阅读说明：本技术 经过遗传改造的紫色杆菌素生物合成基因簇、重组表达载体、工程菌及其应用 (Genetically modified violacein biosynthetic gene cluster, recombinant expression vector, engineering bacterium and application thereof ) 是由 张玉阳 陈红萍 牛志远 杨荣迪 李志坤 李艳娇 于 2020-07-13 设计创作，主要内容包括：本发明公开了经过遗传改造的紫色杆菌素生物合成基因簇、重组表达载体、工程菌及其应用,通过对紫色杆菌素生物合成基因簇的核糖体结合位点进行定点突变和密码子缺失突变改造,促进紫色杆菌素合成蛋白的有效翻译,获得生产潜力显著提高的改造基因簇；通过将上述改造基因簇或者含有改造基因簇的重组载体导入到宿主菌,获得了高产紫色杆菌素的工程菌；进一步对工程菌的发酵条件进行了优化并提供紫色杆菌素的分离纯化方法。本发明构建的基因工程菌株不仅能够显著提高紫色杆菌素的产量,且与常规的紫色杆菌素20℃或25℃低温发酵不同,在25-37℃的温度条件下均可高效生产紫色杆菌素代谢产物,解决了发酵过程中的降温成本高的问题,适用于规模化生产。(The invention discloses a genetically modified violacein biosynthetic gene cluster, a recombinant expression vector, an engineering bacterium and application thereof, wherein the modified gene cluster with remarkably improved production potential is obtained by carrying out site-directed mutation and codon deletion mutation modification on a ribosome binding site of the violacein biosynthetic gene cluster to promote effective translation of violacein synthetic protein; the modified gene cluster or the recombinant vector containing the modified gene cluster is introduced into host bacteria to obtain the engineering bacteria with high violacein yield; further optimizes the fermentation conditions of the engineering bacteria and provides a method for separating and purifying violacein. The genetic engineering strain constructed by the invention can not only obviously improve the yield of violacein, but also can efficiently produce violacein metabolites under the temperature condition of 25-37 ℃ unlike the conventional low-temperature fermentation of violacein at 20 ℃ or 25 ℃, thereby solving the problem of high cooling cost in the fermentation process and being suitable for large-scale production.)

1. A genetically engineered violacein biosynthetic gene cluster, characterized in that its nucleotide sequence is as shown in SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3 or SEQ ID NO: 4, respectively.

2. A recombinant expression vector comprising the genetically engineered violacein biosynthetic gene cluster of claim 1.

3. The method for constructing the recombinant expression vector of claim 2, wherein the genetically engineered violacein biosynthetic gene cluster of claim 1 is ligated to the vector.

4. A genetically engineered bacterium comprising the genetically engineered violacein biosynthetic gene cluster of claim 1 or the recombinant expression vector of claim 2.

5. The method for constructing the genetically engineered bacterium of claim 4, wherein the recombinant expression vector of claim 2 is introduced into a host bacterium to obtain a genetically engineered bacterium with high violacein yield.

6. The method for constructing genetically engineered bacteria of claim 5, wherein the host bacteria is E.coli BL21(DE3) (tnaA)^-) Is a polypeptide as set forth in SEQ ID NO: coli BL21(DE3) genome tnaA gene represented by SEQ ID NO: 7 kanamycin resistance gene replacement.

7. A method for producing violacein, which comprises inoculating the genetically engineered bacterium of claim 4 into a medium containing a carbon source to produce violacein by fermentation.

8. The method for producing violacein according to claim 7, wherein the medium is LB liquid medium: 1% peptone, 0.5% yeast powder, 0.5% sodium chloride; or LB solid medium: 1% of peptone, 0.5% of yeast powder, 0.5% of sodium chloride and 3% of agar powder.

9. The method for producing violacein according to claim 7, wherein the fermentation temperature is 25 ℃ to 37 ℃; the fermentation time is 24-72 hours; preferably, the culture medium is also added with an inducer IPTG with the concentration of 0.01-0.04 mM; preferably, the culture medium is also added with a tryptophan precursor with the concentration of 1-2 mM; more preferably, the fermentation temperature is 30 ℃, the fermentation time is 48h, the concentration of the inducer IPTG added in the culture medium is 0.02mM, and the concentration of the tryptophan precursor fed is 2 mM.

10. A method for separating and purifying violacein, which comprises collecting the fermentation broth obtained by the method for producing violacein according to any one of claims 7 to 9, centrifuging at 12000rpm for 5min, discarding the supernatant to obtain cell precipitate, washing 3 times with one volume of methanol until the cell precipitate is colorless, further vacuum distilling and enriching the washed methanol to obtain violacein crude product, 1) preferably, the method further comprises the step of further purifying the violacein crude product by silica gel column chromatography, wherein 0.2g of violacein crude product is dissolved in 1ml of methanol, the dissolved violacein crude product is applied to a 2cm × 20cm silica gel column, and the eluted product is collected by using 500ml of ethyl acetate/petroleum ether 9/1, and the vacuum dried violacein is obtained, 2) preferably, the method further comprises the step of high performance liquid chromatography, and the conditions of the chromatographic column YMC-Pack-ODS-AQ (4.6 × 250mm, 5 μm), and the mobile phase A: ddH are adopted₂O; mobile phase B: acetonitrile, containing 0.5% formic acid; detection wavelength: UV 575 nm; column temperature: 30 ℃; flow rate: 1.0 mL/min; elution procedure 0-15min, 50% -100% B; the time for the preparation of the medicament is 15-16min,100% of B; 16-17min, 100% -50% B; 17-30min, 50% B; collecting the elution peak with retention time of 7min, and vacuum drying to obtain pure violacein with purity of 99.9%; the elution peak with retention time of 10.5min was collected and dried in vacuo to obtain pure deoxyviolacein of 99.9% purity.

Technical Field

The invention relates to a genetically modified violacein biosynthesis gene cluster, a recombinant expression vector, an engineering bacterium and application thereof, belonging to the technical field of biology.

Background

Violacein is a metabolite produced by microorganisms, belongs to indole derivatives, is formed by oxidative condensation of two tryptophan molecules, has good biological activities of resisting tumors, viruses and staphylococcus aureus infection, resisting oxidation, malaria, regulating immunity and the like, and can be used as a natural pigment due to the bluish purple characteristic of the violacein. Therefore, violacein has potential application value in wide industrial markets of medicine, health care, cosmetics, food additives, insecticides, textiles, toys and the like.

Because the violacein compound has good application potential, people do a great deal of work on improving the yield of the violacein compound in fermentation liquor. However, due to the low productivity and potential conditional pathogenicity of the original strain, violacein biosynthetic gene clusters are currently mainly expressed heterologously in model strains common in laboratories, followed by metabolic engineering and engineering of synthetic biology. In these studies, several strategies are commonly employed. The first strategy is to try different expression strains, such as e.coli, c.freundii, e.aerogenes, c.glutamicum. The second strategy is to optimize the central metabolic pathway for tryptophan precursor supply, including overexpression of tryptophan production and regulatory genes, knock-out of inhibitory and degradative genes, and the like. The third strategy is to engineer the violacein synthetic gene cluster to either overexpress the rate-limiting enzyme or to reorder the five synthetic gene operons of violacein. Although the fermentation yield of the current genetically engineered bacteria is greatly improved, the strict requirements on the cost in future industrial production cannot be met. To further increase the yield of violacein, new strategies should be tried and applied.

Disclosure of Invention

The invention aims to improve the production potential of a violacein synthetic gene cluster by mutating and transforming the corresponding ribosome binding site of the violacein biosynthetic gene from the effective translation angle of the violacein biosynthetic gene, and simultaneously construct a series of genetic engineering strains for high-yield violacein for production and application.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a genetically modified violacein biosynthetic gene cluster. These genetically engineered gene clusters are described in SEQ ID NO: 5 on the basis of the nucleotide sequence of a violacein biosynthesis-related gene shown in the specification. SEQ ID NO: 5 is a nucleotide sequence of a violacein biosynthesis-related gene derived from Chromobacterium violaceum (ATCC 12472) comprising 5 enzyme-related vioA, vioB, vioC, vioD, vioE genes involved in the entire synthesis pathway of violacein.

Specifically, the genetically modified violacein biosynthetic gene cluster is as follows:

1) vioBm: is prepared by mixing SEQ ID NO: 5 the natural ribosome binding site sequence (GGGAAA) of the vioB gene in the violacein biosynthesis-related gene is mutated to (AAGGAG);

2) VioCm: is prepared by mixing SEQ ID NO: 5 in the violacein biosynthesis-related gene, wherein the initiation codon "ATG" of the vioC gene and the termination codon "TGA" of the vioB gene share the base "A", and the mutation shortens the distance between the natural ribosome binding site "GAGAGG" of the vioC and the initiation codon "ATG" thereof to 4 bp;

3) vioDm: is prepared by mixing SEQ ID NO: 5, the codon "GTC" at the 3' end of the vioC gene in the violacein biosynthesis related gene is subjected to deletion mutation, and the mutation shortens the distance between a vioD natural ribosome binding site "AGGGAG" and an initiation codon "ATG" thereof to 6 bp;

4) the vioEm: is prepared by mixing SEQ ID NO: 5 the natural ribosome binding site sequence (AGGAGG) of the vioE gene in the violacein biosynthesis related gene is mutated to (AAGGAG);

wherein the nucleotide sequence of the mutation sites of the vioBm, the vioCm, the vioDm and the vioEm is shown in figure 2.

5) vioBEm: the nucleotide sequence is shown as SEQ ID NO: 1, is a sequence of SEQ ID NO: 5 the natural ribosome binding site sequence (GGGAAA) of the vioB gene in the violacein biosynthesis-related gene is mutated to (AAGGAG), and the natural ribosome binding site sequence (AGGAGG) of the vioE gene is mutated to (AAGGAG);

6) vioBCEm: the nucleotide sequence is shown as SEQ ID NO: 2, is as set forth in SEQ ID NO: 1, further sharing the base of 'A' by the initiation codon 'ATG' of the vioC gene and the termination codon 'TGA' of the vioB gene, wherein the mutation shortens the distance between the natural ribosome binding site 'GAGAGG' of the vioC and the initiation codon 'ATG' to 4 bp;

7) vioBDEm: the nucleotide sequence is shown as SEQ ID NO: 3, is as set forth in SEQ ID NO: 1, further carrying out deletion mutation on a codon GTC at the 3' end of the vioC gene, wherein the deletion mutation shortens the distance between a vioD natural ribosome binding site AGGGAG and an initiation codon ATG thereof to 6 bp;

8) vioBCDEm: the nucleotide sequence is shown as SEQ ID NO: 4, is as set forth in SEQ ID NO: 1, further sharing the base of ' A ' by the initiation codon ' ATG ' of the vioC gene and the termination codon ' TGA ' of the vioB gene, wherein the mutation shortens the distance between the natural ribosome binding site ' GAGAGG ' of the vioC and the initiation codon ' ATG ' of the vioC gene to 4bp, and simultaneously, the deletion mutation of the codon ' GTC ' at the 3 ' end of the vioC gene shortens the distance between the natural ribosome binding site ' AGGGAG ' of the vioD and the initiation codon ' ATG ' of the vioB gene to 6 bp;

in a second aspect, the present invention provides a recombinant expression vector comprising the above-described genetically engineered violacein biosynthetic gene cluster. These recombinant vectors can be constructed by ligating the gene cluster nucleotide sequences of the present invention to various vectors, which may be any vectors conventional in the art, such as plasmids, phage or viral vectors, and the like, preferably high copy number pETduet-1, by methods conventional in the art.

In a third aspect, the present invention provides a genetically engineered bacterium comprising the genetically modified violacein biosynthetic gene cluster, which can be used for the production of violacein. These engineered bacteria can be obtained by transforming the recombinant expression vector of the present invention into a host bacterium. The host bacteria can be various conventional strains in the field, can meet the requirement that the recombinant expression vector can stably and automatically replicate, and the carried violacein biosynthetic gene cluster which is genetically modified can be effectively expressed. Coli BL21(DE3) or E.coli BL21(DE3) (tnaA) are preferred in the present invention^-) The latter is a gene encoding tnaA in the genome of e.coli bl21(DE3) (SEQ ID NO: 6) consisting of the kanamycin resistance gene (seq id NO: 7) and (6) replacing.

In a fourth aspect, the invention provides a method for producing violacein, which comprises inoculating the above engineering bacteria into a culture medium, and fermenting to obtain violacein and deoxyviolacein. The medium may be a medium capable of growing the genetically engineered bacterium and producing violacein in the art, and is preferably LB liquid medium (1% peptone, 0.5% yeast powder, 0.5% sodium chloride) or LB solid medium (1% peptone, 0.5% yeast powder, 0.5% sodium chloride, 3% agar powder). The fermentation conditions are only required to enable the engineering bacteria to normally grow and produce violacein. The invention adopts fermentation conditions: inoculating the overnight cultured seeds of the above genetically engineered bacteria to LB liquid medium at a inoculation amount of 2%, culturing at 37 deg.C at 220rpm/min to OD_600nm0.8, then adjusting to the appropriate fermentation temperature, and simultaneously adding IPTG inducer, continue to ferment for proper time.

Preferably, the fermentation temperature is 25-37 ℃;

preferably, the fermentation time is 24-72 hours;

preferably, the inducer IPTG is added into the culture medium at the concentration of 0.01-0.04 mM;

preferably, the tryptophan precursor can be added into the culture medium at a concentration of 1-2 mM;

the optimal fermentation conditions are obtained through experimental optimization: the fermentation temperature is 30 ℃, the fermentation time is 48h, the concentration of an addition inducer IPTG in the culture medium is 0.02mM, and the concentration of the fed tryptophan precursor is 2 mM.

The fifth aspect of the present invention provides a method for purifying and separating violacein, comprising collecting the fermentation broth obtained by the above-mentioned method for producing violacein, centrifuging at 12000rpm for 5min, discarding the supernatant to obtain cell precipitate, washing several times with methanol of one volume until the cell precipitate is colorless; then carrying out vacuum distillation and enrichment on the washed methanol to obtain a violacein crude product;

preferably, the method further comprises the step of further purifying the obtained crude violacein by silica gel column chromatography: dissolving 0.2g of violacein crude product in 1ml of methanol, adding the methanol into a 2cm multiplied by 20cm silica gel column, eluting by using 500ml of ethyl acetate/petroleum ether which is 9/1, collecting eluent step by step, and drying in vacuum to obtain the violacein;

preferably, the method further comprises a step of high performance liquid chromatography under conditions of a column of YMC-PackODS-AQ (4.6 × 250mm, 5 μm) and a mobile phase A of ddH₂O; mobile phase B: acetonitrile, containing 0.5% formic acid; detection wavelength: UV 575 nm; column temperature: 30 ℃; flow rate: 1.0 mL/min; elution procedure 0-15min, 50% -100% B; 15-16min, 100% B; 16-17min, 100% -50% B; 17-30min, 50% B; collecting the elution peak with retention time of 7min, and vacuum drying to obtain pure violacein with purity of 99.9%; collecting the elution peak with retention time of 10.5min, and vacuum drying to obtain the pure deoxyviolacein product with purity of 99.9%.

The invention has the beneficial effects that:

the invention provides a genetically modified violacein biosynthetic gene cluster, which improves the production potential of the violacein biosynthetic gene cluster by carrying out site-directed mutation and codon deletion mutation modification on a ribosome binding site corresponding to the violacein biosynthetic gene from the effective translation angle of the violacein biosynthetic gene.

The genetic engineering bacteria of high-yield violacein are obtained by introducing the genetically modified violacein biosynthesis gene cluster or a recombinant vector containing the corresponding gene cluster into host bacteria; and carrying out gene modification on the host bacteria on the basis, and knocking out tryptophan degrading tryptophanase (tnaA) gene in E.coli BL21(DE3) genome. The genetic engineering strain constructed by the invention not only can obviously improve the yield of violacein, but also can efficiently produce violacein metabolites under the temperature condition of 25-37 ℃ different from the conventional low-temperature fermentation (20 ℃ or 25 ℃) of violacein, the yield of the violacein metabolites can not be reduced along with the rise of the temperature, the problem of high cooling cost in the fermentation process is solved, and the genetic engineering strain is suitable for large-scale production.

The invention optimizes the fermentation conditions of the genetically engineered bacteria, provides the optimum fermentation temperature, time, inducer concentration and the addition amount of the tryptophan precursor, and further improves the yield of violacein in the fermentation liquor. The violacein genetically engineered bacterium provided by the invention can be used for producing violacein by fermentation in a solid or liquid LB culture medium, the components of the culture medium are simple, the price is low, and a mutant strain BCDEm (tnaA) is provided^-) The yield of violacein is up to 3269.7 mu M/L.

The invention further explores the separation and purification process of violacein in the fermentation liquor, and violacein products with different purity levels are obtained; the violacein and deoxyviolacein are separated by high performance liquid chromatography to respectively obtain pure violacein and deoxyviolacein with the purity of 99.9 percent, thereby laying a foundation for subsequent industrial production or scientific research.

Drawings

FIG. 1 is a schematic diagram of the structure of plasmid Vio12472 for expressing the violacein biosynthetic gene cluster;

FIG. 2 is a graph of sequencing results of RBS mutations corresponding to vioBm, vioCm, vioDm, vioEm in Bm, Cm, Dm, Em strains;

FIG. 3 is a plate culture phenotype map of Vio12472/E.coli BL21(DE3), Bm, Cm, Dm, Em, BEm, BCEm, BDEm;

FIG. 4 shows the host bacterium E.coli BL21(DE3) (tnaA)^-) Constructing and amplifying a verification result graph;

FIG. 5 is a liquid phase assay (UV) of violacein, deoxyviolacein standards and Vio12472/E.coli BL21(DE3) fermentation products_575nm) A result graph;

FIG. 6 is a standard graph of violacein and deoxyviolacein;

FIG. 7 is a graph of fermentation yields of strains Vio12472/E.coli BL21(DE3), Bm, Cm, Dm, Em, BEm, BCEm, BDEm, BCDEm;

FIG. 8 is a graph of fermentation temperature optimization results for BCDEm strains;

FIG. 9 is a graph of the results of optimization of the addition of IPTG as an inducer of BCDEm strains;

FIG. 10 is a graph of the results of optimization of induction fermentation time of BCDEm strain;

FIG. 11 is Vio12472/E.coli BL21(DE3) (tnaA)^-) And BCDEm (tnaA)^-) Results of fermentation with excess tryptophan precursor feeding for both strains are shown.

Detailed Description

The invention will be further described with reference to specific embodiments, but the scope of the invention is not limited thereto: