Promoter and application thereof in enhancing expression of target gene

Abstract

The invention belongs to the technical field of gene engineering, and discloses a promoter and application thereof in enhancing target gene expression. The nucleotide sequence of the promoter P33 is as shown in SEQ ID NO: 2. The promoter P33 is obtained by site-directed mutagenesis of a wild type promoter P06915 of gluconacetobacter xylinum and screening, and has significantly enhanced activity. The promoter P33 provided by the invention can be used for enhancing the expression of a target gene and producing a target compound. For example, the promoter P33 is operably connected with a sucrose hydrolase gene cscA, the expression level of the cscA gene can be remarkably improved, the cscA gene expression cassette driven by the promoter P33 is introduced into gluconacetobacter xylinum, efficient expression of sucrose hydrolase can be promoted, and the gluconacetobacter xylinum efficiently utilizes sucrose to produce bacterial cellulose. Meanwhile, the invention provides a brand new method for industrial production of the bacterial cellulose, and the method not only improves the production efficiency, but also reduces the production cost, and has huge application potential and economic value.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering and discloses a promoter and its application in enhancing the expression of target genes. Background Art

[0002] Bacterial cellulose is a nanoscale biopolymer synthesized by bacteria. It has high purity, high crystallinity, excellent mechanical properties, superb water absorption and moisture retention, and excellent biocompatibility. It is widely used in biomedicine, food industry, environmental protection, electronic devices, cosmetics and other fields. Bacterial cellulose-producing bacterial strains include Agrobacterium, Rhizobium, Pseudomonas and Acetobacter, among which xylindoleacetic acid Bacter ( Komagataeibacter xylinus ) has become the most widely studied bacterial cellulose-producing strain due to its ability to produce high amounts of bacterial cellulose.

[0003] In order to increase the yield of bacterial cellulose, it can be achieved by enhancing the expression of proteases in pathways related to bacterial cellulose metabolism. There are many ways to enhance the expression of enzymes, such as increasing the copy number, replacing strong promoters, etc. However, increasing the copy number may increase the burden on the strain and lead to genomic instability of the strain. In contrast, promoter regulation of gene expression does not have these defects. For xylindole Gluconobacter, there are currently few developments and insufficient knowledge of promoters derived from xylindole Gluconobacter, which leads to more reliance on exogenous promoters in research and application. Exogenous promoters may not be able to fully adapt to the physiological characteristics of xylindole Gluconobacter, thereby affecting their efficiency and stability in bacterial cellulose production. Summary of the invention

[0004] In view of these defects, the present invention provides a promoter and its application in enhancing the expression of target genes. 06915 By performing site-directed mutagenesis on the promoter sequence, multiple mutant promoters were obtained, and a mutant promoter P with significantly enhanced activity was obtained after screening. 33 , which can efficiently drive the expression of target genes, improve the efficiency of metabolic pathway construction, and thus promote the production of target compounds.

[0005] To achieve the above object, the present invention provides the following technical solutions: In a first aspect, the present invention provides a promoter, wherein the promoter P 33 The nucleotide sequence is shown in SEQ ID NO:2.

[0006] The promoter P provided by the present invention 33 The strong promoter P from Gluconacetobacter xylinus 06915 The nucleotide sequence (shown in SEQ ID NO: 1) was modified by directed transformation.06915 The nucleotide segment from position 194 to position 437 of the sequence is used as the basic framework, which is P 06915 The core sequence has high strength. Then, the extracted fragment was subjected to multi-point site-directed mutation: the 36-37 double base was mutated from TC to CT, the 39th base was mutated from G to T, and the 43-44 double base was mutated from GA to TG. After the above transformation, the promoter P was obtained. 33 , whose nucleotide sequence is shown in SEQ ID NO:2.

[0007] In the series of bacterial cellulose-producing recombinant strains constructed in the present invention, the promoter P 33 Driving sucrose hydrolase gene cscA The recombinant strain expressed showed significantly higher bacterial cellulose production than the recombinant strains driven by other promoters and the wild-type strain, indicating that the promoter P 33 Can efficiently drive the expression of target genes.

[0008] In a second aspect, the present invention also provides the above-mentioned promoter P 33 Application in enhancing target gene expression and producing target compounds.

[0009] The promoter P of the present invention 33 It can significantly increase the expression level of the target gene through its transcriptional activation ability, and play an important role in the process of biosynthesis of the target compound. Specifically, the promoter P can be transformed into 33 Connect with the target gene to construct a recombinant expression vector, which is then introduced into host cells (such as bacteria, yeast or plant cells) and expressed under the promoter P 33 Driven by the promoter P, the transcription and translation efficiency of the target gene is significantly improved, thus achieving efficient expression. 33 It is used to regulate the expression of enzyme genes in key metabolic pathways, which can enhance metabolic flux and thus increase the production of target compounds.

[0010] Preferably, the target gene is a gene related to bacterial cellulose synthesis, and the target compound is bacterial cellulose.

[0011] In a third aspect, the present invention also provides an expression cassette, which includes a target gene and the above-mentioned promoter; the expression cassette also includes one or more of a terminator, an SD sequence, an enhancer and a post-transcriptional regulatory element; the promoter is operably connected to the target gene and mediates the transcription of the target gene.

[0012] The "expression cassette" mentioned in the present invention has the meaning generally understood by those skilled in the art, that is, it contains a promoter, a target gene and elements capable of expressing the target gene.

[0013] The "target gene" refers to a gene encoding a target protein in a microorganism, including but not limited to a gene encoding an enzyme related to the biosynthesis of a target compound, a gene encoding an enzyme related to reducing power, a gene encoding an enzyme related to glycolysis or TCA cycle, or a gene encoding an enzyme related to the release of a target compound, etc.

[0014] The term "operably linked" refers to the promoter P of the present invention. 33 Functionally connected to the target gene to initiate and mediate the transcription of the target gene and control the transcriptional activity of the operon gene. The operably connected method can be any method described by those skilled in the art.

[0015] Preferably, the target gene is a gene encoding sucrose hydrolase cscA.

[0016] More preferably, the target gene is a nucleotide sequence of a gene encoding sucrose hydrolase cscA as shown in SEQ ID NO: 9. The encoding gene is derived from Escherichia coli ( Escherichia coli )W, the sucrose hydrolase it expresses can hydrolyze sucrose into fructose and glucose for bacterial use, providing energy for bacterial growth and metabolism.

[0017] In a fourth aspect, the present invention further provides a recombinant vector, which comprises the above-mentioned promoter or the above-mentioned expression cassette.

[0018] The "vector" of the present invention refers to a DNA construct, which comprises a DNA sequence operably linked to a suitable control sequence (such as a promoter, terminator, etc.), capable of introducing a target gene into a host cell, and achieving gene replication, transcription and translation in the host, thereby expressing the target gene. The vector used in the present invention is not particularly limited, and can be any vector known in the art, including but not limited to plasmids and bacteriophages.

[0019] Preferably, the recombinant vector further comprises one or more of a replication origin site, an enhancer, a selection marker, a ribosome binding site and polyA.

[0020] More preferably, the screening marker is one or more of an antibiotic screening marker, a fluorescent protein gene and an anti-screening gene.

[0021] In a fifth aspect, the present invention also provides a recombinant strain comprising the above-mentioned promoter, expression cassette or recombinant vector.

[0022] Preferably, the starting strain of the recombinant strain is Acetobacter ( Acetobacter ), the recombinant strain contains a gene encoding a sucrose hydrolase cscA and the above-mentioned promoter P 33 expression cassette.

[0023] The "starting strain" described in the present invention refers to the original strain used to construct the recombinant strain.

[0024] Acetobacter mainly uses glucose as a substrate to synthesize bacterial cellulose. Currently, metabolic engineering of Acetobacter to increase its bacterial cellulose production includes enhancing the expression of enzymes related to the bacterial cellulose synthesis pathway, weakening the expression of enzymes related to the competitive pathway, etc. However, bacterial cellulose is still produced using glucose as a substrate, which results in a high production cost.

[0025] The present invention involves the promoter P 33 The expression cassette of the gene encoding the sucrose hydrolase cscA driven by the gene expression system was introduced into Acetobacter, so that the gene encoding the sucrose hydrolase cscA was efficiently heterologously expressed in Acetobacter, and the sucrose hydrolase encoded by this gene was used to decompose sucrose into fructose and glucose. This transformation enabled Acetobacter to efficiently utilize sucrose to produce bacterial cellulose, broadening the substrate source for bacterial cellulose production.

[0026] More preferably, the method for constructing the recombinant strain comprises introducing a plasmid containing the expression cassette into the starting strain.

[0027] More preferably, the promoter P in the expression cassette 33 The sucrose hydrolase cscA gene is operably connected to the sucrose hydrolase cscA gene and mediates the sucrose hydrolase gene cscA of transcription.

[0028] More preferably, the starting strain is Gluconobacter xylinacetoacetate ( Komagataeibacter xylinus ).

[0029] Gluconacetobacter xylinum is an important industrial production strain that has been widely studied and applied for its ability to efficiently synthesize bacterial cellulose. The culture conditions of this strain are relatively simple, and as a food safety strain, it has been safely used in large-scale industrial production of bacterial cellulose.

[0030] More preferably, the starting strain is Gluconacetobacter xylinum CGMCC No. 2955.

[0031] In the present invention, Gluconacetobacter xylinum CGMCC No. 2955 was used as the starting strain, and a promoter containing P 33 The plasmid containing the gene expression cassette of the sucrose hydrolase cscA driven by the β-lactamase was used to construct a recombinant strain of xyloglucanacetobacter that can efficiently produce bacterial cellulose using sucrose. Under the culture conditions with sucrose as the substrate, the bacterial cellulose production of the recombinant strain reached 5.60±0.03 g / L after 7 days of cultivation, which was significantly higher than that of the wild-type strain and other recombinant strains.

[0032] In a sixth aspect, the present invention also provides the use of the above-mentioned recombinant strain in the preparation of bacterial cellulose.

[0033] In a seventh aspect, the present invention also provides a method for producing bacterial cellulose, using the above-mentioned recombinant strain to ferment with sucrose or molasses containing sucrose as a substrate.

[0034] Preferably, the substrate is beet molasses. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0036] Figure 1 The recombinant plasmid pRBS-P in Example 2 33 - cscA Schematic diagram of the structure; Figure 2 The recombinant plasmid pRBS-P in Example 2 33 - cscA Gel electrophoresis results. DETAILED DESCRIPTION

[0037] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] The xylinus Gluconobacter CGMCC No. 2955 used in the present invention was screened by the Biochemical Engineering Laboratory of the School of Bioengineering of Tianjin University of Science and Technology. The strain has been used in "A Lambda Red and FLP / FRT-Mediated Site-Specific Recombination System in Komagataeibacter xylinus and Its Application to Enhance the Productivity of Bacterial Cellulose" (Ling-Pu Liu et al., ACSSynthetic Biology . (2020, 9, 3171−3180)).

[0039] The pBla plasmid used in the present invention is constructed based on the pBBR1 plasmid, which contains the kanamycin resistance gene, P bla promoter and rrnB T1 terminator, its own promoter is promoter P bla (nucleotide sequence as shown in SEQ ID NO: 5), the plasmid structure and its construction method have been described in "A Lambda Red and FLP / FRT-Mediated Site-Specific Recombination System in Komagataeibacter xylinus and Its Application to Enhance the Productivity of Bacterial Cellulose" (Ling-Pu Liu et al., ACSSynthetic Biology . (2020, 9, 3171−3180)). The pRBS plasmid used in the present embodiment is a pBla plasmid with the promoter P removed. bla Obtained.

[0040] The culture medium used in the embodiments of the present invention is: LB medium components: tryptone 10 g / L; yeast extract 5 g / L; NaCl 10 g / L; solid medium requires additional 20 g / L agar powder; initial pH 7.0.

[0041] HS solid medium components: glucose 25 g / L; yeast powder 7.5 g / L; peptone 10 g / L; Na2HPO4 10 g / L; agar powder, 20 g / L; initial pH 6.0.

[0042] Seed culture medium components: glucose 25 g / L; yeast powder 7.5 g / L; peptone 10 g / L; Na2HPO4 10 g / L; initial pH 6.0.

[0043] Sucrose fermentation medium components: sucrose 25 g / L; yeast powder 7.5 g / L; peptone 10 g / L; Na2HPO4 10 g / L; initial pH 6.0.

[0044] Sugarcane molasses fermentation medium components: sugarcane molasses 25 g / L (final total sugar content of the medium); yeast powder 7.5 g / L; peptone 10 g / L; Na2HPO4 10 g / L; initial pH 6.0.

[0045] Beet molasses fermentation medium components: beet molasses 25 g / L (final total sugar content of the medium); yeast powder 7.5 g / L; peptone 10 g / L; Na2HPO4 10 g / L; initial pH 6.0.

[0046] If not otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. The test methods in the following embodiments are conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can be obtained commercially. The promoter nucleic acid molecules of the present invention can be isolated or prepared using standard molecular biology techniques. For example, the promoter nucleic acid molecules of the present invention can be isolated by PCR using suitable primer sequences. In addition, the promoter nucleic acid molecules of the present invention can also be prepared by standard synthesis techniques using an automatic DNA synthesizer. The gene sequences involved in the embodiments of the present invention can be encoded and synthesized according to the sequence information of the gene or amplified using the microbial genome of the corresponding source as a template. The primers used in the embodiments of the present invention are shown in Table 1.

[0047] Table 1 Primer sequences used in the examples of the present invention

[0048] Example 1 In order to enhance the promoter P from Gluconacetobacter xylinus 06915 The transcription level of promoter P is obtained by increasing the transcription level of promoter P. 06915 The promoter mutants were designed and transformed. 06915 The nucleotide segment from position 194 to 437 of the nucleotide sequence was used as the basic framework for mutation, and multiple site-directed mutagenesis was performed on this basis: the double base at position 36-37 was mutated from TC to CT, the double base at position 39 was mutated from G to T, and the double base at position 43-44 was mutated from GA to TG. After the above transformation, the promoter mutant P was obtained. 33 , whose nucleotide sequence is shown in SEQ ID NO:2.

[0049] Example 2 This embodiment provides a promoter P 33 Recombinant plasmid of expression cassette driven by the gene and its construction method.

[0050] (1) Using the genome of Glucose Acetobacter xylinum CGMCC No. 2955 as a template, the promoter P was amplified using primers P33-U / P33-D. 33 fragment (nucleotide sequence as shown in SEQ ID NO: 2); using plasmid pRBS as a template, primers PPF / PPR were used to amplify the linearized plasmid fragment of pRBS; then the promoter P 33The linearized plasmid fragment of pRBS was cloned and connected with the one-step recombination kit of Novozyme to obtain a plasmid containing promoter P 33 The recombinant plasmid pRBS-P 33 .

[0051] (2) Synthesized by Jinweizhi Biotechnology Co., Ltd. cscA The gene fragment (nucleotide sequence as shown in SEQ ID NO: 9) was used as a template and amplified using primers cscA-U / cscA-D. The recombinant plasmid pRBS-P 33 As a template, primers PF1 / PR1 were used to amplify pRBS-P 33 The linearized plasmid fragment cscA Gene fragment and pRBS-P 33 The linearized plasmid fragment was cloned and connected by Novozyme's one-step recombination kit to obtain the recombinant plasmid pRBS-P 33 - cscA The schematic diagram of its plasmid structure is shown in Figure 1 shown.

[0052] (3) The recombinant plasmid pRBS-P constructed above was 33 - cscA Transform into competent E. coli DH5α, and after recovery, spread on LB solid medium containing 50 μg / mL kanamycin, and culture at 37°C to obtain positive transformants. Inoculate positive transformants into LB liquid medium containing 50 μg / mL kanamycin, and culture at 37°C overnight to extract plasmids (see gel electrophoresis results for details). Figure 2 ) was sequenced and verified to obtain the recombinant plasmid pRBS-P 33 - cscA , and maintained in Escherichia coli DH5α.

[0053] Example 3 This embodiment provides a recombinant strain for efficiently utilizing sucrose to produce bacterial cellulose and a method for constructing the same.

[0054] The recombinant plasmid pRBS-P provided in Example 2 33 - cscA Electrotransformed into the competent cell of Glucose Acetobacter xylinum CGMCC No.2955, the transformants were spread on HS solid medium containing 50 μg / mL kanamycin after recovery, cultured at 30°C for 3-4 days, and after monoclonal colonies grew out, single colonies were identified by colony PCR using primers PF / PR to screen for overexpression cscA The recombinant strain of the gene was named K11.

[0055] Comparative Example 1 (1) P 06915The nucleotide segment from position 194 to 437 of the nucleotide sequence was used as the basic framework for mutation. Multiple site-directed mutagenesis was performed on this basis: the 9th base was mutated from T to G, and the three bases from position 13 to 15 were mutated from CAT to TTA. After the above transformation, the promoter mutant P was obtained. 20 , whose nucleotide sequence is shown in SEQ ID NO:3.

[0056] (2) Using the genome of Glucose Acetobacter xylinum CGMCC No. 2955 as a template, the promoter P was amplified using primers P20-U / P20-D. 20 fragment; using plasmid pRBS as template, using primers PPF / PPR to amplify the linearized plasmid fragment of pRBS; then the promoter P 20 The linearized plasmid fragment of pRBS was cloned and connected with the one-step recombination kit of Novozyme to obtain a plasmid containing promoter P 20 The recombinant plasmid pRBS-P 20 .

[0057] (3) The recombinant plasmid pRBS-P was constructed using the same primers and methods as in Example 2 and Example 3. 20 - cscA , and introduced it into Gluconacetobacter xylinum CGMCC No. 2955 to screen for overexpression cscA Genetic recombinant strains ， Named K12.

[0058] Comparative Example 2 (1) P 06915 The nucleotide segment from position 194 to 437 of the nucleotide sequence was used as the basic framework for mutation. Multiple site-directed mutagenesis was performed on this basis: the 9th base was mutated from T to G, the 14th-15th bases were mutated from AT to GA, the 37th base was mutated from C to T, and the 39th base was mutated from G to T. After the above transformation, the promoter mutant P was obtained. 22 , whose nucleotide sequence is shown in SEQ ID NO:4.

[0059] (2) Using the genome of Glucose Acetobacter xylinum CGMCC No. 2955 as a template, the promoter P was amplified using primers P22-U / P22-D. 22 fragment (nucleotide sequence as shown in SEQ ID NO: 4); using plasmid pRBS as a template, primers PPF / PPR were used to amplify the linearized plasmid fragment of pRBS; then the promoter P 22 The linearized plasmid fragment of pRBS was cloned and connected with the one-step recombination kit of Novozyme to obtain a plasmid containing promoter P 22 The recombinant plasmid pRBS-P 22 .

[0060] (3) The recombinant plasmid pRBS-P was constructed using the same primers and methods as in Example 2 and Example 3. 22 - cscA , and introduced it into Gluconacetobacter xylinum CGMCC No. 2955 to screen for overexpression cscA Genetic recombinant strains ， Named K13.

[0061] Comparative Example 3 (1) Using plasmid pBla as template, primers PF1 / PR1 were used to amplify the linearized plasmid fragment of pBla; cscA The gene fragment was used as a template and amplified using primers cscA-U / cscA-D. cscA The gene fragment and the linearized plasmid fragment of pBla were cloned and connected using the one-step recombination kit of Novozymes to obtain the recombinant plasmid pBla-P bla - cscA .

[0062] (2) The constructed recombinant plasmid pBla-P bla - cscA Transformed into competent E. coli DH5α, spread on LB solid medium containing 50 μg / mL kanamycin after recovery, and cultured at 37°C to obtain positive transformants. Positive transformants were inoculated into LB liquid medium containing 50 μg / mL kanamycin, cultured at 37°C overnight, and plasmids were extracted for sequencing verification to obtain the recombinant plasmid pBla-P with correct sequencing. bla - cscA , and maintained in Escherichia coli DH5α.

[0063] (3) The recombinant plasmid pBla-P was transformed into bla - cscA Introduced into xylindoleacetobacter CGMCC No. 2955, screened for overexpression cscA Genetic recombinant strains ， Named K1.

[0064] Comparative Example 4 (1) Using the genome of Glucose Acetobacter xylinum CGMCC No. 2955 as a template, the promoter P was amplified using primers P10220-U / P10220-D. 10220 fragment (nucleotide sequence as shown in SEQ ID NO: 6); using plasmid pRBS as a template, primers PPF / PPR were used to amplify the linearized plasmid fragment of pRBS; and the promoter P 10220The linearized plasmid fragment of pRBS was cloned and connected with the one-step recombination kit of Novozyme to obtain a plasmid containing promoter P 10220 The recombinant plasmid pRBS-P 10220 .

[0065] (2) The recombinant plasmid pRBS-P was constructed using the same primers and methods as in Example 2 and Example 3. 10220 - cscA , and introduced it into Gluconacetobacter xylinum CGMCC No. 2955 to screen for overexpression cscA Genetic recombinant strains ， Named K2.

[0066] Comparative Example 5 (1) Using the genome of Glucose Acetobacter xylinum CGMCC No. 2955 as a template, the promoter P was amplified using primers P11970-U / P11970-D. 11970 fragment (nucleotide sequence as shown in SEQ ID NO: 7); using plasmid pRBS as a template, primers PPF / PPR were used to amplify the linearized plasmid fragment of pRBS; then the promoter P 11970 The linearized plasmid fragment of pRBS was cloned and connected with the one-step recombination kit of Novozyme to obtain a plasmid containing promoter P 11970 The recombinant plasmid pRBS-P 11970 .

[0067] (2) The recombinant plasmid pRBS-P was constructed using the same primers and methods as in Example 2 and Example 3. 11970 - cscA , and introduced it into Gluconacetobacter xylinum CGMCC No. 2955 to screen for overexpression cscA Genetic recombinant strains ， Named K3.

[0068] Comparative Example 6 (1) Entrust the synthesis company of Genewise Biotechnology Co., Ltd. to synthesize the base fragment of promoter J23104 according to the nucleotide sequence shown in SEQ ID NO:8; use the plasmid pRBS as a template and use primers PPF / PPR to amplify and obtain the linearized plasmid fragment of pRBS; then clone and connect the promoter J23104 fragment and the linearized plasmid fragment of pRBS through the one-step recombination kit of Novozymes to obtain the recombinant plasmid pRBS-P carrying promoter J23104. J23104 .

[0069] (2) The recombinant plasmid pRBS-P was constructed using the same primers and methods as in Example 2 and Example 3. J23104 - cscA, and introduced it into Gluconacetobacter xylinum CGMCC No. 2955 to screen for overexpression cscA Genetic recombinant strains ， Named K10.

[0070] Verification Example 1 Single colonies were picked from the recombinant strains constructed in Example 3 and Comparative Examples 1-6, respectively, and inoculated into 10 mL of seed culture medium containing 50 μg / mL kanamycin. After culturing at 30°C for 24 h, a pre-cultured bacterial solution was obtained. 1 mL of the pre-cultured bacterial solution was transferred to 30 mL of seed culture medium containing 50 μg / mL kanamycin, and cultured at 30°C for 24 h to obtain a seed solution. The seed solution was transferred to a sucrose fermentation medium containing 50 μg / mL kanamycin, and the initial OD of the fermentation medium was controlled. 600 The concentration of 0.02 was 0.02, and the culture was kept at 30℃ for 7 days. After the fermentation, the bacterial cellulose production of each group was detected, and the results are shown in Table 2. Three parallel samples were set in each group, and the wild type xyloglucanacetobacter CGMCC No. 2955 was used as the control group. During the fermentation process, kanamycin was not added to the culture medium, and the other culture conditions were consistent with the experimental group.

[0071] Bacterial cellulose yield detection: The bacterial cellulose membrane in the final fermentation broth of each group was taken out and repeatedly washed with distilled water to remove the fermentation broth and bacterial residues on the surface of the bacterial cellulose membrane; the bacterial cellulose membrane was soaked in 0.1 M NaOH solution and repeatedly washed until the membrane was milky white to remove impurities and residual protein; it was soaked and repeatedly washed with distilled water until the bacterial cellulose membrane was neutral; the treated bacterial cellulose membrane was placed in a rapid paper sheet forming machine and pressed dry to remove all its moisture, and its mass was weighed. The bacterial cellulose yield is expressed as the mass of bacterial cellulose per liter of fermentation broth (g / L), which is calculated by dividing the mass of dried bacterial cellulose by the volume of fermentation broth.

[0072] Table 2 The yield of bacterial cellulose produced by wild-type and recombinant strains

[0073] The results of the experiment showed that the bacterial cellulose production of all recombinant strains was significantly higher than that of the wild-type strain, indicating that the introduction of cscA Gene, can make the strain produce high bacterial cellulose (BC) using sucrose as substrate. cscA The expression regulation of genes showed significant differences, and this regulatory difference indirectly affected the synthesis efficiency of bacterial cellulose.

[0074] Through comparative studies of different promoters, it was found that the endogenous promoter P from Gluconacetobacter xylinus 10220 , P11970 and P 06915 The mutant (P 20 , P 22 , P 33 ), the BC synthesis efficiency driven by them was better than that of the exogenous promoter P bla and P J23104 , indicating that the host endogenous promoter has significant adaptability advantages in gene expression regulation. 33 for cscA The gene expression regulation ability was optimal, and the BC production driven by it reached 5.60±0.03 g / L, which was about 217% higher than that of the wild-type strain. The standard deviation of BC production was relatively small, indicating that the promoter P 33 In regulation cscA It has high efficiency and stability in gene expression, showing its potential as an efficient regulatory element. 33 It can be used as a regulatory element for the expression of target genes by Gluconacetobacter xylinus, and can be used to enhance the expression of target genes and produce target compounds.

[0075] Verification Example 2 The recombinant strain K11 provided in Example 3 was tested for producing bacterial cellulose by fermentation using sucrose, sugarcane molasses and beet molasses as substrates.

[0076] A single colony of the recombinant strain K11 was picked and inoculated into 10 mL of seed culture medium containing 50 μg / mL kanamycin. After culturing at 30°C for 24 h, a pre-cultured bacterial solution was obtained. 1 mL of the pre-cultured bacterial solution was transferred to 30 mL of seed culture medium containing 50 μg / mL kanamycin and cultured at 30°C for 24 h to obtain a seed solution. The seed solution was transferred to sucrose fermentation medium, sugarcane molasses fermentation medium, and beet molasses fermentation medium containing 50 μg / mL kanamycin, respectively, to control the initial OD value of the fermentation medium. 600 The concentration of 0.02 was 0.02, and the culture was kept at 30℃ for 7 days. After the fermentation, the bacterial cellulose production of each group was detected, and the results are shown in Table 3. Three parallel samples were set in each group, and the wild type xyloglucanacetobacter CGMCC No. 2955 was used as the control group. During the fermentation process, kanamycin was not added to the culture medium, and the other culture conditions were consistent with those of the experimental group.

[0077] Table 3 Comparison of bacterial cellulose production by strains under different substrate conditions

[0078] The results show that the recombinant strain K11 has the highest bacterial cellulose production when using beet molasses as a substrate. Therefore, in actual production, beet molasses can be considered as a substrate.

[0079] In summary, the promoter P provided by the present invention 33 It is a promoter with high transcriptional activity. Compared with other promoters, promoter P 33 It exhibits higher promoter activity and can be used as a new gene expression regulation tool to initiate the expression of target genes in host cells.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A promoter, characterized in that The promoter P 33 The nucleotide sequence is shown in SEQ ID NO:

2.

2. The promoter P of claim 1 33 Application in enhancing target gene expression and producing target compounds.

3. An expression cassette, characterized in that The expression cassette comprises a target gene and the promoter as claimed in claim 1; the expression cassette further comprises one or more of a terminator, an RBS sequence, an enhancer and a post-transcriptional regulatory element; the promoter is operably connected to the target gene and mediates the transcription of the target gene.

4. The expression cassette according to claim 3, characterized in that The target gene is a gene encoding sucrose hydrolase cscA.

5. A recombinant vector, characterized in that: The recombinant vector comprises the promoter according to claim 1 or the expression cassette according to claim 3 or 4.

6. A recombinant strain, characterized in that: It comprises the promoter according to claim 1, the expression cassette according to any one of claims 3 or 4, or the recombinant vector according to claim 5.

7. The recombinant strain according to claim 6, characterized in that The starting strain of the recombinant strain is Acetobacter, and the recombinant strain contains the expression cassette according to claim 4.

8. The recombinant strain according to claim 7, characterized in that The method for constructing the recombinant strain comprises introducing a plasmid containing the expression cassette into the starting strain; and / or The starting strain is Gluconobacter xylinacetobacter ( Komagataeibacter xylinus ); Preferably, the starting strain is Gluconobacter xylinum CGMCC No. 2955.

9. Use of the recombinant strain according to claim 7 or 8 in the preparation of bacterial cellulose.

10. A method for producing bacterial cellulose, characterized in that: The recombinant strain according to claim 7 or 8 is used for fermentation with sucrose or molasses containing sucrose as a substrate.

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