Gene editing system based on SacB, foal bacillus xylosus recombinant bacteria and application of foal bacillus xylosus recombinant bacteria

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a gene editing system based on SacB, a foal bacillus xylosus recombinant bacterium and application. The gene editing system based on SacB is constructed by using the promoter P34 and the chloramphenicol resistance gene, and the system can be used for performing gene editing on different foal bacillus xylosus and is high in editing efficiency. By utilizing the gene editing system, the invention also provides a foal bacillus xylosus recombinant strain and a construction method thereof, and provides a foal bacillus xylosus Kx delta bcsIII with bcs III operon knocked out, the strain is the foal bacillus xylosus recombinant strain, high-crystallinity bacterial cellulose can be produced, and a new way is provided for obtaining high-crystallinity BC.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and specifically relates to a gene editing system based on SacB and a recombinant bacillus xylosus and an application thereof. Background Art

[0002] Bacterial cellulose (BC) is a polysaccharide composed of glucose monomers linearly linked by β-1,4-glycosidic bonds. Compared with plant cellulose, BC has high purity, excellent mechanical strength, excellent crystallinity, good hydrophilicity, and exhibits excellent biocompatibility and biodegradability. Therefore, BC is widely used in the food industry, biomedical materials, cosmetics and other fields.

[0003] Xylella fastidiosa Komagataeibacter xylinus ) is an industrial production strain that can efficiently synthesize BC with simple culture conditions. It is also a food-safe strain suitable for large-scale production of BC. Therefore, it is of great significance to gene-edit Bacillus xylosus to make it more suitable for BC production. However, compared with other important industrial microorganisms Escherichia coli, Corynebacterium glutamicum and Bacillus subtilis, the current genetic tools for editing the genome of Bacillus xylosus are still limited. The lack of efficient and reliable gene editing tools limits the application potential of Bacillus xylosus.

[0004] The gene editing systems at the genome level are the λ-Red and FLP / FRT-mediated site-specific recombination systems and the CRISPR / Cas9 gene editing system, which can be used for gene editing of recombinant strains of Bacillus xylosus. However, the λ-Red recombination method is cumbersome and inefficient, and the CRISPR / Cas9 gene editing system has a certain off-target rate and the loss of plasmids increases the experimental risk and experimental cycle. In many industrial microorganisms, one of the most widely used molecular tools is pK18obsacB, which is capable of genome editing in microorganisms by gene knockout, knock-in or replacement. The system relies on two rounds of homologous recombination selection, using antibiotic resistance markers and counter-selection markers such as levansucrase. Due to sacB The gene encodes fructanase, which converts sucrose into fructans at certain sucrose concentrations. This process disrupts the normal physiological functions of cells and can lead to cell lysis, which is lethal to Gram-negative bacteria. Therefore, it is necessary to tailor new genetic tools for B. xylosus.

[0005] In addition, the high crystallinity of BC gives it good mechanical properties, stability, durability and biocompatibility, thus meeting its basic requirements as medical tissue organs, medical dressings, drug delivery systems, packaging materials and other products. However, the purpose of gene editing of Bacillus xylosus in existing technologies is mostly to increase the yield of BC, and there is little research on improving crystallinity. Summary of the invention

[0006] In view of the above problems, the present invention provides a gene editing system based on SacB and a recombinant bacillus xylosus and its application. The present invention uses promoter P34 and chloramphenicol resistance gene to construct a gene editing system based on SacB, which can be used for gene editing of different bacillus xylosus, and the editing efficiency is high. Using the gene editing system, the present invention also provides a recombinant bacillus xylosus and a construction method thereof, and provides a recombinant bacillus xylosus that can improve the crystallinity of BC, which provides a new way to obtain high-crystallinity BC.

[0007] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical scheme: The first aspect of the present invention provides a gene editing system based on SacB, the gene editing system comprising sacB The invention discloses a plasmid containing a gene, a promoter P34 and a chloramphenicol resistance gene, wherein the nucleotide sequence of the promoter P34 is shown in SEQ ID NO.1.

[0008] When microorganisms use sucrose as a carbon source, sacB The gene has been shown to be lethal in many strains as a reverse screening marker, but there is currently no relevant report on xylosaccharomyces cerevisiae. The present invention first determines that the genome of xylosaccharomyces cerevisiae does not contain sacB gene, and experimental studies have shown that sacB The gene has a lethal effect on Bacillus xylosus. Therefore, the present invention constructs a gene editing system based on SacB as a reverse screening marker.

[0009] The present invention has found through research that Acetobacter xylinum is more sensitive to chloramphenicol, and the promoter P34 can be used to regulate the expression of the chloramphenicol resistance gene. Therefore, the present invention introduces the promoter P34 and the chloramphenicol resistance gene into the gene editing system to facilitate the screening of positive transformants that meet the requirements from the recombinant strains of Acetobacter xylinum obtained by using the gene editing system.

[0010] Experiments have shown that for the genetic modification of Acetobacter xylinum, this gene editing system is capable of knocking out polycistronic gene clusters or constructing a minimal genome, and helps to achieve heterologous expression and multi-copy expression of target genes at the genome level. Therefore, it can be used for the genetic modification of Acetobacter xylinum, providing an effective tool for increasing BC production or regulating BC structure through metabolic engineering.

[0011] In addition, this gene editing system can also be used for genetic modification of other Acetobacter strains in addition to B. xylosus.

[0012] Preferably, the nucleotide sequence of the chloramphenicol resistance gene in the gene editing system is as shown in SEQ ID NO.2.

[0013] Preferably, the gene editing system is to sacB The gene vector skeleton is constructed by introducing the promoter P34 and the chloramphenicol resistance gene.

[0014] More preferably, the sacB The plasmid of the gene is pK18-P 112 -SacB-P 116 -Km R Plasmid.

[0015] The second aspect of the present invention provides a method for constructing the above-mentioned gene editing system, which specifically includes the following operations: The said sacB The vector backbone of the gene is used as a template to obtain the amplified product through PCR amplification; The promoter P34 and the chloramphenicol resistance gene are recombinantly linked to the amplified product to obtain the gene editing system.

[0016] Preferably, the sacB The plasmid of the gene is pK18-P 112 -SacB-P 116 -Km R The plasmid, the amplification product is a gene fragment with a nucleotide sequence as shown in SEQ ID NO.3 and a gene fragment as shown in SEQ ID NO.4, and the nucleotide sequence of the gene editing system is shown in SEQ ID NO.5.

[0017] The third aspect of the present invention provides the use of the above-mentioned gene editing system in constructing recombinant Bacillus xylosus.

[0018] Using the above-mentioned gene editing system to transform Bacillus xylosus can change its performance in synthesizing BC, and may further change the characteristics of BC synthesized by the obtained recombinant Bacillus xylosus.

[0019] The fourth aspect of the present invention provides a method for constructing a recombinant bacterium of Bacillus xylosus using the above gene editing system: using the above gene editing system to knock out a gene on the genome of Bacillus xylosus bcs III Operon, the bcs III The nucleotide sequence of the operon is: the nucleotide sequence with NCBI-locus_tag of CT154_05660 and the nucleotide sequence with NCBI-locus_tag of CT154_05665 and the non-coding region sequence therebetween.

[0020] The bacterial cellulose synthase (BCS)-related operons in the genome of Bacillus xylosus can affect its BC production performance, but not all BCS-related operons can have a positive effect on the performance of xyloglucan acid vinegar in producing BC. The present invention has found through research that bcs III The operon is a BCS-related operon that knocks out the bcs III The operon can significantly improve the crystallinity of BC produced by recombinant Bacillus xylosus. bcs III This role of the operon has not yet been reported.

[0021] Preferably, the method specifically comprises the following operations: Using the genome of Bacillus xylosus as a template, amplifying upstream and downstream homology arms, constructing them into the gene editing system, and transforming the obtained plasmid into Bacillus xylosus to obtain transformants; The transformant is inoculated into a HS liquid culture medium containing chloramphenicol for culture, and the cultured bacterial liquid is spread onto a solid culture medium containing 1% to 7% sucrose, and after inverted culture at 30 to 35° C. for 24 to 36 hours, positive transformants are screened to obtain the product.

[0022] Further preferably, the length of the homology arm is 2000 bp.

[0023] Further preferably, the sucrose concentration in the solid culture medium is 7%.

[0024] More preferably, the inverted culture time is 24 h and the culture temperature is 32°C.

[0025] The fifth aspect of the present invention provides a strain of Bacillus xylosus KxΔ bcsIII , whose taxonomic name is Bacillus xylosus ( Komagataeibacter xylinus ), deposited on November 21, 2024 at the General Microbiology Center of China Microbiological Culture Collection Administration, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101, with the deposit number CGMCC No.32726. This strain is a recombinant strain of Bacillus xylosus.

[0026] The strain is based on Bacillus xylosus with a preservation number of CGMCC No. 2955 (disclosed in a patent with application number "CN201010240130.4" and invention name "A method for preparing Fe3O4 magnetic bacterial cellulose balls") as the starting strain, and is constructed using the gene editing system of the present invention through the above-mentioned construction method, and can synthesize high-crystallinity BC.

[0027] The sixth aspect of the present invention provides the above-mentioned Bacillus xylosus KxΔ bcsIII Application in the production of high-crystallinity bacterial cellulose.

[0028] The seventh aspect of the present invention provides a method for using the above-mentioned Bacillus xylosus KxΔ bcsIII The method for producing high-crystallinity bacterial cellulose specifically comprises the following operations: The xylosaccharomyces cerevisiae KxΔ bcsIII Inoculate into HS liquid culture medium, add cellulase, and culture at 30-40℃ until the bacteria grow logarithmically to obtain seed liquid bacteria; Wash the seed liquid cells with sterile PBS, clean the cellulase and resuspend the cells according to the initial OD 600 The concentration of 0.02-0.2 was inoculated into HS liquid culture medium containing 20-30 g / L glucose, and static fermentation culture was carried out in an incubator at 30-40 ℃; the produced bacterial cellulose was collected.

[0029] Preferably, the HS liquid culture medium is composed of: 7.5-10 g / L yeast powder, 10-15 g / L tryptone, 25-40 g / L glucose, 10-15 g / L Na2HPO4·12H2O, acetic acid as a pH regulator to adjust the pH to 6, and the rest is water.

[0030] The beneficial effects of the present invention are as follows: the present invention constructs a gene editing system based on SacB, which is more diverse than general gene editing systems, can be used for gene editing of different xylosaccharomyces cerevisiae, can achieve knockout, knock-in and replacement of large-fragment genes, has the potential for gene editing in other Acetobacter strains, has high application value, and provides a certain basis for the development of efficient and reliable editing systems. The gene editing system can be used to obtain recombinant xylosaccharomyces cerevisiae bacteria with excellent characteristics.

[0031] The present invention uses the backbone vector pK18-P 112 -SacB-P 116 -Km R , the gene editing vector pK18-P was obtained after modification 112 -SacB-P34-Cm R Under optimized gene editing conditions, using pK18-P 112 -SacB-P34-Cm R Capable of efficient gene editing of Bacillus xylosus.

[0032] The present invention also provides a method for using pK18-P 112 -SacB-P34-Cm R Knockout bcs III Xylosaccharomyces cerevisiae KxΔ bcsIIIThis is a recombinant bacterium of Bacillus xylosus, which can produce BC with high crystallinity and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The results of the SacB-mediated sucrose sensitivity test in Example 1 of the present invention; Figure 2 The experimental results of optimizing the gene editing conditions in Example 3 of the present invention; Figure 3 The genome integration map (up: upstream homology arm of the targeted gene; down: downstream homology arm of the targeted gene) and agarose gel electrophoresis map for verifying the editing diversity of the gene editing system in Example 4 of the present invention; Figure 4 The Sanger sequencing graph (up: upstream homology arm of the targeted gene; down: downstream homology arm of the targeted gene) and the editing efficiency graph for verifying the editing diversity of the gene editing system in Example 4 of the present invention; Figure 5 For Example 5 of the present invention K. intermedius The gene editing efficiency of the SacB-based gene editing system validated in the AF2 strain; Figure 6 The plasmid pK18-Δ bcsIII Convert into K. xylinus Verification results of positive transformants (lanes 2 and 3 are bands with correct fragment sizes, and lanes 1, 4, and 5 are bands with incorrect fragment sizes); Figure 7 The plasmid pK18-Δ bcsIV Convert into K. xylinus Verification results of positive transformants (lanes 1, 2, and 3 are bands with correct fragment sizes, and lanes 4 and 5 are bands with incorrect fragment sizes); Figure 8 In Example 7 of the present invention K. xylinus With KxΔ bcsIII , KxΔ bcsIV The crystallinity of the produced BC. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work fall within the scope of protection of the present invention.

[0035] Xylosaccharomyces cerevisiae is a strain that can efficiently synthesize BC with simple culture conditions and food safety, and is suitable for large-scale production of BC. Gene editing can improve its BC production performance or change the characteristics of the BC it produces. However, the existing λ-Red and FLP / FRT-mediated site-specific recombination systems and CRISPR / Cas9 gene editing systems have shortcomings such as low editing efficiency. The gene editing method based on pK18obsacB can perform genome editing in microorganisms by gene knockout, knock-in or replacement, but sacB The gene encodes the enzyme sucrose glycosyltransferase, which causes cell lysis, which is lethal to Gram-negative bacteria. Therefore, it is necessary to develop new genetic tools suitable for efficient editing of genes in Xylella fastidiosa.

[0036] In addition, BC with high crystallinity has good mechanical properties, stability, durability and biocompatibility, and can be applied in many fields. However, the purpose of gene editing of Bacillus xylosus is mostly to increase the yield of BC, and there is little research on improving crystallinity.

[0037] In view of the above problems, an embodiment of the present invention provides a gene editing system based on SacB: the gene editing system contains sacB The invention discloses a plasmid containing a gene, a promoter P34 and a chloramphenicol resistance gene, wherein the nucleotide sequence of the promoter P34 is shown in SEQ ID NO.1.

[0038] The embodiments of the present invention also provide a method for constructing the above gene editing system.

[0039] The embodiments of the present invention also provide the use of the above gene editing system in constructing recombinant Bacillus xylosus bacteria, and provide a method for constructing recombinant Bacillus xylosus bacteria using the above gene editing system.

[0040] The present invention also provides a method for constructing Bacillus xylosus KxΔ bcsIII .

[0041] The embodiment of the present invention also provides the above-mentioned Bacillus xylosus KxΔ bcsIII Application in the production of high-crystallinity bacterial cellulose, and using the above-mentioned Bacillus xylosus KxΔ bcsIII Method for producing high-crystalline bacterial cellulose.

[0042] The scheme of the present invention is described below through specific embodiments.

[0043] The components of the culture medium used in the following examples are as follows: HS liquid medium: 7.5 g / L yeast extract, 10 g / L tryptone, 10 g / L disodium hydrogen phosphate dodecahydrate, 20 g / L glucose, the solvent is water, and the pH is adjusted to 6.0 with acetic acid.

[0044] HS solid medium: Add agar powder at a rate of 20 g / L to HS liquid medium.

[0045] The xylosaccharomyces cerevisiae with the deposit number of CGMCC No. 2955 was purchased from the General Microbiology Center of China National Microbiological Culture Collection Administration.

[0046] The remaining plasmids used in the following examples are all plasmids obtained from commercial sources or plasmids disclosed in the prior art such as literature, among which pK18-P 112 -SacB-P 116 -Km R For the construction method of the plasmid, please refer to “Systematic metabolic engineering of 2-keto-L-gulonic acid synthesis by Gluconobacter oxydans” (Qin Zhijie, Jiangnan University, DOI: 10.27169 / d.cnki.gwqgu.2022.000472).

[0047] Unless otherwise specified, the raw materials, reagents, drugs or instruments used in the following examples are all conventional commercial products obtained from commercial channels. The methods used in the following examples are all conventional methods in the art unless otherwise specified.

[0048] Example 1 This example provides a sucrose sensitivity test result of Bacillus xylosus.

[0049] The xylosaccharomyces cerevisiae strain (hereinafter referred to as K. xylinus ) was inoculated into HS liquid medium and cultured until the OD 600 Then, the bacterial solution was diluted at a certain multiple and spread on HS solid medium plates without sucrose and with sucrose (5%). K. xylinus Based on the successful single exchange and integration of SacB mutant strains (hereinafter referred to as K. xylinus -SacB) were subjected to the above operation. After incubation for 3 days, all plates were inverted and the colonies on the plates were counted to verify sucrose sensitivity. K. xylinus Chloramphenicol (340 μg / mL) was added during the culture of -SacB.

[0050] The results are as follows Figure 1 As shown, K. xylinus The mutant strains could grow normally on HS solid medium plates with or without 5% sucrose, while K. xylinus-SacB can grow normally on HS solid medium plates without sucrose, but its growth is significantly inhibited on HS solid medium plates with 5% sucrose. This result shows that in the presence of a certain concentration of sucrose, sacB Gene pair K. xylinus It has a lethal effect. K. xylinus A gene editing system based on SacB as a reverse selection marker was constructed.

[0051] Example 2 This example provides a gene editing system based on SacB, pK18-P 112 -SacB-P34-Cm R And its construction method, the specific process is as follows: (1) Primers P1 / P2 were designed and plasmid pSEVA331 was used as a template to obtain the chloramphenicol resistance gene fragment (shown in SEQ ID NO. 2) by PCR amplification.

[0052] (2) Primers P3 / P4 were designed, and the genome of Bacillus xylosus CGMCC 2955 was used as a template to obtain the P34 promoter fragment (shown in SEQ ID NO. 1) by PCR amplification.

[0053] (3) Design primers P5 / P6 and P7 / P8 to generate pK18-P 112 -SacB-P 116 -Km R The plasmid was used as a template, and gene fragments as shown in SEQ ID NO.3 and SEQ ID NO.4 were obtained by PCR amplification.

[0054] (4) The above-mentioned chloramphenicol resistance gene, promoter P34 and linearized pK18-P 112 -SacB-P 116 -Km R The backbone was connected by the recombination kit of Novozymes to obtain pK18-P 112 -SacB-P34-Cm R Vector (shown in SEQ ID NO.5).

[0055] The primer sequences used above are shown in Table 1.

[0056] Table 1 Primers used to construct plasmids

[0057] Example 3 This example provides an optimization process for the editing conditions of the SacB-based gene editing system constructed in Example 2.

[0058] First, the length of the homology arm of the targeted gene was optimized to increase the probability of successful single crossover. K. xylinus ) genome as a template, homology arms of different lengths (500 bp (upstream and downstream homology arm primer pairs are P9 / P10 and P11 / P12, respectively), 1000 bp (upstream and downstream homology arm primer pairs are P13 / P14 and P15 / P16, respectively), 1500 bp (upstream and downstream homology arm primer pairs are P17 / P18 and P19 / P20, respectively) and 2000 bp (upstream and downstream homology arm primer pairs are P21 / P22 and P23 / P24, respectively)) were amplified, and were respectively constructed into the plasmid pK18-P constructed in Example 2 112 -SacB-P34-Cm R These constructed plasmids were electroporated into K. xylinus After recovery, the cells were spread on HS solid medium plates containing chloramphenicol and cultured in a 30°C incubator for 3 days, and then the transformants were counted.

[0059] Table 2 Primers used to amplify homology arms

[0060] Second, optimize the lethal concentration of sucrose. As shown in Example 1, at a certain sucrose concentration, sacB Gene pair K. xylinus The sucrose concentration is too low, the killing efficiency is low, and more single exchange strains appear on the plate after the strain undergoes double exchange; if the sucrose concentration is too high, it may cause sacB Genes are easily mutated by transposon insertion, and the lethality efficiency will be low. Therefore, it is very important to choose the right sucrose concentration. Pick a single colony from the single exchange strain with the best homology arm length and inoculate it into HS liquid medium containing chloramphenicol for cultivation. 600 The concentration was adjusted to 0.5, and 100 μL of bacterial solution was taken and spread on solid culture medium plates containing different concentrations (0%, 1%, 2%, 3%, 4%, 5%, 6% and 7%) of sucrose. After inverted culture for 3 days, the number of colonies was counted and the lethal efficiency was calculated.

[0061] Third, optimize the culture time of the second homologous recombination. When the single exchange strain is inoculated into the antibiotic-free HS liquid medium for cultivation, three strains will be produced: one is a single exchange strain without double exchange, and the other two are strains with double exchange, including mutant strains and reverted mutant strains. If the recovery time is too long, one of the strains will become the dominant strain, which is not conducive to the acquisition of mutant strains, so it is particularly important to optimize the culture time of double exchange. Pick a single colony from the single exchange strain with the optimal homologous arm length, inoculate it into the antibiotic-free HS liquid medium for cultivation, sample it every 12 hours, and spread the bacterial solution on a HS solid medium plate containing 7% sucrose concentration. After inverting and culturing for 3 days, randomly pick a single colony for colony PCR and calculate the editing efficiency.

[0062] Fourth, optimize the culture temperature for the second homologous recombination. The single exchange strain with the best homologous arm length was inoculated into 5 tubes of HS liquid culture medium without antibiotics, and cultured in 25℃, 28℃, 30℃, 32℃ and 35℃ incubators for 24 h. Subsequently, the culture solution was spread on HS solid culture medium plates containing 7% sucrose, and after inverted culture for 3 days, single colonies were randomly picked for colony PCR, and the editing efficiency was calculated.

[0063] Under each optimized condition, 24 colonies were randomly selected and the percentage of positive clones to the total number of colonies was calculated to evaluate the editing efficiency. The gene editing efficiency under each condition was determined three times.

[0064] The experimental results of optimizing the length of homology arms are as follows Figure 2 As shown in Figure a. As can be seen from the figure, as the length of the homology arm increases, the number of single exchange transformants also increases significantly. When the length of the homology arm is 2000 bp, the number of single exchange transformants can reach more than 15. Therefore, the length of the homology arm of the targeted gene is preferably 2000 bp.

[0065] The experimental results of optimizing the lethal concentration of sucrose are as follows Figure 2 As shown in Figure b (Con: no sucrose plate; CFU: colony forming unit). As can be seen from the figure, with the increase of sucrose concentration, SacB K. xylinus The lethal effect gradually increased, therefore, 7% sucrose concentration was the preferred concentration.

[0066] The experimental results of optimizing the culture time of the second homologous recombination are as follows Figure 2 As shown in Figure c. As can be seen from the figure, when the culture time is 24 h, the mutant strain has a higher positive rate in both the dominant and non-dominant cases, and the double exchange culture time is preferably 24 h.

[0067] The experimental results of optimizing the culture temperature for the second homologous recombination are as follows Figure 2As shown in Figure d. As can be seen from the figure, 32°C is more conducive to obtaining mutant strains, and its editing efficiency can reach 87.50%.

[0068] After optimization, the optimal editing conditions of the SacB-based gene editing system provided by the present invention are: homology arm length 2000 bp, sucrose concentration 7%, double exchange culture time 24 h, and double exchange culture temperature 32°C.

[0069] Example 4 This example verifies the editing diversity of the SacB-based gene editing system constructed in Example 2.

[0070] The xylosyl bacillus (hereinafter referred to as K. xylinus ) genome as a template, and primer pairs P25 / P26, P27 / P28, P29 / P30, P31 / P32, P33 / P34, and P35 / P36 were used to amplify K. xylinus The upstream and downstream sequences of the 5 kb fragment were knocked out / the 1 kb fragment was knocked in / the 1.5 kb fragment was replaced. Using the pRedGx plasmid as a template, the sequences of the 1 kb fragment knocked in and the 1.5 kb fragment were amplified using primer pairs P37 / P38 and P39 / P40, respectively, and were constructed into the plasmid pK18-P constructed in Example 2. 112 -SacB-P34-Cm R The plasmids pK18-Δ5k, pK18-I1k and pK18-R1.5k were obtained. The above plasmids were electroporated into K. xylinus After screening, an engineered strain integrating the above plasmid was obtained.

[0071] Table 3 Primers used to amplify each fragment

[0072] The experimental results showed that the SacB-based gene editing system constructed using Example 2 of the present invention successfully knocked out a 5 kb gene fragment (such as Figure 3 and Figure 4 This is of great significance for knocking out polycistronic gene clusters or constructing minimal genomes. In addition, 1 kb gene knock-in was successfully achieved (e.g. Figure 3 and Figure 4 b) and replacement of the 1.5 kb gene (e.g. Figure 3 and Figure 4 This will help achieve heterologous expression and multi-copy expression of target genes at the genome level. The gene editing efficiency calculated based on the agarose gel electrophoresis results was high, indicating that the gene editing system is effective for K. xylinus The transformation can meet the needs.

[0073] Example 5 This example verifies the editing universality of the SacB-based gene editing system constructed in Example 2 of the present invention.

[0074] by Komagataeibacter intermedius ( K. intermedius ) The genome of AF2 was used as a template and primer pairs P41 / P42 and P43 / P44 were used to amplify bcsAI The upstream and downstream sequences of the fragment were constructed into the plasmid pK18-P obtained in Example 2. 112 -SacB-P34-Cm R The plasmid pK18-AF2Δ was obtained bcsAI . Plasmid pK18-AF2Δ bcsAI Electrotransfer K. intermedius AF2, and the engineered strain AF2Δ was obtained after screening bcsAI .

[0075] Table 4 Amplification bcsAI Primers used for upstream and downstream sequences of the fragment

[0076] Then, after screening the obtained K. intermedius The editing effect of the SacB-based gene editing system was verified in the AF2 strain. The results showed that the gene editing system can be used in K. intermedius AF2 plays a role, and the gene editing efficiency is about 25% (such as Figure 5 This suggests that this system has the potential to modify the genomes of various Acetobacter species and could provide an effective tool for improving BC production or regulating BC structure through metabolic engineering.

[0077] Example 6 This example provides the application of the SacB-based gene editing system constructed in Example 2 in the construction of recombinant Bacillus xylosus.

[0078] 1. Recombinant plasmid pK18-Δ bcsIII Construction The xylosyl bacillus (hereinafter referred to as K. xylinus ) genome as a template, and primers P45 / P46 and P47 / P48 were used to amplify bcs III The upstream and downstream homology arms of the operon were inserted into the plasmid pK18-P 112 -SacB-P34-Cm R Linearized by PCR, assembled by Gibson, and transformed into E. coli to obtain the knockout plasmid pK18-Δ bcsIII .

[0079] Table 5 Amplification bcs III Primers used for upstream and downstream homology arms of the operon

[0080] 2. Recombinant Escherichia coli E .coli Construction of DH5D.

[0081] (1) Chemical transformation: The plasmid obtained above was introduced into E.coli In the DH5α competent state, all experimental procedures were performed on ice.

[0082] In a sterile workbench, add the plasmid to the competent E. coli. After standing for 20 minutes, heat shock it in a metal bath at 42°C for 1 minute, cool it on ice for 2 minutes, add 500 μL liquid LB medium (5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl), revive it in a 37°C shaker for 1 hour, centrifuge it at 5000 r / min for 3 minutes, pour out the supernatant, spread it on an LB solid medium plate containing 50 μg / mL chloramphenicol, place it upside down in a 37°C incubator, and culture it overnight.

[0083] (2) Verification by colony PCR: When the transformant grows a single colony on a LB solid medium plate containing 50 μg / mL chloramphenicol, colony PCR is performed. The required primers, reaction system and PCR conditions are as follows: Primer design and synthesis for colony PCR: Upstream primer P23: 5′-acactttatgcttccggctcgt-3′; Downstream primer P24: 5′-ggatgtgctgcaaggcgattaag-3′.

[0084] PCR reaction system (trans fast Taq): ddH2O 6.5 μL; upstream and downstream primers 0.5 μL each; Taq enzyme mix: 7.5 μL; template is colony. A total of 15 μL for one system.

[0085] PCR conditions were as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for X s (30 s for 1 kb), 32 cycles, and a final extension at 72°C for 10 min, followed by insulation at 4°C.

[0086] (3) Extracting plasmids and sequencing: The successfully verified recombinant E. coli was inoculated in an LB test tube, and chloramphenicol was added. The tube was cultured in a shaker at 37°C overnight, and the plasmid was extracted and sequenced.

[0087] If the results of colony PCR and assay are correct, the plasmid is successfully transformed into E.coli Positive transformants of DH5α.

[0088] 3. Construction of engineered Bacillus xylosus (1) The plasmid obtained in the above step was transferred into K. xylinus In competent culture: add 3-5 μL of plasmid into K. xylinus In the competent cells, gently pipette to mix and transfer to the electroporation cup, then place the electroporation cup on ice for 20 min. During this period, turn on the electroporation instrument and preheat it; set the electroporation parameters as follows: voltage 2000 V, resistance 200Ω, capacitance 25 μF, press the electroporation key for electroporation; after the electroporation, immediately add 900 μL of liquid HS medium (7.5g / L yeast extract, 10 g / L tryptone, 10 g / L sodium hydrogen phosphate dodecahydrate, 20 g / L glucose) (containing 1% cellulase) and pipette the electroporation cup to transfer the electroporation liquid to a sterilized 1.5 mL centrifuge tube, and culture at 30 ℃ 180 r / min for 8 hours; centrifuge the bacterial solution, discard part of the supernatant, and apply the remaining about 200 μL of bacterial solution to the HS plate with 200 μg / mL chloramphenicol added, and observe the transformants after inverting and culturing at 30 ℃ for 2-3 days.

[0089] (2) Verification by colony PCR: The transformants were grown on HS solid culture medium plates containing 200 μg / mL chloramphenicol. Colony PCR was performed after obvious single colonies grew.

[0090] (3) The verified transformants were inoculated into antibiotic-free HS liquid medium for culture. Next, 10 μL of the bacterial solution was diluted and spread onto a HS solid medium plate containing 7% sucrose and cultured in a 30°C incubator for 2 days.

[0091] (4) Verification by colony PCR: The transformants were grown on HS solid medium plates containing 7% sucrose. After obvious single colonies were grown, colony PCR was performed. The results of the above colony PCR were detected by nucleic acid electrophoresis. If the results of colony PCR and the target band (4200 bp) were both correct, the plasmid pK18-Δ bcsIII Convert into K. xylinus Positive transformants (such as Figure 6 as shown).

[0092] Screening plasmid pK18-Δ bcsIII Convert into K. xylinus The positive transformants were named as Xylella fastidiosa ( Komagataeibacter xylinus )KxΔ bcsIII, and deposited it with the deposit number CGMCC No.32726.

[0093] Comparative Example 1 This comparative example provides a recombinant strain of Bacillus xylosus.

[0094] 1. Recombinant plasmid pK18-Δ bcsIV Construction The xylosyl bacillus (hereinafter referred to as K. xylinus ) genome as a template, and primers P49 / P50 and P51 / P52 were used to amplify bcs IV The upstream and downstream homology arms of the gene (the NCBI-locus_tag of the nucleotide sequence is CT154_03760) were cloned into plasmid pK18-P 112 -SacB-P34-Cm R Linearized by PCR, assembled by Gibson, and transformed into E. coli to obtain the knockout plasmid pK18-Δ bcsIV .

[0095] Table 6 Amplification bcs IV The primers used for the upstream and downstream homology arms

[0096] 2. Recombinant Escherichia coli E .coli Construction of DH5D Same as Example 6.

[0097] 3. Construction of engineered Bacillus xylosus Same as Example 6. Plasmid pK18-Δ bcsIV Convert into K. xylinus The verification results of the positive transformants are as follows Figure 7 As shown. It is named KxΔ bcsIV .

[0098] Example 7 This example provides the fermentation production of BC by the recombinant Bacillus xylosus constructed in Example 6 and Comparative Example 1 in HS medium containing 25 g / L glucose.

[0099] The deposit number is CGMCC No.2955 K. xylinus and the engineered strain constructed in Example 6 (KxΔ bcsIII , deposit number is CGMCC No.32726) and the positive transformant constructed in comparative example 1 (KxΔ bcsIV) were inoculated into seed culture medium for activation culture, and then further static culture was carried out in fermentation bottles. After fermentation, the fermentation liquid was sampled and centrifuged to obtain the supernatant, and the glucose content was detected by biosensor; the BC membrane was processed, weighed and recorded. Three parallel experiments were performed for each strain.

[0100] (1) Primary seed culture medium: Take a medium sterilized by high pressure steam (test tube plus 10 mL HS liquid culture medium) and pick a single colony from the solid culture medium plate with the target strain in a clean bench, inoculate, culture at 30°C, 180 rpm, for 12 h, add cellulase (Novozymes Celluclast ® 1.5L) to degrade the cellulose fibers until the fibers are no longer visible to the naked eye (about 12 hours) to facilitate subsequent transfer.

[0101] (2) Secondary seed culture: K. xylinus and engineered strain KxΔ bcsIII The first-grade seed solution of (CGMCC No.32726) was inoculated into 100 mL conical flasks containing 30 mL HS liquid medium at a 1% inoculation rate, and cellulase (Novozymes Celluclast ® 1.5L), 30°C, 180 rpm, culture until OD 600 It is 0.6-0.8 (about 48 hours).

[0102] (3) Shake flask fermentation: Wash the cells with sterile PBS, clean the cellulase, resuspend the cells, and measure the OD of the cells. 600 Each strain was adjusted to the same growth state and inoculated into 75 mL of HS liquid medium (with acetic acid to adjust the initial pH to 6) containing 20 g / L glucose as the carbon source to an initial OD of 600 The concentration of 0.02 was used and the static fermentation was carried out in a 30 ℃ incubator for 7 days.

[0103] During the above culture process, the deposit number is CGMCC No.2955 K. xylinus and engineered strain KxΔ bcsIII (CGMCC No.32726) have no resistance and do not need to add antibiotics.

[0104] (4) BC determination: After the fermentation is completed, the BC is taken out and soaked in 0.1 mol / L NaOH to remove the bacteria in the BC, and finally the membrane is soaked until it is milky white and translucent; the BC soaked in the milky white and translucent state is continued to be soaked in distilled water, and fresh distilled water needs to be replaced several times in the middle, and finally the BC is soaked until the pH is neutral; the treated BC is dried at 95°C for 10 min using a sheet machine, and then an X-ray diffraction test is performed to calculate the BC crystallinity of the engineering strain and the starting strain (such as Figure 8 as shown).

[0105] As can be seen from the figure, KxΔ bcsIII The crystallinity of BC produced by the strain was the highest, 63.41%±0.09%, which was 19.85% higher than that of BC produced by the original strain with the deposit number of CGMCC No.2955 (52.91%±0.11%); bcsIV The crystallinity of BC produced by the strain was 50.34%±0.16%, which was slightly lower than that of BC produced by the starting strain with the deposit number CGMCCNo.2955.

[0106] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A gene editing system based on SacB, characterized in that: The gene editing system comprises sacB The invention discloses a plasmid containing a gene, a promoter P34 and a chloramphenicol resistance gene, wherein the nucleotide sequence of the promoter P34 is shown in SEQ ID NO.

1.

2. The gene editing system according to claim 1, characterized in that: The nucleotide sequence of the chloramphenicol resistance gene in the gene editing system is shown in SEQ ID NO.2; and / or The gene editing system is a system that sacB The gene vector skeleton is constructed by introducing the promoter P34 and the chloramphenicol resistance gene.

3. The gene editing system according to claim 2, characterized in that: The said sacB The plasmid of the gene is pK18-P 112 -SacB-P 116 -Km R Plasmid.

4. The method for constructing a gene editing system according to any one of claims 1 to 3, characterized in that: The specific operations include: The said sacB The vector backbone of the gene is used as a template to obtain the amplified product through PCR amplification; The promoter P34 and the chloramphenicol resistance gene are recombinantly linked to the amplified product to obtain the gene editing system.

5. The construction method according to claim 4, characterized in that: The said sacB The plasmid of the gene is pK18-P 112 -SacB-P 116 -Km R The plasmid, the amplification product is a gene fragment with a nucleotide sequence as shown in SEQ ID NO.3 and a gene fragment as shown in SEQ ID NO.4, and the nucleotide sequence of the gene editing system is shown in SEQ ID NO.

5.

6. Use of the gene editing system according to any one of claims 1 to 3 in constructing recombinant bacteria of Bacillus xylosus.

7. A method for constructing a recombinant bacterium of Bacillus xylosus using the gene editing system according to any one of claims 1 to 3: using the gene editing system according to any one of claims 1 to 3 to knock out a gene on the genome of Bacillus xylosus bcs III Operon, the bcs III The nucleotide sequence of the operon is: the nucleotide sequence with NCBI-locus_tag of CT154_05660 and the nucleotide sequence with NCBI-locus_tag of CT154_05665 and the non-coding region sequence therebetween.

8. A strain of Bacillus xylosus KxΔ bcsIII , characterized in that, Its taxonomic name is Bacillus xylosus ( Komagataeibacter xylinus ), deposited on November 21, 2024 in the General Microbiology Center of China Microorganism Culture Collection Administration, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101, with the deposit number CGMCC No.32726.

9. The xylosaccharomyces cerevisiae KxΔ according to claim 8 bcsIII Application in the production of high-crystallinity bacterial cellulose.

10. A method of using the xylosaccharomyces cerevisiae KxΔ described in claim 8 bcsIII A method for producing high-crystallinity bacterial cellulose, characterized in that: The specific operations include: The xylosaccharomyces cerevisiae KxΔ bcsIII Inoculate into HS liquid medium, add cellulase, and culture at 30-40 ℃ until the bacteria grow logarithmically to obtain seed liquid bacteria; Wash the seed liquid cells with sterile PBS, clean the cellulase and resuspend the cells according to the initial OD 600 The concentration of 0.02-0.2 was inoculated into HS liquid culture medium containing 20-30 g / L glucose, and static fermentation culture was carried out in an incubator at 30-40 ℃; the produced bacterial cellulose was collected.

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