Gene cluster synthesized by gas-producing vibrio strain for producing prodigiosin and prodigiosin

Abstract

The invention discloses a gas-producing vibrio strain for producing prodigiosin and a prodigiosin synthetic gene cluster, and belongs to the technical field of microorganisms. The invention relates to a strain B1D10 for producing prodigiosin, the classification name of the strain B1D10 is Vibrio gazogenes, the strain B1D10 is preserved in Guangdong Province Microbial Culture Collection Center, the preservation number is GDMCC NO: 66510, the preservation date is June 13, 2025, and the preservation address is No.59 building, No.100 Courtyard, Xianlie Middle Road, Guangzhou. The strain B1D10 contains a prodigiosin synthetic gene cluster consisting of 16 genes. The strain is rapid in propagation and can be applied to large-scale fermentation production of prodigiosin.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a gas-producing Vibrio strain producing prodigiosin and a prodigiosin synthesis gene cluster. BACKGROUND

[0002] Prodigiosin is a bright red pigment produced by bacteria, and has a methoxy backbone structure of three pyrrole rings. Prodigiosin has attracted much attention due to its wide biological activities, such as bacteriostasis, parasitic inhibition, algaecide, cancer cell inhibition, textile dye, pH indicator and the like.

[0003] The biosynthesis of prodigiosin is regulated by a series of pig gene clusters, and two precursors, 2-methyl-3-pentyl pyrrole (MAP) and 4-methoxy-2,2-dipyrrole-5-carboxymethyl acetaldehyde (MBC), are synthesized through two branch pathways, and then MAP and MBC are condensed to form prodigiosin under the action of condensing enzyme pigC. The biosynthesis of MAP is regulated by pigB (encoding H2MAP oxidoreductase), pigD (encoding 3-acetoxyoctanal synthetase) and PigE (encoding 3-acetoxyoctanal transaminase) genes; and the synthesis of MBC is regulated by pigA (encoding dehydrogenase), pigF (encoding O-methyltransferase), pigG (encoding acyl carrier protein), pigH (encoding synthetase), pigI (encoding ligase), pigJ (encoding ligase), pigK, pigL (encoding transferase), pigM (encoding oxidoreductase) and pigN genes.

[0004] The bacteria capable of producing prodigiosin mainly include Serratia marcescens, Pseudoalteromonas rubra, Zooshikella ganghwensis, Z.rubidus, Z.marina, Hahella chejuensis, Vibrio ruber, Pseudovibrio denitrificans, Streptomyces coelicolor, Streptoverticillium rubrireticuli and Saccharopolyspora sp. However, there are no reports on the prodigiosin-producing Vibrio at present, although there are related patent reports on the prodigiosin-producing Serratia marcescens and Zooshikella. SUMMARY

[0005] The present application aims at a Vibrio gazogenes strain producing prodigiosin and a prodigiosin synthesis gene cluster.

[0006] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions.

[0007] A strain B1D10 derived from Spartina alterniflora root tissue can produce prodigiosin and has a prodigiosin synthesis gene cluster through gene annotation. The strain B1D10 is classified as Vibrio gazogenes, and has been preserved in the Guangdong Microbial Culture Collection Center, with the preservation number GDMCC NO: 66510, the preservation date June 13, 2025, and the preservation address 5th Floor, No. 59 Building, 100 Middle Field Road, Guangzhou.

[0008] The strain B1D10 is used in the production of prodigiosin.

[0009] A prodigiosin synthesis gene cluster derived from the strain B1D10, which contains 16 genes, and the genes are as follows:

[0010] (1) Orf1 gene, the nucleotide sequence is shown in SEQ ID NO. 2, the length is 450 bp, and it encodes an unknown protein with a length of 149 amino acids;

[0011] (2) Orf2 gene, the nucleotide sequence is shown in SEQ ID NO. 3, the length is 318 bp, and it encodes a DUF3634 family protein with a length of 105 amino acids;

[0012] (3) Orf3 gene, the nucleotide sequence is shown in SEQ ID NO. 4, the length is 1821 bp, and it encodes a protein containing a GGDEF domain with a length of 606 amino acids;

[0013] (4) pigB gene, the nucleotide sequence is shown in SEQ ID NO. 5, the length is 2064 bp, and it encodes a protein containing a DUF6041 domain with a length of 687 amino acids;

[0014] (5) pigC gene, the nucleotide sequence is shown in SEQ ID NO. 6, the length is 2661 bp, and it encodes a pigC protein with a length of 886 amino acids;

[0015] (6) pigD gene, the nucleotide sequence is shown in SEQ ID NO. 7, the length is 2589 bp, and it encodes a pigD protein with a length of 862 amino acids;

[0016] (7) pigE gene, the nucleotide sequence is shown as SEQ ID NO. 8, the length is 2553 bp, encoding pigE protein, the length is 850 amino acids;

[0017] (8) pigF gene, the nucleotide sequence is shown as SEQ ID NO. 9, the length is 1035 bp, encoding PigF protein, the length is 344 amino acids;

[0018] (9) pigG gene, the nucleotide sequence is shown as SEQ ID NO. 10, the length is 264 bp, encoding pigG protein, the length is 87 amino acids;

[0019] (10) pigH gene, the nucleotide sequence is shown as SEQ ID NO. 11, the length is 1938 bp, encoding pigH protein, the length is 645 amino acids;

[0020] (11) pigI gene, the nucleotide sequence is shown as SEQ ID NO. 12, the length is 1467 bp, encoding pigI protein length 488 amino acids;

[0021] (12) pigJ gene, the nucleotide sequence is shown as SEQ ID NO. 13, the length is 2370 bp, encoding pigJ protein, the length is 789 amino acids;

[0022] (13) pigK gene, the nucleotide sequence is shown as SEQ ID NO. 14, the length is 315 bp, encoding pigE protein, the length is 104 amino acids;

[0023] (14) pigL gene, the nucleotide sequence is shown as SEQ ID NO. 15, the length is 741 bp, encoding pigL, the length is 246 amino acids;

[0024] (15) pigM gene, the nucleotide sequence is shown as SEQ ID NO. 16, the length is 1050 bp, encoding pigN protein, the length is 349 amino acids;

[0025] (16) pigN gene, the nucleotide sequence is shown as SEQ ID NO. 17, the length is 729 bp, encoding pigN protein, the length is 242 amino acids.

[0026] The gas-producing Vibrio B1D10 provided by the present application can produce a bright red pigment compound, named prodigiosin, when cultured in liquid and solid medium, which is controlled by a gene cluster formed by 16 genes. The Vibrio B1D10 can reproduce rapidly, and can be applied to large-scale fermentation production of prodigiosin. The red pigment has a strong bacteriostatic effect, and has potential application value in the field of medicine research. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is the colony morphology of strain B1D10 cultured on solid plate for 24h.

[0028] Figure 2 is the morphology of strain B1D10 cultured in liquid for 24h.

[0029] Figure 3 is the prodigiosin extracted from strain B1D10 with methanol.

[0030] Figure 4 is the absorption peak of the pigment extracted from strain B1D10.

[0031] Figure 5 is the gene cluster for synthesizing prodigiosin by strain B1D10; the numbers are from left to right, and the gene numbers are Orf1 to pigN in sequence. DETAILED DESCRIPTION

[0032] The present application will be further described in detail in combination with specific implementation cases.

[0033] The experimental methods used in the following implementation cases are all conventional methods unless otherwise specified; and the materials, reagents, etc. used are all available from commercial channels unless otherwise specified.

[0034] Example 1 Isolation and identification of the strain

[0035] 1. Isolation of the strain

[0036] Strain B1D10 was isolated from Spartina alterniflora collected at the dock (north latitude 24.8511, east longitude 118.6529) of Wenhailu, Quanzhou City, Fujian Province.

[0037] A small shovel was used to dig out the Spartina alterniflora growing on the beach and wash the mud attached to the roots with tap water. A small piece of root filament about 1g was cut off with scissors, washed 1-2 times in sterile seawater, then placed in 9ml of sterile seawater and vortexed to prepare a bacterial suspension (10 -1 180uL of Zobell 2216E marine bacterial culture medium (5g / L peptone, 1g / L yeast powder, 1L natural seawater) was sucked into a 96-well cell culture plate with a syringe, covering the entire plate; then 10-1 Dilution gradient of bacterial suspension 1 uL, inoculation; inoculated 96-well cell culture plates were placed in a 30°C incubator for 5 days, and the turbidity of the bacteria in the 96-well plate was observed every day to determine the growth of the bacterial cells. One well was observed to be red in color, and 10 uL of streaked Zobell 2216E medium plates were further purified to obtain the strain, designated as B1D10 strain.

[0038] 2. Morphological characteristics of B1D10 colony

[0039] Strain B1D10, grown on Zobell 2216E marine bacterial culture medium (5 g / L peptone, 1 g / L yeast powder, 1 L natural seawater) for 24 h, the colony was bright red, about 0.1 cm in diameter, round, convex, and the colony was as shown in Figure 1 30°C, 150 rpm liquid culture for 24 h, the bacterial solution was bright red, as shown in Figure 2 .

[0040] 3. Physiological and biochemical characteristics of the strain

[0041] Strain B1D10 was gram-negative, oxidase-negative, and weakly positive for hydrogen peroxide.

[0042] The strain can utilize citric acid, hydrolyze gelatin and esculin ferric citrate, and ferment glucose, mannitol, sucrose, amygdalin, and arabinose to produce acid; it can use dextrose, levorotatory arabinose, dextrose, mannitol, N-acetylglucosamine, malic acid, and potassium citrate as the sole carbon source for growth. The strain showed positive reactions for alkaline phosphatase, lipid esterase (C8), lipidase (C14), leucine aminopeptidase, alpha-galactosidase, beta-galactosidase, and beta-glucosidase; and weakly positive reactions for esterase (C4), acid phosphatase, naphthol-AS-BI-phosphohydrolase, and alpha-glucosidase.

[0043] 4. Identification and phylogenetic analysis of 16S rRNA gene of the strain

[0044] 1 ml of fresh bacterial liquid was taken, and the cells were collected by centrifugation at 13000 rpm, and the genomic DNA of the strain was extracted using a Shanghai SAB Bioscience DNA extraction kit. Then, the 16S rDNA gene sequence of the strain was amplified and sequenced using the Ex Taq PCR system with the genomic DNA as the template and Eub27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3' as primers (as shown in SEQ ID NO. 1). Through sequence similarity comparison analysis and phylogenetic analysis, it was shown that the 16S rRNA gene sequence of the strain B1D10 was 1413 bp in length, and sequence alignment found that the strain had the largest similarity with Vibrio gazogenes ATCC 29988 T , with a similarity of 99.3%. The 16S rRNA gene phylogenetic tree showed that the strain belonged to the genus Vibrio, and was in a branch with Vibrio gazogenes ATCC 29988 T , and it was preliminarily judged that the strain was Vibrio gazogenes.

[0045] 5. Whole genome and genome sequence identification of strain B1D10

[0046] The extracted strain B1D10 was subjected to whole genome sequencing by second-generation whole genome sequencing technology; double-end reads of the strain sequence were obtained, and the whole genome sequence of the strain was obtained after quality control and assembly. The genome size of the strain B1D10 was 4,951,647 bp, containing 53 contigs (sequence length greater than 1000 bp), and the genome GC content was 45.1%. Whole genome comparative analysis found that the average nucleotide similarity of the strain with Vibrio gazogenes ATCC 29988 T was 97.8%, which strongly supported that the strain B1D10 was identified as Vibrio gazogenes.

[0047] In summary, through 16S rRNA gene sequence similarity comparison analysis, phylogenetic analysis, and genome alignment analysis, it was shown that the strain B1D10 belonged to the class Gammaproteobacteria, the order Vibrionales, the family Vibrionaceae, the genus Vibrio, and Vibrio gazogenes.

[0048] Example 2: Production of prodigiosin by using strain B1D10

[0049] In view of the rapid propagation of the strain B1D10, the strain B1D10 strain can be used for large-scale fermentation production of prodigiosin, and the specific steps are as follows:

[0050] (1) Strain culture: use Zobell 2216E marine bacteria solid culture medium to carry out three-zone streaking, and activate the strain. The formula of the 2216E marine bacteria solid culture medium is: 5 g of proteose peptone, 1 g of yeast powder, 15 g of agar powder, 1000 ml of natural seawater, and pH ~ 7.2.

[0051] (2) Fermentation culture: the culture medium is Zobell 2216E marine bacteria liquid culture medium, and the fermentation conditions are 30°C, shaking bed shaking (150-160 rpm), and culturing for 2 days; the formula of the Zobell 2216E marine bacteria liquid culture medium is: 5 g of proteose peptone, 1 g of yeast powder, 1000 ml of natural seawater, and pH ~ 7.2.

[0052] (3) Extraction of prodigiosin: first, centrifuge the fermentation broth at 5000-8000 rpm for 10 minutes; remove the supernatant to obtain the centrifuged cells; then add methanol to dissolve the red pigment in methanol; centrifuge at 5000-8000 rpm for 5-10 minutes to remove the precipitate, and collect the dissolved methanol solution; rotary evaporation is performed on the solution to recover methanol, and the crude extract is obtained.

[0053] (4) Purification of prodigiosin:

[0054] The above crude extract is dissolved in ethyl acetate (as shown in Figure 3 ), and then developed on a preparation plate (the developing agent is acetone: petroleum ether = 1.5:5 by volume). After scraping the red silica gel strip, the prodigiosin dissolved in ethyl acetate is obtained by filtering with filter paper. After drying at 50-60°C, the prodigiosin compound is obtained. The light absorption peak is measured by a UV-visible spectrometer. It is found that the maximum absorbance of the red pigment extracted from the strain is at 530-536 nm, as shown in Figure 4 .

[0055] Example 3 Gene cluster encoding synthesis of prodigiosin in the genome of strain B1D10

[0056] Through the second-generation whole genome sequencing technology, double-end reads of the strain sequence are obtained, and after quality control, assembly, Prodigal gene prediction, and alignment of the nr database annotation, the genome of the strain B1D10 contains a gene cluster for synthesizing prodigiosin, with a length of 25108 bp, and a total of 16 genes Figure 5 .

[0057] (1) Orf1 gene, the nucleotide sequence of which is shown in SEQ ID NO. 2, 450 base pairs (bp) in length, encoding an unknown protein, 149 amino acids in length;

[0058] (2) Orf2 gene, the nucleotide sequence of which is shown in SEQ ID NO. 3, 318 bp in length, encoding a DUF3634 family protein, 105 amino acids in length;

[0059] (3) Orf3 gene, the nucleotide sequence of which is shown in SEQ ID NO. 4, 1821 bp in length, encoding a protein containing a GGDEF domain, 606 amino acids in length;

[0060] (4) pigB gene, the nucleotide sequence of which is shown in SEQ ID NO. 5, 2064 bp in length, encoding a protein containing a DUF6041 domain, 687 amino acids in length;

[0061] (5) pigC gene, the nucleotide sequence of which is shown in SEQ ID NO. 6, 2661 bp in length, encoding a pigC protein, 886 amino acids in length;

[0062] (6) pigD gene, the nucleotide sequence of which is shown in SEQ ID NO. 7, 2589 bp in length, encoding a pigD protein, 862 amino acids in length;

[0063] (7) pigE gene, the nucleotide sequence of which is shown in SEQ ID NO. 8, 2553 bp in length, encoding a pigE protein, 850 amino acids in length;

[0064] (8) pigF gene, the nucleotide sequence of which is shown in SEQ ID NO. 9, 1035 bp in length, encoding a PigF protein, 344 amino acids in length;

[0065] (9) pigG gene, the nucleotide sequence of which is shown in SEQ ID NO. 10, 264 bp in length, encoding a pigG protein, 87 amino acids in length;

[0066] (10) pigH gene, the nucleotide sequence of which is shown in SEQ ID NO. 11, 1938 bp in length, encoding a pigH protein, 645 amino acids in length;

[0067] (11) pigI gene, the nucleotide sequence of which is shown in SEQ ID NO. 12, 1467 bp in length, encoding a pigI protein, 488 amino acids in length;

[0068] (12) pigJ gene, the nucleotide sequence is shown as SEQ ID NO. 13, 2370 bp in length, encoding pigJ protein, 789 amino acids in length;

[0069] (13) pigK gene, the nucleotide sequence is shown as SEQ ID NO. 14, 315 bp in length, encoding pigE protein, 104 amino acids in length;

[0070] (14) pigL gene, the nucleotide sequence is shown as SEQ ID NO. 15, 741 bp in length, encoding pigL, 246 amino acids in length;

[0071] (15) pigM gene, the nucleotide sequence is shown as SEQ ID NO. 16, 1050 bp in length, encoding pigN protein, 349 amino acids in length;

[0072] (16) pigN gene, the nucleotide sequence is shown as SEQ ID NO. 17, 729 bp in length, encoding pigN protein, 242 amino acids in length.

Claims

1. A prodigiosus-producing strain B1D10, characterized in that, The strain B1D10 is classified as Vibrio gazogenes, which has been preserved in Guangdong Microbial Culture Collection Center, with the preservation number of GDMCC NO: 66510, the preservation date of June 13, 2025, and the preservation address of 5th floor, No. 59, Building 100, Xianlie Middle Road, Guangzhou.

2. Application of the strain B1D10 in production of prodigiosin according to claim 1.

3. A prodigiosin synthesis gene cluster derived from the strain B1D10 of claim 1, characterized in that, The gene cluster contains 16 genes, which are as follows: (1) Orf1 gene, the nucleotide sequence of which is shown in SEQ ID NO. 2, with a length of 450 bp, encoding an unknown protein with a length of 149 amino acids; (2) Orf2 gene, the nucleotide sequence of which is shown in SEQ ID NO. 3, with a length of 318 bp, encoding a DUF3634 family protein with a length of 105 amino acids; (3) Orf3 gene, the nucleotide sequence of which is shown in SEQ ID NO. 4, with a length of 1821 bp, encoding a protein containing a GGDEF domain with a length of 606 amino acids; (4) pigB gene, the nucleotide sequence of which is shown in SEQ ID NO. 5, with a length of 2064 bp, encoding a protein containing a DUF6041 domain with a length of 687 amino acids; (5) pigC gene, the nucleotide sequence of which is shown in SEQ ID NO. 6, with a length of 2661 bp, encoding a pigC protein with a length of 886 amino acids; (6) pigD gene, the nucleotide sequence of which is shown in SEQ ID NO. 7, with a length of 2589 bp, encoding a pigD protein with a length of 862 amino acids; (7) pigE gene, the nucleotide sequence of which is shown in SEQ ID NO. 8, with a length of 2553 bp, encoding a pigE protein with a length of 850 amino acids; (8) pigF gene, the nucleotide sequence of which is shown in SEQ ID NO. 9, with a length of 1035 bp, encoding a PigF protein with a length of 344 amino acids; (9) pigG gene, the nucleotide sequence of which is shown in SEQ ID NO. 10, with a length of 264 bp, encoding a pigG protein with a length of 87 amino acids; (10) pigH gene, the nucleotide sequence of which is shown in SEQ ID NO. 11, with a length of 1938 bp, encoding a pigH protein with a length of 645 amino acids; (11) pigI gene, the nucleotide sequence of which is shown in SEQ ID NO. 12, with a length of 1467 bp, encoding a pigI protein with a length of 488 amino acids; (12) pigJ gene, the nucleotide sequence of which is shown in SEQ ID NO. 13, with a length of 2370 bp, encoding a pigJ protein with a length of 789 amino acids; (13) pigK gene, the nucleotide sequence of which is shown in SEQ ID NO. 14, with a length of 315 bp, encoding a pigE protein with a length of 104 amino acids; (14) pigL gene, the nucleotide sequence is shown as SEQ ID NO. 15, the length is 741 bp, encoding pigL, the length is 246 amino acids; (15) pigM gene, the nucleotide sequence is shown as SEQ ID NO. 16, the length is 1050 bp, encoding pigN protein, the length is 349 amino acids; (16) pigN gene, the nucleotide sequence is shown as SEQ ID NO. 17, the length is 729 bp, encoding pigN protein, the length is 242 amino acids.

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