Pigment and construction method of engineering bacteria for producing the same

Abstract

The application discloses a pigment and a construction method of an engineering bacterium for producing the pigment. A high-expression protein is identified in a blue jellyfish umbrella epidermis through a proteomics method. It is found through sequence alignment and domain analysis that the protein is similar to a lung-shaped root mouth jellyfish blue pigment protein precursor, has a curled cysteine-rich domain and a ring cake domain, and conforms to the properties of a root mouth jellyfish order specific pigment family. Therefore, the protein is named as a jellyfish blue pigment. A jellyfish blue pigment gene expression vector is constructed, and a round red blastoconidium yeast is reformed through a genetic engineering method to construct an industrial yeast strain for producing the blue pigment. The blue pigment synthesized by the constructed strain is non-toxic and has no side effects, and can be applied to the printing and dyeing industry in the future.

Description

TECHNICAL FIELD

[0001] The application relates to a pigment and a construction method of an engineering bacterium for producing the pigment, and belongs to the technical field of biotechnology. BACKGROUND

[0002] The pigment is widely used in food, textile, cosmetics, medicine and health care, and feed industries, and is an important component of food additives and printing and dyeing industries. The pigment includes a synthetic pigment and a natural pigment. Compared with the synthetic pigment, the natural pigment has the advantages of natural color tone, high safety, nutrition, health care, antibacterial, and antitumor effects, and is widely concerned in the world. In the natural pigment, red and yellow pigments are relatively widely sourced, and blue pigments are relatively rare.

[0003] It has been reported that natural blue pigments include spirulina blue pigment, gardenia blue pigment, indigo, actinomycin, and violacein, and the gardenia blue pigment is a main source of the natural blue pigment. The gardenia blue pigment is prepared from tropical plant gardenia fruits through microbial fermentation or enzyme biotransformation, and has the advantages of stable physical and chemical properties, good coloring power, and food safety, and the like. However, the source of the raw material cannot be guaranteed. From the economic point of view, the microorganism has the advantages of fast growth, easy cultivation, and no limitation of resources, environment, and space, and the like. Meanwhile, the production of the blue pigment by using the microorganism has the advantages of low cost and less pollution. SUMMARY

[0004] The application first identifies a new blue pigment from jellyfish, and then expresses the blue pigment in the round red winter spore yeast through a genetic engineering method to construct an engineering yeast strain for producing the blue pigment. The blue pigment of the jellyfish in the application is a high-expression protein identified in the umbrella epidermis of the blue jellyfish. Through NCBI-blast sequence comparison and analysis, it is found that the protein is similar to the blue protein precursor of the lung-shaped root mouth jellyfish, and therefore the protein is the blue pigment (Blup) of the jellyfish.

[0005] The application adopts the following technical scheme:

[0006] A pigment, wherein the pigment contains a pigment protein, and the amino acid sequence of the pigment protein is shown as SEQ ID NO: 1.

[0007] The application further provides an engineering bacterium, which can synthesize the pigment protein according to claim 1.

[0008] The application further provides a construction method of the engineering bacterium, which includes the following steps:

[0009] The vector containing the gene for synthesizing the pigment protein is introduced into a recipient bacterium to make the gene for synthesizing the pigment protein express, so that the engineering bacterium is obtained.

[0010] Optionally, the acceptor bacteria is Rhodosporidium sphaerocarpum.

[0011] Optionally, the nucleotide sequence of the gene for synthesizing the pigment protein is shown as SEQ ID NO: 3.

[0012] Optionally, the vector comprises a promoter XYL and a terminator HSP, both of which are cloned from Rhodosporidium sphaerocarpum.

[0013] Optionally, the vector further comprises a nourseothricin resistance gene, and positive transformants are screened by nourseothricin.

[0014] Optionally, the vector is pZPK-pGK-Nrs-Tnos-Pxyl-Blup-tHSP, wherein Blup represents a coding gene for synthesizing the pigment protein.

[0015] The method for constructing the engineering strain involved in the present application comprises codon optimization, synthesis, expression cassette construction and yeast genetic transformation of the blue pigment protein coding gene. The blue pigment gene is designed according to the codon preference of Rhodosporidium sphaerocarpum, and the GC content is increased from 47.46% to 60.18%. The optimized gene is synthesized by Suzhou Hongxin Biotechnology Co., Ltd. The expression cassette construction involves a promoter XYL and a terminator HSP, both of which are cloned from Rhodosporidium sphaerocarpum. The blue pigment gene expression cassette pXYL-Blup-tHSP is constructed with pZPK as the vector.

[0016] Optionally, the vector is first transformed into Agrobacterium tumefaciens by electroporation, and then the gene for synthesizing the pigment protein is transformed into Rhodosporidium sphaerocarpum by Agrobacterium-mediated method.

[0017] The present application also proposes the application of the above-mentioned pigment and engineering bacteria in dyeing and weaving, medicine.

[0018] The beneficial effects that can be produced by the present application include:

[0019] The present application identifies a high expression protein in the umbrella epidermis of blue jellyfish by comparative proteomics method. Through sequence alignment and domain analysis, it is found that the protein is similar to the blue pigment protein precursor of lung-shaped root mouth jellyfish, has a curled cysteine-rich domain and a ring cake domain, and meets the properties of the root mouth jellyfish family. Therefore, the protein is named jellyfish blue pigment. The jellyfish blue pigment gene expression vector is constructed, and Rhodosporidium sphaerocarpum is modified by genetic engineering method to construct an industrial yeast strain producing blue pigment. The blue pigment synthesized by the constructed strain has no toxic side effects and can be applied to the printing and dyeing industry in the future.

[0020] In nature, the blue pigment-producing microorganisms are rare. The reported blue pigment-producing microorganisms include Streptomyces, Pseudomonas and Duganella, but the production of blue pigment is low and cannot meet the demand of human beings. Therefore, it has broad application prospects to use metabolic engineering or synthetic biology strategy to modify industrial microorganisms to produce blue pigment. Researchers have modified prokaryotic Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum to produce blue pigment. Although the prokaryotic expression system has the advantages of easy operation, low cost and easy industrial production, it has the disadvantages of insufficient secretion capacity and lack of post-translational modification function, and the expressed eukaryotic proteins cannot be glycosylated or properly folded. The use of yeast expression system can overcome the shortcomings of prokaryotic expression system. At present, there are few studies on the synthesis of blue pigment by using yeast. The red-pigmented yeast has a wide range of engineering modification basis and is often used in the construction of engineering strains for pigment and protein expression. Therefore, the red-pigmented yeast is selected as the expression system to synthesize blue pigment in the patent.

[0021] Compared with the extraction of blue pigment from microorganisms, plants and animals, the use of engineered yeast strains to produce blue pigment can reduce environmental pollution and effectively reduce production cost. According to the existing research reports, blue pigment can be used as a natural pigment in food and dyeing industry, and can be applied in pharmaceutical and health care industry due to its antibacterial and antitumor activities. Therefore, the constructed blue pigment-producing engineered yeast strains can be applied in dyeing and pharmaceutical and health care industry in the future. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is blue jellyfish.

[0023] Figure 2 is the NCBI-blastp homology alignment result diagram of the amino acid sequence of sea cucumber blue pigment.

[0024] Figure 3 is the alignment diagram of the amino acid sequence of sea cucumber blue pigment and the blue pigment precursor of lung-shaped stoma jellyfish.

[0025] Figure 4 is the domain analysis result of sea cucumber blue pigment.

[0026] Figure 5 is the schematic diagram of the expression vector for constructing blue pigment engineered yeast strain.

[0027] Figure 6 is the microscope observation diagram of blue pigment-producing engineered yeast strain. DETAILED DESCRIPTION

[0028] The red-pigmented yeast involved in the examples Rhodosporidium toruloides It belongs to Sporidiobolales, Sporidiobolaceae and Rhodotorula Rhodotorula The strain preservation number is CGMCC 2.1389.

[0029] The construction method of the engineering strain involved in the embodiment includes codon optimization, synthesis, expression cassette construction and yeast genetic transformation of the blue pigment protein coding gene. The blue pigment gene is designed according to the codon preference of Rhodosporidium sphaerocarpum, and the GC content is increased from 47.46% to 60.18%. The optimized gene is synthesized by Suzhou Hongxin Biotechnology Co., Ltd. The expression cassette construction involves the promoters XYL and terminators HSP, which are cloned from Rhodosporidium sphaerocarpum. The blue pigment gene expression cassette pXYL-Blup-tHSP is constructed with pZPK as the vector.

[0030] Example 1 Jellyfish blue pigment protein analysis

[0031] (1) Through comparative proteomic analysis of the umbrella epidermis of red and blue jellyfish, one high-expression protein was identified in the umbrella epidermis of blue jellyfish, and the amino acid sequence is shown as sequence 1. Through online software NCBI-blastp (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) comparison analysis, the results show that the protein sequence is similar to the blue pigment protein of Rhopilema esculentum Rhizostoma pulmo blue pigment precursor, with a similarity of 54.96% ( Figure 2 and Figure 3 ). It is inferred that the protein is related to the formation of blue pigment of jellyfish, and is named as blue pigment of jellyfish;

[0032] (2) The structure domain of blue pigment protein was analyzed by CD-domain (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi) online software, and the results show that the blue pigment protein has a curled cysteine-rich domain (Fz domain) and a ring cake domain (Kringle domain) ( Figure 4 ), which is consistent with the structural characteristics of the specific pigment family of Rhopilema (Lawley, J. W. et al. Front Mar Sci, 2021, 8: 752949.);

[0033] (3) According to the amino acid sequence and domain characteristics of blue pigment protein, the blue pigment protein coding gene sequence is searched in the jellyfish genomic sequence (BioProjects: PRJNA505074), and the sequence information is shown as sequence 2.

[0034] Example 2 Construction of blue pigment engineering yeast strain

[0035] Rhodosporidium tortum R. toruloides CGMCC 2.1389 was purchased from China General Microbiological Culture Collection Center. The synthetic blue pigment engineering yeast strain is a blue pigment protein coding gene overexpressing engineering yeast strain R. toruloides CGMCC 2.1389 strain, the construction process is as follows:

[0036] (1) According to the codon preference of Rhodosporidium tortum, the blue pigment coding gene sequence is optimized, and the optimized sequence information is shown in sequence 3;

[0037] (2) The optimized blue pigment gene is synthesized by Suzhou Hongxin Biotechnology Co., Ltd.;

[0038] (3) Blue pigment expression vector construction. With the vector pZPK-pGK-Nrs-Tnos-Pxyl-tHSP as the skeleton (Lin X et al. FEMS Yeast Res, 2014, 14(4): 547-555.), the synthesized blue pigment gene is inserted by enzyme digestion and ligation method, and the blue pigment expression vector pZPK-pGK-Nrs-Tnos-Pxyl-Blup-tHSP is constructed. Figure 5 ). The vector is a constitutive expression vector, and the blue pigment expression frame is Pxyl-Blup-tHSP, and the resistance gene is nourseothricin. The xylose reductase promoter Pxyl and the shock protein 70 terminator tHSP sequence are referred to the published literature (Díaz T et al. Appl Microbiol Biotechnol, 2018, 102(7):3287-3300);

[0039] (4) Blue pigment expression vector transformation Rhodosporidium tortum, construction of blue pigment engineering yeast strain. The constructed blue pigment expression vector is first transformed into Agrobacterium tumefaciens by electroporation, and then the blue pigment gene is transformed into Rhodosporidium tortum R. toruloides CGMCC 2.1389 by Agrobacterium tumefaciens mediated method. The positive transformants are obtained by screening on YEPD solid plate containing 50 mg / L nourseothricin, and the Rhodosporidium tortum engineering strain overexpressing blue pigment is obtained, named Rhodosporidium tortum Blup. The method of Agrobacterium tumefaciens mediated transformation of Rhodosporidium tortum is referred to the method of Lin XP et al. (Lin XP et al. FEMS Yeast Res., 2014, 14(6), 547-555.), and the specific steps are as follows:

[0040] a. Rhodosporidium tortum was picked from the activated plate and inoculated in 5 mL YEPD medium, and cultured at 30 °C and 200 rpm overnight.

[0041] b. Pick a single colony of Agrobacterium from the activated plate and inoculate into 5 mL LB medium, incubate at 30 °C and 200 rpm overnight.

[0042] c. Take several milliliters of the overnight culture of Rhodosporidium and Agrobacterium, wash once with sterile water, then adjust OD 600nm to 0.6 with sterile water.

[0043] d. Mix 100 μL of each of the yeast and Agrobacterium with OD 600nm of 0.6, then directly spread on the IM induction plate, and incubate at 24 °C for 2-3 days.

[0044] e. Directly transfer the filter paper membrane on the IM plate to the YEPD resistance plate with 50 mg / L nourseothricin, and incubate at 30 °C until the transformants grow.

[0045] (5) Select positive transformants as blue pigment-producing engineering yeast strains. The specific process is as follows:

[0046] a. Inoculate the transformants into 5 mL YPD medium, and incubate at 30 °C and 200 rpm overnight for 24 h;

[0047] b. Transfer about 1 mL of the overnight culture into 10 mL of YPD medium, and incubate at 30 °C and 200 rpm for expansion culture;

[0048] c. Since the blue pigment is a pigment protein, the transformant cells can be directly observed for cell morphology and color using a German Lecia DM 6B in bright field and dark field. Figure 6 ).

[0049] The above is only a few embodiments of the present application, and does not limit the present application in any form. Although the present application is disclosed with the preferred embodiments, it is not intended to limit the present application. Any skilled person in the art can make some changes or modifications to the above disclosed technical content without departing from the scope of the technical solution of the present application, and such changes or modifications are equivalent to equivalent embodiments, and all belong to the scope of the technical solution.

Claims

1. A pigment, characterized in that, The pigment contains a pigment protein, the amino acid sequence of which is shown in SEQ ID NO:

1.

2. An engineered bacterium, characterized in that, The engineered bacteria can synthesize the pigment protein as described in claim 1.

3. The method for constructing engineered bacteria as described in claim 2, characterized in that, Includes the following steps: A vector containing a gene for synthesizing the pigment protein is introduced into a recipient bacterium, causing the gene for synthesizing the pigment protein to be expressed, thus obtaining the engineered bacterium.

4. The construction method according to claim 3, characterized in that, The recipient bacteria is *Rhodotorula buergerianum*.

5. The construction method according to claim 4, characterized in that, The nucleotide sequence of the gene that synthesizes the pigment protein is shown in SEQ ID NO:

3.

6. The construction method according to claim 3, characterized in that, The vector contains a promoter XYL and a terminator HSP, both of which are cloned from Rhodotorula buergerianum.

7. The construction method according to claim 3, characterized in that, The vector also contains a Norsmin resistance gene, and positive transformants are screened using Norsmin.

8. The construction method according to claim 3, characterized in that, The vector was first transferred into *Agrobacterium tumefaciens* via electroporation, and then the gene for synthesizing the pigment protein was transferred into *Rhodotorula buergerianum* via *Agrobacterium tumefaciens*-mediated transformation.

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