A new resource-recycling technology for converting crustacean waste into purple bacteriocin

Abstract

The present invention provides a novel resource-recycling technology for converting crustacean waste into violacein. Specifically, an engineered strain of Viola viride that efficiently metabolizes chitin and synthesizes violacein is disclosed. The engineered strain can be used for crustacean waste treatment and chitin resource conversion.

Description

Technical Field

[0001] The invention belongs to the fields 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 bioconversion technology for crustacean waste. Background Art

[0002] Nearly 10 million tons of crustacean waste are generated annually worldwide, often landfilled or dumped into the ocean. This waste contains a large amount of chitin, a biopolymer composed of N-acetylglucosamine (GlcNAc) linked by β-1,4-glycosidic bonds. Consequently, the release of crustacean waste into the environment can lead to environmental and health problems such as eutrophication and the spread of pathogens. Improper disposal of crustacean waste not only pollutes the environment but also wastes resources. Utilizing crustacean waste as a resource offers a cost-effective solution to these challenges.

[0003] Currently, the most established resource recovery approach for crustacean waste is to extract chitin from the waste through chemical treatment, which is then processed into chitosan products. However, this production process requires significant amounts of acid and alkali, resulting in high heterogeneity in the resulting chitosan, limiting the product's application scenarios. While complex chemical treatments can convert chitin into high-value compounds such as 3-acetylamino-5-acetylfuran and 1-O-methyl-N-acetylglucosamine, these processes are limited by stringent operating conditions, significant energy consumption, and reagent consumption, making them difficult to scale up. Rapidly advancing synthetic biology technologies have shown promise in addressing these challenges. For example, researchers have developed a technique that couples mechanochemical hydrolysis with enzymatic hydrolysis and microbial synthesis to convert chitin into tyrosine and dopamine compounds. However, mechanical ball milling and hydrolysis of chitin consumes significant amounts of electricity, and the primary hydrolysis products are oligosaccharides that cannot be directly metabolized by the microorganisms in the chassis. The high cost of the conversion process makes it difficult to scale up. Chitin waste urgently requires new, environmentally friendly, green, and efficient resource recovery technologies.

[0004] Viola viridis has the ability to hydrolyze chitin and synthesize high-value bioactive substances, making it an ideal microorganism for converting chitin into high-value resources. Viola viridis synthesizes the biopigment violacein, a high-value pharmaceutical molecule with antibacterial, antitumor, and antiviral properties, valued at 1,600-2,000 yuan per mg. However, the production of violacein by wild-type Viola viridis suffers from low and unstable yields, hindering its industrial production and application. Summary of the Invention

[0005] The present invention aims to establish a novel method for recycling crustacean waste. This method reduces the cost of the crustacean waste recycling process and increases the economic value of the recycled products. Through genetic engineering of Chromobacterium violaceum, an engineered strain capable of efficiently metabolizing chitin to produce violacein was obtained, achieving efficient, fully biological conversion of waste chitin into violacein.

[0006] The present invention provides a novel, high-yield strain that can efficiently metabolize chitin to synthesize high-value compounds. This invention uses the violaceous bacteria as the base microorganism. Through genetic engineering, the base microorganism's ability to metabolize and grow using chitin as its sole carbon and nitrogen source is enhanced, significantly increasing the yield of violaceous bacteriocin synthesized by the bacteria when chitin is used as the sole carbon and nitrogen source.

[0007] The present invention provides a purple bacterium engineered strain WT / pYYDT-Chi, which is characterized in that wild-type purple bacterium is used as a starting bacterium, purple bacterium cv_1440, cv_2935 and cv_4240 genes are overexpressed, and the purple bacterium 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 plasmid pYYDT.

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

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

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

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

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

[0014] The method for synthesizing violacein 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 broth and fermented at 30°C.

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

[0018] The present invention provides a novel method for efficiently synthesizing violacein from crustacean waste using the aforementioned novel engineered strain. The method comprises utilizing a pretreatment technique to obtain chitin from the crustacean waste, and using chitin as the sole carbon and nitrogen source for the novel engineered strain to achieve 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 violacein. The constructed expression vector includes:

[0020] Using the pYYDT plasmid vector and an arabinose-inducible promoter to regulate gene expression, the key chitinase and violacein biosynthesis gene clusters were co-expressed to generate the recombinant expression plasmid pYYDT-ChiVio. This resulted in an engineered strain of Viola viride 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 synthesis gene cluster (cv_vioA, cv_vioB, cv_vioC, cv_vioD, cv_vioE) in the genome of Bacillus violaceus. In addition, the arabinose-inducible promoter P was amplified from the pYYDT plasmid. araBAD The plasmid backbone DNA fragments were synthesized and integrated into the expression plasmid pYYDT-ChiVio using seamless cloning method.

[0023] (2) The pYYDT-ChiVio plasmid was transfected 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 purple bacteriocin, comprising the following steps:

[0025] (1) Extracting chitin from crustacean waste. Crab shells from food waste were collected, cleaned, and dried. After three rounds of pretreatment with dilute hydrochloric acid, sodium hydroxide solution, and hydrogen peroxide, the chitin was dried and sheared into fragments using a grinder. A fermentation broth was prepared using chitin as the sole carbon and nitrogen source.

[0026] (2) After the engineered strains were cultured to the stationary phase, they were transferred to chitin fermentation broth. After four days of culture at 30°C, the white chitin was completely hydrolyzed and the broth became darker purple. The cells were collected by centrifugation and purple bacteriocin was extracted with anhydrous ethanol.

[0027] Chromobacterium violaceum includes strains such as ATCC12472.

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

[0029] Genetic engineering has endowed the microorganisms in the chassis with the biotransformation ability to utilize chitin as a sole carbon and nitrogen source, sustaining growth and metabolism, and synthesizing violacein, thus creating a novel resource-based pathway. This invention uses synthetic biology tools to construct genetically engineered strains, successfully achieving the production of violacein in a culture medium containing crystalline chitin as the sole carbon and nitrogen source, establishing a green, efficient, and low-cost new resource-based approach for chitin waste.

[0030] This application has the following advantages: first, it increases the value of the conversion product; second, it achieves resource conversion of chitin in a gentle and green manner; third, it provides an economically viable method for recycling waste, avoiding the environmental and health risks associated with indiscriminate waste disposal; and finally, it broadens the types of substrates for microbial production of violacein. With process improvements, the cost of extracting chitin from waste will be reduced and its quality will be improved, making chitin a promising low-cost 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 yield of violacein produced by the engineered strain WT / pYYDT-ChiVio and the control strain WT / pYYDT in CM-2, a culture 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 purple bacteriocin by the engineered strain WT / pYYDT-ChiVio in CM-3 and CM-4. CM-3 is a culture medium containing crystalline chitin extracted from crustacean waste in the laboratory as the sole carbon and nitrogen source, and CM-4 is a culture medium containing crystalline chitin extracted from crustaceans by the company as the sole carbon and nitrogen source. DETAILED DESCRIPTION

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

[0039] The wild type purple bacillus described in this 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 Biotechnology.

[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 plates were prepared with 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, and 15 g / L agar. The culture medium was sterilized by autoclaving at 121°C for 20 min and then poured into sterile Petri dishes in a clean bench to create LB plates.

[0043] The primers used for plasmid construction are shown in Table 1. All other reagents not otherwise specified 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 cassette for the Cas9 fusion protein nCas9(D10A)-rAPOBEC1 driven by the J23119 promoter and the sgRNA expression cassette driven by the trc promoter (prepared as described in Komor, A., Kim, Y., Packer, M. et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420-424 (2016)), the plasmid pRK2-Geo2i (Addgene) was used as a template, and primers pRK2-F and pRK2-R were used, respectively, to amplify the plasmid backbone DNA fragment containing the replicon, resistance gene, and other elements, i.e., the pRK2-Geo2i vector fragment. PCR products were subjected to agarose gel electrophoresis, and the gel containing the target fragment was cut and purified using a gel extraction kit.

[0049] The nCas9(D10A)-rAPOBEC1 expression cassette, the sgRNA expression cassette, and the pRK2-Geo2i vector fragment were ligated using a seamless cloning assembly method. The ligated plasmids were transfected into competent Escherichia coli Turbo cells (Tolo Biotech) using heat shock chemical transfection. After incubation at 37°C and 200 rpm for 1 hour, the bacterial suspension was evenly plated onto 50 μg / mL kanamycin-resistant LB plates. After incubation at 37°C for 24 hours, single colonies were isolated and verified by PCR amplification to obtain positive plasmid pRK2-BE.

[0050] 2. Identification of key chitinases

[0051] Based on a search of the KEGG database (https: / / www.genome.jp / kegg / ), six possible chitin hydrolase genes from Chromobacterium violaceum were identified: cv_1440, cv_1897, cv_2935, cv_3316, cv_3489, and cv_4240. A protospacer sequence was incorporated into a single-base editing plasmid using the Golden Gate assembly method. The specific process is as follows: the pRK2-BE plasmid was digested with BsaI to create a cohesive end nick. Impurities in the digested plasmid were removed using a purification kit. A protospacer sequence was designed that met the following requirements: a 20-bp protospacer sequence with a PAM sequence of NGG and cytosine located at positions 3-8 distal to the PAM. Finally, 4-bp oligonucleotide sequences were added to the 5' end of the selected protospacer sequence in both directions, forming cohesive ends with the restriction enzyme cleavage site of the plasmid. The sequences were annealed to form double-stranded DNA fragments, and the two 24-nt protospacer sequences were phosphorylated using T4 polynucleotide kinase. The protospacer sequences were ligated to the digested plasmid pRK2-BE using Rapid Ligase and then chemically transformed into E. coli Turbo competent cells (Tolo Biotech). Plates were plated on 50 μg / mL kanamycin-resistant LB plates. Single colonies were isolated to obtain E. coli cells harboring the positive plasmids pRK2-BE-1440, pRK2-BE-1897, pRK2-BE-2935, pRK2-BE-3316, pRK2-BE-3489, and pRK2-BE-4240. After expansion, the plasmids were extracted and used for further analysis.

[0052] The six plasmids were transfected into wild-type Bacillus purpurogenus using the sucrose wash electrofection method. The steps for the sucrose wash electrofection method are as follows:

[0053] (1) Prepare electrocompetent cells of Bacillus purpurogenus. Follow the following procedure. Inoculate Bacillus purpurogenus from the culture tube and culture to the stationary phase. Centrifuge 3 mL of the bacterial solution at 12,000 g for 2 min to collect the cells. Add 1 mL of 300 mM sucrose solution in a clean bench and resuspend the cells by slowly pipetting. 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 purpurogenus. 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 cuvette with an electrode spacing of 2 mm. Place the electroporation cuvette in an electroporator, set the voltage to 2.5 kV, and perform electroporation under 5 ms conditions. Immediately add 1 mL of fresh LB to the electroporation cuvette, pipette to mix, and carefully transfer the liquid to a sterile 1.5 mL EP tube. After incubation in a shaker at 30°C and 200 rpm for 4 h, take the bacterial solution and spread it on an LB plate containing 100 μg / mL kanamycin resistance.

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

[0056] Key chitinases were identified by visual detection of chitin hydrolysis ability.

[0057] Prepare colloidal chitin as follows. Weigh 20 g of chitin, cut into 5 mm pieces, and add 400 mL of concentrated hydrochloric acid. Stir at room temperature for 2 h until some of the solid becomes translucent. Pour the solution into 2 L of pre-chilled (4°C) deionized water. Shake well and let stand at 4°C for 24 h. Collect the colloidal chitin by centrifugation at 8000 rpm for 10 min. Rinse the solution 5-6 times with pre-chilled (4°C) deionized water until the pH of the colloidal solution reaches 5.5. Resuspend the solution in 400 mL of 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 / L MgSO4·7H2O, 1.18 g / L K2HPO4·3H2O, 0.2 g / L KCl, 1.0 g / L NH4NO3, 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 plates were prepared in a clean bench.

[0059] The six mutant strains and the wild-type strain were cultured in LB broth for 48 hours until the plateau phase was reached. A 4 μL droplet was placed on the surface of a CM-1 medium plate. After 4 days of incubation at 30°C, chitin hydrolysis zones appeared. The plates were photographed and processed using ImageJ software to determine the hydrolysis zone area. The area ratio of the hydrolysis zones produced by the mutant strains to that produced by the wild-type strain was then calculated.

[0060] The results are as follows Figure 3 As shown, mutant strain Δ3316 BE The hydrolysis zone area was only reduced by 19.8% compared with the wild strain, 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 to the wild-type strain, the hydrolysis zone areas decreased by 88.1%, 60.8%, and 37.8%, respectively, demonstrating that chitinase genes 1440, 2935, and 4240 play a key role in chitin hydrolysis in Bacillus purpurogenus. Therefore, overexpressing chitinases cv_1440, cv_2935, and cv_4240 in Bacillus purpurogenus is a potential option for constructing engineered strains capable of efficiently hydrolyzing chitin.

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

[0062] 1. Construction of plasmid pYYDT-Chi

[0063] A seed solution of wild-type Chromobacterium violaceum ATCC12472 was inoculated into LB medium and cultured overnight at 30°C, 200 rpm to obtain 5 ml of bacterial culture. The Chromobacterium violaceum genome was extracted using a rapid bacterial genomic DNA extraction kit (Sangon Biotech). Using this as a template, three chitinase gene fragments, cv_1440, cv_2935, and cv_4240, were amplified using Primestar HS DNase (SEQ ID NOs. 1-3) using primers 1440-F and 1440-R, 2935-F and 2935-R, and 4240-F and 4240-R.

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

[0065] Add loading buffer to the DNA fragment product solution and slowly pipette into the agarose gel channels. Run electrophoresis at 100 V for 1 hour. Confirm the location of the target DNA fragment using a gel imager and cut the gel containing the target DNA fragment. Recover the target DNA fragment using a gel recovery kit (Sangon Biotechnology).

[0066] The Gibson assembly method was used to react at 50°C for 1 h, and the above four gene fragments (SEQ ID NO.1-4) were connected into a plasmid. 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, respectively. 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 to perform PCR amplification using Novamix enzyme. A positive single clone in which all three pairs of primers amplified the target fragment 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 electroporated into Bacillus violaceus to obtain a high-efficiency expression strain WT / pYYDT-Chi. Using the above method, the plasmid pYYDT was electroporated into Bacillus violaceus to obtain the strain WT / pYYDT as a control strain.

[0069] 3. Testing the chitin hydrolysis performance of the engineered strain

[0070] Prepare CM-1 medium according to the method described in step 2 of Example 1. After autoclaving at 115°C for 30 min, add kanamycin and arabinose inducer to a final concentration of 100 μg / mL and 0.4%, respectively, when the temperature reaches approximately 45°C. Prepare solid culture medium plates in a clean bench.

[0071] WT / pYYDT and WT / pYYDT-Chi were inoculated into LB medium containing 100 μg / mL kanamycin. After incubation at 30°C, 200 rpm for 12 hours, 0.4% (w:v) L-arabinose was added for induction for 36 hours. A 4 μl droplet of each bacterial suspension was placed on the surface of the solid medium and incubated in a 30°C incubator for 4 days. The plates were photographed and processed using ImageJ software to determine the hydrolysis zone area. The area ratio of the hydrolysis zone produced by the test strain to the control strain was calculated.

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

[0073] Example 3 Construction of an engineered strain capable of metabolizing chitin and synthesizing violacein

[0074] 1. Construction of plasmid pYYDT-ChiVio

[0075] The violacein genome prepared in Example 2 was used as a template, Vio-F and Vio-R were used as primers, and the high-fidelity enzyme Primestar HS DNase (Takara) was used to PCR amplify the violacein biosynthesis gene cluster vioABCDE (sequence shown in SEQ ID NO. 5). The plasmid pYYDT extracted in Example 2 was used as a template, pYYDT-F and pYYDT-R were used as primers, and the high-fidelity enzyme Primestar HS DNase was used to PCR amplify the pYYDT plasmid backbone DNA fragment containing the replicon, amplicon, kanamycin marker gene, and arabinose-inducible promoter (sequence 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 enzyme 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 was selected in which all three pairs of primers amplified the target fragment. The single colony contained the successfully connected positive plasmid pYYDT-ChiVio. The plasmid schematic diagram is shown in FIG. Figure 2 .

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

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

[0079] 3. Testing the chitin hydrolysis performance of the engineered strain

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

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

[0082] Prepare medium CM-2 with colloidal chitin as the sole carbon and nitrogen source as follows: 5 g / L colloidal chitin, 0.2 g / L MgSO4·7H2O, 1.18 g / L K2HPO4·3H2O, 0.2 g / L KCl, pH = 7.2. Autoclave at 121°C for 20 min.

[0083] WT / pYYDT and WT / pYYDT-ChiVio were inoculated into LB medium and cultured at 30°C and 200 rpm for 12 hours. Then, 0.4% (w:v) L-arabinose was added for induction for 36 hours. The culture was transferred to CM-2 medium at a volume of 10% of the CM-2 volume. Fermentation was continued at 30°C and 200 rpm in a shaker for 4 days. The fermentation broth was collected on days 0 and 4 and assayed for violacein concentration.

[0084] The method for detecting violacein was as follows: 500 μL of fermentation broth was centrifuged and the supernatant discarded. 500 μL of anhydrous ethanol was added and the cells were redispersed by pumping. The cells were disrupted by ultrasonication until complete decolorization and then centrifuged at 10,000 × g for 5 min. The supernatant contained crude violacein. Violet violacein was detected using a high-performance liquid chromatography (HPLC) instrument (Shimadzu Instrument LC-16 Co.). The sample was separated using a ZORBAX SB-C18 column. 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, with a characteristic wavelength set to 580 nm. Each sample was detected for 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 four days of fermentation in CM-2 medium, the engineered strain WT / pYYDT-ChiVio produced 125 mg / L of violacein, while the control strain WT / pYYDT produced 37 mg / L of violacein after four days of fermentation, a mere 1 mg / L increase from day 0. The engineered strain WT / pYYDT-ChiVio's ability to produce violacein was 79 times greater than that of the control strain WT / pYYDT. Wild-type violacein bacteria cannot synthesize violacein using chitin as the sole carbon and nitrogen source. However, the present invention has modified violacein bacteria through genetic engineering, achieving efficient production of the high-value compound violacein using chitin as the sole carbon and nitrogen source. After four days of culture in CM-2 medium, the chitin concentration in the WT / pYYDT-ChiVio culture medium dropped to 0 g / 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, indicating that 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 concentrations 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] Discarded crab shells were collected, cleaned, and dried for later use. The shells were soaked in 0.5 M hydrochloric acid solution and stirred overnight at room temperature to remove calcium carbonate. The shells were then washed to a pH of 7 and evaporated to dryness at 65°C. The shells were then placed in a 0.3 M NaOH solution and stirred at 80°C for 1 hour to remove protein. The shells were washed to a pH of 7 and evaporated to dryness at 65°C. The shells were then placed in a 5% H₂O₂ solution and stirred at 90°C for 1 hour to decolorize the shells. Chitin was obtained by heat drying and mechanically sheared into fragments using a mill. The culture medium was prepared according to the following composition: 0.2 g / L MgSO₄·7H₂O, 1.18 g / L K₂HPO₄·3H₂O, 0.2 g / L KCl, and 5 g / L of the chitin fragments obtained above. The pH of the solution was adjusted to 6.8. The culture medium was sterilized by autoclaving at 121°C for 20 minutes to prepare the fermentation medium, which was then used to prepare the culture medium CM-3.

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

[0094] Crystalline chitin from Sangon is extracted from crustaceans. Commercially available crystalline chitin (Sangon) was cut into approximately 3 mm x 3 mm fragments. A culture medium was prepared with the following composition: 0.2 g / L MgSO₄·7H₂O, 1.18 g / L K₂HPO₄·3H₂O, 0.2 g / L KCl, and 5 g / L commercial crystalline chitin fragments. The pH of the solution was adjusted to 6.8. The culture medium was sterilized by autoclaving at 121°C for 20 minutes to prepare the fermentation medium, which was then used to prepare medium CM-4.

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

[0096] Transplant WT / pYYDT-ChiVio into LB medium and culture at 30°C, 200 rpm, for 12 hours. Add 0.4% (w:v) L-arabinose as inducer and induce for 36 hours. Transfer the culture to CM-3 or CM-4 medium, adding 10% of the volume of the CM-3 or CM-4 medium. Ferment for 4 days in a shaker at 30°C, 200 rpm. Samples of fermentation broth were collected on days 0 and 4 and assayed for violacein concentrations using the same assay method as described in Example 3.

[0097] The results are as follows Figure 7 As shown, it was verified that when different types of chitin were used as the sole carbon source and nitrogen source, WT / pYYDT-ChiVio could efficiently synthesize purple bacteriocin. 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 could 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 could 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 establish 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 saccharin synthesis gene cluster vioABCDE

[0108]

[0109] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify 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 purple bacterium as the starting bacteria, purple bacterium cv_1440, cv_2935 and cv_4240 genes were overexpressed. The gene sequences of the purple bacterium cv_1440, cv_2935 and cv_4240 are 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 used an arabinose-inducible promoter and plasmid pYYDT.

3. The engineered strain of Bacillus violaceus WT / pYYDT-ChiVio, characterized in that: Using wild-type Bacillus violaceus as the starting bacteria, the cv_1440, cv_2935 and cv_4240 genes of Bacillus violaceus and the violacein synthesis gene cluster vioABCDE were overexpressed. The gene sequences of the Bacillus violaceus cv_1440, cv_2935 and cv_4240 were shown in SEQ ID NO.1, NO.2 and NO.3, respectively, and the sequence of the violacein synthesis gene cluster vioABCDE was shown in SEQ ID NO.

5.

4. The engineered strain WT / pYYDT-ChiVio of claim 3, characterized in that The construct used an arabinose-inducible promoter and plasmid pYYDT.

5. Use of the engineered strain WT / pYYDT-Chi of Bacillus purpurogenus 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 according to claim 1 or 2 and / or the purple bacillus engineered strain WT / pYYDT-ChiVio according to 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 using L-arabinose as an induction treatment.

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