Novel recycling technology for converting crustacean waste into violacein

Abstract

The invention provides a novel recycling technology for converting crustacean waste into violacein. Specifically, the invention discloses a purpuribacillus engineering strain capable of efficiently metabolizing chitin and synthesizing violacein, and the engineering strain can be used for treatment of crustacean waste and resource conversion of chitin.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and waste resource utilization, and specifically relates to an engineering strain that efficiently metabolizes chitin to synthesize violacein and a high-value biological transformation technology for crustacean waste. Background Art

[0002] Nearly 10 million tons of crustacean waste are generated every year in the world, which are usually landfilled or dumped into the sea. This type of waste contains a large amount of chitin, a biopolysaccharide composed of N-acetylglucosamine (GlcNAc) through β-1,4-glycosidic bonds. Therefore, crustacean waste entering the environment will cause environmental and health problems such as eutrophication of water bodies and the spread of pathogenic bacteria. Improper disposal of crustacean waste not only causes environmental pollution, but also wastes resources. Realizing the resource utilization of crustacean waste provides an economically valuable solution to the above problems.

[0003] At present, the more mature resource utilization approach for crustacean waste is to extract chitin from the waste by chemical treatment methods and then process it into chitosan products. However, this production process requires the consumption of a large amount of acid and alkali, and the obtained chitosan has high heterogeneity, which limits the application scenarios of the product. Although chitin can be converted into high-value compounds such as 3-acetylamino-5-acetylfuran and 1-O-methyl-N-acetylglucosamine through complex chemical treatment processes, this process is difficult to scale up due to harsh operating conditions, huge energy consumption and reagent consumption. The rapid development of synthetic biology technology in recent years is expected to solve the above challenges. For example, researchers have developed a technology that uses mechanical chemistry to couple enzymatic hydrolysis and microbial synthesis to convert chitin into tyrosine and dopamine compounds. However, mechanical ball milling hydrolysis of chitin consumes a lot of electricity, and the main hydrolysis product is oligosaccharides that cannot be directly metabolized and utilized by chassis microorganisms. The cost of the conversion process is expensive, so it is difficult to apply on a large scale. Chitin waste urgently needs a new environmentally friendly, green and efficient resource technology.

[0004] Viola has a certain ability to hydrolyze chitin and synthesize high-value bioactive substances, and is an ideal chassis microorganism for realizing high-value resource conversion of chitin. Viola can synthesize the biological pigment violacein, which is a high-value drug molecule with antibacterial, anti-tumor and antiviral properties, with a value of up to 1,600-2,000 yuan / mg. The production of violacein by wild-type Viola generally has defects such as low yield and unstable yield, which restricts the industrial production and application of violacein. Summary of the invention

[0005] The purpose of the present invention is to establish a new resource recovery method for crustacean waste, reduce the cost of the crustacean waste recovery process, and improve the economic value of the recovered product. Through genetic engineering of Chromobacterium violaceum, an engineered strain that can efficiently metabolize chitin to synthesize violaceum is obtained, and the efficient conversion of waste chitin to violaceum is achieved by the whole biological method.

[0006] The present invention provides a novel high-yield strain that can efficiently metabolize chitin to synthesize high-value compounds. The present invention selects Vibrio purpurogenus as the chassis microorganism, and through genetic engineering modification, strengthens the ability of the chassis microorganism to use chitin as the sole carbon source and nitrogen source for metabolic growth, and significantly improves the yield of Vibrio purpurogenus synthesized when chitin is the sole carbon source and nitrogen source.

[0007] The invention provides a purple bacillus engineered strain WT / pYYDT-Chi, which is characterized in that a wild type purple bacillus is used as a starting bacterium, purple bacillus cv_1440, cv_2935 and cv_4240 genes are overexpressed, and the purple bacillus cv_1440, cv_2935 and cv_4240 gene sequences are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively.

[0008] The purple bacillus engineered strain WT / pYYDT-Chi of the present invention is characterized in that it is constructed using an arabinose-inducible promoter and a plasmid pYYDT.

[0009] The present invention also provides a purple bacterium engineering strain WT / pYYDT-ChiVio, characterized in that a wild-type purple bacterium is used as a starting bacterium, the cv_1440, cv_2935 and cv_4240 genes of the purple bacterium and the purple bacterium synthesis gene cluster vioABCDE are overexpressed, the purple bacterium cv_1440, cv_2935 and cv_4240 gene sequences are shown in SEQ ID NO.1, NO.2 and NO.3, respectively, and the purple bacterium synthesis gene cluster vioABCDE sequence is shown in SEQ ID NO.5.

[0010] The purple bacillus engineered strain WT / pYYDT-ChiVio described in the present invention is characterized in that it is constructed using an arabinose-inducible promoter and plasmid pYYDT.

[0011] The present invention also provides the use of the purple bacillus engineered strain WT / pYYDT-Chi in hydrolyzing chitin and / or treating crustacean waste.

[0012] The present invention also provides the use of the purple bacillus engineered strain WT / pYYDT-ChiVio in hydrolyzing chitin and / or treating crustacean waste and / or synthesizing purple bacillus.

[0013] The present invention provides a method for synthesizing violacein by utilizing chitin and / or crustacean waste, characterized in that the violacein engineering strain WT / pYYDT-Chi and / or the violacein engineering strain WT / pYYDT-ChiVio processes chitin and / or crustacean waste.

[0014] The method for synthesizing violacein by using chitin and / or crustacean waste according to the present invention is characterized in that the method comprises the steps of:

[0015] (a) preparing a fermentation broth using the chitin and / or chitin obtained from the crustacean waste as a carbon source and / or a nitrogen source; and

[0016] (b) The engineered strain of Bacillus violaceus WT / pYYDT-Chi and / or the engineered strain of Bacillus violaceus WT / pYYDT-ChiVio were transferred into chitin fermentation culture medium and fermented at 30°C.

[0017] The method for synthesizing violacein by using chitin and / or crustacean wastes of the present invention further comprises using L-arabinose induction treatment.

[0018] The present invention provides a novel method for efficiently synthesizing violacein from crustacean waste using the novel engineered strain. The method comprises obtaining chitin from crustacean waste using pretreatment technology, using chitin as the sole carbon source and nitrogen source for the novel engineered strain, and realizing efficient synthesis of the high-value compound violacein.

[0019] The present invention provides a set of expression vector tools suitable for Bacillus violaceus, which enhances the ability of the engineered strain to hydrolyze chitin and synthesize Bacillus violaceus. The constructed expression vector includes:

[0020] The pYYDT plasmid vector and the arabinose-inducible promoter were used to regulate gene expression, and the key chitinase and violacein synthesis gene clusters were co-expressed to obtain the recombinant expression plasmid pYYDT-ChiVio, thereby obtaining the violacein engineering strain that metabolizes chitin and synthesizes violacein.

[0021] In an embodiment of the present invention, the method for constructing the engineered strain comprises:

[0022] (1) PCR was used to amplify the three chitinase genes (cv_4240, cv_1440, cv_2935) and the violacein biosynthesis gene cluster (cv_vioA, cv_vioB, cv_vioC, cv_vioD, cv_vioE) in the genome of Viola viride. In addition, the arabinose-inducible promoter P was amplified from the pYYDT plasmid. araBAD The plasmid backbone DNA fragments were integrated into the expression plasmid pYYDT-ChiVio using seamless cloning method.

[0023] (2) Transfecting the pYYDT-ChiVio plasmid into Bacillus violaceus to obtain the engineered strain WT / pYYDT-ChiVio of the present invention.

[0024] Furthermore, the present invention also provides a method for converting crustacean waste into violacein, comprising the following steps:

[0025] (1) Extracting chitin from crustacean waste. Collect crab shells from food waste, wash the shell waste and then dry it. After three rounds of pretreatment with dilute hydrochloric acid, sodium hydroxide solution and hydrogen peroxide, dry the chitin and cut it into pieces with a grinder. Prepare a fermentation culture medium with chitin as the sole carbon and nitrogen source.

[0026] (2) After the engineered strains are cultured to the stationary phase, they are transferred to chitin fermentation culture medium. After four days of culture at 30°C, it can be seen that the white chitin is completely hydrolyzed and the culture medium turns dark purple. The cells are collected by centrifugation and purple bacteriocin is extracted with anhydrous ethanol.

[0027] Chromobacterium violaceum includes strains such as ATCC12472.

[0028] The present invention can achieve the following effects:

[0029] Genetic engineering modification endows chassis microorganisms with the bioconversion ability to use chitin as the sole carbon and nitrogen source, maintain growth metabolism and synthesize violacein, thus realizing a new resource-based pathway from scratch. The present invention uses synthetic biology tools to construct genetically engineered strains, successfully achieving the production of violacein in a culture medium with crystalline chitin as the sole carbon and nitrogen source, and establishing a new green, efficient and low-cost resource-based method for chitin waste.

[0030] This application has the following advantages: first, it increases the value of the conversion product; second, it realizes the resource conversion of chitin in a gentle and green way; third, it provides an economically feasible resource method for waste, avoiding the environmental and health risks caused by the arbitrary disposal of waste; and finally, it broadens the types of substrates for microbial production of purple bacteriocin. With the improvement of the process, the cost of extracting chitin from waste will be reduced and the quality will be improved. Chitin is expected to provide a cheap nutrient source for microbial fermentation production. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Flowchart of the conversion of crustacean waste into violacein.

[0032] Figure 2 Schematic diagram of plasmids WT / pYYDT-Chi and pYYDT-ChiVio.

[0033] Figure 3 Mutant strain (Δ3489 BE , Δ1440 BE , Δ1897 BE , Δ2935 BE , Δ3316 BE , Δ4240 BE ) and the wild-type strain (WT) on CM-1 chitin plates.

[0034] Figure 4 Comparison of the relative areas of hydrolysis zones produced by the engineered strains WT / pYYDT-Chi, WT / pYYDT-ChiVio and the control strain WT / pYYDT on CM-1 chitin plates.

[0035] Figure 5 The production of violacein by the engineered strain WT / pYYDT-ChiVio and the control strain WT / pYYDT in CM-2, a medium containing colloidal chitin as the sole carbon and nitrogen source.

[0036] Figure 6 Comparative photos of the engineered strain WT / pYYDT-ChiVio and the control strain WT / pYYDT on day 0 and day 4 of fermentation in CM-2.

[0037] Figure 7 The production of violacein by the engineered strain WT / pYYDT-ChiVio in CM-3 and CM-4, CM-3 is a culture medium with crystalline chitin extracted from crustacean waste in the laboratory as the sole carbon and nitrogen source, and CM-4 is a culture medium with crystalline chitin extracted from crustaceans by the company as the sole carbon and nitrogen source. DETAILED DESCRIPTION

[0038] The present invention is further described below with reference to examples and drawings, which may help researchers in the field understand, but the embodiments of the present invention are not limited thereto.

[0039] The wild type purple bacillus described in the present application is purple bacillus ATCC12472, which was purchased from Beijing Baozang Biotechnology Co., Ltd.

[0040] Bacterial genomic DNA rapid extraction kit, plasmid extraction kit, gel recovery kit, chitin and colloidal chitin are all from Sangon Biotech.

[0041] LB culture medium, composed of 5 g / L yeast extract, 10 g / L tryptone, and 10 g / L NaCl, was prepared by high-pressure steam sterilization at 121°C for 20 min.

[0042] LB plate, composed of 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, 15 g / L agar. The culture medium was sterilized by high pressure steam at 121°C for 20 min, and poured into a sterile culture dish in a clean bench to prepare an LB plate.

[0043] The primers used for plasmid construction are shown in Table 1. All other reagents not specifically described were commercially available.

[0044] Table 1. Primers used for plasmid construction

[0045]

[0046] Example 1 Preferred high-efficiency chitinase

[0047] 1. Build the editing tool pRK2-BE

[0048] Using the expression frame of the Cas9 fusion protein nCas9(D10A)-rAPOBEC1 driven by the promoter J23119 and the sgRNA expression frame driven by the promoter trc (for preparation methods, see Komor, A., Kim, Y., Packer, M. et al. Programmable editing of a target base in genomic DNA without double-strandedDNA cleavage. Nature 533,420-424 (2016)), plasmid pRK2-Geo2i (Addgene) was used as a template, and pRK2-F and pRK2-R were used as primers to amplify the plasmid backbone DNA fragment containing elements such as replicons and resistance genes, i.e., the pRK2-Geo2i vector fragment. The PCR products were subjected to agarose gel electrophoresis, the gel containing the target fragment was cut, and the purified DNA fragment was obtained using a gel recovery kit.

[0049] The nCas9(D10A)-rAPOBEC1 expression frame, sgRNA expression frame, and pRK2-Geo2i vector fragment were connected using the seamless cloning assembly method. The connected plasmid was transfected into Escherichia coli Turbo competent (Tolo Harbor Biotech) using the heat shock chemical transfection method. After incubation at 37°C and 200 rpm for 1 h, the bacterial solution was evenly spread on a 50 μg / mL kanamycin resistance LB plate. After culturing at 37°C for 24 h, a single clone was picked and verified by PCR amplification to obtain the positive plasmid pRK2-BE.

[0050] 2. Identification of key chitinases

[0051] According to the KEGG database (https: / / www.genome.jp / kegg / ), six possible chitin hydrolase genes in Chromobacterium violaceum were inferred, namely cv_1440, cv_1897, cv_2935, cv_3316, cv_3489, and cv_4240. The Golden Gate assembly method was used to add the pre-spacer sequence to the single-base editing plasmid. The specific process is as follows: the pRK2-BE plasmid was digested with BsaI to form a sticky end gap, and the impurities in the digested plasmid were removed using a purification kit. The pre-spacer sequence that meets the following requirements was designed: the length of the pre-spacer sequence was 20 bp, the PAM sequence was NGG, and the cytosine was located at positions 3-8 of the far PAM end sequence. Finally, a 4 bp oligonucleotide sequence was added to the bidirectional 5' ends of the selected pre-spacer sequence to form a sticky end with the restriction site of the plasmid. The sequences were annealed to form double-stranded DNA fragments, and two 24-nt protospacer sequences were phosphorylated using T4 polynucleotide kinase. The protospacer sequence and the restriction plasmid pRK2-BE were ligated using a quick ligase, and then introduced into E. coli Turbo competent cells (Tolo Biotech) using a chemical transformation method. The plasmids were spread on 50 μg / mL kanamycin-resistant LB plates. Single clones were picked to obtain E. coli containing positive plasmids pRK2-BE-1440, pRK2-BE-1897, pRK2-BE-2935, pRK2-BE-3316, pRK2-BE-3489, and pRK2-BE-4240. After amplification, the plasmids were extracted for later use.

[0052] The 6 plasmids were transfected into wild-type Bacillus violaceus using sucrose washing electro-transfection method. The steps of sucrose washing electro-transfection method are as follows:

[0053] (1) Prepare electrocompetent cells of Bacillus violaceus. Follow the following procedure. Inoculate Bacillus violaceus from the culture tube and culture to the stationary phase. Take 3 mL of the bacterial solution and centrifuge at 12,000 g for 2 min to collect the cells. Add 1 ml of 300 mM sucrose solution in a clean bench and slowly pipette to resuspend the cells. Centrifuge at 12,000 g for 2 min and discard the supernatant. Repeat the previous step and add 100 μL of 300 mM sucrose solution to prepare a cell suspension to obtain electrocompetent cells.

[0054] (2) Electrotransfect the plasmid into Bacillus violaceus. Follow the following procedure. Take 1 μg of the edited plasmid and add it to the above 100 μL competent cells, pipette to mix, and transfer it to an electroporation cup with an electrode spacing of 2 mm. Place the electroporation cup in an electroporator, set the voltage to 2.5 kV, and perform electroporation at 5 ms. Immediately add 1 mL of fresh LB to the electroporation cup, pipette to mix, and carefully transfer the liquid to a sterile 1.5 mL EP tube. After incubating in a shaker at 30 °C and 200 rpm for 4 h, take the bacterial solution and spread it on a 100 μg / mL kanamycin resistance LB plate.

[0055] After selecting a single clone and amplifying the target gene using PCR technology, the mutant strain Δ1440 was obtained by Sanger sequencing. BE , Δ1897 BE , Δ2935 BE , Δ3316 BE , Δ4240 BE , Δ3489 BE (i.e., mutant strains of Bacillus violaceus in which cv_1440, cv_1897, cv_2935, cv_3316, cv_3489, and cv_4240 were inactivated, respectively).

[0056] Identification of key chitinases by visual detection of chitin hydrolysis ability.

[0057] Prepare colloidal chitin as follows. Weigh 20 g chitin, cut into 5 mm pieces, and add 400 mL concentrated hydrochloric acid. Stir at room temperature for 2 h until some of the solids become translucent. Pour the solution into 2 L pre-cooled (4°C) deionized water. After shaking, let stand at 4°C for 24 h. Collect the colloidal chitin by centrifugation at 8000 rpm for 10 min. Wash 5-6 times with pre-cooled (4°C) deionized water until the pH of the colloidal solution is 5.5. Resuspend in 400 mL deionized water to obtain a colloidal chitin stock solution. Weigh 1.5 mL of the stock solution, dry it at 65°C, and weigh it to quantify the concentration of the colloidal chitin stock solution.

[0058] Chitin solid medium plate medium CM-1 was prepared as follows. Its composition was 0.2 g / LMgSO 4 7H 2 O, 1.18 g / LK 2 HPO 4 ·3H 2 O, 0.2 g / L KCl, 1.0 g / L NH 4 NO 3 , 15 g / L glucose, 10% LB (v:v), 0.002 g / L Fe 2+, 0.002 g / L Mn 2+ , 0.002 g / L Zn 2+ , 5 g / L colloidal chitin, 15 g / L agar, pH = 6.8. After autoclaving at 115°C for 30 min, solid culture medium plates were prepared in a clean bench.

[0059] The above six mutant strains and wild-type strain were cultured in LB medium for 48 h to reach the stationary phase. 4 μL of bacterial liquid was dropped on the surface of the CM-1 medium plate. Chitin hydrolysis circles appeared after culturing at 30°C for 4 days. The plate was photographed, and the images were processed using ImageJ software to obtain the area of ​​the hydrolysis circle, and the area ratio of the hydrolysis circle produced by the mutant strain and the wild-type strain was calculated.

[0060] The results are as follows Figure 3 As shown, mutant strain Δ3316 BE The hydrolysis zone area of ​​the wild strain was only reduced by 19.8%, Δ1897 BE and Δ3489 BE There was no significant decrease compared with the wild-type strain, indicating that the chitin hydrolase genes 1897, 3489 and 3316 did not play a significant role in the hydrolysis of chitin. BE , Δ1440 BE , Δ2935 BE Compared with the wild strain, the hydrolysis zone area was reduced by 88.1%, 60.8% and 37.8% respectively, proving that chitin hydrolase genes 1440, 2935 and 4240 play a key role in the hydrolysis of chitin by Bacillus violaceus. Therefore, in the construction of an engineered strain that efficiently hydrolyzes chitin, it is considered to choose to overexpress chitinase cv_1440, cv_2935 and cv_4240 in Bacillus violaceus.

[0061] Example 2 Construction of an engineered strain capable of efficiently hydrolyzing chitin

[0062] 1. Construction of plasmid pYYDT-Chi

[0063] The seed liquid of wild-type Chromobacterium violaceum ATCC12472 was transferred to LB medium and cultured overnight at 30°C and 200rpm to obtain 5 ml of bacterial liquid. The genome of Chromobacterium violaceum was extracted using a bacterial genomic DNA rapid extraction kit (Sangon Biotech), and the genome was used as a template. 1440-F and 1440-R, 2935-F and 2935-R, 4240-F and 4240-R were used as three pairs of primers, and the three gene fragments of chitinase cv_1440, cv_2935, and cv_4240 were amplified by PCR using the high-fidelity enzyme Primestar HS DNA enzyme (sequences are shown in SEQ ID NO.1-3).

[0064] The strain E. coli / pYYDT (Tolo Biotech) was transferred to 5 ml LB medium and cultured in a shaker at 37°C for 24 h. Plasmid pYYDT was extracted using a plasmid extraction kit (Sangon Biotech). Using pYYDT-F and pYYDT-R as primers and plasmid pYYDT as a template, the plasmid pYYDT backbone DNA fragment containing the replicon, amplicon, kanamycin marker gene, and arabinose-inducible promoter was PCR amplified using the high-fidelity enzyme Primestar HS DNA enzyme (the sequence is shown in SEQ ID NO.4).

[0065] Add loading buffer to the DNA fragment product solution and slowly add it to the agarose gel channel using a pipette. Electrophoresis was performed at 100V for 1 hour. The location of the target DNA fragment was confirmed using a gel imager, and the gel containing the target DNA fragment was cut. The target DNA fragment was recovered using a gel recovery kit (Sangon Biotech).

[0066] Using the Gibson assembly method, the four gene fragments (SEQ ID NO.1-4) were connected into a plasmid at 50°C for 1 h. The obtained plasmid was introduced into Escherichia coli Turbo competent cells by heat shock chemical transfection. After incubation at 37°C, 200 rpm shaking for 1 h, it was spread on a 50 μg / mL kanamycin resistance plate. Several single clones were picked and used as templates. Three pairs of verification primers CK-1-F and CK-1-R, CK-2-F and CK-2-R, CK-3-F and CK-3-R were used for PCR amplification using Novamix enzyme. A positive single clone that amplified the target fragment with all three pairs of primers was selected. The single colony contained the successfully connected positive plasmid pYYDT-Chi. The plasmid pYYDT-Chi was extracted using a plasmid extraction kit (Sangon Biotech). See the plasmid schematic diagram for details. Figure 2 .

[0067] 2. Construction of an engineered strain of Bacillus violaceus that overexpresses chitin hydrolase

[0068] According to the method described in 2 of Example 1, the plasmid pYYDT-Chi was electrotransformed into Bacillus violaceus to obtain the highly efficient expression strain WT / pYYDT-Chi. Using the above method, the plasmid pYYDT was electrotransformed into Bacillus violaceus to obtain the strain WT / pYYDT as a control strain.

[0069] 3. Testing the performance of engineered strains in hydrolyzing chitin

[0070] Prepare CM-1 medium according to the method described in 2 of Example 1. After high pressure steam sterilization at 115°C for 30 min, add kanamycin and arabinose inducer when the temperature reaches about 45°C, so that the final concentrations of the two are 100 μg / mL and 0.4%, respectively. Prepare solid culture medium plates on a clean bench.

[0071] WT / pYYDT and WT / pYYDT-Chi were transferred to LB medium containing 100 μg / mL kanamycin. After culturing at 30℃ and 200rpm for 12 h, 0.4% (w:v) L-arabinose inducer was added and induced for 36 h. 4 μl of each bacterial solution was dropped on the surface of the solid culture medium and placed in a 30 ℃ incubator for 4 days. The plate was photographed and the image was processed using ImageJ software to obtain the hydrolysis zone area, and the area ratio of the hydrolysis zone produced by the test strain and the control strain was calculated.

[0072] The results are as follows Figure 4 As shown, the chitin hydrolysis ability of the engineered strain WT / pYYDT-Chi is 15.2 times that of the control strain WT / pYYDT, which indicates that the chitin hydrolysis performance of Bacillus violaceus has been successfully and significantly improved through genetic engineering.

[0073] Example 3 Construction of an engineered strain capable of metabolizing chitin to synthesize violacein

[0074] 1. Construction of plasmid pYYDT-ChiVio

[0075] Using the purple bacillus genome prepared in Example 2 as a template, using Vio-F and Vio-R as primers, and using the high-fidelity enzyme Primestar HS DNA enzyme (Takara) PCR amplification of the purple bacillus synthesis gene cluster vioABCDE (sequence as shown in SEQ ID NO.5). Using the plasmid pYYDT extracted in Example 2 as a template, using pYYDT-F and pYYDT-R as primers, and using the high-fidelity enzyme Primestar HS DNA enzyme PCR amplification of the pYYDT plasmid backbone DNA fragment containing the replicon, amplicon, kanamycin marker gene, and arabinose inducible promoter (sequence as shown in SEQ ID NO.4).

[0076] According to the method described in Example 2, the above DNA fragments were obtained by agarose gel electrophoresis and gel recovery kit, and the SEQ ID NO.1, NO.2, NO.3, NO.4, and NO.5 fragments were connected into plasmids using the Gibson assembly method. Heat shock transfection was performed in E. coli Turbo competent cells. PCR amplification was performed using Novamix enzymes using CK-4-F and CK-4-R, CK-5-F and CK-5-R, CK-6-F and CK-6-R. A positive single clone that amplified the target fragment with all three pairs of primers was selected. The single colony contained the successfully connected positive plasmid pYYDT-ChiVio. See the schematic diagram of the plasmid. Figure 2 .

[0077] 2. Construction of an engineered strain overexpressing chitinase and violacein biosynthesis gene clusters

[0078] Plasmid pYYDT-ChiVio was extracted using a plasmid extraction kit, and the plasmid pYYDT-ChiVio was electro-transfected into Bacillus violaceus according to the method described in Example 2 to obtain the engineered strain WT / pYYDT-ChiVio that metabolizes chitin and synthesizes Bacillus violaceus. The strain WT / pYYDT constructed in Example 2 was used as a control strain.

[0079] 3. Testing the performance of engineered strains in hydrolyzing chitin

[0080] The test method was as described in Example 2, 3, and the performance of the engineered strain WT / pYYDT-ChiVio in hydrolyzing chitin was tested on CM-1 medium. Figure 4 As shown, the chitin hydrolysis ability of the engineered strain WT / pYYDT-ChiVio is 14.63 times that of the control strain WT / pYYDT, which indicates that the chitin hydrolysis performance of Bacillus violaceus has been successfully and significantly improved through genetic engineering.

[0081] 4. Verify the strain's ability to metabolize chitin to produce violacein

[0082] The medium CM-2 with colloidal chitin as the sole carbon and nitrogen source was prepared as follows. 5 g / L colloidal chitin, 0.2 g / L MgSO 4 7H 2 O, 1.18 g / LK 2 HPO 4 ·3H 2 O, 0.2 g / L KCl, pH = 7.2. High-pressure steam sterilization at 121 °C for 20 min.

[0083] Transfer WT / pYYDT and WT / pYYDT-ChiVio to LB medium, culture at 30℃, 200rpm for 12 h, add 0.4% (w:v) L-arabinose inducer, and induce for 36 h. Take the bacterial liquid and transfer it to CM-2 medium, the amount of bacterial liquid added is 10% of the volume of CM-2 medium. Ferment in a shaker at 30℃, 200rpm for 4 days. Take the fermentation liquid on day 0 and day 4 to detect the concentration of purple bacteriocin.

[0084] The method for detecting violacein was as follows: 500 μL of fermentation broth was taken, centrifuged and the supernatant was discarded, 500 μL of anhydrous ethanol was added, and the bacteria were dispersed again by pumping. Ultrasonic disruption was performed until the cells were completely decolorized, and then centrifuged at 10,000×g for 5 min. The supernatant was the crude violacein. Violet bacteriocin was detected using a high performance liquid chromatography instrument (Shimadzu Instrument LC-16 Co.). The samples were separated using a chromatographic column ZORBAX SB-C18. The column oven was set to 35°C. The mobile phase was 75% methanol, and the flow rate was set to 0.5 mL / min. A UV detector was selected, and the characteristic wavelength was set to 580 nm. The detection time for each sample was 20 min. The instrument injection volume was set to 10 μL.

[0085] The results are shown in Table 2, Table 3, Figure 5 , Figure 6 As shown. After 4 days of fermentation in CM-2 medium, the purple bacteriocin content of the engineered strain WT / pYYDT-ChiVio reached 125 mg / L, while the purple bacteriocin content of the control strain WT / pYYDT was 37 mg / L after 4 days of fermentation, which was only 1 mg / L higher than that on day 0. The ability of the engineered strain WT / pYYDT-ChiVio to produce purple bacteriocin was 79 times higher than that of the control strain WT / pYYDT. Wild-type purple bacterium cannot synthesize purple bacteriocin using chitin as the only carbon and nitrogen source, while the present invention transforms purple bacterium through genetic engineering means to achieve efficient production of high-value compound purple bacteriocin using chitin as the only carbon and nitrogen source. After four days of culture, in CM-2 medium, the chitin concentration in the WT / pYYDT-ChiVio bacterial solution dropped to 0g / L, indicating that WT / pYYDT-ChiVio can effectively metabolize chitin. The chitin concentration in the WT / pYYDT bacterial solution was 4.83±0.497 g / L, and chitin was almost not degraded.

[0086] Table 2. Production of violacein by engineered strain WT / pYYDT-ChiVio and control strain WT / pYYDT in CM-2

[0087]

[0088] Table 3. Changes in chitin concentration of engineered strain WT / pYYDT-ChiVio and control strain WT / pYYDT after 4 days of fermentation in CM-2

[0089]

[0090] Example 4 Construction of a new resource-based pathway for biosynthesis of violacein from crustacean waste

[0091] 1. Preparation of CM-3 culture medium with chitin waste as the sole carbon and nitrogen source

[0092] Collect the crab shells discarded from the kitchen, clean them and dry them for later use. Soak the crab shell waste in 0.5 M hydrochloric acid solution and stir at room temperature overnight to remove calcium carbonate. Wash until pH=7 and evaporate the water at 65℃. Then, place it in 0.3 M NaOH solution and stir at 80℃ for 1 hour to remove protein. Wash until pH=7 and evaporate the water at 65℃. Then, place it in 5% H 2 O 2 The shells were decolorized by stirring at 90°C for 1 hour, and chitin was obtained by heating and drying. The chitin was mechanically sheared into pieces using a grinder. The culture medium was prepared according to the following ingredients: 0.2 g / L MgSO 4 7H 2 O, 1.18 g / LK 2 HPO 4 ·3H 2 O, 0.2 g / L KCl, 5 g / L chitin fragments obtained above. The pH of the solution was adjusted to 6.8. The culture solution was sterilized by high pressure steam at 121°C for 20 min to prepare a fermentation culture solution, and further prepared a culture medium CM-3.

[0093] 2. Preparation of CM-4 medium with commercial crystalline chitin as the sole carbon and nitrogen source

[0094] Crystalline chitin from Sangon is extracted from crustaceans. The purchased commercial crystalline chitin (Sangon) was cut into pieces of about 3 mm × 3 mm in size, and the culture medium was prepared according to the following composition: 0.2 g / L MgSO 4 7H 2 O, 1.18 g / LK 2 HPO 4 ·3H 2 O, 0.2 g / L KCl, 5 g / L commercial crystalline chitin fragments. The pH of the solution was adjusted to 6.8. The culture solution was sterilized by high pressure steam at 121°C for 20 min to prepare a fermentation culture solution, and further prepared a culture medium CM-4.

[0095] 3. Testing the performance of engineered strains in metabolizing chitin waste to synthesize violacein

[0096] Transfer WT / pYYDT-ChiVio to LB medium, culture at 30°C, 200rpm for 12 h, add 0.4% (w:v) L-arabinose inducer, and induce for 36 hours. Take the bacterial solution and transfer it to CM-3 or CM-4 medium, and the amount of bacterial solution added is 10% of the volume of CM-3 or CM-4 medium. Ferment in a shaker at 30°C, 200rpm for 4 days. Take the fermentation broth on day 0 and day 4 to detect the concentration of purple bacteriocin. The purple bacteriocin detection method is as described in Example 3.

[0097] The results are as follows Figure 7 As shown, it was verified that WT / pYYDT-ChiVio can efficiently synthesize purple bacteriocin when different types of chitin are used as the sole carbon source and nitrogen source. After four days of culture, in the culture medium CM-4 with commercial crystalline chitin as the sole carbon source and nitrogen source, WT / pYYDT-ChiVio can synthesize 70.87 mg / L purple bacteriocin within 4 days. Crustacean waste was collected, and crystalline chitin was extracted in the laboratory. In the culture medium CM-3 with it as the sole carbon source and nitrogen source of the culture medium, WT / pYYDT-ChiVio can synthesize 54.29 mg / L purple bacteriocin within 4 days. This proves that the engineered strain WT / pYYDT-ChiVio can convert chitin extracted from crustacean waste into the high-value compound purple bacteriocin, and successfully establishes a new resource technology for converting crustacean waste into purple bacteriocin. Resource technology such as Figure 1 The process shown.

[0098] Sequence Listing

[0099] SEQ ID NO.1 Purple Bacillus cv_1440 gene

[0100]

[0101] SEQ ID NO.2 Purple Bacillus cv_2935 gene

[0102]

[0103] SEQ ID NO.3 Purple Bacillus cv_4240 gene

[0104]

[0105] SEQ ID NO.4 Plasmid pYYDT backbone DNA fragment

[0106]

[0107] SEQ ID NO.5 Viola bacteriocin synthesis gene cluster vioABCDE

[0108]

[0109] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. The purple bacillus engineered strain WT / pYYDT-Chi, characterized in that: Using wild type Vibrio purpurogenus as the starting bacteria, Vibrio purpurogenus cv_1440, cv_2935 and cv_4240 genes were overexpressed, and the gene sequences of Vibrio purpurogenus cv_1440, cv_2935 and cv_4240 were shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively.

2. The purple bacillus engineered strain WT / pYYDT-Chi according to claim 1, characterized in that The construct was constructed using an arabinose-inducible promoter and plasmid pYYDT.

3. The purple bacillus engineered strain WT / pYYDT-ChiVio, characterized in that Using wild-type Viola villosa as the starting bacteria, the cv_1440, cv_2935 and cv_4240 genes of Viola villosa and the Viola bacteriocin synthesis gene cluster vioABCDE were overexpressed. The gene sequences of Viola villosa cv_1440, cv_2935 and cv_4240 are shown in SEQ ID NO.1, NO.2 and NO.3, respectively, and the sequence of the Viola bacteriocin synthesis gene cluster vioABCDE is shown in SEQ ID NO.

5.

4. The purple bacillus engineered strain WT / pYYDT-ChiVio according to claim 3, characterized in that The construct was constructed using an arabinose-inducible promoter and plasmid pYYDT.

5. Use of the engineered strain WT / pYYDT-Chi of Bacillus violaceus according to claim 1 or 2 in hydrolyzing chitin and / or treating crustacean waste.

6. Use of the engineered strain WT / pYYDT-ChiVio of claim 3 or 4 in hydrolyzing chitin and / or treating crustacean waste and / or synthesizing violacein.

7. A method for synthesizing violacein using chitin and / or crustacean waste, characterized in that: Chitin and / or crustacean waste is treated using the purple bacillus engineered strain WT / pYYDT-Chi described in claim 1 or 2 and / or the purple bacillus engineered strain WT / pYYDT-ChiVio described in claim 3 or 4.

8. The method according to claim 7, characterized in that The method comprises the steps of: (a) preparing a fermentation broth using the chitin and / or chitin obtained from the crustacean waste as a carbon source and / or a nitrogen source; and (b) The engineered strain WT / pYYDT-Chi of claim 1 or 2 and / or the engineered strain WT / pYYDT-ChiVio of claim 3 or 4 is transferred into a chitin fermentation broth and fermented at 30°C.

9. The method according to claim 8, characterized in that The method further comprises inducing the treatment with L-arabinose.

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