ethylmalonyl-CoA mutase of the present invention derived from M. extorquens TK 0001 DSM 1337) combination promotes the production of glycolic acid and lactic acid in the present invention.

[0389] Figure 10 Shows the construction used to generate the expression ghrA eco -ecmmea Vector map of Methylobacteriaceae cells, having the following elements: lacI gene, lacI promoter, PL / O4 / A1 promoter → -33 region → -10 region → transcription start point, ribosome binding site (RBS) of PL / O4 / A1 promoter, ghrA eco (codon-optimized), ecm mea (native), lambdaT0 terminator, kanamycin resistance, mobilization genes mobS and mob, regulatory protein RepA, origin of replication colE1.

[0390] Using the transgenic cell M. radiotolerans DSM 760 of the present invention, a mixture of glyoxylate and lactate can be produced, and the total concentration of glyoxylate plus lactate can be as high as 0.39 g L -1 (M. radiotolerans DSM 760 + pTE1887-ghrA eco -ecm mea ).

[0391] These experimental data indicate that it is possible for Methylobacteriaceae cells according to the present invention to produce glyoxylate and lactate.

[0392] Figure 17 Shows the screening results of the production of glyoxylate and lactate in the recombinant Methylobacterium organophilum DSM 18172 strain expressing glyoxylate reductase, based on the corresponding codon-optimized gene. The first bar from the left represents the methanol concentration in the minimal medium at the start of the culture. pTE1887 was used as the expression vector and also as a negative control in the form of an empty vector of the reference strain M. organophilum DSM 18172 + pTE1887 (the second bar from the left on the x-axis).

[0393] Surprisingly, neither the reference strain M. organophilum DSM 18172 + pTE1887 (the second bar from the left) nor the transgenic Methylobacteriaceae cells showed the production of glyoxylate and lactate (the third to tenth bars and the twelfth to eighteenth bars from the left), with the exceptions including: the transgenic cells of the present invention ( Figure 17 the solid black bars in) M. organophilum DSM 18172 + pTE1887-ghrA eco (codon-optimized nucleic acid form of SEQ ID No: 3), that is, the transgenic Methylobacteriaceae cells of the present invention contain at least one exogenous nucleic acid sequence encoding glyoxylate reductase derived from Escherichia bacteria (the eleventh bar from the left); the transgenic cells M. organophilum DSM 18172 + pTE1887-ghrA eco -ecm mea, comprising an exogenous codon-optimized nucleic acid sequence encoding glyoxylate reductase derived from Escherichia coli K-12 MG1655 (SEQ ID No: 3) and an exogenous native nucleic acid sequence encoding ethylmalonyl-CoA mutase derived from Methylobacterium extorquens TK 0001 DSM 1337 (SEQ ID No: 4), that is, the transgenic Methylobacteriaceae cell of the present invention comprises at least one exogenous nucleic acid sequence encoding glyoxylate reductase derived from a bacterium of the genus Escherichia (the 17th from the left); transgenic cell M. organophilum DSM 18172 + pTE1887-ghrA eco -ecm rsh , comprising an exogenous codon-optimized nucleic acid sequence encoding glyoxylate reductase derived from Escherichia coli K-12 MG1655 (SEQ ID No: 3) and an exogenous codon-optimized nucleic acid sequence encoding ethylmalonyl-CoA mutase derived from Rhodobacter sphaeroides ATCC 17029 (SEQ ID No: 8), that is, the transgenic Methylobacteriaceae cell of the present invention comprises at least one exogenous nucleic acid sequence encoding glyoxylate reductase derived from a bacterium of the genus Escherichia (the 18th from the left).

[0394] Figure 9 Shows the vector map for generating these Methylobacteriaceae cells, having the following elements: lacI gene, lacI promoter, PL / O4 / A1 promoter → -33 region → -10 region → transcription start point, PL / O4 / A1 promoter ribosome binding site (RBS), ghrA eco -c-optimized, LambdaT0 terminator, kanamycin resistance, mobilization genes mobS and mob, regulatory protein RepA, replication origin colE1.

[0395] Figure 10 Shows the vector map for generating Methylobacteriaceae cells expressing ghrA eco -ecm mea having the following elements: lacI gene, lacI promoter, PL / O4 / A1 promoter → -33 region → -10 region → transcription start point, PL / O4 / A1 promoter ribosome binding site (RBS), ghrA eco (codon-optimized), ecm mea (native), lambdaT0 terminator, kanamycin resistance, mobilization genes mobS and mob, regulatory protein RepA, replication origin colE1.

[0396] Figure 11 Shows for generating Methylobacteriaceae cells expressing ghrA eco -ecm rshVector map of Methylobacteriaceae cells, having the following elements: lacI gene, lacI promoter, PL / O4 / A1 promoter → -33 region → -10 region → transcription start point, PL / O4 / A1 promoter ribosome binding site (RBS), ghrA eco (codon-optimized), rsh-ecm (codon-optimized), lambdaT0 terminator, kanamycin resistance, mobilization genes mobS and mob, regulatory protein RepA, origin of replication colE1.

[0397] Using the transgenic cell M. organophilum DSM 18172 of the present invention, a mixture of glyoxylate and lactate can be produced, wherein the total concentration of glyoxylate plus lactate can be as high as 0.13 g L -1 (M. organophilum DSM 18172 + pTE1887-ghrA eco ), at least 0.10 g L -1 . (M. organophilum DSM 18172 + pTE1887-ghrA eco -ecm mea ), at least 0.04 g L -1 (M. organophilum DSM 18172 + pTE1887-ghrA eco -ecm rsh ).

[0398] These experimental data indicate that it is possible for Methylobacteriaceae cells according to the present invention to produce glyoxylate and lactate.

[0399] Figure 18 Shows the screening results of the recombinant Methylobacterium extorquens PA1 DSM 23939 strain for the production of glyoxylate and lactate. The first lane from the left represents the methanol concentration in the minimal medium at the start of cultivation. pTE1887 was used as the expression vector and also as a negative control in the form of an empty vector of the reference strain M. extorquens PA1 DSM 23939 + pTE1887 (the second lane from the left on the x-axis).

[0400] Surprisingly, the reference strain M. extorquens PA1 DSM 23939 + pTE1887 (the second lane from the left) did not show the production of glyoxylate and lactate, in contrast to the transgenic cell of the present invention M. extorquens PA1 DSM 23939 + pTE1887-ghrA eco (codon-optimized nucleic acid form of SEQ ID No: 3), that is, the transgenic Methylobacteriaceae cell of the present invention contains at least one exogenous nucleic acid sequence encoding glyoxylate reductase derived from Escherichia bacteria ( Figure 18the solid black bar (the third bar from the left).

[0401] Figure 9 shows a vector map for generating these Methylobacteriaceae cells, having the following elements: the lacI gene, the lacI promoter, the PL / O4 / A1 promoter → -33 region → -10 region → transcription start point, the ribosome binding site (RBS) of the PL / O4 / A1 promoter, ghrA eco -c-optimized, LambdaT0 terminator, kanamycin resistance, mobilization genes mobS and mob, regulatory protein RepA, replication origin colE1.

[0402] Using the transgenic cell M. extorquens PA1 DSM 23939 of the present invention, a mixture of glyoxylic acid and lactic acid can be produced, wherein the total concentration of glyoxylic acid plus lactic acid can be as high as 1.50 g L -1 (M. extorquens PA1 DSM 23939 + pTE1887-ghrA eco (M. extorquens GA17).

[0403] These experimental data indicate that it is possible for Methylobacteriaceae cells according to the present invention to produce glyoxylic acid and lactic acid.

[0404] Figure 19 shows the screening results of the production of glyoxylic acid and lactic acid in the recombinant M. extorquens AM1Δcel strain expressing glyoxylate reductase, based on the corresponding codon-optimized gene. The first bar from the left represents the methanol concentration in the minimal medium at the start of the culture. pTE1887 is used as an expression vector and also as a negative control in the form of an empty vector of the reference strain M. extorquens AM1Δcel + pTE1887 (the second bar from the left on the x-axis).

[0405] Surprisingly, the reference strain M. extorquens AM1Δcel + pTE1887 (the second bar from the left) did not show the production of glyoxylic acid and lactic acid, except for the transgenic cells of the present invention ( Figure 19 the solid black bar in) M. extorquens AM1Δcel + pTE1887-ghrA eco (in the form of the codon-optimized nucleic acid of SEQ ID No: 3), that is, the transgenic Methylobacteriaceae cells of the present invention contain at least one exogenous nucleic acid sequence encoding glyoxylate reductase derived from Escherichia bacteria (the 11th bar from the left).

[0406] Figure 9Shows the vector map for generating these Methylobacteriaceae cells, with the following elements: lacI gene, lacI promoter, PL / O4 / A1 promoter → -33 region → -10 region → transcription start point, PL / O4 / A1 promoter ribosome binding site (RBS), ghrA eco -c-optimized, LambdaT0 terminator, kanamycin resistance, mobilization genes mobS and mob, regulatory protein RepA, replication origin colE1.

[0407] Using the transgenic cell M. extorquens AM1Δcel of the present invention, a mixture of glycolic acid and lactic acid can be produced, where the total concentration of glycolic acid plus lactic acid can be as high as 0.60 g L -1 (M. extorquens AM1Δcel + pTE1887-ghrA eco ).

[0408] These experimental data indicate that it is possible for Methylobacteriaceae cells according to the present invention to produce glycolic acid and lactic acid. Deletion of the cellulase gene (Δcel) does not affect the metabolism of glyoxylate - glycolic acid - lactic acid.

[0409] A summary of the research results is shown in Table 5 (see below).

[0410] Further study was conducted on methylotrophic microorganisms that do not belong to Methylobacteriaceae. For this purpose, transgenic strains of Methylomonas methanica DSM 25384 (Gammaproteobacteria), Methylophilus methylotrophus DSM 6330 (Betaproteobacteria), and Methanobacterium DSM 16454 (Firmicutes) were cultivated according to the strain construction process of Example 1. In each case, the strains used were unable to achieve this. In the strain construction process described in Example 1, for all vectors pTE1887, pTE1887-ghrA eco , pTE1887-ghrA eco -ecm mea and pTE1887-ghrA eco -ecm rsh , the tested strains did not grow (Table 5).

[0411] Table 5: Overview of glycolic acid and lactic acid titers obtained for Methylobacteriaceae test strains and control examples (microorganisms that do not belong to Methylobacteriaceae). Abbreviations: GS: glycolic acid; MS: lactic acid.

[0412]

[0413] 1Temperatures used for growth tests: Methylophilus methylotrophus DSM 6330 (37 °C), Methanobacterium sp. DSM 16454 (45 °C).

Claims

1. A transgenic Methylobacteriaceae cell comprising at least one exogenous nucleic acid sequence encoding a glyoxylate reductase derived from a bacterium of the genus Escherichia.

2. The transgenic Methylobacteriaceae cell according to claim 1, wherein, the Methylobacteriaceae cell is a Methylorubrum cell, in particular Methylobacterium extorquens, especially Methylobacterium extorquens AM1, Methylobacterium extorquens TK 0001, Methylobacterium extorquens PA1, Methylobacterium rhodesianum or Methylobacterium zatmanii, or a Methylobacterium cell, in particular Methylobacterium organophilum or Methylobacterium radiotolerans.

3. The transgenic Methylobacteriaceae cell according to claim 1 or 2, wherein, the bacterium is Escherichia coli, especially Escherichia coli K-12 MG1655.

4. The transgenic Methylobacteriaceae cell according to any one of the preceding claims, wherein, the glyoxylate reductase derived from a bacterium of the genus Escherichia is encoded by the nucleic acid sequence of SEQ ID No: 3 or a functionally equivalent nucleic acid sequence, and the nucleic acid sequence identity of the functionally equivalent nucleic acid sequence with the nucleic acid sequence of SEQ ID No: 3 is 30.0% to 99.9%, or wherein, the glyoxylate reductase contains the amino acid sequence of SEQ ID No: 2 or a functionally equivalent amino acid sequence, and the amino acid sequence identity of the functionally equivalent amino acid sequence with the amino acid sequence of SEQ ID No: 2 is 30.0% to 99.9%.

5. The transgenic Methylobacteriaceae cell according to any one of the preceding claims, comprising: at least one exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase, especially derived from at least one bacterium selected from the group consisting of Methylobacterium extorquens, especially Methylobacterium extorquens TK 0001 DSM 1337; and Rhodobacter sphaeroides, especially Rhodobacter sphaeroides ATCC 17029.

6. The transgenic Methylobacteriaceae cell according to claim 5, wherein, the ethylmalonyl-CoA mutase is encoded by the nucleic acid sequence of SEQ ID No: 8 or SEQ ID No: 13 or a functionally equivalent nucleic acid sequence, and the nucleic acid sequence identity of the functionally equivalent nucleic acid sequence with the nucleic acid sequence of SEQ ID No: 8 or SEQ ID No: 13 is 30.0% to 99.9%, or wherein, the ethylmalonyl-CoA mutase contains the amino acid sequence of SEQ ID No: 5 or SEQ ID No: 7 or a functionally equivalent amino acid sequence, and the amino acid sequence identity of the functionally equivalent amino acid sequence with the amino acid sequence of SEQ ID No: 5 or SEQ ID No: 7 is 30.0% to 99.9%.

7. The transgenic Methylobacteriaceae cell according to any one of the preceding claims, wherein, 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 exists extrachromosomally, especially integrated into the cell as an episomal expression vector.

8. The transgenic Methylobacteriaceae cell according to any one of the preceding claims, wherein, The transgenic Methylobacteriaceae cells are cells of Methylobacterium extorquens Mea-GA1 strain DSM 34286, Methylobacterium extorquens Mea-GA2 strain DSM 34287, or Methylobacterium extorquens Mea-GA3 strain DSM 34288 deposited at the DSMZ - German Collection of Microorganisms and Cell Cultures in Braunschweig, Germany on June 10, 2022, or derivatives thereof; or wherein the transgenic Methylobacteriaceae cells are cells of Methylobacterium rhodesianum Mrh-GA4 strain DSM34697, Methylobacterium rhodesianum Mrh-GA5 strain DSM 34698, Methylobacterium zatmanii Mza-GA14 strain DSM 34701, Methylobacterium extorquens Mea-GA17 strain DSM 34702, Methylobacterium radiotolerans Mra-GA12 strain DSM 34700, or Methylobacterium organophilum Mor-GA8 strain DSM 34699 deposited at the DSMZ - German Collection of Microorganisms and Cell Cultures in Braunschweig, Germany on July 19, 2023, or derivatives thereof.

9. The transgenic 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 to at least one other regulatory unit to form an expression cassette, in particular a promoter, especially an inducible promoter, a derepressible promoter or a constitutive promoter, an enhancer, a ribosome binding site and / or a terminator.

10. A method for preparing a transgenic Methylobacteriaceae cell according to any one of claims 1 to 9, comprising the following steps: (a) providing a Methylobacteriaceae cell, especially a wild-type cell, and an expression vector or a genome editing system comprising at least one exogenous nucleic acid sequence encoding glyoxylate reductase derived from a bacterium of the genus Escherichia, especially an expression cassette comprising the nucleic acid sequence; (b) transforming the Methylobacteriaceae cell with the expression vector or the genome editing system under conditions allowing uptake and optionally stable integration of the at least one exogenous nucleic acid sequence into the Methylobacteriaceae cell; and (c) obtaining at least one exogenous transgenic Methylobacteriaceae cell containing glyoxylate reductase.

11. The method according to claim 10, wherein, in process step (a), at least one exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase is provided, especially derived from at least one bacterium selected from the group consisting of Methylobacterium extorquens, especially Methylobacterium extorquens TK 0001 DSM 1337; and Rhodobacter sphaeroides, especially Rhodobacter sphaeroides ATCC 17029, especially providing an expression cassette comprising the nucleic acid sequence; in process step (b), the Methylobacteriaceae cell is transformed with the exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase, especially the expression cassette comprising the nucleic acid sequence; in process step (c), at least one exogenous transgenic Methylobacteriaceae cell containing glyoxylate reductase is obtained, which additionally contains at least one exogenous nucleic acid sequence encoding ethylmalonyl-CoA mutase.

12. A transgenic Methylobacteriaceae cell according to any one of claims 1 to 9 or a transgenic Methylobacteriaceae cell prepared by the method according to claim 10 or 11, wherein, the cell is a live cell, a dead cell, a cryopreserved cell, or in the form of a cell lysate or cell extract obtained from a transgenic Methylobacteriaceae cell according to any one of claims 1 to 9 or a transgenic Methylobacteriaceae cell prepared by the method according to claim 10 or 11.

13. A biocatalyst comprising a transgenic Methylobacteriaceae cell according to any one of claims 1 to 9 or 12 or a transgenic Methylobacteriaceae cell prepared by the method according to claim 10 or 11, wherein, the transgenic Methylobacteriaceae cell is arranged on a carrier.

14. A bioreactor comprising a transgenic Methylobacteriaceae cell according to any one of claims 1 to 9 or 12 or a transgenic Methylobacteriaceae cell prepared by the method according to claim 10 or 11 or the biocatalyst according to claim 12.

15. A method for producing a glyoxylic acid-containing product from a reactant containing at least one Cx compound, comprising the following steps: (x) providing a transgenic Methylobacteriaceae cell according to any one of claims 1 to 9 or 12 or the biocatalyst according to claim 13, a reaction medium, and a reactant containing at least one Cx compound; (y) reacting the reactant under conditions allowing the Cx compound to form glyoxylic acid; and (z) obtaining the glyoxylic acid-containing product from the reaction medium.

16. The method 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.

17. The method according to claim 15 or 16, wherein, the glyoxylic acid-containing product is a product containing glyoxylic acid and lactic acid.

18. The method according to any one of claims 15 to 17, wherein, The Cx compound is produced from CO 2 , especially from synthesis gas containing CO 2 , a mixture of CO and H 2 , especially by means of chemical heterogeneous catalysis.

19. The method according to claim 18, wherein, The CO 2 , in particular the synthesis gas, is produced by the chemical conversion of organic substances or materials, in particular sludge and other biological residues and waste materials.

20. A method for preparing polyglycolic acid, polylactic acid or a polylactic acid-glycolic acid copolymer, comprising: performing the method according to any one of claims 15 to 19, and then polymerizing the glyoxylic acid, lactic acid or a mixture of glyoxylic acid and lactic acid obtained by this method.

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