CN114703185A - Promoter function identification of acetobacter xylinum source sequence and application of promoter function identification in promotion of bacterial cellulose synthesis - Google Patents
Promoter function identification of acetobacter xylinum source sequence and application of promoter function identification in promotion of bacterial cellulose synthesis Download PDFInfo
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Abstract
The invention discloses a sequence with a promoter function derived from acetobacter xylinum and application thereof in promoting bacterial cellulose synthesis. The sequence has stronger promoter activity than a pBla plasmid promoter. Therefore, the expression of a target gene can be enhanced, for example, the target gene is operably connected with a phosphofructokinase gene, the expression strength of phosphofructokinase pfkA is enhanced, and the bacterial cellulose production efficiency of the strain is improved.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to identification of functions of a promoter of an acetobacter xylinum source sequence and application of the promoter in promoting bacterial cellulose synthesis.
Background
Bacterial cellulose is a nano-scale biopolymer synthesized by bacteria. It has high crystallinity, high polymerization degree, high tensile strength, high water holding capacity, biocompatibility, degradability and other excellent quality, and may be used widely in biomedicine, food, textile, functional film, cosmetics, audio equipment and other industries. The cellulose-producing bacterial strains include Agrobacterium, Rhizobium, Pseudomonas, Acetobacter, and the like. Among them, gluconacetobacter xylinus is the most widely studied bacterial cellulose-producing strain due to its ability to produce bacterial cellulose at high yield. With the continuous development of biotechnology, reports of metabolic engineering of acetobacter to improve the yield of bacterial cellulose are increasing, and the engineering includes enhancing the expression of enzymes related to the synthetic pathway of bacterial cellulose, weakening the expression of enzymes related to competitive pathways, and the like.
The acetobacter xylinum is an important production strain of industrial products, can efficiently synthesize bacterial cellulose, has simple culture conditions, is a food-safe strain, and is safely used for large-scale industrial production of the bacterial cellulose. However, the lack of efficient expression elements limits their value. At present, the promoter from the acetobacter xylinum is developed less, the cognition of the promoter is insufficient, and more exogenous promoters are utilized, including lac promoter and J promoter23104Promoter, PblaPromoters, and the like.
Due to complexity and diversity of a fermentation process, the existing constitutive promoter and inducible promoter have a plurality of problems in application, such as higher cost of an inducer, incapability of opening the constitutive promoter under specific conditions and the like, but the promoter from the gluconacetobacter xylinus does not have the problems, and the promoter can be well used for genetic engineering transformation of a strain and accurate opening of gene expression. At present, the function of the promoter of the sequence derived from the gluconacetobacter xylinus is not clear. The acetobacter xylinum is used as an excellent bacterial cellulose production strain, and has great significance for developing expression elements from the strain and expanding a promoter library of the strain.
Phosphofructokinase pfkA is a key enzyme of the Embden Meyerhof Parnas (EMP) pathway, and the pfkA gene is absent in Acetobacter xylinum, and encodes phosphofructokinase, and the deletion of the pfkA gene is a very well-known genotype characteristic of the Komagataeibacter species. Research shows that the expression of pfkA gene generates more ATP in the complete glycolysis process, and provides energy for the growth of thalli and the production of bacterial cellulose. The pfkA gene expression Komagataeibacter mutant strain is used as a basic production strain, so that the yield of bacterial cellulose can be further improved.
The invention content is as follows:
in view of the above-mentioned deficiencies and needs of the prior art, the present inventors have studied and identified a sequence derived from Acetobacter xylinum, and have completed the present invention.
In a first aspect, the invention identifies a gluconacetobacter xylinus source sequence which has the function of a strong promoter, and the nucleotide sequence of the sequence is shown as SEQ ID NO. 1.
The specific sequence of SEQ ID NO.1 is:
5’-TTCCCTGCCCTGAGGGAGGGGCTGCAATCTTTAGTAATGTTTCAGTTCTTGAGATTGTGGTCCCCTTGCGGCAAGTAGATGATGCTTTCCATAAATTTAGAAATTTTAATTCGAGACAACCCTCCTGCGGCATGTTACAGCATGGGGGTCGGTTCGGGTCTGGCGATTCGCCCCCTGATAGACATATTGGGATGAGAAGTCCC-3’
furthermore, the sequence is derived from the genome of acetobacter xylinum.
Further, the acetobacter xylinum is Komagataeibacter xylinus CGMCC 2955.
Furthermore, the above sequence has complete promoter function.
Further, the above sequence has a strong promoter activity.
Furthermore, the expression intensity peak value of the strong promoter of the sequence is not lower than that of the pBla plasmid promoter Pbla255 times higher than the first.
An expression vector comprises the sequence with the function of the promoter from the gluconacetobacter xylinus.
Preferably, the expression vector constructs a sequence based on the function of the promoter from the gluconacetobacter xylinus on the expression vector, so that the replication and expression in a microorganism are facilitated.
Preferably, the expression vector is pRBS.
Further, an expression vector pRBS backbone plasmid was constructed based on a pBla vector from which a promoter, a Ribosome Binding Site (RBS) and a terminator were removed.
A genetically engineered bacterium having a sequence of the above-mentioned acetobacter xylinum-derived promoter function or the above-mentioned expression vector.
Furthermore, the gene engineering bacteria realize the function of opening the expression of the red fluorescent protein by utilizing the sequence of the acetobacter xylinum source promoter function.
Preferably, the red fluorescent protein is RFP.
Furthermore, the genetically engineered bacteria are derived from acetobacter xylinum.
In a second aspect, the invention provides an expression cassette and a recombinant vector comprising the sequence of the promoter function derived from the acetobacter xylinum.
The expression cassette has the meaning commonly understood by those skilled in the art, i.e., contains a promoter, a gene of interest, and elements capable of expressing the gene of interest.
The term "vector" refers to a DNA construct containing a DNA sequence operably linked to suitable control sequences for expression of a gene of interest in a suitable host. The vector used in the present invention is not particularly limited, and may be any vector known in the art as long as it can replicate in a host. That is, the vector includes, but is not limited to, a plasmid, a phage, such as the pRBS plasmid used in the embodiments of the present invention. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or in some cases, integrate into the genome itself.
In a third aspect, the present invention provides a recombinant host cell comprising a mutant of the sequence functional with the promoter derived from acetobacter xylinum.
Recombinant host cells are specifically realized, for example, by transformation. "transformation" herein has the meaning generally understood by those skilled in the art, i.e., the process of introducing exogenous DNA into a host. The method of transformation includes any method of introducing a nucleic acid into a cell including, but not limited to, electroporation, transduction, calcium phosphate (CaPO)4) Precipitation method, calcium chloride (CaCl)2) Precipitation, microinjection, polyethylene glycol (PEG), DEAE-dextran, cationic liposome, and lithium acetate-DMSO.
The "host cell" according to the invention has the meaning generally understood by a person skilled in the art, i.e.a cell into which a sequence according to the invention having promoter activity can be introduced, is subsequently referred to as recombinant host cell. In other words, the present invention can utilize any host cell as long as the cell contains the sequence having promoter activity of the present invention and is operably linked to a gene to mediate transcription of the gene. The host cell of the present invention may be a prokaryotic cell or a eukaryotic cell, preferably a bacillus aceticus, including but not limited to komagataibacterium xylinus CGMCC2955, and bacterial cellulose-producing mutants or strains prepared therefrom.
In the present invention, the culture of the host cell may be performed according to a conventional method in the art, including, but not limited to, a well plate culture, a shake flask culture, a stationary culture, and the like, and various culture conditions such as temperature, time, pH of a medium, and the like may be appropriately adjusted according to actual circumstances.
In a fourth aspect, the present invention provides a method for enhancing the expression of a gene of interest, said method comprising operably linking a sequence that functions as a promoter derived from acetobacter xylinum to a gene of interest, preferably to enhance the expression of genes associated with bacterial metabolism and bacterial cellulose synthesis.
The term "operably linked" in the present invention means that the sequence of the strong promoter function derived from Acetobacter xylinum of the present invention is functionally linked to the coding gene to initiate and mediate the transcription of the gene, indicating that the sequence of the strong promoter function derived from Acetobacter xylinum of the present invention is operably linked to the coding gene to control the transcriptional activity of the operon gene. The means of operably linking may be any means described by one skilled in the art. The method for enhancing the expression of a target gene of the present invention is carried out by a method generally used by those skilled in the art, including the method using a sequence having a strong promoter function derived from Acetobacter xylinum of the present invention.
In a fifth aspect, the present invention provides a method for producing bacterial cellulose with enhanced bacterial energy metabolism, the method comprising culturing a host cell comprising a sequence having a strong promoter function derived from acetobacter xylinum operably linked to a gene associated with enhanced bacterial energy metabolism, and collecting the bacterial cellulose produced. The genes related to enhancing the energy metabolism of the thallus include, but are not limited to phosphofructokinase pfkA.
In one embodiment, the acetobacter xylinum is modified in such a way that a phosphofructokinase coding gene in the acetobacter xylinum is operably linked to a sequence having a strong promoter function derived from the acetobacter xylinum as described above and introduced into the acetobacter xylinum to obtain a bacterial cellulose-producing strain.
In the production method for improving the yield of the bacterial cellulose by enhancing the energy metabolism of the thalli, a method commonly used by a person skilled in the art is adopted on the basis of utilizing the sequence of the function of the strong promoter derived from the acetobacter xylinum, and the method also comprises a step of recovering a target product from a culture solution. Methods for recovering a product of interest from a cell or culture medium are well known in the art and include, but are not limited to: filtering, soaking with NaOH, washing, drying, tabletting, freeze-drying and the like.
The beneficial technical effects of the invention are as follows:
the invention overcomes the problems that part of constitutive promoters can not be expressed in the existing fermentation process and the like, and provides a sequence with strong promoter function derived from acetobacter xylinum and application thereof in promoting bacterial cellulose synthesis. The invention providesSupply P01095The function of the promoter sequence, the gene sequence, the construction method of each component, the method for transferring engineering bacteria, the work application and the specific application of the acetobacter xylinum expression system; the method specifically comprises the following steps:
1. the invention specifically constructs an excellent high-efficiency expression system through a strong promoter function sequence derived from acetobacter xylinum and an expression vector pRBS.
2. The sequence with strong promoter function derived from the acetobacter xylinum has the characteristic of strong promoter expression strength.
3. The sequence with strong promoter function derived from the acetobacter xylinum has the promoter expression intensity peak value not lower than that of a pBla plasmid promoter Pbla255 times higher.
Compared with the prior art, the invention has the following advantages:
the sequence with strong promoter expression strength is found in the acetobacter xylinum and developed into an expression system, so that the cognition of the regulation and control of the acetobacter xylinum is increased, the cellulose synthesis of bacterial fiber is promoted, and a certain basis is provided for the development of excellent standardized biological elements.
The functional sequence of the strong promoter from the acetobacter xylinum shows higher promoter activity than that of a common constitutive promoter, can be used for expressing a target gene, especially for expressing a gene related to bacterial cellulose production, for example, the functional sequence is operably connected with a pfkA gene, and can enhance the expression strength of phosphofructokinase, so that the bacterial cellulose production efficiency of a recombinant strain is improved, and the application value is higher.
Drawings
FIG. 1 shows pRBS-P01095-mRFP and pRBS-PblaSingle-restriction map of-mRFP, P01095Gene sequence and pRBS-P01095Plasmid map of mRFP plasmid.
FIG. 2 shows a fluorescent screening plate having a sequence functioning as a promoter derived from Acetobacter xylinum.
The present invention will be described in detail with reference to the accompanying drawings and examples.
All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The proper amount of the invention is determined by ordinary technicians in the field according to national technical specifications and actual production conditions. The starting materials described in the present invention are all commercially available unless otherwise specified.
Example 1
The promoter strength of the gluconacetobacter xylinus derived sequence characterizes the construction of the plasmid.
In order to characterize the promoter expression strength of the sequence derived from the acetobacter xylinum, a characterization vector is firstly constructed, and a promoter P is used on the basis of a pRBS plasmid framework01095Expressing the red fluorescent protein gene. First, a backbone plasmid pBRS was constructed for subsequent studies. The skeleton plasmid is formed by adding BamHI enzyme cutting site, RBS, mRFP and TrrnB terminator based on pBla vector with promoter, Ribosome Binding Site (RBS) and terminator removed. According to the published genome sequence and P of the gluconacetobacter xylinus CGMCC295501095Promoter annotation information, design of primer P01095F/R, using CGMCC2955 genome as template, obtaining P by PCR amplification01095A promoter fragment. The vector pRBS is digested by BamHI to obtain a linear vector, and the two fragments are cloned and connected by a one-step recombination kit of Novozam to obtain a pRBS-characterization vector, wherein a plasmid map is shown in figure 1. The primer sequences used above are shown in Table 1.
TABLE 1
Example 2
Construction and strength characterization of promoter screening plasmid of the acetobacter xylinum source sequence.
Red fluorescent protein gene mRFP is used as a reporter gene, and the reference is P on a commonly used pBla plasmidblaPromoter, survey tableTo the element P01095Fluorescent protein expression profile, and fluorescence intensity at 96h was determined.
1. Fluorescent protein expression intensity assay
The method for measuring the expression of the fluorescent protein comprises the following steps: single colonies were picked from the solid medium plate with an inoculating loop and cultured in 5mL of liquid medium at 180r/min at 30 ℃ for 24 h. Transferring 1mL of the fermentation broth to 75mL of seed culture medium containing 4 ‰ (v/v) cellulase (Celluclast 1.5L, Novozymes), culturing at 180r/min and 30 deg.C to OD6000.5 to 0.6. 750 μ L of seed solution was transferred to 75mL of liquid medium (initial pH6.0, initial glucose concentration 10g/L) to obtain the initial OD600Culturing at 30 deg.C for 4 days at 0.05 deg.C, centrifuging 400 μ L of the bacterial solution at 4000r/min for 10min, discarding the supernatant, suspending in 400 μ L of PBS, placing 200 μ L of the sample in a black 96-well plate, placing the 96-well plate in a TECAN microplate reader, setting the excitation wavelength at 590nm and the emission wavelength at 630nm, and measuring the fluorescence intensity value RFU of the strain expressing mRFP protein1Simultaneously measuring the fluorescence intensity value RFU of the strain not expressing mRFP protein0. In addition, OD was measured for fresh medium and bacterial suspension, respectively600Value OD0And OD1。
The fluorescence intensity of mRFP red fluorescent protein is expressed as:
2. the fluorescent protein expression characteristics were (fig. 2): as can be seen from Table 2, P01095The fluorescence intensity of the starter group thallus is far larger than PblaStarting the fluorescence intensity of the subgroup of bacteria, starting from the logarithmic growth phase, P01095The fluorescence intensity of the starter group thallus rapidly increases along with time, and relatively slowly increases in a stationary phase, and is consistent with a thallus growth curve. Final fluorescence intensity and OD600The ratio of (a) reaches 1021. Finally, the present invention identifies the sequence P01095Has strong promoter function of gluconacetobacter xylinus source, which is not less than pBla plasmid promoter Pbla255 times higher than the first.
TABLE 2
| Bacterial strains | Fluorescence intensity/OD600 |
| K.xylinus-Pbla-mRFP | 4±0.21 |
| K.xylinus-P01095-mRFP | 1021±13 |
Example 3
The sequence of the acetobacter xylinum source with strong promoter function is applied to the synthesis of bacterial cellulose in the acetobacter xylinum.
1. And (3) constructing a recombinant vector of a sequence with a strong promoter function derived from the acetobacter xylinum in the acetobacter xylinum.
According to the reported genome sequence of the gluconacetobacter xylinus CGMCC2955, the CGMCC2955 genome is respectively taken as a template, and P is taken as01095-pfkA-F/R、PblaTaking pfkA-F/R as a primer, amplifying target gene pfkA by PCR, recovering fragments, cloning and connecting by a one-step recombination kit of nunoprazan to obtain a recombinant vector pRBS-P01095-pfkA and pRBS-Pbla-pfkA. The primer sequences used above are shown in Table 3.
TABLE 3
2. Construction of bacterial cellulose producing bacteria having a sequence with a strong promoter function derived from acetobacter xylinum.
The recombinant vector pR constructed as described aboveBS-P01095-pfkA and pRBS-Pblathe-pfkA was transformed into E.coli DH 5. alpha. and plated on LB solid medium containing 50. mu.g/mL of kanamycin, followed by culture at 37 ℃ to obtain recombinant transformants. The correct recombinant transformant was inoculated into LB medium containing 50. mu.g/mL kanamycin, cultured overnight, the plasmid was extracted for sequencing, the plasmid with the correct sequencing was electrically transformed into Acetobacter xylinum, plated on HS solid medium containing 25g/L glucose and 50. mu.g/mL kanamycin, and cultured at 30 ℃ to obtain a recombinant transformant. The correct strain is the correct transformant K.xylinus-P verified by colony PCR01095pfkA and K.xylinus-Pbla-pfkA。
3. Evaluation of cellulose productivity of gluconacetobacter xylinus bacteria having a sequence with a strong promoter function derived from gluconacetobacter xylinus.
In order to test the influence of the sequence of the strong promoter function derived from the acetobacter xylinum in the acetobacter xylinum on the bacterial cellulose produced by the strain, on K.xylinus-P01095-pfkA、K.xylinus-Pbla-pfkA and wild bacteria were subjected to fermentation tests, the seed medium composition being: glucose, 10 g/L; yeast powder, 7.5 g/L; peptone, 10 g/L; na (Na)2HPO410 g/L; initial pH 6.0. The fermentation medium comprises the following components: glucose, 25 g/L; yeast powder, 7.5 g/L; peptone, 10 g/L; na (Na)2HPO410 g/L; initial pH 6.0. Inoculating the two recombinant transformants and wild strain obtained in the previous step into HS seed culture medium containing 50 μ g/mL kanamycin and seed culture medium not containing kanamycin, culturing at 30 deg.C for 24 hr, transferring into fermentation culture medium containing 50 μ g/mL kanamycin and fermentation culture medium not containing kanamycin, respectively, and starting OD600Controlling the temperature to be 0.05, and standing, fermenting and culturing at 30 ℃ to a stationary phase. Each strain is parallel to 3 strains, and after fermentation is finished, the bacterial cellulose membrane is processed and the yield is detected, wherein the specific processing mode is as follows: and (3) standing and culturing the bacterial cellulose to a stationary phase, stopping fermentation, taking out the bacterial cellulose, and repeatedly cleaning the bacterial cellulose by using distilled water to remove fermentation liquor and thallus residues on the surface of the cellulose membrane. Then the membrane is soaked in 0.1M NaOH solution and repeatedly washed until the membrane is milky white. Soaking in distilled water and reactingAnd then washing to be neutral. And finally, placing the bacterial cellulose in a constant-temperature drying box at 80 ℃ for drying until the weight is constant. The bacterial cellulose yield is expressed as: g/L (cellulose/medium). As a result, as shown in Table 4, the strains having the sequences functioning as promoters derived from Acetobacter xylinum were found to be PblaThe bacterial cellulose yield of the starter group strain and the wild strain is obviously improved.
TABLE 4
| Strain of bacillus | Bacterial cellulose yield (g/L) | Conversion of substrate% |
| Wild type | 3.12±0.13 | 12.48±0.52 |
| K.xylinus-Pbla-pfkA | 4.35±0.16 | 17.4±0.64 |
| K.xylinus-P01095-pfkA | 5.13±0.06 | 20.52±0.24 |
By combining the above examples 1-3, it can be found that the identification of the function of the promoter derived from the gluconacetobacter xylinus and the application thereof in promoting the synthesis of bacterial cellulose can be applied to the expression of different target genes in the gluconacetobacter xylinus and the production of bacterial cellulose. Book (I)The invention provides P01095Promoter gene sequence and function identification, construction method of each component, method for transferring engineering bacteria, working way and specific application based on an acetobacter xylinum expression system; the method specifically comprises the following steps:
1. the invention specifically constructs an excellent high-efficiency expression system through the identification of the function of the acetobacter xylinum source sequence promoter and the expression vector pRBS.
2. The identification of the promoter function of the acetobacter xylinum source sequence has a strong promoter function.
3. The identification of the function of the acetobacter xylinum source sequence promoter and the application of the promoter in promoting the synthesis of bacterial cellulose have the characteristic of opening the mass expression of target genes, and greatly promote the synthesis of the bacterial cellulose.
The above are only preferred embodiments of the present invention, and the scope of the present invention should not be limited thereby, and all the simple equivalent changes and modifications made by the claims and the summary of the invention should be included in the scope of the present invention.
Claims (11)
1. The identification of the promoter function of the gluconacetobacter xylinus-derived sequence and the application of the gluconacetobacter xylinus-derived sequence in promoting the synthesis of bacterial cellulose are characterized in that the gluconacetobacter xylinus-derived sequence has the promoter function.
2. The identification of the function of the promoter of the sequence derived from the acetobacter xylinum and the application of the promoter in promoting the synthesis of bacterial cellulose are characterized in that the promoter is named as P from 232298 th site to 232501 th site of a Komagataeibacter xylinus CGMCC2955 genome01095。
3. The identification of the function of the promoter of the sequence derived from acetobacter xylinum as claimed in claim 1 and the application thereof in promoting the synthesis of bacterial cellulose, wherein the promoter has strong promoter expression strength. Compared with wild type, the method can promote the yield of bacterial cellulose to be improved by 1.64 times, and the yield reaches 5.13 g/L.
4. The identification of the function of the promoter of the sequence derived from acetobacter xylinum and the application of the promoter in promoting the synthesis of bacterial cellulose as claimed in claim 1, wherein the nucleotide sequence of the sequence is as follows: TTCCCTGCCCTGAGGGAGGGGCTGCAATCTTTAGTAATGTTTCAGTTCTTGAGATTGTGGTCCCCTTGCGGCAAGTAGATGATGCTTTCCATAAATTTAGAAATTTTAATTCGAGACAACCCTCCTGCGGCATGTTACAGCATGGGGGTCGGTTCGGGTCTGGCGATTCGCCCCCTGATAGACATATTGGGATGAGAAGTCCC are provided.
5. The method for identifying the function of the promoter of the sequence derived from acetobacter xylinum as claimed in claim 1 or 2, and the application thereof in promoting the synthesis of bacterial cellulose, wherein the expression intensity of the promoter is stronger than that of the pBla plasmid promoter Pbla。
6. The method for identifying the function of the promoter of the sequence derived from acetobacter xylinum and the application of the promoter in promoting the synthesis of bacterial cellulose as claimed in claim 3, wherein the peak value of the expression intensity of the promoter is not lower than that of the pBla plasmid promoter Pbla255 times higher than the first.
7. An expression vector comprising the Acetobacter xylinum-derived sequence according to any one of claims 1 to 6.
8. A genetically engineered bacterium comprising an expression vector for the sequence derived from Acetobacter xylinum according to any one of claims 1-6 or claim 7.
9. The genetically engineered bacterium of claim 8, wherein the genetically engineered bacterium utilizes a sequence derived from Acetobacter xylinum, and is capable of expressing a red fluorescent protein.
10. The genetically engineered bacterium of claim 9, wherein the genetically engineered bacterium is derived from acetobacter xylinum.
11. Use of an acetobacter xylinum-derived sequence according to any one of claims 1-6 for the genetic engineering of acetobacter xylinum.
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| US20190119657A1 (en) * | 2017-10-19 | 2019-04-25 | Samsung Electronics Co., Ltd. | Genus komagataeibacter recombinant microorganism, method of producing cellulose using the same, and method of producing the microorganism |
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