Applications of xanthan gum-producing strains constructed using UDP-glucose dehydrogenase, xanthan gum-producing strains, their construction methods and applications

Abstract

This invention provides the application of xanthan gum-producing strains constructed from UDP-glucose dehydrogenase, the xanthan gum-producing strains and their construction methods and applications. Single copies of UDP-glucose dehydrogenase encoding genes from different sources, driven by the Ptac promoter, were integrated into the hrpF site on the chromosome of *Xanthomonas javanica*, and engineered strains with SeUgdG as the preferred enzyme were screened. Furthermore, expression cassettes of the water-producing NADH oxidase SmNox-2, driven by different promoters, were integrated into the XC_3033 site. The results show that the combined expression of SmNox-2 and SeUgdG driven by the Plac promoter can regulate intracellular NAD+. + This invention improves the xanthan gum yield by adjusting the NADH ratio. It provides a new technical solution for constructing high-yield xanthan gum engineered strains through the synergistic regulation of xanthan gum precursor synthesis and intracellular coenzyme regeneration.

Description

Technical Field

[0001] This invention belongs to the field of microbial engineering and fermentation engineering technology, and relates to the application of xanthan gum production strains constructed from UDP-glucose dehydrogenase, xanthan gum production strains and their construction methods and applications. Specifically, it relates to the screening of UDP-glucose dehydrogenase, the expression regulation of water-producing NADH oxidase, and the construction methods and applications of xanthan gum production strains. Background Technology

[0002] Xanthan gum is an extracellular heteropolysaccharide produced by the fermentation of Xanthomonas species. It possesses excellent thickening, suspending, emulsifying stability, and pseudoplasticity, and is widely used in food, oil extraction, daily chemical, pharmaceutical, and agricultural industries. With the expanding applications of xanthan gum, improving the synthesis capacity and fermentation yield of the producing strains is crucial for reducing production costs and increasing production efficiency.

[0003] Xanthan gum repeating units are mainly composed of glucose, mannose, and glucuronic acid, and their biosynthesis requires activated sugar precursors such as UDP-glucose, GDP-mannose, and UDP-glucuronic acid. UDP-glucuronic acid, produced by UDP-glucose dehydrogenase, is one of the essential precursors for the assembly of xanthan gum repeating units. Therefore, improving the catalytic efficiency of UDP-glucose dehydrogenase enhances the supply of UDP-glucuronic acid and can further promote xanthan gum synthesis.

[0004] UDP-glucose dehydrogenases from different sources may differ in amino acid composition, catalytic performance, and host adaptability. The effects of introducing UDP-glucose dehydrogenases from different sources into Xanthomonas bacteria on xanthan gum synthesis are not entirely the same. Therefore, simply increasing the expression level of endogenous UDP-glucose dehydrogenase is insufficient to fully realize the enzymatic performance and host adaptability advantages gained from source selection; systematic screening of UDP-glucose dehydrogenases from different sources remains necessary.

[0005] UDP-glucose dehydrogenase uses NAD+ in the process of catalyzing the conversion of UDP-glucose to UDP-glucuronic acid. + It acts as a coenzyme and generates NADH. Increasing UDP-glucose dehydrogenase activity can promote the formation of target precursors and also alter intracellular NAD+. + The conversion relationship between UDP-glucuronic acid and NADH. Therefore, by enhancing UDP-glucuronic acid synthesis and moderately regulating the intracellular coenzyme regeneration process, it is beneficial to maintain the continuous catalysis of UDP-glucose dehydrogenase.

[0006] Aqueous NADH oxidases can oxidize NADH to NAD. + It uses water as the main reaction product and can be used to regulate intracellular NAD.+ / NADH state. However, too low an expression level of NADH oxidase may be insufficient to exert an effective coenzyme regulatory function, while too high an expression level may affect the host's normal metabolism. Therefore, it is necessary to rationally match the expression level of NADH oxidase according to the expression and catalytic level of UDP-glucose dehydrogenase.

[0007] Current technologies still lack a technical solution to synergistically enhance xanthan gum synthesis capacity in Xanthomonas by screening UDP-glucose dehydrogenases from different sources and further combining this with NADH oxidase expression intensity regulation. Therefore, it is necessary to develop a method for constructing xanthan gum-producing strains that simultaneously provides UDP-glucuronic acid precursors and facilitates intracellular coenzyme regeneration. Summary of the Invention

[0008] In one aspect, the present invention provides the application of UDP-glucose dehydrogenase SeUgdG or its encoded nucleic acid molecule in the construction of xanthan gum production strains.

[0009] In some implementations, the application includes the recombination and integration of the expression cassette encoding the nucleic acid molecule of the UDP-glucose dehydrogenase SeUgdG into the Xanthomonas genome.

[0010] In some implementations, the application includes recombinantly integrating the expression cassette into the hrpF site of the Xanthomonas genome.

[0011] In some embodiments, the UDP-glucose dehydrogenase SeUgdG is derived from *Elodea sphingosine monocytogenes* (…). Sphingomonas elodea ).

[0012] In some implementations, the Ptac promoter is used in the expression cassette of the UDP-glucose dehydrogenase gene.

[0013] In some embodiments, the amino acid sequence of the UDP-glucose dehydrogenase SeUgdG is as shown in SEQ ID NO: 8, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 8 and has UDP-glucose dehydrogenase activity.

[0014] In some embodiments, the nucleic acid molecule encoding the UDP-glucose dehydrogenase SeUgdG is as shown in SEQ ID NO:7, or has at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO7 and encodes the UDP-glucose dehydrogenase SeUgdG.

[0015] In some implementations, the starting strain used is Xanthomonas brasiliensis.

[0016] On the other hand, the present invention provides a method for constructing a xanthan gum-producing strain, characterized by comprising recombinantly integrating the expression cassette of the heterologous UDP-glucose dehydrogenase SeUgdG gene into the Xanthomonas genome.

[0017] In some embodiments, the method further includes recombinantly integrating the expression cassette of the water-producing NADH oxidase SmNox-2 gene into the Xanthomonas genome.

[0018] In some implementations, the expression cassette of the UDP-glucose dehydrogenase gene is integrated into the hrpF site of the Xanthomonas genome.

[0019] In some embodiments, the expression cassette of the water-producing NADH oxidase SmNox-2 gene is integrated into the XC_3033 site of the Xanthomonas genome.

[0020] In some implementations, the Ptac promoter is used in the expression cassette of the UDP-glucose dehydrogenase gene.

[0021] In some embodiments, the Plac promoter is used in the expression cassette of the aqueous NADH oxidase SmNox-2 gene.

[0022] In some embodiments, the UDP-glucose dehydrogenase SeUgdG is derived from *Elodea sphingosine monocytogenes* (…). Sphingomonas elodea ).

[0023] In some embodiments, the amino acid sequence of the UDP-glucose dehydrogenase SeUgdG is as shown in SEQ ID NO: 8, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 8 and has UDP-glucose dehydrogenase activity.

[0024] In some embodiments, the nucleic acid molecule encoding the UDP-glucose dehydrogenase SeUgdG is as shown in SEQ ID NO:7, or has at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:7 and encodes the UDP-glucose dehydrogenase SeUgdG.

[0025] In some embodiments, the amino acid sequence of the aqueous NADH oxidase SmNox-2 is as shown in SEQ ID NO:20, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:20 and has aqueous NADH oxidase activity.

[0026] In some embodiments, the nucleotide sequence of the aqueous NADH oxidase SmNox-2 gene is as shown in SEQ ID NO: 19, or has at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 19 and encodes the aqueous NADH oxidase SmNox-2.

[0027] In some embodiments, the Xanthomonas is Xanthomonas brasiliensis.

[0028] On the other hand, the present invention provides a xanthan gum producing strain constructed by the above method.

[0029] On the other hand, the present invention provides the application of the above-mentioned xanthan gum producing strain in the production of xanthan gum or in increasing xanthan gum yield.

[0030] This invention, through systematic screening of UDP-glucose dehydrogenases from different sources, obtained SeUgdG, which can enhance the xanthan gum synthesis capacity of Xanthomonas, thus expanding the functional enzyme resources that can be used for the modification of xanthan gum-producing strains. Compared with wild-type strains or other control strains, the xanthan gum-producing strain constructed in this invention significantly increases the xanthan gum yield produced by fermentation. Attached Figure Description

[0031] Figure 1 This is a schematic diagram illustrating the principle of the combined regulation of xanthan gum precursor synthesis by UDP-glucose dehydrogenase and aqueous NADH oxidase according to the present invention. In this diagram, Ugd represents UDP-glucose dehydrogenase, Nox represents aqueous NADH oxidase, UDP-Glc represents UDP-glucose, and UDP-GlcA represents UDP-glucuronic acid. Detailed Implementation

[0032] Unless otherwise stated, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art. As used herein, "encoding nucleic acid molecule" refers to a nucleic acid molecule that follows the rules of the genetic code, directs the synthesis of a specific protein or polypeptide chain, and determines its amino acid sequence. Encoding nucleic acid molecules can be DNA, cDNA, or RNA. For DNA-encoding nucleic acid molecules, their boundaries are typically defined by open reading frames, which typically begin with the ATG start codon or alternative start codons such as GTG and TTG, and end with a stop codon such as TAA, TAG, or TGA.

[0033] The term "expression cassette" as used in this article refers to a DNA molecule or fragment thereof that contains functional elements capable of driving the transcription and expression of a target gene. Typically, an expression cassette includes at least a promoter and a target gene, and may further include a ribosome binding site, a transcription termination signal, and other regulatory sequences.

[0034] The term “amino acid sequence” as used in this article is synonymous with the terms “polypeptide,” “protein,” and “peptide” and is used interchangeably. A standard single-letter or three-letter code for the amino acid residues is used, where the amino acid sequence is presented with a standard amino-to-carboxyl terminus orientation (i.e., N→C).

[0035] When referring to amino acid or nucleotide sequences, the term "sequence identity" (also known as "sequence uniformity") refers to the degree of similarity between two amino acid or nucleotide sequences (e.g., a query sequence and a reference sequence), typically expressed as a percentage. Commonly used sequence alignment algorithms or software include DANMAN, CLUSTALW, MAFFT, BLAST, MUSCLE, etc. For the purposes of this invention, the publicly available alignment software BLAST (available at https: / / www.ncbi.nlm.nih.gov / ) can be used to obtain the optimal sequence alignment and calculate the sequence identity between the two amino acid or nucleotide sequences using default settings.

[0036] The term "or" refers to a single element among the listed optional elements, unless the context explicitly indicates otherwise. The term "and / or" refers to any one, any two, any three, any more, or all of the listed optional elements.

[0037] The terms “comprising,” “containing,” “having,” and similar expressions used herein do not exclude elements not listed. These terms also include cases where the text consists only of the listed elements.

[0038] Compared with the prior art, the present invention has the following beneficial effects: By systematically screening UDP-glucose dehydrogenases from different sources, SeUgdG, which can enhance the xanthan gum synthesis capacity of Xanthan Bacillus, was obtained, thus expanding the functional enzyme resources that can be used to modify xanthan gum production strains.

[0039] By combining UDP-glucose dehydrogenase-mediated enhancement of precursor synthesis with water-producing NADH oxidase-mediated coenzyme regeneration, the supply of xanthan gum precursors and the intracellular coenzyme status were regulated.

[0040] By using promoters of different strengths to regulate SmNox-2 expression, a NADH oxidase expression mode adapted to SeUgdG was determined, avoiding the mismatch in regulatory effects caused by simply increasing the expression intensity of NADH oxidase.

[0041] The expression cassette is site-specifically integrated into the Xanthomonas chromosome in a single-copy form, without relying on free plasmids for maintenance. This is beneficial for improving the genetic stability of the expression element during continuous culture and can be used for xanthan gum fermentation production.

[0042] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0043] The methods for preparing Xanthomonas campestris competent cells, electroporation, and gene replacement used in this invention are based on the literature (Wang XL, Zheng DN, Liang RB. An Efficient Electro-Competent Cells Generation Method of Xanthomonas campestris pv. campestris: Its Application for Plasmid Transformation and Gene Replacement[J]. Advances in Microbiology , 2016, 6(2):79-87.), and made appropriate adjustments according to the strains and recombinant vectors used in this invention.

[0044] Example 1: Preliminary screening and sequence determination of candidate UDP-glucose dehydrogenase and NADH oxidase To obtain a UDP-glucose dehydrogenase suitable for the Xanthomonas xanthan gum synthesis system, the inventors searched and preliminarily screened UDP-glucose dehydrogenases and their homologous enzymes described in published literature and sequence databases. The screening of candidate enzymes mainly considered the following factors: candidate sequences had reported UDP-glucose dehydrogenase activity or corresponding functional annotations; the coding region was complete and did not contain frameshift mutations or premature stop codons; different candidate enzymes showed certain differences in microbial origin and amino acid sequence to increase the likelihood of obtaining a target enzyme suitable for the Xanthomonas expression system.

[0045] Based on the above screening principles, eight enzymes from different sources—BsUglF, LbjUGD, RkpK, SeUgdG, SpHasB, SsanUgdG, SzHasB, and ZmUGD—were identified as candidate UDP-glucose dehydrogenases. Simultaneously, the endogenous UDP-glucose dehydrogenase XccUGDH from the *Xanthomonas* strain used in this invention was used as a control to subsequently compare the effects of candidate enzymes from different sources on xanthan gum synthesis, and to screen for candidate enzymes that are beneficial for increasing xanthan gum yield through functional verification.

[0046] To regulate the intracellular redox balance involved in the catalytic process of UDP-glucose dehydrogenase, this invention further selected the water-producing NADH oxidase SmNox-2 as a redox regulatory element for subsequent investigation of the effects of combined expression of SmNox-2 and candidate UDP-glucose dehydrogenases on Xanthomonas growth, intracellular redox status, and xanthan gum synthesis. A candidate enzyme sequence library was thus established, consisting of eight exogenous candidate UDP-glucose dehydrogenases, one endogenous XccUGDH control, and one SmNox-2.

[0047] Open reading frame (ORF) identification, coding region integrity verification, and amino acid sequence translation analysis were performed on the nucleotide sequences of the nine UDP-glucose dehydrogenases and one SmNox-2 enzyme. The results showed that each nucleotide sequence possessed a complete ORF, containing both a start codon and a stop codon, and no frameshift mutations or premature stop codons were found within the ORF. The corresponding complete amino acid sequences were obtained through theoretical translation according to the standard genetic code table.

[0048] Except for endogenous XccUGDH, the coding sequences of the other eight candidate UDP-glucose dehydrogenases were obtained by artificial synthesis, with codon optimization based on the codon usage preferences of Xanthomonas, while keeping the corresponding protein amino acid sequences unchanged.

[0049] The SmNox-2 coding sequence was also optimized based on the codon usage preferences of Xanthomonas, while maintaining its amino acid sequence. During gene synthesis, the P0935 promoter was placed upstream of the SmNox-2 coding sequence to form the P0935-SmNox-2 expression cassette, which was then synthesized as a whole. The SmNox-2 nucleotide sequence listed in the sequence listing is its coding region.

[0050] The nucleotide sequences and corresponding amino acid sequences of the above candidate enzymes are as follows: The nucleotide sequence of BsUglF is shown in SEQ ID NO: 1, and its encoded amino acid sequence is shown in SEQ ID NO: 2; the nucleotide sequence of LbjUGD is shown in SEQ ID NO: 3, and its encoded amino acid sequence is shown in SEQ ID NO: 4; the nucleotide sequence of RkpK is shown in SEQ ID NO: 5, and its encoded amino acid sequence is shown in SEQ ID NO: 6; the nucleotide sequence of SeUgdG is shown in SEQ ID NO: 7, and its encoded amino acid sequence is shown in SEQ ID NO: 8; the nucleotide sequence of SpHasB is shown in SEQ ID NO: 9, and its encoded amino acid sequence is shown in SEQ ID NO: 10; the nucleotide sequence of SsanUgdG is shown in SEQ ID NO: 11, and its encoded amino acid sequence is shown in SEQ ID NO: 12; the nucleotide sequence of SzHasB is shown in SEQ ID NO: 13, and its encoded amino acid sequence is shown in SEQ ID NO: 14; the nucleotide sequence of ZmUGD is shown in SEQ ID NO: 15, and its encoded amino acid sequence is shown in SEQ ID NO: 16. The nucleotide sequence of XccUGDH is shown in SEQ ID NO: 16; the nucleotide sequence of XccUGDH is shown in SEQ ID NO: 17, and the amino acid sequence it encodes is shown in SEQ ID NO: 18; the nucleotide sequence of SmNox-2 is shown in SEQ ID NO: 19, and the amino acid sequence it encodes is shown in SEQ ID NO: 20.

[0051] Example 2 Construction of UDP-glucose dehydrogenase expression strains from different sources This embodiment uses the integration of the XccUGDH encoding gene driven by the Ptac promoter at the hrpF site of the Xanthomonas campestris pv. campestris8004 genome as an example to illustrate the construction method of UDP-glucose dehydrogenase expression strains from different sources.

[0052] 2.1 Construction of Recombinant Plasmids Based on the hrpF gene and its flanking sequences in the genome of Xanthomonas javanica 8004, upstream and downstream homologous arms were designed to enable the Ptac-XccUGDH expression cassette to be site-specifically integrated into the hrpF site via double crossover homologous recombination. The functional fragments in the recombinant plasmid were linked in the following order: "hrpF upstream homologous arm - Ptac promoter - XccUGDH coding sequence - hrpF downstream homologous arm".

[0053] Using the genomic DNA of the Xanthomonas oryzae originating strain used in this invention as a template, the upstream homologous arm of hrpF was amplified using primer pair hrpF-UF / hrpF-UR, and the downstream homologous arm of hrpF was amplified using primer pair hrpF-DF / hrpF-DR; the coding sequence of XccUGDH was amplified using primer pair XccUGDH-F / XccUGDH-R, and its nucleotide sequence is shown in SEQ ID NO: 17.

[0054] The Ptac promoter sequence is shown in SEQ ID NO: 22. This promoter sequence is introduced by extending the 5′ end of hrpF-UR and XccUGDH-F. An overlap region is set between the promoter fragments carried by the two primers, thereby forming a complete Ptac promoter during homologous splicing and placing it upstream of the XccUGDH coding sequence.

[0055] Using plasmid pK18mobsacB as a template, linearization amplification was performed using primer pair pK18-line-F / pK18-line-R. The upstream homologous arm of hrpF, the XccUGDH coding sequence fragment, the downstream homologous arm of hrpF, and the linearized vector fragment were recovered and ligated according to the homologous splicing kit instructions. During the splicing process, a complete Ptac-XccUGDH expression unit was formed, yielding the recombinant plasmid pK18-hrpF-Ptac-XccUGDH. The primers used are listed in Table 1.

[0056] The DNA fragments were amplified using high-fidelity DNA polymerase. The PCR reaction system and reaction conditions are shown in Tables 2 and 3, respectively.

[0057] Table 1 Primers Table 2. Xanthomonas oryzae gene PCR system Table 3. PCR conditions for Xanthomonas oryzae gene. The annealing temperature of different primer pairs can be adjusted within the range of 60 to 65°C, based on the melting temperature of their effective annealing zone.

[0058] The splicing product was transformed into *E. coli* Top10 competent cells and plated on LB agar containing kanamycin. Single colonies were picked for colony PCR identification, and clones with expected identification results were sequenced to confirm the correct ligation sequence and ligation site of the upstream and downstream homologous arms of hrpF, the Ptac promoter, and the XccUGDH coding sequence. The recombinant plasmid was extracted from the correctly sequenced clones to obtain pK18-hrpF-Ptac-XccUGDH.

[0059] 2.2 Homologous recombination and integration of the Ptac-XccUGDH expression cassette at the hrpF site The recombinant plasmid pK18-hrpF-Ptac-XccUGDH was introduced into competent cells of the originating strain Xanthomonas oryzae 8004 via electroporation. After resuscitation, the electroporated cells were plated on LB agar containing 25 μg / mL kanamycin and cultured at 30°C to screen for single-crossover clones that had undergone the first homologous recombination.

[0060] Single-recombination clones were selected and inoculated into LB liquid medium without antibiotics and cultured overnight. The culture was then appropriately diluted and spread onto LB solid medium containing 10% sucrose and cultured at 30°C to screen for candidate clones that underwent a second homologous recombination.

[0061] Clones that underwent a second homologous recombination and lost the pK18mobsacB plasmid backbone no longer carried the sacB gene and the kanamycin resistance gene. Therefore, they were able to grow on medium containing 10% sucrose and revert to the kanamycin-sensitive phenotype. Sucrose-resistant clones were selected and seeded on LB solid medium containing and without kanamycin, respectively. Candidate double-crossover clones that could grow on antibiotic-free medium but not on kanamycin-containing medium were screened.

[0062] 2.3 PCR and sequencing identification of recombinant strains Using genomic DNA from candidate double-crossover clones as templates, PCR amplification was performed using the identification primers hrpF-VF / hrpF-VR, located outside the hrpF integration region. PCR products were detected by agarose gel electrophoresis, and candidate clones whose amplified fragment size matched the theoretical value were screened. The colony PCR system and reaction conditions are shown in Tables 4 and 5, respectively.

[0063] Table 4 Conventional PCR System Table 5. Conventional PCR conditions Candidate clones correctly identified by PCR were sequenced to confirm that the connection sites between the upstream homologous arm of hrpF, the Ptac promoter, the XccUGDH coding sequence, and the downstream homologous arm of hrpF were all consistent with the designed sequence, and that no base deletions, insertions, or other unexpected mutations occurred in the XccUGDH coding region. The correctly identified recombinant strain was named Xc-XccUGDH.

[0064] 2.4 Construction of UDP-glucose dehydrogenase expression strains from different sources Using the same construction method as the aforementioned Ptac-XccUGDH expression strain, the candidate enzyme coding sequences were amplified using the artificially synthesized sequences BsUglF, LbjUGD, RkpK, SeUgdG, SpHasB, SsanUgdG, SzHasB, and ZmUGD as templates. The primer pairs listed in Table 1 were used to amplify the XccUGDH coding sequences, and the resulting fragments replaced the XccUGDH coding sequences, while maintaining the Ptac promoter, hrpF integration site, upstream and downstream homologous arms, and vector backbone unchanged. The promoter sequence was introduced via hrpF-UR and the 5′ extension sequences of primers BsUglF-F, LbjUGD-F, RkpK-F, SeUgdG-F, SpHasB-F, SsanUgdG-F, SzHasB-F, and ZmUGD-F. An overlap region was established between the promoter fragments carried by the two primers, thereby forming a complete Ptac promoter during homologous splicing and positioning it upstream of the corresponding coding sequence.

[0065] Recombinant plasmids were constructed, and each Ptac-candidate UDP-glucose dehydrogenase expression cassette was integrated into the hrpF site of the Xanthomonas genome via double crossover homologous recombination. After sucrose reverse screening, kanamycin sensitivity verification, integration site PCR, and sequencing identification, UDP-glucose dehydrogenase expression strains from different sources were obtained and named Xc-BsUglF, Xc-LbjUGD, Xc-RkpK, Xc-SeUgdG, Xc-SpHasB, Xc-SsanUgdG, Xc-SzHasB, and Xc-ZmUGD, respectively.

[0066] Example 3: Fermentation screening of UDP-glucose dehydrogenase expression strains from multiple sources 3.1 Test strains and experimental design To compare the effects of UDP-glucose dehydrogenases from different sources on xanthan gum synthesis, nine recombinant strains constructed in Example 2 and verified to be correct were screened by shake-flask fermentation. The recombinant strains were Xc-XccUGDH, Xc-BsUglF, Xc-LbjUGD, Xc-RkpK, Xc-SeUgdG, Xc-SpHasB, Xc-SsanUgdG, Xc-SzHasB, and Xc-ZmUGD.

[0067] The UDP-glucose dehydrogenase expression units in the above recombinant strains were all integrated into the hrpF site of the Xanthomonas chromosome in single-copy form and expressed by the Ptac promoter. Except for the different sources of the expressed UDP-glucose dehydrogenase, the expression promoters, chromosome integration sites, and culture conditions of the recombinant strains were consistent, and no additional inducers were added during fermentation.

[0068] Wild-type strain WT was used as the baseline yield control. This wild-type strain retained only the natural expression state of the endogenous UDP-glucose dehydrogenase gene and did not contain any additional UDP-glucose dehydrogenase expression units. Meanwhile, Xc-XccUGDH was used as the enzyme source control under the same promoter and chromosomal integration site conditions to compare the effects of UDP-glucose dehydrogenases from different sources. Three independent biological replicates were set up for each strain.

[0069] 3.2 Strain activation and shake-flask fermentation The seed culture medium consisted of 10 g / L sucrose, 10 g / L peptone, and 5 g / L yeast extract. The pH of the medium was 6.8–7.2, and it was sterilized at 121°C for 20 min.

[0070] The fermentation medium consisted of 45 g / L starch, 6 g / L soybean protein powder, 0.2 g / L light calcium carbonate, and 0.2 g / L organosilicon defoamer. The pH of the medium was 6.8–7.2, and it was sterilized at 121°C for 20 min.

[0071] Take 100 μL of glycerol preservation solution for each tested strain and spread it evenly on LB solid medium. Incubate at 30℃ for 48 h. Pick single colonies with consistent growth status and inoculate them into 500 mL Erlenmeyer flasks containing 50 mL of seed culture medium. Incubate at 30℃ and 250 rpm for 16–24 h until the seed culture reaches OD500. 600 It reaches 1.5 to 2.0.

[0072] At an inoculum rate of 10% (v / v), the seed culture was inoculated into a 500 mL baffled Erlenmeyer flask containing 90 mL of fermentation medium and fermented at 30 °C and 250 rpm for 60 h. After fermentation, the OD600 of the washed cells and the amount of gel produced at oven dryness were measured.

[0073] 3.3 Determination of biomass and oven-dry rubber yield After fermentation, an equal volume of fermentation broth was taken, appropriately diluted, and centrifuged to collect the bacterial cells. The cells were washed three times with sterile physiological saline and then resuspended with an equal volume of sterile physiological saline. The resuspended bacterial solution was diluted to the linear detection range of the spectrophotometer, and the absorbance was measured at a wavelength of 600 nm. The OD of the washed bacterial cells was calculated based on the dilution factor.600 .

[0074] Take another 10 mL of fermentation broth, dilute it 5 times with deionized water, boil it in a water bath for 15 min, and centrifuge it at 8000 r / min and 4℃ for 30 min to remove the cell precipitate. Collect the supernatant, add 3 volumes of anhydrous ethanol, and let it stand overnight at 4℃. Then centrifuge it at 8000 r / min and 4℃ for 10 min, collect the resulting precipitate on pre-dried and weighed aluminum foil, and dry it at 80℃ to constant weight. Calculate the oven-dry yield of xanthan gum based on the mass of the dried product and the volume of the original fermentation broth, in g / L.

[0075] 3.4 Fermentation Data Analysis Results for each group are expressed as mean ± standard deviation. One-way ANOVA was used to compare overall differences among different strains. When overall differences were statistically significant, Dunnett's multiple comparison method was used to compare each recombinant strain with the wild-type strain WT. For the dominant strains identified in the initial screening, further multiple comparisons were performed using Xc-XccUGDH, which has the same promoter and integration site, as a control to evaluate the efficacy of UDP-glucose dehydrogenases from different sources. P < 0.05 indicated statistical significance.

[0076] Table 6. Fermentation screening results of UDP-glucose dehydrogenase expression strains from different sources. 3.5 Fermentation Screening Results Significant differences were found in the oven-dry xanthan gum production of UDP-glucose dehydrogenase expression strains from different sources. A one-way ANOVA was performed based on the data shown in Table 6, and the result was F(9, 20) = 24.62, P < 0.0001, indicating that there were significant differences in xanthan gum synthesis capacity among UDP-glucose dehydrogenase expression strains from different sources.

[0077] The oven-dry gel yield of wild-type strain WT was 10.97±0.22 g / L. The oven-dry gel yields of Xc-XccUGDH and Xc-BsUglF were 12.00±0.29 g / L and 12.61±0.32 g / L, respectively, representing increases of 9.4% and 15.0% compared to WT. The oven-dry gel yields of Xc-RkpK and Xc-ZmUGD were 11.70±0.28 g / L and 11.80±0.30 g / L, respectively, representing increases of 6.7% and 7.6% compared to WT. The oven-dry gel yields of Xc-LbjUGD, Xc-SpHasB, and Xc-SzHasB were 2.1%–4.9% higher than WT.

[0078] Among the UDP-glucose dehydrogenases from different sources screened, Xc-SsanUgdG and Xc-SeUgdG showed more outstanding gum-producing capacity. The oven-dry gum production of Xc-SsanUgdG reached 13.50±0.32 g / L, which was 23.1% higher than that of WT; the oven-dry gum production of Xc-SeUgdG reached 13.10±0.31 g / L, which was 19.5% higher than that of WT. The differences between the two and WT were statistically significant (P<0.0001).

[0079] Compared with Xc-XccUGDH, which has the same promoter and integration site, the oven-dry xanthan gum production of Xc-SsanUgdG and Xc-SeUgdG was increased by 12.5% ​​and 9.2%, respectively, with differences of P < 0.0001 and P = 0.0011, respectively. These results indicate that under the conditions of the same promoter, the same chromosomal integration site, and single-copy expression, both SsanUgdG and SeUgdG can effectively enhance the xanthan gum synthesis capacity of Xanthomonas, with the average oven-dry xanthan gum production of Xc-SsanUgdG being slightly higher than that of Xc-SeUgdG.

[0080] The mean OD600 values ​​of the washed bacterial cells of each tested strain ranged from 3.39 to 3.78. A one-way ANOVA was performed based on the data shown in Table 6, and the results were F(9, 20) = 0.78, P = 0.6375, indicating no statistically significant difference between groups. This suggests that single-copy expression of UDP-glucose dehydrogenase from different sources did not significantly affect bacterial growth. Xc-SsanUgdG and Xc-SeUgdG achieved higher oven-dry gel yields without a significant increase in bacterial biomass, indicating that their increased yield was not primarily due to increased bacterial growth.

[0081] Considering the overall dry xanthan gum production, yield increase, and cell growth, Xc-SsanUgdG and Xc-SeUgdG exhibited superior comprehensive fermentation performance. Therefore, SsanUgdG and SeUgdG were identified as the preferred UDP-glucose dehydrogenases for increasing xanthan gum production by Xanthomonas.

[0082] Example 4 Construction of combined expression strains for NADH oxidase driven by different promoters 4.1 Design of Combined Expression Scheme To further improve the intracellular redox imbalance that may be caused by NADH accumulation during the catalytic process of UDP-glucose dehydrogenase, the dominant strains Xc-SsanUgdG and Xc-SeUgdG obtained in Example 3 were used as starting strains to further express water-producing NADH oxidase.

[0083] The expression of the NADH oxidase gene SmNox-2 was driven by the Ptac, P0935, and Plac promoters, respectively. The three promoters have different sequences and can have different effects on the SmNox-2-mediated intracellular coenzyme regulation. Apart from the different promoter sequences, the SmNox-2 coding sequence and other components used in each expression unit were consistent, and no additional inducing agents were added during culture.

[0084] The expression units Ptac-SmNox-2, P0935-SmNox-2, and Plac-SmNox-2 were integrated in single-copy form into the XC_3033 site of chromosomes Xc-SsanUgdG and Xc-SeUgdG, respectively, to obtain UDP-glucose dehydrogenase and NADH oxidase co-expression strains driven by different promoters.

[0085] 4.2 Construction of recombinant plasmids Using Xc-SsanUgdG or Xc-SeUgdG genomic DNA as templates, the upstream and downstream homologous arms of the XC_3033 site were amplified; using the artificially synthesized P0935-SmNox-2 sequence as a template, the P0935-SmNox-2 expression unit or the SmNox-2 coding sequence fragment was amplified. When constructing the Ptac-SmNox-2 and Plac-SmNox-2 integration plasmids, the corresponding promoter sequences were introduced by the 5′ extension sequences of the reverse primer of the XC_3033 upstream homologous arm and the forward primer of the SmNox-2, respectively, and formed complete promoters during homologous splicing.

[0086] When constructing the Ptac-SmNox-2 integration plasmid, the upstream homologous arm of XC_3033 containing partially overlapping Ptac sequences was amplified using primer pair XC_3033-UF / XC_3033-U-tac-R, and the SmNox-2 coding sequence fragment containing partially overlapping Ptac sequences was amplified using primer pair tac-Nox-F / Nox-R. The two fragments were homologously spliced ​​to form a complete Ptac-SmNox-2 expression unit.

[0087] When constructing the P0935-SmNox-2 integration plasmid, primer pair XC_3033-UF / XC_3033-U-0935-R was used to amplify the upstream homologous arm of XC_3033 containing the P0935 overlapping sequence, and primer pair 0935-nox-F / Nox-R was used to amplify the P0935-SmNox-2 expression unit.

[0088] When constructing the Plac-SmNox-2 integration plasmid, the upstream homologous arm of XC_3033 containing partially overlapping Plac sequences was amplified using primer pair XC_3033-UF / XC_3033-U-lac-R, and the SmNox-2 coding sequence fragment containing partially overlapping Plac sequences was amplified using primer pair lac-Nox-F / Nox-R. The two fragments were homologously spliced ​​to form a complete Plac-SmNox-2 expression unit.

[0089] All three recombinant plasmids were amplified using primer pair XC_3033-DF / XC_3033-DR to amplify the downstream homologous arm of XC_3033. Linearization amplification was performed using the suicide vector pK18mobsacB as a template, with primer pair pK18-line-F / pK18-line-R.

[0090] The upstream homologous arm of XC_3033, the SmNox-2 expression cassette driven by different promoters, the downstream homologous arm of XC_3033, and the linearized pK18mobsacB vector were seamlessly assembled to obtain recombinant plasmids pK18-XC3033-Ptac-Nox, pK18-XC3033-P0935-Nox, and pK18-XC3033-Plac-Nox.

[0091] The assembly products were transformed into E. coli Top10 competent cells and screened on LB agar containing kanamycin. Transformants were picked for colony PCR and sequencing verification to obtain recombinant plasmids with correct sequences.

[0092] Except for the template, primers, and expression cassette specifically described in this embodiment, the PCR amplification system, amplification conditions, vector assembly method, E. coli transformation method, and recombinant plasmid verification method are all performed in accordance with the corresponding methods in Example 2, and will not be repeated here.

[0093] 4.3 Chromosomal Integration of SmNox-2 Expression Cassette The recombinant plasmids pK18-XC3033-Ptac-Nox, pK18-XC3033-P0935-Nox, and pK18-XC3033-Plac-Nox were introduced into Xc-SsanUgdG and Xc-SeUgdG competent cells by electroporation, respectively.

[0094] The transformation products were plated on LB solid medium containing 25 μg / mL kanamycin and incubated at 30°C to screen for single-crossover clones that underwent the first homologous recombination. Single-crossover clones verified by PCR were selected, inoculated into antibiotic-free LB liquid medium, and then plated on LB solid medium containing 10% sucrose to screen for clones that underwent the second homologous recombination.

[0095] Candidate clones obtained from sucrose plates were inoculated onto LB solid medium containing and without kanamycin for resistance verification. Candidate clones that could grow normally on the non-resistance medium but not on the kanamycin medium were retained. After two homologous exchanges, the candidate clones lost the pK18mobsacB plasmid backbone but retained the SmNox-2 expression cassette integrated at the XC_3033 site.

[0096] The preparation of electrotransformation competent cells, the first homologous recombination screening, the second homologous recombination screening, and the sucrose reverse screening were all carried out according to the corresponding methods in Example 2.

[0097] 4.4 Validation of recombinant strains The validation primer pair XC_3033-VF / XC_3033-VR, located outside the integration region of XC_3033, was used to amplify the target region of the candidate double-crossover clone by PCR. The size of the amplified product was used to preliminarily determine whether the SmNox-2 expression cassette was correctly integrated, and the PCR product was sequenced.

[0098] Sequencing results showed that the Ptac-SmNox-2, P0935-SmNox-2, and Plac-SmNox-2 expression cassettes were all integrated into the XC_3033 site in the expected direction, and the ligation sequences at both ends of the expression cassettes were correct. No unexpected base deletions, insertions, or mutations were found.

[0099] Simultaneously, the original UDP-glucose dehydrogenase expression cassette was verified using primer pair hrpF-VF / hrpF-VR to confirm that the SsanUgdG or SeUgdG expression cassette was still completely preserved at the hrpF site. This resulted in the six UDP-glucose dehydrogenase and NADH oxidase co-expression strains listed in Table 7.

[0100] Table 7. Combination expression strains regulated by different promoters Example 5: Effects of different promoters driving SmNox-2 on the fermentation performance of xanthan gum and intracellular coenzyme status of dominant engineered bacteria 5.1 Fermentation Culture To investigate the effects of different promoters driving SmNox-2 on the xanthan gum synthesis ability of dominant engineered bacteria, Xc-SsanUgdG and Xc-SeUgdG were used as starting strains. The combined expression strains Ptac-SmNox-2, P0935-SmNox-2, and Plac-SmNox-2 constructed in Example 4 were selected for fermentation evaluation. Xc-SsanUgdG and Xc-SeUgdG, which did not integrate the SmNox-2 expression unit, served as corresponding reference controls. The SmNox-2 coding sequence and XC_3033 integration site were identical in the three combined expression strains; only the promoters used differed, allowing for comparison of the regulatory effects of different promoters driving SmNox-2.

[0101] Each strain was cultured in seed culture and shake-flask fermented according to the method described in Example 3, with three independent biological replicates for each group. Intracellular NAD was measured after 30 h of fermentation. + The NADH ratio was used to evaluate the effect of different promoters driving SmNox-2 expression on the intracellular coenzyme status of the bacteria; cell OD was measured at 60 h of fermentation. 600 And xanthan gum oven-dry yield.

[0102] 5.2 Detection Methods The oven-dry yield of xanthan gum was determined according to the method described in Example 3.

[0103] Determination of bacterial cell OD 600 At that time, take an equal volume of fermentation broth, dilute appropriately, centrifuge to collect the bacterial cells, wash three times with sterile physiological saline to remove xanthan gum and culture medium residues adhering to the bacterial cell surface, and then resuspend the bacterial cells and measure the OD. 600 .

[0104] Intracellular NAD + The / NADH ratio uses enhanced NAD. + The NADH assay was performed using the WST-8 method with a NADH detection kit. After 30 hours of fermentation, an equal volume of fermentation broth was collected by centrifugation, and the cells were washed three times with sterile physiological saline. The washed cells were normalized to the same amount, and sample lysis and total NAD were performed according to the kit instructions. + NADH and NADH content were measured, and intracellular NAD+ was calculated. + / NADH ratio.

[0105] 5.3 Data Processing Experimental results are expressed as mean ± standard deviation. One-way ANOVA was performed against the backgrounds of Xc-SsanUgdG and Xc-SeUgdG strains, and the Dunnett method was used to compare each SmNox-2 co-expression strain with its corresponding non-SmNox-2 reference strain. Tukey's method was also used for multiple comparisons of SmNox-2 co-expression strains driven by three different promoters. P < 0.05 indicated statistical significance.

[0106] 5.4 Evaluation results of SmNox-2 fermentation driven by different promoters Different promoters drive the effects of SmNox-2 on the oven-dry yield of xanthan gum in engineered bacteria, cell growth, and intracellular NAD. + The effect of the / NADH ratio is shown in Table 8.

[0107] Table 8. Fermentation performance and intracellular NAD of engineered bacteria driven by different promoters of SmNox-2 + The effect of the / NADH ratio Note: Within the same strain background, compared with the corresponding control that does not express SmNox-2, This indicates that P < 0.05. This indicates that P < 0.01. This indicates P < 0.001; OD of the washed bacterial cells in each group 600 There were no statistically significant differences.

[0108] 5.5 Results Analysis The results showed that, in both Xc-SsanUgdG and Xc-SeUgdG strain backgrounds, SmNox-2 driven by different promoters could increase intracellular NAD in cells fermented for 30 h to varying degrees. + / NADH ratio. Among them, the intracellular NAD+ / NADH ratio is driven by the Ptac promoter. + The / NADH ratio was the highest, reaching 2.82±0.14 and 2.76±0.13, respectively; followed by the P0935 promoter-driven group; the Plac promoter-driven group had the highest intracellular NAD ratio. + The SmNox / NADH ratios were 1.96±0.09 and 1.93±0.09, respectively. These results indicate that SmNox-2 driven by different promoters exerts varying degrees of regulatory effects on intracellular coenzyme status.

[0109] In the Xc-SsanUgdG strain background, the xanthan gum oven-dry yield of the Plac-SmNox-2 co-expression strain reached 15.31±0.33 g / L, an increase of 13.4% compared to Xc-SsanUgdG; in the Xc-SeUgdG strain background, the xanthan gum oven-dry yield of the Plac-SmNox-2 co-expression strain reached 14.95±0.33 g / L, an increase of 14.1% compared to Xc-SeUgdG, and the differences were statistically significant. Further comparison of the three promoter combinations showed that, in both strain backgrounds, the xanthan gum oven-dry yield of the Plac-SmNox-2 co-expression strain was significantly higher than that of the Ptac-SmNox-2 co-expression strain (P<0.01) and the P0935-SmNox-2 co-expression strain (P<0.05).

[0110] Following P0935 promoter-driven SmNox-2 expression, the oven-dry xanthan gum yields of the two strains increased by 6.9% and 6.5%, respectively; while the oven-dry xanthan gum yields of the Ptac promoter-driven group increased by only 1.9% and 1.7%, respectively, with no statistically significant difference compared to the corresponding controls. OD values ​​of washed bacterial cells in each group... 600 The results were largely consistent, indicating that different SmNox-2 expression combinations did not have a significant impact on bacterial growth, and the changes in xanthan gum production were mainly related to the regulation of intracellular metabolic state.

[0111] Combined xanthan gum oven-dry yield, cell OD 600 and intracellular NAD + The NADH ratio indicates that the Ptac-SmNox-2 combination achieved a higher intracellular NAD ratio. + The / NADH ratio was optimized, but the highest xanthan gum yield was not achieved. In contrast, the Plac-SmNox-2 combination showed better compatibility with SsanUgdG or SeUgdG, promoting xanthan gum synthesis while maintaining normal cell growth.

[0112] Therefore, Plac-SmNox-2 was determined to be the preferred NADH oxidase expression combination for Xc-SsanUgdG and Xc-SeUgdG.

[0113] The nucleotide and amino acid sequences involved in this article are as follows.

[0114] BsUglF comes from Bacillus subtilis subsp. subtilis 168. The BsUglF nucleotide sequence is SEQ ID NO: 1 (DNA, 1323 bp in length). BsUglF amino acid sequence SEQ ID NO: 2 (PRT, length 440 aa) MNITVIGTGYVGLVTGVSLSEIGHHVTCIDIDAHKIDEMRKGISPIFEPGLEELMRKNTADGRLNFETSYEKGLAQADIIFIAVGTPQKSDGHANLEQITDAAKRIAKHVKRDTVVVTKSTVPVGTNDLINGLITEHLAEPVSISVASNPEFLREGSAIYDTFHGDRIVIGTADEKTANTLEELFRPFQIPIYQTDIRSAEMIKYASNAFLATKISFINEISNICEKVGADIEAVAYGMGQDKRIGSQFLKAGIGYGGSCFPKDTNALVQIAGNVEHDFELLKSVIKVNNNQQAMLVDKALNRLGGVTGKTIALLGLSFKPNTDDMREAPSIVIADRLAALDARIRAYDPIAVSHAKHVLPQAVEYKETIEEAVKGSDAVMILTDWADIKQFPLAAYQDLMETPLIFDGRNCYTLDEALAAGVEYYSVGRKAVVPSGAIQ LbjUGD is derived from Lactobacillus johnsonii LbjUGD nucleotide sequence SEQ ID NO: 3 (DNA, length 1248 bp) The amino acid sequence of LbjUGD is SEQ ID NO: 4 (PRT, length 415 aa). MNSYKIAVAGTGYVGLSLATLLSQHNEVTAVDIVPEKVDLINNHKSPIVDKEIEDFLANKDLNLKATTDAESAYKDADFVIIATPTNYDSEKNFFDTSAVEAV IKLVKQYNPQAIMIIKSTIPVGYTNHVRKEFETENIIFSPEFLREGHALYDNLHPSRIVVGTDKENTKLVEAAHTFASLLQEGAEDKDIATLFMGFTEAEAVKL FANTYLALRVSYFNELDTYAESKGLNTQEIIDGVGLDPRIGSHYNNPSFGYGGYCLPKDTKQLLANYKDVPENLIEAIVKSNDTRKDFIADQVLSKAGYYDYDD NNTYDPSEEKQVTIGVYRLTMKSNSDNFRQSSVQGVMKRVKAKGAKIIIFEPTLENGSTFFGSEVVNDLDEFKNKSDAIIANRYNTILDDVKDKVYTRDIFRKD RkpK comes from *Sinorhizobium meliloti* (a type of rhizobium in alfalfa). RkpK nucleotide sequence SEQ ID NO: 5 (DNA, 1314 bp in length) RkpK amino acid sequence SEQ ID NO: 6 (PRT, length 437 aa) MKITMIGAGYVGLVSGVCFADFGHDVVCVDKDEGKISALKKGQIPIFEPGLDHLVASNVASGRLNFTDDLKTAVAASDVVFIAVGTPSRRGDGHADLSYVYAAAREIASNLQGFTVVVTKSTVPVGTGDEVERIIRETNPAVDVTVVSNPEFLREGAAIEDFKRPDRIVIGVDGSDGRAREVMTEVYRPLYLNQSPLVFTTRRTSELIKYAGNAFLAMKITFINEIADLCEKVGANVQDVARGIGLDGRIGSKFLHAGPGYGGSCFPKDTLALVKTAQDHDTPVRLVETTVAVNDNRKRAMGRKVIAAAGGDIRGSKYAVLGLTFKPNTDDMRDTPAIAVVQALQDAGARVTGYDPEGMENARKLIEGLDCARDPYEAAAEADALVIITEWNEFRALDFDRLKSTMKTPLLVDLRNIYRKDEVAKHGFRYASIGRPD SeUgdG is derived from Sphingomonas elodea SeUgdG nucleotide sequence SEQ ID NO: 7 (DNA, length 1317 bp) SeUgdG amino acid sequence SEQ ID NO: 8 (PRT, length 438 aa) MRIAMIGTGYVGLVSGACFSDFGHEVVCVDKDARKIELLHQNVMPIYEPGLDALVASNVKAGRLSFTTDLAEGVKDADAVFIAVGTPSRRGDGHADLSYVFAAAREIAENLTKPSVIVTKSTVPVGTGDEVERIIAEVAPNSGAKVVSNPEFLREGAAIEDFKRPDRVVVGTEDEFARQVMREIYRPLSLNQSAPVLFTGRRTSELIKYAANAFLAVKITFINEIADLCEQVGADVQEVSRGIGMDNRIGGKFLHAGPGYGGSCFPKDTLALMKTAADNETPLRIVEATVQVNDARKRAMGRKVIKAMGGDVRGKTVGILGLTFKPNTDDMRDAPSLSIIAALQDAGATVKAYDPEGVEQASKMLTDVEFVENPYAAADGADALVIVTEWDAFRALDLTRIKNSLKSPVLVDLRNIYPPAELERAGLQYTGVGKPSRD SpHasB is derived from Streptococcus pyogenes SpHasB nucleotide sequence SEQ ID NO: 9 (DNA, length 1209 bp) SpHasB amino acid sequence SEQ ID NO: 10 (PRT, length 402 aa) MKIAVAGSGYVGLSLGVLLSLQNEVTIVDILPSKVDKINNGLSPIQDEYIEYYLKSKQLSIKATLDSKAAYKEAELVIIATPTNYNSRINYFDTQHVETVIKEVLSVNSHATLIIKSTIPIGFITEMRQKFQTDRIIFSPEFLRESKALYDNLYPSRIIVSCEENDSPKVKADAEKFALLLKSAAKKNNVPVLIMGASEAE AVKLFANTYLALRVAYFNELDTYAESRKLNSHMIIQGISYDDRIGMHYNNPSFGYGGYCLPKDTKQLLANYNNIPQTLIEAIVSSNNVRKSYIAKQIINV LKEQESPVKVVGVYRLIMKSSNSDNFRESAIKDVIDILKSKDIKIIIYEPMLNKLESEDQSVLVNDLENFKKQANIIVTNRYDNELQDVKNKVYSRDIFGRD SsanUgdG comes from Sphingomonas sanxanigenens NX02 (a strain of Sphingomonas sanxanigenens NX02). SsanUgdG nucleotide sequence SEQ ID NO: 11 (DNA, 1365 bp in length) SsanUgdG amino acid sequence SEQ ID NO: 12 (PRT, 454 aa in length) MKIVMIGAGYVGLVSGACLADFGHDVICVDKDAGKIADLEANRMPIYEPGLAQLVKSNVETGRLSFSLDLPAAVADADAIFIAVGTPSRRGDGHADLSYVFAATREIGAALTGPTVVVTKSTVPVGTGDQVERILRDVAPDIEAAVVSNPEFLREGAAIGDFKRPDRIVIGTTDERARAVMRAIYRPLYLNEAPLLFTARRTAELIKYAANAFLATKITFINEIADLCEQVDADVKDVARGIGLDNRIGPKFLHAGPGFGGSCFPKDTLALLKTAEDHESPMRIVEAVVQVNEQRKRAMGRKVIQAAGGDVRGKRIGVLGLTFKPNTDDMRDAPALAIVQTLLDGGAEVRAFDPEGMEAARPMLPDVTLVGDPYAAAEGADALVIVTEWDLFRALDLKRVARLMALPILVDLRNIYPPEEALAAGFSYTSVGRSAVSGDFAAVQPKMAEDAATG SzHasB is derived from Streptococcus equi subsp. zooepidemicus SzHasB nucleotide sequence SEQ ID NO: 13 (DNA, 1206 bp in length) SzHasB amino acid sequence SEQ ID NO: 14 (PRT, length 401 aa) MKISVAGSGYVGLSLSILLAQHNDVTVVDIIDEKVRLINQGISPIKDADIEEYLKNAPLNLTASLDGASAYSNADLIIIATPTNYDSERNYFDTRHVEEVIEQVLDLNASATIVIKSTIPLGFIKHVREKYQTDRIIFSPEFLRESKALYDNLYPSRIIVSYEKDDSPRVIQAAKAFAGLLKEGAKSKDTPVLFMGSQEA EAVKLFANTFLAMRVSYFNELDTYSESKGLDAQRVIEGVCHDQRIGNHYNNPSFGYGGYCLPKDSKQLLANYRGIPQSLMSAIVESNKIRKSYLAEQILH MASNQKQPGLPLTIGFYRLIMKSNSDNFRESAIKDIIDIINDYGVNIVIYEPMLGEDIGYRVVKDLEQFKNESTIIVSNRFEDDLADVIDKVYTRDVFGRD ZmUGD comes from Zymomonas mobilis ZmUGD nucleotide sequence SEQ ID NO: 15 (DNA, 1314 bp in length) (Modular fermentation bacteria) ZmUGD amino acid sequence SEQ ID NO: 16 (PRT, length 437 aa) MRITMIGSGYVGLVSGACFSDFGHNVICVDHDQEKIALLQKGVMPIYEPGLADMVANNVKAGRLSFSNDLASSVKNADAVFIAVGTPSRRGDGHADLSYVFAAAKEIAA SVSDNTVVVDKSTVPVGTGDEVERIIREIAPDKKIWVASNPEFLREGAAIGDFKRPDRIVIGTENPEAREIMQEIYRPLYLNQSPILFTSRRSAELIKYASNAFLATKI TFINEMADLCEAVGGDVQDIARGIGADNRIGSKFLHAGPGYGGSCFPKDTLALLKTAENFATPLRVVESVVTANNIRKRAMARKVLQAIGKDMRGQKIGLLGLTFKPNT DDMRDAPSLALIQGLEDAGAIIHAYDPEGMTQARPLLPNVIFEDEPYKVAEKAEALVIVTEWDAFRALDLKRIAGLLKTPLLLDLRNIYTEEEAKKAGLVYHGIGRPNAG XccUGDH comes from Xanthomonas campestris pv. campestris 8004. The XccUGDH nucleotide sequence is SEQ ID NO: 17 (DNA, 1347 bp in length). XccUGDH amino acid sequence SEQ ID NO: 18 (PRT, length 448 aa) MRVAIFGTGYVGLVTGTCLAEVGHHVICVDIDQAKVDGLNRGVIPIYEPGLEPMVKANHASGRLRFTTDAAEAIAHGEITFIAVGTPPDEDGSADLQYVLAVARTVGRHIDG PSVIVNKSTVPVGTADKVRAAIQEELDARGVDHEFDVVSNPEFLKEGDAVADCMRPDRIVIGAKKPAAIARMRRLYAPFNRNRDRIVEMDVRSAELTKYAANAMLATKISFM NEIANIAERVGADVEHVRNGIGSDPRIGWHFIYPGAGYGGSCFPKDVQALAKTAQQYGMQPTLLNAVESVNNAQKGHLFELVQRHYADGNGSIAGKTFAVWGLAFKPNTDDM RAASSRRLMAQLWEAGAKVRAYDPEATHEAKRIFGERDDLAFCDDAFAALEGADALVVVTEWKQFRSPDFGKIKQALKDDVVFDGRNLYDPQEIEAAGLAYYAIGRGRSLHA SmNox-2 comes from Streptococcus mutans. SmNox-2 nucleotide sequence SEQ ID NO: 19 (DNA, 1374 bp in length) SmNox-2 amino acid sequence SEQ ID NO: 20 (PRT, length 457 aa) MSKIVIVGANHAGTAAINTILDNYGSENEVVVFDQNSNISFLGCGMALWIGKQISGPQGLFYADKESLEAKGAKIYMESPVTAIDYDAKRVTALVNGQEHVESYEKLILATGSTPILPPIKGAAIKEGSRDFEATLKNLQFVKLYQNAEDVINKLQDKSQNLNRIAVVGAGYIGVELAEAFKRLGKEVILIDVVDTCLAGYYDQDLSEMMRQNLEDHGIELAFGETVKAIEGDGKVERIVTDKASHDVDMVILAVGFRPNTALGNAKLKTFRNGAFLVDKKQETSIPDVYAIGDCATVYDNAINDTNYIALASNALRSGIVAGHNAAGHKLESLGVQGSNGISIFGLNMVSTGLTQEKAKRFGYNPEVTAFTDFQKASFIEHDNYPVTLKIVYDKDSRLVLGAQMASKEDMSMGIHMFSLAIQEKVTIERLALLDYFFLPHFNQPYNYMTKAALKAK P0935 promoter SEQ ID NO: 21 (DNA, length 442 bp) GGTAATGGGTGAGGGCGACGGCTTTCATCGGAGCGCTCCTGGTGGGGAGTTCAGGGTGCGTTGTGTGGGAAGTTCCCCGCAACCACCAACCCCACAATTCAGGTCGGGCGGGCCTTTCCGTTCACCAACGTGCACTTGGACACGGTCTTCACGCACCTGCCCAAGGCGTTCAACCGACGTATTCAACTGGATAAATTGGGGTGGCGTTCATCTGCACTGCATGTCCGAACTGGGCGCCCGGTCGCACTCCTCATAAAGCTGTATACGTAGGATTAGCAGCGTTTGCGGCGGGCGGCATTTGCTTTCACATGTTATATGACCGTTAACGCGATCTTCACTAAGCCCGGCTTAATCTGCGCCCCGATGGCTCTGGATGTGACGAGCATCTTTTACCAAGAACAACGCCTCTATCGGTTTATCTCCCAAAGAAGGAGCTGTATAC Ptac promoter SEQ ID NO: 22 (DNA, 75 bp in length) TTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGAATTC Plac promoter SEQ ID NO: 23 (DNA, 82 bp in length) TTTACACTTTATGCTTCCGGCTCGTATGTTGACTATCTAGTCATTCACTCATGCGTACCACGAGACAAAGGAGGATTACTAT

Claims

1. The application of UDP-glucose dehydrogenase SeUgdG or its encoded nucleic acid molecule in the construction of xanthan gum production strains, characterized in that, The amino acid sequence of the UDP-glucose dehydrogenase SeUgdG is shown in SEQ ID NO: 8, and the producing strain is Xanthomonas.

2. The application as described in claim 1, characterized in that, The expression cassette, including the UDP-glucose dehydrogenase SeUgdG encoding nucleic acid molecule, was recombined and integrated into the genome of Xanthomonas.

3. The application as described in claim 2, characterized in that, The expression cassette is recombined and integrated into the hrpF site of the genome.

4. The application as described in any one of claims 1-3, characterized in that, The starting strain used in the application was Xanthomonas oryzae.

5. A method for constructing a xanthan gum production strain, characterized in that, This includes the recombinant integration of the expression cassette of the heterologous UDP-glucose dehydrogenase SeUgdG gene into the Xanthomonas genome, the amino acid sequence of which is shown in SEQ ID NO:

8.

6. The method as described in claim 5, characterized in that, It also includes the recombinant integration of the expression cassette of the water-producing NADH oxidase SmNox-2 gene into the Xanthomonas genome.

7. The method as described in claim 6, characterized in that, The expression cassette of the UDP-glucose dehydrogenase gene is integrated into the hrpF site of the Xanthomonas genome, and / or the expression cassette of the water-producing NADH oxidase SmNox-2 gene is integrated into the XC_3033 site of the Xanthomonas genome.

8. The method as described in claim 6, characterized in that, The Ptac promoter is used in the expression cassette of the UDP-glucose dehydrogenase gene, and / or the Plac promoter is used in the expression cassette of the aqueous NADH oxidase SmNox-2 gene.

9. The xanthan gum producing strain constructed by the method of any one of claims 5-8.

10. The use of the xanthan gum producing strain according to claim 9 in the production of xanthan gum.

Images