Engineered strains of *Fermentomonas motilityis* used for xyloic acid production, their construction methods and applications

Abstract

This invention belongs to the field of genetic engineering technology, and particularly relates to an engineered strain of *Fermentomonas motilityis* for the production of xylose, its construction method, and its applications. The engineered strain uses *Fermentomonas motilityis* ZM4 as the starting strain, and xylose dehydrogenase gene expression elements are inserted into at least two gene loci in its genome. These xylose dehydrogenase gene expression elements include the Ppdc promoter and the xylose dehydrogenase gene. This invention achieves the introduction and copy number increase of the exogenous gene encoding xylose dehydrogenase by dispersing and integrating the xylose dehydrogenase gene at specific loci in the *Fermentomonas motilityis* ZM4 genome. Through the strong promoter Ppdc regulating gene expression, the resulting engineered strain can efficiently convert D-xylose into D-xylose, exhibiting advantages such as high xylose conversion efficiency, strong D-xylose production capacity, and high genetic stability. This provides a new approach for the industrial production of D-xylose and the full utilization of xylose hydrolysate.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to an engineered strain of *Mammotrophic motility-fermenting* for the production of xylosonic acid, its construction method, and its application. Background Technology

[0002] Xylic acid is a five-carbon organic acid with wide applications in the food industry. Due to its similar physical properties to gluconic acid, which is primarily derived from starch, xylic acid, being of "non-food origin," can serve as a cost-effective alternative. Xylic acid also has significant industrial applications, acting as a high-efficiency cement adhesive, metallurgical rust remover, textile bleaching agent, and cleaning agent. It can also serve as a precursor for the synthesis of various compounds, such as polyamides, polyesters, and hydrogels, as well as high-value chemicals like 1,2,4-butanetriol, ethylene glycol, and glycolic acid.

[0003] Currently, xylan acid is mainly produced using chemical and biological methods. Chemical synthesis typically employs high-temperature or alkaline systems and often requires heavy or precious metal catalysts, such as platinum or gold, making this method costly and causing significant environmental pollution. Compared to chemical synthesis, microbial transformation offers the promising potential of high specificity and low production costs. Using sustainable lignocellulosic biomass rich in glucose and xylose as raw material to produce xylan acid not only effectively utilizes agricultural waste such as straw but also promotes green biomanufacturing and improves overall socio-economic benefits.

[0004] The metabolic pathway from xylose to xylonic acid in microorganisms has been largely elucidated. In the xylose oxidation pathway, xylose is converted to xylose lactone by xylose dehydrogenase (XDH), and subsequently to xylose acid by xylose lactonease (XL). Xylose lactone can also undergo spontaneous hydrolysis to form xylonic acid. Some natural strains, such as *Gluconobacterium tumefaciens* and *Burkholderia gravidarum*, can be used to produce xylonic acid, but these strains have low efficiency in xylose metabolism, resulting in low xylonic acid yields. With the rapid development of modern molecular biology and synthetic biology techniques, significant progress has been made in constructing microbial cell factories to synthesize high-value-added compounds from xylose. Some recombinant strains, such as *Saccharomyces cerevisiae* and *Escherichia coli*, enhance xylonic acid fermentation capacity through heterologous expression of the XDH gene. However, most of them have some drawbacks, such as the need to use complex culture media and limited production capacity. In particular, when using plant waste hydrolysate containing xylose to produce D-xylose, there are problems such as low xylose conversion efficiency and low xylose yield. Undoubtedly, these will increase the production cost of xylose and, to some extent, limit the widespread application of xylose as a raw material for preparing xylose. Summary of the Invention

[0005] To address the aforementioned problems in the prior art, this invention provides a novel engineered strain of *Fermentomonas motilityis*, its construction method, and its applications. The engineered strain possesses advantages such as high xylose conversion efficiency, strong D-xylonic acid production capacity, and good genetic stability, providing a new approach for the industrial production of D-xylonic acid and the full utilization of xylose hydrolysate. This invention is specifically achieved through the following technical solutions:

[0006] The first aspect of the present invention provides an engineered strain of *M. motile fermentum* for the production of xylonic acid. The engineered strain is based on *M. motile fermentum* ZM4 as the starting strain, and at least two xylose dehydrogenase gene expression elements are inserted into the gene loci ZMO0038, ZMO1650, ZMO1094, ZMO1547 and ZMO1577 in its genome. The xylose dehydrogenase gene expression elements include, from upstream to downstream, a pyruvate decarboxylase promoter and a xylose dehydrogenase gene.

[0007] Furthermore, the engineered strain uses *Z. motile fermentum* ZM4 as the starting strain, and inserts xylose dehydrogenase gene expression elements at least three of the ZMO0038, ZMO1650, ZMO1094, ZMO1547, and ZMO1577 gene loci in its genome; even further, it inserts xylose dehydrogenase gene expression elements at at least four of the ZMO0038, ZMO1650, ZMO1094, ZMO1547, and ZMO1577 gene loci in its genome; still further, the engineered strain uses *Z. motile fermentum* ZM4 as the starting strain, and inserts xylose dehydrogenase gene expression elements at all of the ZMO0038, ZMO1650, ZMO1094, ZMO1547, and ZMO1577 gene loci in its genome.

[0008] Furthermore, the nucleotide sequence of the xylose dehydrogenase gene is shown in SEQ ID NO.1, and the nucleotide sequence of the pyruvate decarboxylase promoter is shown in SEQ ID NO.2.

[0009] A second aspect of the present invention provides a method for constructing an engineered strain of *Syntrophus muscarinii* for producing xylosonic acid as described above, comprising the following steps:

[0010] Guide RNAs and donor DNA were designed based on the ZMO0038, ZMO1650, ZMO1094, ZMO1547, and ZMO1577 gene loci to be edited. The guide RNA targets a sequence 32 bp downstream of the CCC motif adjacent to the original spacer sequence of the gene to be edited. The nucleotide sequences of the guide RNAs targeting the ZMO0038, ZMO1650, ZMO1094, ZMO1547, and ZMO1577 gene loci are shown in SEQ ID NO. 7-11, respectively. The donor DNA includes upstream and downstream homologous arms of the guide RNA targeting the sequence, and a xylose dehydrogenase gene expression element located between the upstream and downstream homologous arms. The nucleotide sequences of the upstream and downstream homologous arms targeting the ZMO0038 gene locus are shown in SEQ ID NO. 22-23, respectively, and the nucleotide sequences of the upstream and downstream homologous arms targeting the ZMO1650 gene locus are shown in SEQ ID NO. 22-23, respectively. As shown in NO.24-25, the nucleotide sequences of the upstream and downstream homologous arms targeting the ZMO1094 gene site are shown in SEQ ID NO.26-27, the nucleotide sequences of the upstream and downstream homologous arms targeting the ZMO1547 gene site are shown in SEQ ID NO.28-29, and the nucleotide sequences of the upstream and downstream homologous arms targeting the ZMO1577 gene site are shown in SEQ ID NO.30-31.

[0011] The guide RNA and the donor DNA targeting the ZMO0038, ZMO1650, ZMO1094, ZMO1547 and ZMO1577 gene loci were ligated into the expression vector to obtain the first to fifth editing plasmids. The editing plasmids of each gene locus were transformed into *Bacillus mobilis* ZM4, and the *Bacillus mobilis* engineered strain of the present invention was obtained by screening.

[0012] A third aspect of the present invention provides the use of the engineered strain of *M. motile fermentum* for producing xylan acid as described above in the preparation of xylan acid.

[0013] A fourth aspect of the present invention provides a method for preparing xylosonic acid, comprising the following steps: inoculating an engineered strain of *Fermentomonas motilityis* for producing xylosonic acid as described above into a culture medium containing D-xylose and D-glucose for fermentation; and harvesting the D-xylosonic acid from the fermentation product.

[0014] The advantages and positive effects of this invention are as follows: This invention uses Zymomonas mobilis ZM4 as the starting strain, and by dispersing and integrating the xylose dehydrogenase (XDH) gene at a specific site in its genome, it achieves the introduction and copy number increase of the exogenous gene encoding xylose dehydrogenase. The expression of the gene is regulated by the strong promoter—the pyruvate decarboxylase promoter Ppdc. The resulting engineered strain, especially the engineered strain with four to five copies, can efficiently convert D-xylose into D-xylonic acid. It has the advantages of high xylose conversion efficiency and strong D-xylonic acid production capacity. Moreover, the engineered strain has high genetic stability, providing a new approach for the industrial production of D-xylonic acid and the full utilization of xylose hydrolysate. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments are briefly introduced below.

[0016] Figure 1 This is a schematic diagram illustrating the construction principle of engineered strains of *Bacillus simulans* with different XDH gene copy numbers in embodiments of the present invention.

[0017] Figure 2 This is a schematic diagram illustrating the construction process of an engineered strain of *Bacillus simulans* with a single copy of the XDH gene, as described in an embodiment of the present invention.

[0018] Figure 3 This is a schematic diagram illustrating the construction process of an engineered strain of *Bacillus simulans* with a double copy of the XDH gene, as described in an embodiment of the present invention.

[0019] Figure 4 This is a schematic diagram illustrating the construction process of the *Mammotrophic Fermentation Monoclonal* engineered strain with a copy of the XDH gene in Embodiment 3 of the present invention.

[0020] Figure 5 This is a schematic diagram illustrating the construction process of the *Mammotrophic Fermentation Monoclonal* engineered strain with a copy of the XDH gene in Embodiment 4 of the present invention.

[0021] Figure 6 This is a schematic diagram illustrating the construction process of the *Mammotrophic Fermentation Monoclonal* engineered strain with copied XDH gene in Example 5 of the present invention.

[0022] Figure 7 The figure shows the fermentation performance test results of engineered strains of *Bacillus hygroscopicus* with different XDH gene copy numbers in embodiments of the present invention.

[0023] Figure 8 The graph shows the performance test results of xylonic acid production by the engineered strain of *Bacillus simulans* with a copy of the XDH gene in RM medium with different glucose contents in Example 5 of this invention.

[0024] Figure 9This is a graph showing the performance test results of the engineered strain of *Bacillus simulans* with a copy of the XDH gene in lignocellulose hydrolysate for the production of xyloic acid, as described in Example 5 of this invention.

[0025] Figure 10 The figure shows the performance test results of the *Mammotrophic Fermentatosporium* engineered strain with a copy of the XDH gene in Example 5 of this invention, after 15 generations of subculture to produce xyloic acid. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The embodiments described herein are for illustrative purposes only and are not intended to limit the invention. Based on the information contained in this invention, various changes to the precise description of the invention can be readily made by those skilled in the art without departing from the spirit and scope of the appended claims. For a better understanding of the invention and not to limit its scope, all figures indicating quantities, percentages, and other numerical values ​​used in this application should be understood in all cases to be modified by the word "approximately." Unless otherwise stated, the numerical parameters listed in the specification and appended claims are approximate values ​​and may be varied depending on the desired properties being sought. Each numerical parameter should at least be considered as obtainable by conventional rounding methods. Furthermore, it should be noted that, unless otherwise defined, scientific and technical terms used in the context of this invention should have the meaning commonly understood by those skilled in the art.

[0027] The terms “comprising,” “including,” “containing,” “having,” and similar words are non-restrictive and can include other steps and components that do not affect the result. The term “and / or” should be considered as a specific disclosure of each of the two specified features or components, with or without the other. For example, “A and / or B” would be considered to include (i) A, (ii) B, and (iii) A and B. The terms “first” and “second” are used for descriptive purposes only and should not be construed as indicating or implying relative importance or sequence.

[0028] The term "gene" refers to the complete nucleotide sequence required to produce a polypeptide chain or functional RNA. The terms "chimeric gene" or "fusion gene" have the same meaning and refer to any gene that is not a naturally occurring gene, containing regulatory and coding sequences that are not naturally present together. Therefore, a fusion gene can include regulatory and coding sequences originating from different organisms, or regulatory and coding sequences originating from the same organism but arranged in a manner different from their naturally occurring form.

[0029] The term "heterologous" or "exogenous" refers to a source different from the native (original) organism, for example, an organism derived from another species. For example, "heterologous gene" or "exogenous gene" refers to a gene that is not naturally present in the host organism and is introduced into the host organism through gene transfer. The XDH gene expression element of this invention is an exogenous gene that is not naturally present in *Zygomorpha motileis* ZM4.

[0030] The term "vector" refers to a self-replicating DNA molecule, often in the form of a circular double-stranded DNA molecule, used to transfer a foreign target gene into a host organism. Vectors containing foreign genes are called recombinant vectors. Typical vectors include plasmids, viruses, bacteriophages, granules, and minichromosomes. Plasmids are the most common form of vector; therefore, in the context of this invention, plasmids and vectors are used interchangeably.

[0031] The term "import" refers to the transfer of a foreign gene into a host organism, resulting in stable genetic inheritance. The imported gene can be in plasmid form retained in the host organism, or some imported genes can be integrated into the host organism's genome. The target gene and vector can be transferred into the host organism through methods such as "transfection," "transformation," or "transduction." A host organism containing the imported gene is called a "transgenic," "recombinant," "transformed," or "engineered" organism. Vector introduction into the host organism can be performed using conventional techniques well known to those skilled in the art.

[0032] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below.

[0033] Zymomonas mobilis (Z. mobilis), a naturally occurring facultative anaerobic Gram-negative bacterium that produces ethanol, possesses a unique ED metabolic pathway and high sugar fermentation efficiency, making it an ideal choice for industrial applications. Zymomonas mobilis is generally considered a safe (GRAS) strain; it exhibits high glucose uptake and tolerance; ethanol tolerance is as high as 16% (v / v); and it has a wide pH tolerance range, growing under conditions from 3.5 to 7.5. Furthermore, Zymomonas mobilis shows high tolerance to lignocellulose hydrolysates, and the mechanisms related to tolerance to inhibitors in the hydrolysate are well-established. Given these characteristics, Zymomonas mobilis can be considered a potential platform for producing organic acids from inexpensive lignocellulose hydrolysates. However, naturally occurring Zymomonas mobilis has a low metabolic utilization capacity for xylose, resulting in low xylose conversion efficiency and low xylose acid yield during fermentation.

[0034] To address this issue, this invention uses Zymomonas mobilis ZM4 as the starting strain and integrates the xylose dehydrogenase (XDH) gene into different sites in its genome (ZMO0038, ZMO1650, ZMO1094, ZMO1547, and / or ZMO1577) using gene editing technology. This achieves the introduction and copy number increase of the exogenous gene encoding xylose dehydrogenase. Furthermore, the expression of the gene is regulated by the strong promoter—the pyruvate decarboxylase promoter Ppdc—resulting in engineered strains with two to five copies of the XDH gene. The construction principle of the engineered strain is described in [link to documentation]. Figure 1 This engineered strain can efficiently convert D-xylose into D-xylonic acid. In particular, the four- to five-copy strains exhibit high xylose conversion efficiency and strong D-xylonic acid production capacity, providing a new approach for the industrial production of D-xylonic acid and the full utilization of xylose hydrolysate. Furthermore, the gene copy number of this invention is distributed in a dispersed manner, and the XDH gene integration sites are verified and relatively stable gene editing sites. The resulting engineered strain has high genetic stability, significantly reducing the problem of copy number loss and decreased fermentation capacity caused by homologous recombination of the same gene during multiple-copy gene passages.

[0035] The genome sequence of ZM4, a motile fermentation monoclonal strain used in this invention, can be found in Genbank (GCA_003054575).

[0036] Based on this, one embodiment of the present invention provides an engineered strain of Zymomonas mobilis for the production of xylosonic acid. The engineered strain is based on Zymomonas mobilis ZM4 as the starting strain, and at least two of the following gene loci in its genome—ZMO0038, ZMO1650, ZMO1094, ZMO1547, and ZMO1577—contain xylose dehydrogenase gene expression elements. The xylose dehydrogenase gene expression elements, from upstream to downstream, include a pyruvate decarboxylase promoter (Ppdc) and a xylose dehydrogenase (XDH) gene.

[0037] According to experimental verification of the present invention, the ability of the above-mentioned engineered strain to produce acetic acid through fermentation increases with the increase of the XDH gene copy number. Therefore, in a preferred embodiment of the present invention, at least three of the ZMO0038, ZMO1650, ZMO1094, ZMO1547 and ZMO1577 sites of the engineered strain genome contain xylose dehydrogenase gene expression elements, that is, it contains at least three xylose dehydrogenase gene copies; more preferably, at least four sites contain xylose dehydrogenase gene expression elements, that is, it contains at least four xylose dehydrogenase gene copies; even more preferably, all five sites contain xylose dehydrogenase gene expression elements, in which case it contains five xylose dehydrogenase gene copies.

[0038] Optionally, the nucleotide sequence of the xylose dehydrogenase gene is shown in SEQ ID NO.1, the XDH gene is derived from the strain *Paraburkholderia xenovorans*, and the nucleotide sequence of the pyruvate decarboxylase promoter is shown in SEQ ID NO.2.

[0039] Another embodiment of the present invention provides a method for constructing an engineered strain of *Syntrophus muscarinii* for producing xylosonic acid as described above, comprising the following steps:

[0040] Guide RNAs and donor DNA were designed based on the gene loci to be edited: ZMO0038, ZMO1650, ZMO1094, ZMO1547, and ZMO1577. The guide RNAs target a 32 bp sequence downstream of the CCC motif adjacent to the original spacer sequence of the gene to be edited. The nucleotide sequences of the guide RNAs targeting the ZMO0038, ZMO1650, ZMO1094, ZMO1547, and ZMO1577 gene loci (named gr0038, gr1650, gr1094, gr1547, and gr1577, respectively) are shown in SEQ ID. As shown in NO.7-11; the donor DNA includes the upstream and downstream homologous arm sequences of the guide RNA targeting sequence and the xylose dehydrogenase gene expression element located between the upstream and downstream homologous arm sequences, that is, the donor DNA sequence is composed of: upstream homologous arm of the targeting sequence—xylose dehydrogenase gene expression element—downstream homologous arm of the targeting sequence, wherein the nucleotide sequences of the upstream homologous arm (0038US) and downstream homologous arm (0038DS) targeting the ZMO0038 gene site are shown in SEQ ID NO.22-23, the nucleotide sequences of the upstream homologous arm (1650US) and downstream homologous arm (1650DS) targeting the ZMO1650 gene site are shown in SEQ ID NO.24-25, and the nucleotide sequences of the upstream homologous arm (1094US) and downstream homologous arm (1094DS) targeting the ZMO1094 gene site are shown in SEQ ID NO. As shown in NO.26-27, the nucleotide sequences of the upstream homologous arm (1547US) and downstream homologous arm (1547DS) targeting the ZMO1547 gene site are shown in SEQ ID NO.28-29, and the nucleotide sequences of the upstream homologous arm (1577US) and downstream homologous arm (1577DS) targeting the ZMO1577 gene site are shown in SEQ ID NO.30-31, respectively.

[0041] The guide RNA and the donor DNA targeting the ZMO0038, ZMO1650, ZMO1094, ZMO1547 and ZMO1577 gene loci were ligated into the expression vector to obtain the first to fifth editing plasmids. The editing plasmids of each gene locus were transformed into *Bacillus mobilis* ZM4, and the *Bacillus mobilis* engineered strain of the present invention was obtained by screening.

[0042] Specifically, the method for constructing the first editing plasmid includes: annealing the primer pair of the guide RNA targeting the ZMO0038 gene site; ligating the annealed product with the expression vector pEZ15Asp digested with restriction endonuclease BsaⅠ to obtain the first targeting plasmid, wherein the nucleotide sequence of the primer pair of the guide RNA targeting the ZMO0038 gene site is shown in SEQ ID NO. 12-13; obtaining the upstream homologous arm 0038US and the downstream homologous arm 0038DS sequences of the ZMO0038 site by PCR amplification; ligating the xylose dehydrogenase gene expression element between the 0038US and 0038DS sequences to construct the first donor DNA, wherein the nucleotide sequence of the primer pair for amplifying the 0038US sequence is shown in SEQ ID NO. 32-33, and the nucleotide sequence of the primer pair for amplifying the 0038DS sequence is shown in SEQ ID NO. 34-35; and ligating the first donor DNA into the first targeting plasmid using Gibson assembly technology to obtain the first editing plasmid.

[0043] Specifically, the method for constructing the second editing plasmid includes: annealing the primer pair of the guide RNA targeting the ZMO1650 gene site; ligating the annealed product with the expression vector pEZ15Asp digested with restriction endonuclease BsaⅠ to obtain the second targeting plasmid, wherein the nucleotide sequence of the primer pair of the guide RNA targeting the ZMO1650 gene site is shown in SEQ ID NO. 14-15; obtaining the upstream homologous arm 1650US and the downstream homologous arm 1650DS sequences of the ZMO1650 site by PCR amplification; ligating the xylose dehydrogenase gene expression element between the 1650US and 1650DS sequences to construct the second donor DNA, wherein the nucleotide sequence of the primer pair for amplifying the 1650US sequence is shown in SEQ ID NO. 36-37, and the nucleotide sequence of the primer pair for amplifying the 1650DS sequence is shown in SEQ ID NO. 38-39; and ligating the second donor DNA into the second targeting plasmid using Gibson assembly technology to obtain the second editing plasmid.

[0044] Specifically, the method for constructing the third editing plasmid includes: annealing the primer pair of the guide RNA targeting the ZMO1094 gene site; ligating the annealed product with the expression vector pEZ15Asp digested with restriction endonuclease BsaⅠ to obtain the third targeting plasmid, wherein the nucleotide sequence of the primer pair of the guide RNA targeting the ZMO1094 gene site is shown in SEQ ID NO. 16-17; obtaining the upstream homologous arm 1094US and the downstream homologous arm 1094DS sequences of the ZMO1094 site by PCR amplification; ligating the xylose dehydrogenase gene expression element between the 1094US and 1094DS sequences to construct the third donor DNA, wherein the nucleotide sequence of the primer pair for amplifying the 1094US sequence is shown in SEQ ID NO. 40-41, and the nucleotide sequence of the primer pair for amplifying the 1094DS sequence is shown in SEQ ID NO. 42-43; and ligating the third donor DNA into the third targeting plasmid using Gibson assembly technology to obtain the third editing plasmid.

[0045] Specifically, the method for constructing the fourth editing plasmid includes: annealing the primer pair of the guide RNA targeting the ZMO1547 gene site; ligating the annealed product with the expression vector pEZ15Asp digested with restriction endonuclease BsaⅠ to obtain the fourth targeting plasmid, wherein the nucleotide sequence of the primer pair of the guide RNA targeting the ZMO1547 gene site is shown in SEQ ID NO. 18-19; obtaining the upstream homologous arm 1547US and the downstream homologous arm 1547DS sequences of the ZMO1547 site by PCR amplification; ligating the xylose dehydrogenase gene expression element between the 1547US and 1547DS sequences to construct the fourth donor DNA, wherein the nucleotide sequence of the primer pair for amplifying the 1547US sequence is shown in SEQ ID NO. 44-45, and the nucleotide sequence of the primer pair for amplifying the 1547DS sequence is shown in SEQ ID NO. 46-47; and ligating the fourth donor DNA into the fourth targeting plasmid using Gibson assembly technology to obtain the fourth editing plasmid.

[0046] Specifically, the fifth editing plasmid construction method includes: annealing the primer pair of the guide RNA targeting the ZMO1577 gene site; ligating the annealed product with the expression vector pEZ15Asp digested with restriction endonuclease BsaⅠ to obtain the fifth targeting plasmid, wherein the nucleotide sequence of the primer pair of the guide RNA targeting the ZMO1577 gene site is shown in SEQ ID NO. 20-21; obtaining the upstream homologous arm 1577US and the downstream homologous arm 1577DS sequences of the ZMO1577 site by PCR amplification; ligating the xylose dehydrogenase gene expression element between the 1577US and 1577DS sequences to construct the fifth donor DNA, wherein the nucleotide sequence of the primer pair for amplifying the 1577US sequence is shown in SEQ ID NO. 48-49, and the nucleotide sequence of the primer pair for amplifying the 0038DS sequence is shown in SEQ ID NO. 50-51; and ligating the fifth donor DNA into the fifth targeting plasmid using Gibson assembly technology to obtain the fifth editing plasmid.

[0047] The method described above for ligating donor DNA to a target plasmid using Gibson assembly technology includes: amplifying the target plasmid using primer pairs 15Afk-F (nucleotide sequence as shown in SEQ ID NO. 52) and 15Afk-R (nucleotide sequence as shown in SEQ ID NO. 53) to obtain a linearized plasmid; then mixing the linearized plasmid with donor DNA at a molar ratio of 1:3; adding T5 exonuclease; and ligating the linearized plasmid with the donor DNA to obtain the editing plasmid. It should be noted that the above method is used for ligation of different donor DNAs and different target plasmids.

[0048] Additionally, it should be noted that the order in which the first to fifth edited plasmids are transformed into ZM4 of *Bacillus mobilis* is not particularly limited. For example, the first edited plasmid can be transformed first to integrate XDH into the ZMO0038 gene locus, and then the second edited plasmid can be transformed to integrate the XDH gene into the ZMO1650 gene locus. Alternatively, the second edited plasmid or other edited plasmids can be transformed first, followed by the first edited plasmid, etc., to screen for the target engineered strain.

[0049] The above-mentioned expression vector pEZ15Asp was constructed by referring to the literature "Yang S, Mohagheghi A, Franden MA, et al. Metabolic engineering of Zymomonas mobilis for 2,3-butanediol production from lignocellulosic biomass sugars[J]. Biotechnol Biofuels, 2016, 9(1):189." and the patent "CN110408642A", and will not be described in detail in this invention.

[0050] Optionally, the method for preparing the xylose dehydrogenase gene expression element includes: using the primer pair shown in SEQ ID NO.3-4, amplifying the xylose dehydrogenase gene sequence using the *Paraburkholderia xenovorans* xylose dehydrogenase gene as a template; using the primer pair shown in SEQ ID NO.5-6, amplifying the pyruvate decarboxylase promoter sequence using the *ZM4* genome as a template; and ligating the pyruvate decarboxylase promoter and the xylose dehydrogenase gene sequence to obtain the xylose dehydrogenase gene expression element.

[0051] Based on the excellent fermentation ability of the engineered strain of the present invention, another embodiment of the present invention provides the application of the above-described engineered strain of *Mammotrophic motility-fermenting* for the production of xylan acid in the preparation of xylan acid, and provides a specific application method.

[0052] Specifically, a method for preparing xylosonic acid includes the following steps: inoculating an engineered strain of *M. molybdenum* for producing xylosonic acid, as described above, into a culture medium containing D-xylose and D-glucose for fermentation; and harvesting the D-xylosonic acid from the fermentation product.

[0053] Optionally, the culture containing D-xylose and D-glucose can be a lignocellulose fermentation broth.

[0054] In the context of this invention, upstream refers to the 5' end of the gene, downstream refers to the 3' end of the gene, and from upstream to downstream is from the 5' end to the 3' end.

[0055] The invention is further illustrated below with reference to specific embodiments. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in *Molecular Cloning: A Laboratory Manual (Fourth Edition)* published by Cold Spring Harbor Laboratory, or as recommended by the manufacturer.

[0056] 1. Construction of Xylose Dehydrogenase (XDH) Gene Expression Elements

[0057] The xylose dehydrogenase (XDH) gene expression element consists of the XDH gene and a strong promoter located upstream of the XDH gene—the pyruvate decarboxylase promoter Ppdc. This promoter is more effective in expressing the xylose dehydrogenase gene XDH in motile fermentation monoclonal bacteria. The xylose dehydrogenase gene XDH is derived from the strain Paraburkholderia xenovorans.

[0058] The XDH gene sequence was amplified using the Burkholderia xylose dehydrogenase gene as a template, with primer pairs XDH-F and XDH-R. The Ppdc promoter sequence was amplified using the genome of *Fermentomonas motilityis* ZM4 as a template, with primer pairs Ppdc-F and Ppdc-R. The PCR system consisted of 20 μL of the following: 0.5 μL each of 10 μM forward and reverse primers (XDH-F / R or Ppdc-F / R), 5–10 μg template, and 10 μL of high-fidelity enzyme PrimerSTAR DNA Polymerase (purchased from Takara, catalog number R050A), with water added to a final volume of 20 μL. The PCR amplification program was set as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 55℃ annealing for 10 s, and 72℃ extension (set at 10 s / kb according to fragment length), for a total of 30 cycles; after the cycle reaction, the product was held at 72℃ for 5 min; the purified product was stored at -20℃. The Ppdc and XDH gene sequences were ligated using overlap PCR to obtain the XDH gene expression element. Overlap PCR procedure: 5-10 μg template (XDH and Ppdc), 25 μL PrimerSTAR DNA Polymerase, add water to make up to 46 μL; PCR amplification program set as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 55℃ annealing for 10 s, 72℃ extension (set according to fragment length at 10 s / kb), for a total of 10-12 cycles; add 2 μL each of 10 μM upstream and downstream primers (XDH-F and Ppdc-R), PCR amplification for 20-25 cycles, after which hold at 72℃ for 5 min; after purification, store at -20℃.

[0059] 2. Construction of editing plasmids for xylose dehydrogenase (XDH) gene expression elements

[0060] This invention uses Zymomonas mobilis ZM4 (ATCC31821) as the starting strain. Using its endogenous IF-type CRISPR-Cas gene editing system, exogenous editing plasmids containing guide RNA (gRNA) and donor DNA with targeted insertion sites are introduced into the bacterial cells. This allows for the insertion of xylose dehydrogenase (XDH) gene expression elements at the ZMO0038, ZMO1650, ZMO1094, ZMO1547, and / or ZMO1577 gene sites, thereby introducing and increasing the copy number of the exogenous gene encoding xylose dehydrogenase, resulting in an engineered strain that efficiently produces D-xylonic acid.

[0061] The construction process of the editing plasmid and the gene editing principle are detailed in patent "CN110408642A Efficient Deletion Method for Large Genomic Fragments Based on Endogenous CRISPR-Cas System of *Cytozoa motifosa* and Its Application (Publication Date: 2019-11-05)". The editing plasmid uses pEZ15Asp as its vector backbone and constructs an artificial CRISPR expression unit. This artificial CRISPR expression unit includes a promoter (leader) sequence, a CRISPR cluster, and a terminator. The CRISPR cluster consists of two repeat sequences (R) and two BsaI restriction sites located between the two repeat sequences. The sequence composition is: promoter leader—R—BsaI—BsaI—R—terminator. A gRNA gene is inserted between the two BsaI restriction sites. The gRNA guides the CRISPR-Cas system to cut the target insertion site. Guide RNA is obtained by annealing a guide RNA primer pair with an upstream BsaI restriction site. The annealed primer pair is then ligated to the BsaI-digested pEZ15Asp vector to obtain a target plasmid containing an artificial CRISPR expression unit. Further insertion of donor DNA into the target plasmid yields the target gene editing plasmid. The donor DNA includes upstream and downstream homologous arm sequences of the insertion site (i.e., the gRNA target sequence) and an XDH gene expression element located between these sequences. The donor DNA serves as a template for homologous recombination repair, enabling homologous recombination with the ZM4 genome through upstream and downstream homologous sequences, thus inserting the XDH gene expression element at the guide RNA target sequence.

[0062] The plasmid pEZ15Asp was obtained by inserting a spectinomycin-encoding gene as a marker gene into pEZ15A. Its construction method followed the literature "Yang S, Mohagheghi A, Franden MA, et al. Metabolic engineering of Zymomonas mobilis for 2,3-butanediol production from lignocellulosic biomass sugars[J]. Biotechnol Biofuels, 2016, 9(1):189." and the patent "CN110408642A". The plasmid pEZ15Asp (pEZ) contains only the essential elements: the origin of replication for *Escherichia coli* and *Zymomonas mobilis*, the antibiotic marker spectinomycin resistance gene addA, and a multiple cloning site. The multiple cloning site includes restriction enzyme sites such as EcoRI, XbaI, SpeI, and PstI. It has a simplified structural composition and can replicate in *Zymomonas mobilis* and *Escherichia coli*, serving as a Z. mobilis-E. coli shuttle vector. To obtain pEZ15A with different coding genes (e.g., resistance genes), different resistance genes can be inserted into it, referring to "Construction and Application of Plasmid pUC19-CM-D [J]. Anhui Agricultural Sciences, 2010, Published Method, No. 19".

[0063] The specific construction methods for the relevant editing plasmids are as follows:

[0064] (1) Guide RNA and primer design: Guide RNA gene sequences were designed based on the xylose dehydrogenase (XDH) gene expression element insertion sites genes ZMO0038, ZMO1650, ZMO1094, ZMO1547 and ZMO1577. The sequence 32 bp downstream of the protospacer adjacent motif (PAM) CCC inside the insertion site gene was used as the target sequence of the gRNA to guide the nuclease to cleave the target site, and guide RNA encoding genes gr0038, gr1650, gr1094, gr1547 and gr1577 targeting ZMO0038, ZMO1650, ZMO1094, ZMO1547 and ZMO1577 were designed.

[0065] Primer sequences were designed based on the gRNA sequence. The primer pairs for gr0038 were gr0038-F and gr0038-R, the primer pairs for gr1650 were gr1650-F and gr1650-R, the primer pairs for gr1094 were gr1094-F and gr1094-R, the primer pairs for gr1547 were gr1547-F and gr1547-R, and the primer pairs for gr1577 were gr1577-F and gr1577-R.

[0066] (2) Construction of target plasmids: The gr0038, gr1650, gr1094, gr1547 and gr1577 of the target insertion site genes ZMO0038, ZMO1650, ZMO1094, ZMO1547 and ZMO1577 were respectively ligated between the two BsaⅠ restriction sites of the pEZ15Asp vector to obtain the ZMO0038, ZMO1650, ZMO1094, ZMO1547 and ZMO1577 target plasmids, which were named the first to the fifth target plasmids.

[0067] The first target plasmid construction procedure is as follows: The vector pEZ15Asp was digested with the restriction endonuclease BsaⅠ to obtain linearized pEZ15Asp. Then, the gr0038 primer pair was annealed. The annealing system (10 μL) contained 1 μL each of 10 μM upstream and downstream primers (gr0038-F and gr0038-R), with water added to bring the total volume to 10 μL. The mixture was denatured at 95°C for 5 min and then cooled to room temperature. The annealed product was ligated to the linearized vector using T4 DNA ligase. The ligation system (10 μL) contained 2 μL of the annealed product, 20-40 ng of the linearized vector, 0.5 μL of T4 DNA ligase, 1 μL of T4 ligase buffer, and the remainder ddH2O. Ligation was performed at 22°C for 3-6 h. The ligation product was then transferred into the E. coli clone strain DH5α using conventional methods for plasmid construction. Screening was performed using spectinomycin plates, and single colonies were picked. Recombinants were screened by colony PCR using primer pairs pEZ15A-F and gr0038-R. Finally, the first target plasmid was obtained by sequencing verification.

[0068] The construction process of the second to fifth target plasmids is the same as that of the first target plasmid, the only difference being the annealing primers and the downstream primers for colony PCR, which will not be described in detail here.

[0069] (3) Ligation of donor DNA: In this embodiment of the invention, the upstream homologous arm sequences (US) and downstream homologous arm sequences (DS) of the ZMO0038, ZMO1650, ZMO1094, ZMO1547 and ZMO1577 sites of the ZM4 genome are amplified, and the XDH gene expression element is placed between the US and DS sequences to obtain the donor DNA of ZMO0038, ZMO1650, ZMO1094, ZMO1547 and ZMO1577, respectively, which are named the first to fifth donor DNA.

[0070] The ZM4 genome extraction method includes: collecting 2 mL of overnight ZM4 bacterial culture and extracting the genome using a bacterial genome extraction kit (purchased from Beijing TransGen Biotech Co., Ltd., catalog number EE10101R30722-V2R137).

[0071] The first donor DNA ligation procedure is as follows: Using the ZM4 genome as a template, the ZMO0038 US sequence (referred to as 0038US) is amplified using primer pairs 0038US-F and 0038US-R, and the ZMO0038 DS sequence (referred to as 0038DS) is amplified using primer pairs 0038DS-F and 0038DS-R. 0038US, the XDH gene expression element, and 0038US are then sequentially ligated using overlap PCR to form the first donor DNA fragment. The overlap PCR process is consistent with the method used to construct the XDH gene expression element.

[0072] The ligation process for the second to fifth donor DNA is the same as that for the first donor DNA, except that the US and DS amplification primers are different, which will not be repeated here. Specifically, the primer pair for amplifying the upstream homologous arm sequence (1650US) of ZMO1650 is 1650US-F and 1650US-R, and the primer pair for amplifying the downstream homologous arm sequence (1650DS) is 1650DS-F and 1650DS-R. Similarly, the primer pair for amplifying the upstream homologous arm sequence (1094US) of ZMO1094 is 1094US-F and 1094US-R, and the primer pair for amplifying the downstream homologous arm sequence (1094DS) is 1094DS-F and 1094DS-R. The primer pairs for amplifying the upstream homologous arm sequence (1547US) of ZMO1547 are 1547US-F and 1547US-R, and the primer pairs for amplifying the downstream homologous arm sequence (1547DS) are 1547DS-F and 1547DS-R. The primer pairs for amplifying the upstream homologous arm sequence (1577US) of ZMO1577 are 1577US-F and 1577US-R, and the primer pairs for amplifying the downstream homologous arm sequence (1577DS) are 1577DS-F and 1577DS-R.

[0073] (4) Construction of editing plasmids: The DNA from the first to the fifth donors was ligated into the first to the fifth target plasmids respectively, resulting in ZMO0038, ZMO1650, ZMO1094, ZMO1547 and ZMO1577 target plasmids, which were named the first to the fifth editing plasmids.

[0074] The first plasmid construction procedure is as follows: The first target plasmid constructed in the previous step was reverse-amplified by PCR using primers 15Afk-F and 15Afk-R to obtain a linearized first target plasmid. The linearized first target plasmid and the first donor DNA were then assembled and ligated using Gibson at a molar ratio of 1:3. The ligation product was then transformed into competent *E. coli* cells. The Gibson assembly reaction system consisted of: 0.12 pM donor DNA, 0.03 pM vector, 0.5 μL of 10×Buffer 4 (purchased from NEB, catalog number M0633L), and T5 Exonuclease (purchased from NEB, catalog number 7004S), with ddH2O added to a final volume of 5 μL. The two fragments were ligated using a one-step Gibson assembly reaction. The ligation product was then transferred into the E. coli clone strain DH5α using conventional methods for plasmid construction. Screening was performed using spectinomycin plates, and single colonies were picked. Recombinants were screened by colony PCR using primer pairs pEZ15A-F and pEZ15A-R. Finally, the results were verified by sequencing to obtain the first edited plasmid (pl2r-ZMO0038-XDH).

[0075] The construction process of the second to fifth editing plasmids (pl2r-ZMO1650-XDH, pl2r-ZMO1094-XDH, pl2r-ZMO1547-XDH and pl2r-ZMO1577-XDH) is the same as that of the first editing plasmid, the only difference being the different donor DNA, which will not be described in detail here.

[0076] 3. Preparation of competent cells of *Mammotrophic Fermentation Monoclonal*

[0077] 100 μL of the cryopreserved Zymomonas mobilis ZM4 (Z. mobilis subsp. mobilis ZM4, ATCC 31821) strain was inoculated into a cryovial containing 1 mL of RM liquid medium and incubated statically at 30°C to activate the strain. The strain was then cultured until it reached the logarithmic growth phase (OD2). 600nm =1.5-2.0) and then transferred to 20 mL of RM liquid medium, and incubated statically at 30°C. Once the strain reached the logarithmic growth phase, it was transferred to 200 mL of RM liquid medium, and the initial OD was controlled. 600nm=0.025-0.05, cultured at 100 rpm and 30℃. When the OD600nm is in the range of 0.3-0.4, the cells are collected by centrifugation at 400 rpm at room temperature. Then, the cells are washed once with sterile water and twice with 10% glycerol. Finally, the cells are resuspended in 400 μL of 10% glycerol to obtain ZM4 competent cells, which are then dispensed into 50 μL portions for use.

[0078] 4. Preparation of engineered strains of *Morphozoa motilityis*

[0079] Construction of engineered strain ZM4-XDH: A schematic diagram of the construction of this engineered strain is shown below. Figure 2 As shown, the xylose dehydrogenase XDH expression element was integrated into the ZMO0038 site in the genome of the starting strain, *Z. motile fermentomonas* ZM4, using an endogenous CRISPR-Cas gene editing system. The specific procedure was as follows: 1 μg of the first edited plasmid (pl2r-ZMO0038-XDH) was added to 50 μL of competent ZM4 cells, mixed well, and then transferred to a 0.1 cm electroporation cuvette. The cuvette was placed in an electroporator for electroporation. The electroporator program settings were: 200 Ω, 25 μF, and 1800 V. After electroporation, the culture was transferred to 1 mL of RM medium and incubated statically at 30°C for 4-6 h. Then, 200 μL of the culture was evenly spread onto an RM plate containing 100 μg / mL spectinomycin (spectinomycin resistant) and incubated upside down at 30°C for 2-3 days. After colonies grew, colony PCR was performed on the engineered strain using the verification primers 0038check-F and 0038check-R. The strain with the band size consistent with the expectation was verified by sequencing, and a single-copy engineered strain ZM4-XDH capable of producing D-xyloic acid was obtained. The correct strain was stored for later use.

[0080] Construction of the engineered strain ZM4-2XDH: The second editing plasmid (pl2r-ZMO1650-XDH) was electrotransformed into ZM4-XDH competent cells to introduce the XDH gene expression element into the ZMO1650 site of the engineered strain ZM4-XDH genome, resulting in a double-copy engineered strain ZM4-2XDH capable of producing D-xyloic acid. A schematic diagram of its construction is shown below. Figure 3 As shown. The verification primer pairs are 1650check-F and 1650check-R.

[0081] Construction of engineered strain ZM4-3XDH: The third editing plasmid (pl2r-ZMO1094-XDH) was electrotransformed into ZM4-2XDH competent cells to introduce the XDH gene expression element into the ZMO1094 site of the engineered strain ZM4-2XDH genome, resulting in a three-copy engineered strain ZM4-3XDH capable of producing D-xyloic acid. A schematic diagram of its construction is shown below. Figure 4As shown. The verification primer pairs are 1094check-F and 1094check-R.

[0082] Construction of the engineered strain ZM4-4XDH: The fourth editing plasmid (pl2r-ZMO1547-XDH) was electrotransformed into ZM4-3XDH competent cells to introduce the XDH gene expression element into the ZMO1547 site of the engineered strain ZM4-3XDH genome, resulting in a four-copy engineered strain ZM4-4XDH capable of producing D-xyloic acid. A schematic diagram of its construction is shown below. Figure 5 As shown. The verification primer pairs are 1547check-F and 1547check-R.

[0083] Construction of engineered strain ZM4-5XDH: The fifth editing plasmid (pl2r-ZMO1577-XDH) was electrotransformed into ZM4-4XDH competent cells to introduce the XDH gene expression element into the ZMO1577 site of the engineered strain ZM4-4XDH genome, resulting in a five-copy engineered strain ZM4-5XDH capable of producing D-xyloic acid. A schematic diagram of its construction is shown below. Figure 6 As shown. The verification primer pairs are 1577check-F and 1577check-R.

[0084] The gene and primer sequences involved in this embodiment are shown in Table 1.

[0085] Table 1. Gene and primer sequences used in the embodiments of the present invention.

[0086]

[0087]

[0088]

[0089] 5. Performance testing of engineered strains of *Bacillus simulans*

[0090] 5.1. Growth and fermentation performance testing of engineered strains

[0091] The engineered strains were inoculated into 100 mL Erlenmeyer flasks containing 20% ​​RMG2X5, RMG5X5, or lignocellulose hydrolysate, respectively, and 40 g / L calcium carbonate was added. The flasks were then incubated at 100 rpm and 30 °C. Samples were taken at intervals during fermentation for HPLC analysis. Samples from different time points were centrifuged at 12000 rpm for 2 min, and the supernatant was used as the detection sample. The RMG2X5 enrichment medium contained 20 g / L glucose, 50 g / L xylose, 10 g / L yeast extract, and 2 g / L KH2PO4. The RMG5X5 enrichment medium contained 50 g / L glucose, 50 g / L xylose, 10 g / L yeast extract, and 2 g / L KH2PO4. The lignocellulose hydrolysate, containing 102 g / L glucose and 48 g / L xylose, was diluted 3 times and used for xylosinic acid fermentation.

[0092] The method for determining the content of xylose, xylose acid, glucose, and ethanol in the fermentation supernatant included: using a Shimadzu Prominence Plus series high-performance liquid chromatograph; detecting the content of xylose, glucose, and ethanol using a differential refractive index detector (RID-20A); detecting the content of xylose acid using a UV detector (SPD-20A); using an organic acid column (Bio-Rad Aminex HPX-87H, 300mm × 7.8mm); maintaining a cell temperature of 40℃ and a column oven temperature of 60℃; using 5mM sulfuric acid as the mobile phase at a flow rate of 0.5mL / min, initially setting the flow rate to 0.2mL / min and gradually increasing it to 0.5mL / min after the column pressure stabilized; and injecting a volume of 20μL. The mobile phase preparation was 0.005mol / L H₂SO₄ at a flow rate of 0.5mL / min. It can be used after returning to room temperature. After filtering the supernatant through a 0.22 μL filter, load 20 μL for detection.

[0093] The D-xylose and D-xylonic acid contents in the fermentation supernatant were detected using the above-mentioned detection method, and the carbon conversion rate was calculated. Carbon conversion rate = D-xylonic acid yield / D-xylose consumption / 1.10, where "1.10" represents the theoretical value of D-xylose conversion to D-xylonic acid, that is, 1.00 g / L D-xylose can theoretically be completely converted to 1.10 g / L D-xylonic acid.

[0094] The ability of engineered strains with different XDH gene copy numbers to produce D-xyloic acid in RMG2X5 medium is as follows: Figure 7 As shown, it can be seen that as the gene copy number increases, the strain's ability to metabolize xylose and its xylonic acid production also increase, reaching a maximum at five copies.

[0095] The five-copy engineered strain ZM4-5XDH was selected to further determine the D-xyloic acid fermentation performance under different initial glucose concentrations. The xyloic acid production performance in RMG2X5, RMG5X5 media, or lignocellulose hydrolysate was as follows: Figure 8-9 As shown, the relevant results are summarized in Table 2. The results show that the engineered strain ZM4-5XDH was able to consume all the D-xylose in RMG2X5, RMG5X5 and lignocellulose hydrolysate within 72 h, and almost all of the D-xylose was converted into D-xylonic acid, with a carbon conversion rate of nearly 100%.

[0096] Table 2. Fermentation performance of the engineered strain ZM4-5XDH in Example 5 of this invention.

[0097]

[0098] 5.2 Stability test of engineered strains in communication culture

[0099] Multi-copy genes are often lost due to homologous recombination of identical gene sequences. This invention employs commonly used or validated, relatively stable gene editing sites, expressing each copy of the gene at multiple sites, which makes gene editing easier and better ensures genetic stability. To verify the genetic stability of the multi-copy engineered strain in this embodiment, the engineered strain ZM4-5XDH was selected and cultured for 15 generations. Its xylose production performance was compared with that of the 0th generation parent to evaluate its stability.

[0100] The subculture process included: inoculating the engineered strain ZM4-5XDH into a cryovial containing 1 mL of RMG5 liquid medium and incubating at 30°C until the culture became turbid, approximately 12 hours later. Then, 50 μL of the culture was inoculated into a new cryovial containing 1 mL of RMG5 liquid medium and incubated at 30°C until the culture became turbid. This process was repeated until the 15th generation strain was obtained. th ) strain and generation 0 (0 th The fermentation performance of the strain was tested, and the results are as follows: Figure 10 As shown, the relevant results are summarized in Table 3. The results show that the fermentation performance of the engineered strain ZM4-5XDH did not decrease after 15 generations of subculturing, demonstrating good subculturing stability.

[0101] Table 3 Comparison of fermentation performance of the fifth copy engineered strain ZM4-5XDH before and after subculturing in Example 5 of this invention.

[0102]

[0103] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An engineered strain of *Syntrophus muscarinii* for the production of xylosonic acid, characterized in that, The engineered strain uses *Zeromonas motileis* ZM4 as the starting strain, and xylose dehydrogenase gene expression elements are inserted into the ZMO0038, ZMO1650, ZMO1094, ZMO1547, and ZMO1577 gene loci in its genome. The xylose dehydrogenase gene expression elements include a pyruvate decarboxylase promoter and a xylose dehydrogenase gene from upstream to downstream. The nucleotide sequence of the xylose dehydrogenase gene is shown in SEQ ID NO.1, and the nucleotide sequence of the pyruvate decarboxylase promoter is shown in SEQ ID NO.

2.

2. The method for constructing an engineered strain of *Syntrophus muscarinii* for producing xylosonic acid as described in claim 1, characterized in that, Includes the following steps: Guide RNA and donor DNA were designed based on the ZMO0038, ZMO1650, ZMO1094, ZMO1547 and ZMO1577 gene loci to be edited. The guide RNA targets a sequence 32 bp downstream of the motif CCC adjacent to the original spacer sequence of the gene to be edited. The nucleotide sequences of the guide RNA targeting the ZMO0038, ZMO1650, ZMO1094, ZMO1547 and ZMO1577 gene sites are shown in SEQ ID NO.7-11, respectively. The donor DNA includes upstream and downstream homologous arm sequences of the guide RNA's target sequence and a xylose dehydrogenase gene expression element located between the upstream and downstream homologous arm sequences. The nucleotide sequences of the upstream and downstream homologous arms targeting the ZMO0038 gene site are shown in SEQ ID NO. 22-23, the upstream and downstream homologous arms targeting the ZMO1650 gene site are shown in SEQ ID NO. 24-25, the upstream and downstream homologous arms targeting the ZMO1094 gene site are shown in SEQ ID NO. 26-27, the upstream and downstream homologous arms targeting the ZMO1547 gene site are shown in SEQ ID NO. 28-29, and the upstream and downstream homologous arms targeting the ZMO1577 gene site are shown in SEQ ID NO. 30-31. The guide RNA and donor DNA targeting the ZMO0038, ZMO1650, ZMO1094, ZMO1547 and ZMO1577 gene loci were ligated into the expression vector to obtain the first to fifth editing plasmids. The editing plasmids of each gene locus were transformed into *Syntrophus motilityis* ZM4, and the engineered strains of *Syntrophus motilityis* were screened to obtain the *Syntrophus motilityis* strains.

3. The method for constructing an engineered strain of *Xylaria motility-fermenting monofilament* for producing xylosonic acid according to claim 2, characterized in that, The method for constructing the first editing plasmid includes: annealing the primer pair of the guide RNA targeting the ZMO0038 gene site; ligating the annealed product with the expression vector pEZ15Asp digested with restriction endonuclease BsaⅠ to obtain the first targeting plasmid, wherein the nucleotide sequence of the primer pair of the guide RNA targeting the ZMO0038 gene site is shown in SEQ ID NO. 12-13; obtaining the upstream homologous arm 0038US and the downstream homologous arm 0038DS sequences of the ZMO0038 site by PCR amplification; ligating the xylose dehydrogenase gene expression element between the 0038US and 0038DS sequences to construct the first donor DNA, wherein the nucleotide sequence of the primer pair for amplifying the 0038US sequence is shown in SEQ ID NO. 32-33, and the nucleotide sequence of the primer pair for amplifying the 0038DS sequence is shown in SEQ ID NO. 34-35; and ligating the first donor DNA into the first targeting plasmid using Gibson assembly technology to obtain the first editing plasmid. The method for constructing the second editing plasmid includes: annealing the primer pair of the guide RNA targeting the ZMO1650 gene site; ligating the annealed product with the expression vector pEZ15Asp digested with restriction endonuclease BsaⅠ to obtain the second targeting plasmid, wherein the nucleotide sequence of the primer pair of the guide RNA targeting the ZMO1650 gene site is shown in SEQ ID NO.14-15; obtaining the upstream homologous arm 1650US and the downstream homologous arm 1650DS of the ZMO1650 site by PCR amplification; ligating the xylose dehydrogenase gene expression element between the 1650US and 1650DS sequences to construct the second donor DNA, wherein the nucleotide sequence of the primer pair for amplifying the 1650US sequence is shown in SEQ ID NO.36-37, and the nucleotide sequence of the primer pair for amplifying the 1650DS sequence is shown in SEQ ID NO.38-39; and ligating the second donor DNA into the second targeting plasmid using Gibson assembly technology to obtain the second editing plasmid. The method for constructing the third editing plasmid includes: annealing the primer pair of the guide RNA targeting the ZMO1094 gene site; ligating the annealed product with the expression vector pEZ15Asp digested with restriction endonuclease BsaⅠ to obtain the third targeting plasmid, wherein the nucleotide sequence of the primer pair of the guide RNA targeting the ZMO1094 gene site is shown in SEQ ID NO.16-17; obtaining the upstream homologous arm 1094US and the downstream homologous arm 1094DS sequences of the ZMO1094 site by PCR amplification; ligating the xylose dehydrogenase gene expression element between the 1094US and 1094DS sequences to construct the third donor DNA, wherein the nucleotide sequence of the primer pair for amplifying the 1094US sequence is shown in SEQ ID NO.40-41, and the nucleotide sequence of the primer pair for amplifying the 1094DS sequence is shown in SEQ ID NO.42-43; and ligating the third donor DNA into the third targeting plasmid using Gibson assembly technology to obtain the third editing plasmid. The method for constructing the fourth editing plasmid includes: annealing the primer pair of the guide RNA targeting the ZMO1547 gene site; ligating the annealed product with the expression vector pEZ15Asp digested with restriction endonuclease BsaⅠ to obtain the fourth targeting plasmid, wherein the nucleotide sequence of the primer pair of the guide RNA targeting the ZMO1547 gene site is shown in SEQ ID NO. 18-19; obtaining the upstream homologous arm 1547US and the downstream homologous arm 1547DS of the ZMO1547 site by PCR amplification; ligating the xylose dehydrogenase gene expression element between the 1547US and 1547DS sequences to construct the fourth donor DNA, wherein the nucleotide sequence of the primer pair for amplifying the 1547US sequence is shown in SEQ ID NO. 44-45, and the nucleotide sequence of the primer pair for amplifying the 1547DS sequence is shown in SEQ ID NO. 46-47; and ligating the fourth donor DNA into the fourth targeting plasmid using Gibson assembly technology to obtain the fourth editing plasmid. The fifth editing plasmid construction method includes: annealing the primer pair of the guide RNA targeting the ZMO1577 gene site; ligating the annealed product with the expression vector pEZ15Asp digested with restriction endonuclease BsaⅠ to obtain the fifth targeting plasmid, wherein the nucleotide sequence of the primer pair of the guide RNA targeting the ZMO1577 gene site is shown in SEQ ID NO. 20-21; obtaining the upstream homologous arm 1577US and the downstream homologous arm 1577DS of the ZMO1577 site by PCR amplification; ligating the xylose dehydrogenase gene expression element between the 1577US and 1577DS sequences to construct the fifth donor DNA, wherein the nucleotide sequence of the primer pair for amplifying the 1577US sequence is shown in SEQ ID NO. 48-49, and the nucleotide sequence of the primer pair for amplifying the 0038DS sequence is shown in SEQ ID NO. 50-51; and ligating the second donor DNA into the fifth targeting plasmid using Gibson assembly technology to obtain the fifth editing plasmid.

4. The method for constructing an engineered strain of *Syntrophus motilityis* for producing xylosonic acid according to claim 2, characterized in that, The method for preparing the xylose dehydrogenase gene expression element includes: using the primer pair shown in SEQ ID NO.3-4, amplifying the xylose dehydrogenase gene sequence using the Burkholderia xylose dehydrogenase gene as a template; using the primer pair shown in SEQ ID NO.5-6, amplifying the pyruvate decarboxylase promoter sequence using the genome of *Fermentomonas motilityis* ZM4 as a template; and ligating the pyruvate decarboxylase promoter and the xylose dehydrogenase gene sequence to obtain the xylose dehydrogenase gene expression element.

5. The application of the engineered strain of *Fermentomonas motilityis* for producing xylan acid as described in claim 1 in the preparation of xylan acid.

6. A method for preparing xylan acid, characterized in that, Includes the following steps: The engineered strain of *M. motile fermentum* for producing xyloic acid as described in claim 1 is inoculated into a medium containing D-xylose and D-glucose for fermentation; and D-xyloic acid is harvested from the fermentation product.

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