Promoter of gluconacetobacter xylinum source sequence and application of promoter in efficiently utilizing mannitol to synthesize bacterial cellulose

Abstract

The invention belongs to the technical field of gene engineering, and discloses a promoter and application thereof in enhancing target gene expression and promoting mannitol metabolism to synthesize bacterial cellulose. The nucleotide sequence of the promoter P00300 is as shown in SEQ ID NO: 2. The promoter is obtained by site-directed mutagenesis of a wild type promoter P06915 of gluconacetobacter xylinum and screening. The promoter provided by the invention can be used for enhancing the expression of a target gene and producing a target compound. For example, the expression level of fk, mdh1 and mdh2 three-gene combination is remarkably improved, the recombinant strain can efficiently synthesize BC by using mannitol, and the yield reaches 6.1 g / L, is about 2.87 times of that of a wild strain, and is 1.27 times of that of a bla promoter in pBla. The result lays a foundation for further understanding mannitol metabolism of gluconacetobacter xylinum and for high-value conversion and utilization of marine carbon resource mannitol and BC synthesis.

Description

Technical Field

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

[0002] Bacterial cellulose, a nanoscale biopolymer synthesized by bacteria, boasts high purity, high crystallinity, excellent mechanical properties, exceptional water absorption and moisture retention, and outstanding biocompatibility. It is widely used in biomedicine, the food industry, environmental protection, electronics, and cosmetics. Bacterial cellulose-producing strains include Agrobacterium, Rhizobia, Pseudomonas, and Acetobacter. Among them, Komagataeibacter xylinus is the most widely studied bacterial cellulose-producing strain due to its high bacterial cellulose production.

[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 enzyme expression, 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 Gluconacetobacter xylanis, there are currently few developments and insufficient knowledge of promoters derived from Gluconacetobacter xylanis. This has led to greater reliance on exogenous promoters in research and applications, and exogenous promoters may not be able to fully adapt to the physiological characteristics of Gluconacetobacter xylanis, 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 after screening, a mutant promoter P with significantly enhanced activity was obtained. 00300 , 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 objectives, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides a promoter, wherein the promoter P 00300 The nucleotide sequence is shown in SEQ ID NO: 2.

[0007] The promoter P provided by the present invention 00300 The strong promoter P from Gluconacetobacter xylophilus is 06915The nucleotide sequence (shown in SEQ ID NO: 1) was modified to obtain the nucleotide sequence. 06915 The nucleotide segment from position 194 to position 437 of the sequence is used as the basic framework, which is the P 06915 The core sequence has high strength. Then, multiple site-directed mutagenesis was performed on the truncated fragment: the 37th base was mutated from C to T, the 39th base was mutated from G to T, and the 43-44th bases were mutated from GA to CT. After the above transformation, the promoter P was obtained. 00300 , whose nucleotide sequence is shown in SEQ ID NO:2.

[0008] In the series of bacterial cellulose-producing recombinant strains constructed in the present invention, the promoter P 00300 The recombinant strain driving the expression of the multi-gene combination of mannitol metabolism-related genes fk, mdh1 and mdh2 showed a significantly higher bacterial cellulose production than the recombinant strains driven by other promoters and the wild-type strain, indicating that the promoter P 00300 Can efficiently drive the expression of target genes.

[0009] In a second aspect, the present invention also provides the above-mentioned promoter P 00300 Application in enhancing target gene expression and producing target compounds.

[0010] The promoter P of the present invention 00300 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 00300 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 00300 Driven by the promoter P, the transcription and translation efficiency of the target gene is significantly improved, thereby achieving efficient expression. 00300 Used to regulate the expression of enzyme genes in key metabolic pathways, it can enhance metabolic flux and thus increase the production of target compounds.

[0011] Preferably, the target gene is a gene related to bacterial cellulose synthesis, and the target compound is bacterial cellulose.

[0012] 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.

[0013] 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.

[0014] The "target gene" refers to a gene encoding a target protein in a microorganism, including but not limited to genes encoding enzymes related to the biosynthesis of the target compound, genes encoding enzymes related to reducing power, genes encoding enzymes related to glycolysis or the TCA cycle, or genes encoding enzymes related to the release of the target compound, etc.

[0015] The term "operably linked" refers to the promoter P of the present invention. 00300 Functionally linked to the target gene to initiate and mediate transcription of the target gene and control the transcriptional activity of the operon gene. The operably linked manner can be any manner described by those skilled in the art.

[0016] Preferably, the target gene is a gene related to mannitol metabolism, such as fk, mdh1 and mdh2 genes.

[0017] More preferably, the target gene is a gene related to mannitol metabolism, such as the fk, mdh1, and mdh2 genes, whose nucleotide sequences are shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively. The encoding genes are derived from Gluconacetobacter xylinum, and overexpression of the genes can improve mannitol metabolism for bacterial use, providing energy for bacterial growth and metabolism.

[0018] In a fourth aspect, the present invention further provides a recombinant vector comprising the above-mentioned promoter or the above-mentioned expression cassette.

[0019] The term "vector" as used herein refers to a DNA construct comprising a DNA sequence operably linked to appropriate control sequences (e.g., 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.

[0020] Preferably, the recombinant vector further comprises one or more of a replication origin, an enhancer, a selection marker, a ribosome binding site and polyA.

[0021] More preferably, the screening marker is one or more of an antibiotic screening marker, a fluorescent protein gene and an anti-screening gene.

[0022] In a fifth aspect, the present invention also provides a recombinant strain comprising the above-mentioned promoter, expression cassette or recombinant vector.

[0023] Preferably, the starting strain of the recombinant strain is Acetobacter, and the recombinant strain contains mannitol metabolism-related genes fk, mdh1 and mdh2 and the above-mentioned promoter P 00300 expression cassette.

[0024] The "starting strain" described in the present invention refers to the original strain used to construct the recombinant strain.

[0025] Acetobacter mainly uses glucose as a substrate to synthesize bacterial cellulose. Currently, metabolic engineering modifications of Acetobacter to increase its bacterial cellulose production include strengthening the expression of enzymes related to the bacterial cellulose synthesis pathway, weakening the expression of enzymes related to the competitive pathway, etc. However, the production of bacterial cellulose still uses glucose as a substrate, which poses a problem of competing with the people for food.

[0026] The present invention is to promote the 00300 The expression cassettes of mannitol metabolism-related genes fk, mdh1 and mdh2 driven by the gene were introduced into Acetobacter, causing the genes fk, mdh1 and mdh2 to be overexpressed in Acetobacter. By overexpressing these three genes, mannitol was efficiently metabolized. This transformation enabled Acetobacter to efficiently utilize mannitol to produce bacterial cellulose, broadening the substrate source for bacterial cellulose production.

[0027] More preferably, the method for constructing the recombinant strain comprises introducing a plasmid containing the expression cassette into the starting strain.

[0028] More preferably, the promoter P in the expression cassette 00300 It is operably connected to the mannitol metabolism-related gene to improve the mannitol metabolism efficiency.

[0029] More preferably, the starting strain is Komagataeibacter xylinus.

[0030] Gluconacetobacter xylonitrile is an important industrial production strain, widely studied and applied for its ability to efficiently synthesize bacterial cellulose. The strain's cultivation conditions are relatively simple, and as a food-safe strain, it has been safely used in large-scale industrial production of bacterial cellulose.

[0031] More preferably, the starting strain is Gluconacetobacter xyloniae CGMCC No.2955.

[0032] In the present invention, Gluconobacter xylonic acid acetobacter CGMCC No.2955 was used as the starting strain, and the promoter P 00300A recombinant strain of Glucose Acetobacter xylonitrile, driven by a plasmid expressing a gene expression cassette for mannitol metabolism, was constructed that can efficiently produce bacterial cellulose using mannitol. After seven days of cultivation using mannitol as a substrate, the recombinant strain achieved a bacterial cellulose yield of 6.13±0.18g / L, significantly higher than that of the wild-type strain and other recombinant strains.

[0033] In a sixth aspect, the present invention also provides the use of the above-mentioned recombinant strain in the preparation of bacterial cellulose.

[0034] In a seventh aspect, the present invention also provides a method for producing bacterial cellulose, using the above-mentioned recombinant strain to ferment with mannitol as a substrate.

[0035] Preferably, the substrate is mannitol. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. 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 any creative work.

[0037] Figure 1 is the recombinant plasmid P in Example 2 00300 -Schematic diagram of the structure of mdh1-2:fk.

[0038] Figure 2 The expression intensity of the RFP gene connected to different promoters.

[0039] Figure 3 is the bacterial cellulose yield of different xylindoleacetic acid bacteria. DETAILED DESCRIPTION

[0040] 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 embodiments described 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 making creative efforts are within the scope of protection of the present invention.

[0041] The xylinobacter CGMCC No. 2955 used in the embodiments of the present invention was screened by the Biochemical Engineering Laboratory, School of Bioengineering, Tianjin University of Science and Technology. The strain has been disclosed 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)).

[0042] 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 (The nucleotide sequence is shown in SEQ ID NO: 6). The plasmid structure and its construction method have been disclosed 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 embodiment of the present invention is a pBla plasmid with the promoter P removed. bla Obtained.

[0043] The culture medium used in the embodiments of the present invention is:

[0044] 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.

[0045] 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.

[0046] 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.

[0047] Mannitol fermentation medium components: mannitol 25 g / L; yeast powder 7.5 g / L; peptone 10 g / L; Na2HPO4 10 g / L; initial pH 6.0.

[0048] Unless 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, unless otherwise specified, are all conventional methods. 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.

[0049] Table 1 Primer sequences used in the examples of the present invention

[0050]

[0051] Example 1

[0052] In order to enhance the expression of the promoter P from Acetobacter xylophilus 06915 The transcription level of promoter P is obtained. 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 37th base was mutated from C to T, the 39th base was mutated from G to T, and the 43-44th bases were mutated from GA to CT. After the above transformation, the promoter mutant P was obtained. 00300 , whose nucleotide sequence is shown in SEQ ID NO:2.

[0053] Example 2

[0054] This embodiment provides a promoter P 00300 The invention relates to a recombinant plasmid of an expression cassette driven by the gene and a method for constructing the same.

[0055] (1) Using the genome of xylinacetobacter CGMCC No. 2955 as a template, the promoter P was amplified using primers P00300-U / P00300-D.00300 fragment (nucleotide sequence as shown in SEQ ID NO: 2); using plasmid pBla as template, primers KXM-U / KXM-D were used to amplify the linearized plasmid fragment of pBla; and the promoter P 00300 The fragment was cloned and connected with the linearized plasmid fragment of pBla by using the one-step recombination kit of Novozymes to obtain a plasmid containing promoter P 00300 The recombinant plasmid pBla-P 00300 .

[0056] (2) Using the mdh1, mdh2, and fk gene fragments synthesized by Jinweizhi Biotechnology Co., Ltd. as templates, the fragments were amplified; the recombinant plasmid pBla-P 00300 As a template, pBla-P 00300 Linearized plasmid fragments; mdh1, mdh2, fk gene fragments and pBla-P 00300 The linearized plasmid fragment was cloned and connected by Novozymes' one-step recombination kit to obtain the recombinant plasmid pBla-P 00300 -mdh1-mdh2-fk, the plasmid structure diagram is as follows Figure 1 shown.

[0057] (3) The recombinant plasmid pBla-P constructed above was 00300 -mdh1-mdh2-fk was transformed into competent E. coli DH5α, and after recovery, it was spread on LB solid medium containing 50μg / mL kanamycin and cultured at 37℃ to obtain positive transformants. The positive transformants were inoculated into LB liquid medium containing 50μg / mL kanamycin, cultured at 37℃ overnight, and the plasmid was extracted for sequencing verification to obtain the recombinant plasmid pBla-P with correct sequencing. 00300 -mdh1-mdh2-fk and maintained in Escherichia coli DH5α.

[0058] Example 3

[0059] Construction of a bacterial strain that overexpresses mdh1, mdh2, and fk gene sequences and can efficiently utilize mannitol and produce high bacterial cellulose.

[0060] The recombinant plasmid pBla-P provided in Example 2 was 00300 -mdh1-mdh2-fk was electroporated into competent cells of Gluconic Acetobacter xylinus CGMCC No.2955. After recovery, the transformants were plated on HS solid medium containing 50 μg / mL kanamycin and cultured at 30°C for 3-4 days. After the monoclonal colonies grew, the single colonies were identified by colony PCR. The recombinant strain that overexpressed the mdh1-2-fk gene was obtained and named K. xylinus-P 00300 -mdh1-2:fk.

[0061] Example 4

[0062] Synthesis of bacterial cellulose by gluconic acid acetobacter xylinum that efficiently metabolizes mannitol.

[0063] In order to test the effect of MDH1, MDH2 and FK gene sequences on bacterial cellulose production, K. xylinus-P 00300 -mdh1-2:fk strain and wild bacteria and other modified strains (other promoters simultaneously overexpressed the three gene fragments of the modified bacteria, named K.xylinus-P bla -mdh1-2:fk, K.xylinus-P 01095 -mdh1-2:fk, K.xylinus-P 06915 -mdh1-2:fk; At the same time, using this promoter, three transformed strains overexpressing mdh1, mdh2, and fk were named K.xylinus-P 00300 -mdh1, K.xylinus-P 00300 -mdh2, K.xylinus-P 00300 :fk; and the modified strains overexpressing mdh1 and mdh2 at the same time, and the modified strains overexpressing mdh1 and fk at the same time were named K.xylinus-P 00300 -mdh1-2, K.xylinus-P 00300Fermentation tests were performed using a 1:100-mdh1:fk (mdhl-mdh1:fk) culture medium. The seed culture medium consisted of 25 g / L glucose, 7.5 g / L yeast extract, 10 g / L peptone, and 10 g / L Na2HPO4; the initial pH was 6.0. The fermentation culture medium consisted of 25 g / L mannitol, 7.5 g / L yeast extract, 10 g / L peptone, and 10 g / L Na2HPO4; the initial pH was 6.0. After thawing, 20 μL of the culture suspension was transferred to HS liquid culture medium and shaken. The suspension was then transferred to a centrifuge tube and centrifuged to collect the pellet. The pellet was washed twice with sterile saline and resuspended in 3 mL of sterile saline, and the cell density was measured. An appropriate amount of the resuspension was added to 75 mL of fermentation medium, with an initial OD600 of 0.02. The culture was then incubated at 30°C until the stationary phase. Each strain was cultured in parallel for two times. After the fermentation was completed, the bacterial cellulose membrane was treated and the yield was detected. The specific treatment method was as follows: the bacterial cellulose was cultured statically until the stable phase, the fermentation was terminated, the bacterial cellulose was taken out and repeatedly washed with distilled water to remove the fermentation liquid and bacterial residues on the surface of the cellulose membrane. It was then soaked in a 0.1M NaOH solution and repeatedly washed until the membrane turned milky white. It was then soaked in distilled water and repeatedly washed until neutral. Finally, the bacterial cellulose was placed in an 80°C constant temperature drying oven and dried to constant weight. The bacterial cellulose yield was expressed as: g / L (cellulose / culture medium). The results are shown in Table 2. The bacterial cellulose production in mannitol of the strain expressing the gene related to metabolism of mannitol with the sequence of the promoter function derived from Gluconobacter xylinum was significantly improved compared to the wild-type strain.

[0064] Table 2 The yield of bacterial cellulose produced by fermentation of wild type and recombinant strains

[0065] strain Bacterial cellulose yield (g / L) Wild type (WT) 2.08±0.11 <![CDATA[K.xylinus-P 00300 -mdh1-2:fk(K1)]]> 6.13±0.18 <![CDATA[K.xylinus-P bla -mdh1-2:fk(K2)]]> 4.79±0.06 <![CDATA[K.xylinus-P 01095 -mdh1-2:fk(K3)]]> 4.65±0.07 <![CDATA[K.xylinus-P 06915 -mdh1-2:fk(K4)]]> 4.58±0.10 <![CDATA[K.xylinus-P 00300 -mdh1(K5)]]> 4.13±0.18 <![CDATA[K.xylinus-P 00300 -mdh2(K6)]]> 2.53±0.18 <![CDATA[K.xylinus-P 00300 ::fk(K7)]]> 4.31±0.04 <![CDATA[K.xylinus-P 00300 -mdh1-2(K8)]]> 3.41±0.27 <![CDATA[K.xylinus-P 00300 -mdh1:fk(K9)]]> 4.63±0.18

[0066] Based on the above examples 1-4, it can be found that the present invention utilizes a strong promoter P 00300 The invention also provides sequences of genes that overexpress mannitol metabolism and their application in promoting bacterial cellulose synthesis. The invention provides sequences and functional identification of the mdh1, mdh2, and fk genes, methods for constructing each component and transferring it into engineered bacteria, and a working approach and specific applications of an expression system based on gluconic acid acetobacter xylinum. Specifically,

[0067] 1. The present invention specifically utilizes a strong promoter P 00300 And overexpressing mdh1 gene, mdh2 gene and fk gene in xylinobacter and expression vector pBla to construct an excellent and efficient expression system.

[0068] 2. The present invention utilizes strong promoter P 00300As well as overexpression of mdh1, mdh2 and fk genes in gluconic acid acetobacter xylariae and their application in promoting bacterial cellulose synthesis, the synthesis of bacterial cellulose is greatly promoted.

[0069] 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 scope of protection of the present invention.

Claims

1. A promoter, characterized in that The promoter P 00300 The nucleotide sequence is shown in SEQ ID NO:

2.

2. The promoter P according to claim 1 00300 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 according to 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 linked to the target gene and mediates transcription of the target gene.

4. The expression cassette according to claim 3, wherein The target gene is a gene related to mannitol metabolism.

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 includes introducing a plasmid containing the expression cassette into a starting strain, and / or the starting strain is Komagataeibacter xylinus; preferably, the starting strain is Komagataeibacter xylinus 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 mannitol as a substrate.

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