CN120192995A - A method for transforming Bacillus subtilis to produce phycocyanin - Google Patents
A method for transforming Bacillus subtilis to produce phycocyanin Download PDFInfo
- Publication number
- CN120192995A CN120192995A CN202510227764.2A CN202510227764A CN120192995A CN 120192995 A CN120192995 A CN 120192995A CN 202510227764 A CN202510227764 A CN 202510227764A CN 120192995 A CN120192995 A CN 120192995A
- Authority
- CN
- China
- Prior art keywords
- phycocyanin
- gene
- pcya
- bacillus subtilis
- vector
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
- C12N9/0083—Miscellaneous (1.14.99)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
- C12Y103/07—Oxidoreductases acting on the CH-CH group of donors (1.3) with an iron-sulfur protein as acceptor (1.3.7)
- C12Y103/07005—Phycocyanobilin:ferredoxin oxidoreductase (1.3.7.5)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
- C12Y114/99—Miscellaneous (1.14.99)
- C12Y114/99003—Heme oxygenase (1.14.99.3)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to the technical field of phycocyanin, and particularly discloses a method for modifying bacillus subtilis to produce phycocyanin, which comprises the following steps of preparing a culture medium, synthesizing a key gene for phycocyanin biosynthesis, constructing a carrier, constructing a high-efficiency coenzyme factor cycle for phycocyanin synthesis, synthesizing an ancestral sequence of a ferredoxin oxidoreductase gene PcyA through calculation biology, adopting a bacillus subtilis expression system to express a heterologous synthetic phycocyanin gene, improving the yield of phycocyanin by increasing the synthesis of coenzyme, improving the yield of 20.42mg/L in a shake flask, improving the yield of phycocyanin in the bacillus expression system, and simplifying an endotoxin removal step for the later purification of the phycocyanin by using the bacillus expression system, so that the purification and the use of the phycocyanin are safer.
Description
Technical Field
The invention belongs to the technical field of phycocyanin, and particularly relates to a method for modifying bacillus subtilis to produce phycocyanin.
Background
Phycocyanin (also called phycocyanin or phycobiliprotein) is a pigment protein complex mainly existing in red algae, blue algae and the like, has the capability of capturing blue-green light, and has wide application in the fields of foods, cosmetics, health products, medicines and the like. It consists of apoprotein and chromophore, and has fluorescent activity, and its synthesis process involves the action of several enzymes, such as porphobilinogen synthase, uroporphyrinogen III synthase, etc.
At present, the method for obtaining phycocyanin mainly adopts a high-temperature methanol decomposition method to extract the phycocyanin from spirulina. The method has a plurality of problems:
(1) The high-temperature pyrolysis of the methanol needs to consume a large amount of energy, so that energy waste is caused;
(2) Other pigments in spirulina that coexist with phycocyanin, such as chlorophyll, lutein and a series of precursors, have adverse effects on the purification of the later-stage products;
(3) The spirulina has longer growth period, and limits the production efficiency and application of phycocyanin. Thus, extracting high purity PCB from natural algae is a complex and uneconomical method.
To this problem, jiang Nada, zhao Xinrui et al have conducted studies on phycocyanin synthesis, heterologously expressed specific genes in E.coli and enhanced heme synthesis, realized conversion of protoheme to phycocyanin synthesis intermediate biliverdin, realized yield of phycocyanin 28.32mg/L in a 5L fermenter, and university of Jiangnan Zhou Jingwen et al realized conversion of protoheme to phycocyanin synthesis intermediate biliverdin by heterologously expressing specific genes in E.coli and enhanced heme synthesis, reduced accumulation of intermediate biliverdin, produced phycocyanin by fermentation, and finally produced 147mg/L.
The use of the above technique increases the biosynthesis of phycocyanin by a large step, but the yield is difficult to meet the requirement of industrial production, and in addition, the problem of endotoxin detection is also faced after the purification of phycocyanin due to the adoption of an escherichia coli expression system.
Disclosure of Invention
The invention aims to provide a method for modifying bacillus subtilis to produce phycocyanin, which aims to solve the problems in the background technology.
In order to achieve the above purpose, the present invention provides the following technical solutions:
A method for modifying bacillus subtilis to produce phycocyanin comprises the following steps:
S1, preparing a culture medium:
The LB culture medium comprises 5g/L Angel yeast extract powder, 10g/L peptone and 10g/L sodium chloride, and 10g/L agar is added when preparing LB solid culture medium;
TB medium comprises peptone 12g/L, yeast extract 24g/L, glycerol 0.4%, potassium dihydrogen phosphate 2.313g/L, which is the product of separate autoclaving;
The fermentation medium comprises 10g of yeast powder, 30g of glycerol, 3g/L of dipotassium hydrogen phosphate, 1.5g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate, 0.35g/L of ammonium sulfate, 0.2g/L of yeast extract powder, 0.2g/L of manganese sulfate, 0.1g/L of ferrous sulfate and 0.1g/L of calcium carbonate;
s2, synthesis of phycocyanin biosynthesis key genes and vector construction
The successful pMATE-HO 1-T2A-PcyA vector is constructed by chemically synthesizing the ancestral sequences of the heme oxygenase Ho1 gene, the ferredoxin oxidoreductase gene PcyA and the self-cleaving peptide tag T2A and loading the sequences into the pMATE vector;
the nucleotide sequence of the heme oxygenase Ho1 gene (YP_ 214522.1) is shown as SEQ ID NO. 1;
The ancestral sequence of the ferredoxin oxidoreductase gene PcyA has a nucleotide sequence shown as SEQ ID NO. 2;
The nucleotide sequence of the self-cleaving peptide tag T2A is shown as SEQ ID NO. 3;
s3, constructing phycocyanin synthesized efficient coenzyme factor circulation
Under the induction of heme oxidase and ferredoxin oxidoreductase, phycocyanin is successfully produced, however, the step of converting heme into biliverdin is a speed limiting step, and the participation of coenzyme NADPH is needed, so that the circulation of the coenzyme factor is accelerated and the production of phycocyanin is increased by knocking out the bacillus subtilis outer membrane channel protein Tolc and introducing the nad gene.
Preferably, the pMATE-HO 1-T2A-PcyA vector which is successfully constructed is transferred into an expression strain in an electrotransformation mode to obtain a recombinant strain containing the HO1-T2A-PcyA gene.
Preferably, the recombinant strain is cultured in LB medium or TB medium.
Preferably, the recombinant strain is fermented using a fermentation medium and induced using maltose.
Compared with the prior art, the invention has the beneficial effects that:
According to the invention, an ancestral sequence of the ferredoxin oxidoreductase gene PcyA is synthesized through calculation biology, a bacillus subtilis expression system is adopted to express heterologous synthetic phycocyanin genes, the yield of phycocyanin is improved through increasing the synthesis of coenzyme, the yield of phycocyanin reaches 20.42mg/L in a shake flask, the yield of phycocyanin in a bacillus expression system is improved, the use of the bacillus expression system simplifies endotoxin removal steps for the subsequent purification of phycocyanin, and the purification and use of phycocyanin are safer.
Drawings
FIG. 1 shows a plasmid map of the recombinant vector pMAT-HO1-T2A-pcyA of the invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
A method for modifying bacillus subtilis to produce phycocyanin comprises the following steps:
Wherein the culture medium is as follows:
LB culture medium, 10g/L peptone, 10g/L sodium chloride, 5g/L yeast extract, and sterilizing at 121 ℃ for 15min.
TB medium 6g/L peptone, 5g/L glycerol, 12g/L yeast extract, 17mM potassium dihydrogen phosphate, 72mM dipotassium hydrogen phosphate, and sterilizing at 115℃for 20min.
Embodiment one:
An emerging technique for generating stable enzymes is Ancestral Sequence Reconstruction (ASR), a bioinformatic method that predicts evolutionary ancestors based on a collection of homologous sequences.
ASR-produced enzymes typically have improved biocatalytic properties, such as enhanced thermostability, solvent tolerance, and substrate diversity.
The ancestral sequence was subjected to library analysis by Mafft using pcyA derived from Synechocystis (Synechocystis sp.) PCC6803 as a template from NCBI alignment to identify >60%, and codon optimization was performed using Bacillus subtilis genome as a model after the ancestral sequence was obtained.
Embodiment two:
The method comprises the steps of entrusting a certain of the Enteromorpha proliferator-activated enzyme (HO-214522.1) gene of a heme oxygenase from Prochlorococcus phage P-SSM2 to be subjected to total gene chemical synthesis, wherein the nucleotide sequence is shown as SEQ ID NO.1, synthesizing an ancestral sequence of a ferredoxin oxidoreductase gene PcyA, the nucleotide sequence is shown as SEQ ID NO.2, synthesizing a self-cleaving peptide tag T2A shown as SEQ ID NO.3, loading the synthesized sequence into a pMATE carrier through bridge PCR, and constructing a successful pMATE03-HO1-T2A-PcyA carrier. Transferring the vector into a WB600 expression strain by electrotransformation to obtain a recombinant strain S1 containing HO1-T2A-PcyA genes. The recombinant strain was shake-flask fermented using LB culture, and when the recombinant Bacillus was cultured at 37℃to an OD 600 value of 0.6-0.8, the culture was continued at 30℃for 24 hours after adding 3% maltose. The results of the fermentation protein expression detection are shown in the figure, the recombinant strain is fermented, maltose is used for induction, and the phycocyanin content reaches 14.52mg/L.
Embodiment III:
The genome of B.subtilis 168 is used as a template, the primers tolc-1-F/tolc-1-R and kan-F/kan-R are used for respectively amplifying the gene fragment tolc-1 with the length of the first 500bp of the outer membrane channel protein, and the Kanamycin (KANAMYCIN) resistance gene fragment kan is obtained by amplification, the two fragments are connected by using an overlap PCR technology, and a specific electrophoresis band is found to appear at about 2000bp by 1% agarose gel electrophoresis detection, and the size accords with the theoretical value 2002bp, namely the two fragments are connected into tolc-1-kan fragments. After the fragment is recovered by gel and BamHI restriction enzyme, the amyE-1-kan after the fragment is digested is converted into B.subtilis WB600 by the above electric conversion method, a positive clone is selected, PCR is verified to verify kan gene fragment, a specific electrophoresis band is found to appear at about 1500bp through 1% agarose gel electrophoresis detection, and the size accords with the theoretical value 1502bp, which means that the outer membrane channel protein tolc is successfully knocked out from the B.subtilis WB600 genome by a single-crossover interchange mode, thus obtaining the recombinant strain S2.
Embodiment four:
The bacillus subtilis with the outer membrane protein Tolc knocked out is taken as a chassis strain, pMATE03-HO1-T2A-PcyA vectors are introduced in an electrotransduction mode, a recombinant strain S3 is constructed, the recombinant strain is subjected to shake flask fermentation by using a TB culture medium, and when the recombinant bacillus is cultured at 37 ℃ to an OD 600 value of 1.2, 3% maltose is added, and then the culture is continued for 24 hours at 30 ℃. Fermenting the recombinant strain, and inducing with maltose to obtain phycocyanin content of 20.42mg/L
It should be appreciated that in the development of any such actual implementation, as in any engineering or design project, numerous implementation-specific decisions may be made. Such a development effort might be complex and time consuming, but would nevertheless be a routine undertaking of design, fabrication, and manufacture for those of ordinary skill having the benefit of this disclosure.
It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered in the scope of the claims of the present invention.
SEQ ID NO.1
HO-1 gene:
ATGACCGTTGCTGATTTCTCCGTTCAGATCAAAGAAGGTACTAAGAAGAGCCACTCTGCTGCGGAGAACACCAGCTTCGTTGCGAGCTTCTTGCGTGGTGTGGTTTCTAAAGAATCTTACAAAGCGCTGGTTAAAGACCTGTACTACGTTTACCGTACTCTGGAAGAAGAGTTCGAGAAACACAAAGACCATCCGGTTGTAGGTAAACTGTACCTGCCGGAACTGAACCGTGTTAACGCACTGGAACGTGATCTGCGTTTCTACTACGGTCCGATCTGGCGTTCTCTGATCATGCCATCCGAAGCGTGCGCGAACTACGTTTCTCGTATCAAATGCTGCTCTATCGAAGATCCAACTCTGCTGGTTGGTCACCACTACACTCGTTACCTGGGCGACCTGTCTGGTGGTCAGATCTTGAAAGGCATCGCGGAGAAAGCGATGGGTCTGAAAGATGAAGGTCTGTACTTCTACGACTTCGACAAGATCGAGAACGCGAAGAAATACAAAGATGGTTACCGTGCGATTCTGAACGGTCTGGACGTTGACCAGCACCAGGTAGACGCTATCATCGTTGAAGCGAACTACGCGTTCCGTCTGAACATGTATATGTTCGACACCTTGGAAGGTAACTGGTTCCAGTCCCTGATCCAGATGATCTTCGGTTTCATCAAATCTATCTTGAAGAAACGTAAATCCGAA.
SEQ ID NO.2:
pcya ancestral sequence genes:
ATGAGAGGAAGAATATGCATTCTGTTAGCAGCTCCACCGGCAATGACGAGCAGCAAGAGCTCAAAAACAACGTTAGAACCGGTAAGAGCGCCTCAAGTTCCGGGAGTCGGCTCCATGATGCCGGTAAGAATGATTGTTGATCAGAACAAAAAGACCTCTATGAACAGCGTGAAACTGGAAGATGCGGAGGGCTTATGCAGCGAACTGGCAAACGTGATACGAAATGCCATCGAGTCTCTGGATGATGTCAATGAACCTCTTGAATGCGATCCGGATTTAGAGGCGATTTATGAGGAAATTGAGAAAGATGATCTTGTTATCAAGAACGAAATGTACAAATGCAAGGGCTTAAGAAAGATACACTTAGAAACGGCTAAAACAGGCAAGAATCTCGATGTTCTCCACTGCGTATTTTTCCCAGATTTCGAGTATAACATTCCGATATTCGGTTGCGATATTGTAGCTACACGCAACATCGTGACAGCAGCGATTGTGGACATTTCCCCAGTCCATGGATGTAGTCCAGAAGAAATCTATTATGGTTACGATAAAATCGCTCCTATCTCCAACAGTTATCATTTCAAGGAAAAAAGACCACTTCCACTGTGGGGTGATGAGATTTTTTCCCCGTATTGCAAATTCGTTCGTCTCCGGAACGAGGAAGAACAAGATGACTTCGTCAGAATCGTTAAAGAATATTTGAACATCTTTTGCGATTGGGTTCGTAAAGCAGAAAAGGATTCCAATCATGGTACATTAGATAACTGGGTAAATACTATGTTAAGACTTGACGATCAAATCTGGTACTGTAAGCAGCAGCGGAAAAATAAAAAGACGCTTGCCGTACTGTCTCAATGGTTCGACAAAGAATGGGCGGATAACTACATCAACAACATTCTTTTTGACAAACCTCCGAAGCTGAAGGAACAGCAGGATTATAACCCATTTGATTACGTGAACAGCATTAACCTTAAAACGGCTGACTACACCTCTGACGAAGGATACATGTCCCAGTTACCGAGCTTTCAGCATCAGCGGGAAGCCCGGTATTTTATTGACACCGAAACACATACGCACCAGATGAATCGCCTTGGAGCCAGTTTGGATAAGGACATGCAATATAACTTCTTGTGGTTTATGTTAGGCAACCTTCGGGGCTTTTTGGTAGCGCAAAAATCACTGTTGACACTGATC.
SEQ ID NO.3
self-cleaving peptide tag T2A
GAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCA
Acetyl-coa synthase gene:
ATGTCTCAGATCCACAAACACACCATCCCGGCTAACATCGCGGATCGTTGCCTGATCAATCCGCAGCAGTACGAAGCGATGTACCAGCAGTCTATCAACGTTCCGGACACCTTCTGGGGTGAACAGGGTAAGATTCTGGACTGGATCAAACCGTACCAGAAAGTTAAGAACACCTCTTTCGCTCCAGGTAACGTTTCCATCAAATGGTACGAAGATGGTACTCTGAACCTGGCAGCAAACTGCCTGGACCGTCATCTGCAAGAGAACGGTGATCGTACCGCGATCATCTGGGAAGGTGACGATGCGTCTCAGTCCAAACACATCTCTTACAAAGAACTGCACCGTGACGTTTGCCGTTTCGCTAACACCTTGCTGGAACTGGGTATCAAGAAAGGTGACGTTGTTGCGATCTACATGCCAATGGTTCCGGAAGCGGCTGTTGCAATGCTGGCGTGTGCTCGTATCGGTGCGGTTCACTCTGTTATCTTCGGTGGTTTCTCTCCAGAAGCTGTTGCTGGTCGTATCATCGACTCTAACAGCCGTCTGGTAATCACCTCTGATGAAGGTGTTCGTGCAGGTCGTTCTATTCCGCTGAAGAAGAACGTGGATGACGCACTGAAGAATCCGAACGTGACCTCTGTTGAACACGTTGTTGTTCTGAAACGTACCGGTGGTAAGATCGACTGGCAAGAAGGTCGTGACCTGTGGTGGCACGATCTGGTTGAACAGGCGTCTGATCAGCACCAGGCTGAAGAAATGAACGCTGAGGACCCGCTGTTCATTCTGTACACTTCTGGTTCTACCGGTAAACCGAAAGGTGTTCTGCACACTACCGGTGGTTACCTGGTTTACGCTGCGCTGACCTTCAAATACGTGTTCGACTACCATCCAGGTGACATCTACTGGTGTACCGCGGACGTTGGTTGGGTTACCGGTCACTCTTACCTGCTGTACGGTCCGCTGGCGTGCGGTGCGACTACCTTGATGTTCGAAGGTGTTCCAAACTGGCCGACTCCGGCACGTATGGCACAGGTTGTTGACAAACACCAGGTTAACATTCTGTATACCGCTCCAACCGCTATCCGTGCACTGATGGCAGAAGGTGACAAAGCGATCGAAGGTACTGACCGTTCTTCTCTGCGTATCTTGGGTTCTGTTGGTGAACCAATCAATCCGGAAGCATGGGAATGGTACTGGAAGAAGATCGGTAACGAGAAATGTCCGGTTGTTGATACCTGGTGGCAGACCGAAACTGGTGGTTTCATGATCACTCCGCTTCCAGGTGCTACTGAACTGAAAGCGGGTTCTGCGACTCGTCCGTTCTTCGGTGTTCAGCCGGCTCTGGTTGACAACGAAGGTAACCCGCTGGAAGGTGCTACCGAAGGTTCTCTGGTTATCACCGACAGCTGGCCAGGTCAGGCGCGTACCTTGTTCGGTGACCACGAACGTTTCGAACAGACCTACTTCTCTACCTTCAAGAACATGTACTTCTCTGGTGACGGTGCACGTCGTGACGAAGATGGTTACTACTGGATCACCGGTCGTGTTGACGACGTTCTGAACGTTTCTGGTCATCGTCTGGGTACTGCGGAAATCGAATCTGCGCTGGTTGCACATCCGAAGATCGCGGAAGCTGCTGTTGTTGGTATCCCACACAACATCAAAGGTCAGGCAATCTACGCGTACGTTACGCTGAACCACGGTGAGAAACCGTCTCCGGAACTGTACGCAGAAGTTCGTAACTGGGTTCGTAAAGAAATCGGTCCGTTGGCTACTCCGGACGTTCTGCACTGGACCGACTCTCTGCCAAAGACTCGTTCCGGTAAGATCATGCGTCGTATCTTGCGTAAGATCGCTGCTGGTGACACCTCTAACCTGGGTGACACTTCTACTCTGGCTGATCCGGGTGTTGTTGAGAAACTGCTGGAAGAGAAACAGGCGATCGCTATGCCGAGCTAA.
Claims (4)
1. The method for modifying bacillus subtilis to produce phycocyanin is characterized by comprising the following steps:
S1, preparing a culture medium:
The LB culture medium comprises 5g/L Angel yeast extract powder, 10g/L peptone and 10g/L sodium chloride, and 10g/L agar is added when preparing LB solid culture medium;
TB medium comprises peptone 12g/L, yeast extract 24g/L, glycerol 0.4%, potassium dihydrogen phosphate 2.313g/L, which is the product of separate autoclaving;
The fermentation medium comprises 10g of yeast powder, 30g of glycerol, 3g/L of dipotassium hydrogen phosphate, 1.5g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate, 0.35g/L of ammonium sulfate, 0.2g/L of yeast extract powder, 0.2g/L of manganese sulfate, 0.1g/L of ferrous sulfate and 0.1g/L of calcium carbonate;
s2, synthesis of phycocyanin biosynthesis key genes and vector construction
The successful pMATE-HO 1-T2A-PcyA vector is constructed by chemically synthesizing the ancestral sequences of the heme oxygenase Ho1 gene, the ferredoxin oxidoreductase gene PcyA and the self-cleaving peptide tag T2A and loading the sequences into the pMATE vector;
the nucleotide sequence of the heme oxygenase Ho1 gene (YP_ 214522.1) is shown as SEQ ID NO. 1;
The ancestral sequence of the ferredoxin oxidoreductase gene PcyA has a nucleotide sequence shown as SEQ ID NO. 2;
the nucleotide sequence of the self-cleaving peptide tag T2A is shown as SEQ ID NO. 3;
s3, constructing phycocyanin synthesized efficient coenzyme factor circulation
Under the induction of heme oxidase and ferredoxin oxidoreductase, phycocyanin is successfully produced, however, the step of converting heme into biliverdin is a speed limiting step, and the participation of coenzyme NADPH is needed, so that the circulation of the coenzyme factor is accelerated and the production of phycocyanin is increased by knocking out the bacillus subtilis outer membrane channel protein Tolc and introducing the nad gene.
2. The method for modifying Bacillus subtilis phycocyanin production according to claim 1, wherein the pMATE-HO 1-T2A-PcyA vector which is successfully constructed is transferred into an expression strain by electrotransformation to obtain a recombinant strain containing HO1-T2A-PcyA gene.
3. The method for modifying Bacillus subtilis to produce phycocyanin according to claim 1, wherein the recombinant strain is cultured in LB medium or TB medium.
4. The method of claim 1, wherein the recombinant strain is fermented using a fermentation medium and induced using maltose.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202510227764.2A CN120192995A (en) | 2025-02-28 | 2025-02-28 | A method for transforming Bacillus subtilis to produce phycocyanin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202510227764.2A CN120192995A (en) | 2025-02-28 | 2025-02-28 | A method for transforming Bacillus subtilis to produce phycocyanin |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN120192995A true CN120192995A (en) | 2025-06-24 |
Family
ID=96066009
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202510227764.2A Pending CN120192995A (en) | 2025-02-28 | 2025-02-28 | A method for transforming Bacillus subtilis to produce phycocyanin |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN120192995A (en) |
-
2025
- 2025-02-28 CN CN202510227764.2A patent/CN120192995A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Asada et al. | Photobiological hydrogen production | |
| CN118207172B (en) | Bifunctional glutathione synthase mutant and application thereof | |
| CN113604472B (en) | A CRISPR/Cas gene editing system applied to Trichoderma reesei | |
| CN118421574A (en) | 4-Hydroxyphenylacetic acid-3-hydroxylase mutant and its application | |
| CN117625504A (en) | A set of engineering bacteria for synthesizing 6,6’-dibromoindigo and its preparation method and application | |
| CN102382790B (en) | A kind of recombined bacillus subtilis of high yield catalase and its construction method and application | |
| CN115927332B (en) | A promoter for overexpressing protease, Streptomyces recombinant bacteria and its construction method and application | |
| CN104789516B (en) | A kind of genetic engineering bacterium of cytoalgae 6803 for producing trihydroxy propionic acid and construction method and application | |
| CN101250539B (en) | Method for preparing recombinant heat-proof beta-glucuronic acid enzyme | |
| CN120026012B (en) | Keratinase mutant, encoding gene and expression vector thereof and application of keratinase mutant | |
| CN113462628B (en) | Gene engineering bacterium for producing heme as well as construction method and application thereof | |
| CN120192995A (en) | A method for transforming Bacillus subtilis to produce phycocyanin | |
| CN110760533B (en) | Gene for coding glutamate decarboxylase, recombinant engineering bacterium and application thereof | |
| CN117535217B (en) | A recombinant Bacillus subtilis engineered strain and its application in biological preparation of ursodeoxycholic acid | |
| CN108060203B (en) | Method for producing 1, 3-propylene glycol by whole-cell mixed transformation of glycerol | |
| CN113061563B (en) | Method for synthesizing L-malic acid by utilizing recombinant escherichia coli whole cell catalysis | |
| CN101892228B (en) | A kind of highly acrylamide and acrylonitrile tolerance engineering bacteria producing nitrile hydratase and its application | |
| CN118256529A (en) | Recombinant bacterium for producing canthaxanthin and construction method and application thereof | |
| CN111394396B (en) | Method for producing 1, 3-propylene glycol by using glycerol fermentation by microorganisms | |
| CN102061290B (en) | Method for realizing over expression of thermostable laccase gene through location transformation | |
| CN114934057B (en) | IsDNA sequence suitable for high-efficiency expression of escherichia coli, preparation method, recombinant expression plasmid and engineering bacteria | |
| CN115991761B (en) | A pigment and its engineered production bacteria construction method | |
| CN121320403A (en) | Method for improving expression intensity of bacillus subtilis self-induction expression system | |
| CN120424896A (en) | A multi-enzyme combination for synthesizing sesamin, recombinant engineering bacteria and applications | |
| CN119464175A (en) | Escherichia coli producing capsanthin and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |