CN101705265B - Method for producing phenazine-1-carboxylic acid by using engineering bacterial strain M18G to carry plasmid pME6032Phz - Google Patents
Method for producing phenazine-1-carboxylic acid by using engineering bacterial strain M18G to carry plasmid pME6032Phz Download PDFInfo
- Publication number
- CN101705265B CN101705265B CN2009101986642A CN200910198664A CN101705265B CN 101705265 B CN101705265 B CN 101705265B CN 2009101986642 A CN2009101986642 A CN 2009101986642A CN 200910198664 A CN200910198664 A CN 200910198664A CN 101705265 B CN101705265 B CN 101705265B
- Authority
- CN
- China
- Prior art keywords
- carboxylic acid
- pme6032phz
- phenazine
- plasmid
- bacterial strain
- 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.)
- Active
Links
Abstract
The invention relates to a method for producing phenazine-1-carboxylic acid by using engineering bacterial strain M18G to carry plasmid pME6032Phz. The method is implemented by amplifying coding gene segment phzA1-G1 from cerebiogen antagonist antibiosis M18 genome for biosynthesis of phenazine-1-carboxylic acid, inserting the gene segment into expression plasmid pME6032, placing under control of strong promoten Ptac to construct a recombinant plasmid pME6032Phz; then introducing the recombinant plasmid in the derivative bacterial strain M18G of cerebiogen antagonist antibiosis M18 to construct an engineering bacterial strain M18G/pME6032Phz. The engineering bacterial strain can realize efficient and stable expression of coding genes while amplifying reproduction number of coding genes for biosynthesis of phenazine-1-carboxylic acid. Finally the engineering bacterial strain M18G/pME6032Phz is cultured in soybean meal culture solution to efficiently and stably produce phenazine-1-carboxylic acid with vastly enhanced yield. By using the high yield engineering bacterial strain to produce phenazine-1-carboxylic acid, the production cost can meet the agricultural requirement for application in preventing and curing rice sheath blight disease, and the invention is applicable to preparation of bactericide phenazino-1-carboxylic acid and can be used for preventing and curing plant diseases in large scale.
Description
Technical field
The present invention relates to a kind of production method of microbial source sterilant, relate in particular to a kind of growth-promoting antagonistic bacterium derivative strain M18G that utilizes, make up the engineering strain M18G/pME6032Phz that carries plasmid pME6032Phz, the method for High-efficient Production phenazine-1-carboxylic acid.Belong to the microbial pesticide production technical field.
Background technology
The loss severity that corps diseases causes accounts for 25～75% of ultimate production, with the main food crop paddy rice of China is example, about 400,000,000 mu of annual cultivated area, by 400 kilograms of per mu yields, rice disease on average causes the underproduction 10%, cause every year financial loss to reach more than 300 hundred million yuans, the safety in production of serious threat food crop.At present, the controlling plant disease is except selecting breeding for use and improving the cultivation step in the production, and main dependence sprayed chemical bactericide.On producing, the most chemical bactericide that uses has in various degree toxic action to human body and animal at present, and the oxious component that remains in the plant edible portion can cause potential threat to HUMAN HEALTH, has caused the concern of government and all orders of society; Moreover, some chemical pesticide is difficult to decompose, and can be accumulated in chronically in the ecosystem, causes the pollution to environment, is unfavorable for society and economic Sustainable development; And existing chemical pesticide is not in full force and effect to the certain plants disease.Therefore, make great efforts that development a new generation is efficient, safety, low toxicity, to the good chemical pesticide of Environmental compatibility in, also need research and development biogenic pesticide energetically.
1992, world environments once proposed with the development conference, and before the new millennium, the usage quantity of biogenic pesticide will reach the target that agricultural chemicals uses total amount 60%.Yet, even to this day, also only account for about 15% as U.S. of developed country, and China accounts for 5%.At present, compare with chemical pesticide, the biogenic pesticide of on producing, having promoted the use of, its kind and quantity are all less, and some kind then owing to use year for a long time, has caused the resistance of phytopathogen, and prevention effect is not ideal enough.With the rice sheath blight disease is example, and this disease and rice blast, bacterial leaf-blight are classified the three big main diseases that rice crop process of growth median year takes place as, and the medicament of preventing and treating this disease mainly depends on old brand biogenic pesticide jinggangmeisu.But through the use for prolonged period of time in nearly 40 years, the fusion group of portion water Rhizoctonia solani Kuhn developed immunity to drugs; And jinggangmeisu is only effective to Rhizoctonia solani Kuhn, and other pathogenic bacterias are not had tangible prevention effect, and use range has significant limitation.
Growth-promoting antagonistic bacterium M 18 for biologic agricultural chemical, to Plant diseases have efficiently, the germicidal action of safety, wide spectrum, good with Environmental compatibility, be easy in environment, degrade.This growth-promoting antagonistic bacterium M18 on June 27th, 2000 in specified depositary institution of Patent Office of the People's Republic of China: Beijing, China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preserving number is CGMCC NO.0462.The preparation of growth-promoting antagonistic bacterium M 18 for biologic agricultural chemical and application have obtained national inventing patent, and the patent No. is 00119857.2.But, growth-promoting antagonistic bacterium M 18 for biologic agricultural chemical is a kind of viable bacteria microbial inoculum, its mechanism of action mainly is to realize crop plants pathogeny bacterium is implemented restraining effect by the activeconstituents of the synthetic anti-Plant diseases of M18 viable bacteria, and its synthetic active component content is subjected to the influence of envrionment conditions easily.Thereby, the prevention effect of Plant diseases is had instable defective, be difficult in agriculture production, apply on a large scale.
Find out, the main active ingredient of growth-promoting antagonistic bacterium M18 controlling plant diseases is a phenazine-1-carboxylic acid, from the fermented liquid of growth-promoting antagonistic bacterium M18, extract phenazine-1-carboxylic acid, utilize activeconstituents rather than viable bacteria to the control of corps diseases have equally efficiently, safety, wide spectrum, with features such as Environmental compatibility is good; Simultaneously, can overcome and utilize growth-promoting antagonistic bacterium M18 to prevent and treat the instable defective of disease effect.But, utilize wild-type growth-promoting antagonistic bacterium strain M18, the output of fermentative production phenazine-1-carboxylic acid only is every liter of 200-300 milligram, the production cost of activeconstituents is too high, does not possess the value of using in agriculture production.In recent years, the molecular biological technology of existing utilization to the regulatory mechanism of the synthetic phenazine-1-carboxylic acid of growth-promoting antagonistic bacterium M18, is carried out research in depth.The utilization genetic engineering means, two-pack regulatory gene gacA in the growth-promoting antagonistic bacterium M18 genome has been carried out the targeted inactivation sudden change, obtained M18 derivative strain M18G, improved the output of phenazine-1-carboxylic acid greatly, its fermentation titer is reached about every liter of 1500-1700 milligram.The technological method of this achievement in research is open the 761st～765 page of " microorganism journal " 44 volume in 2004, and thesis topic is " pseudomonas gacA inserts sudden change to pyoluteorin and the anabolic otherness regulation and control of phenazine-1-carboxylic acid ".
With the phenazine-1-carboxylic acid is the microbial source sterilant of main component, and unit confirms through the name of China agricultural chemicals, name to be Shen piperazine mycin, and (registration card number is: LS20031381) to have obtained the interim registration card of agricultural chemicals that the Ministry of Agriculture issues.Be called (the patent No.: 200610023459.9) in the Chinese invention patent of " utilizing antagonizing bacteria M 18 strain to prepare the method for sterilant " in name, providing a kind of utilizes antagonizing bacteria M 18 strain M18G and M18R to prepare the method for sterilant, utilize the meta-bolites of microorganism and the non-microorganism live body prepares sterilant, meta-bolites by two kinds of derivative strains composite, reach the purpose that improves prevention effect, can be more stable, effective controlling plant disease, melon dish cash crop, the particularly control to pimento eqpidemic disease and watermelon blight have been obtained prominent achievement.But, China and area, Asia crowd's topmost food crop rice, be a kind of low value-added commodity, if by present fermentation level production Shen piperazine mycin, the cost that is used to prevent and treat the rice crop disease is still higher, still is difficult to be accepted and carry out large-area applying by the grain farmer.
Summary of the invention
The objective of the invention is at the defective that exists in the prior art, a kind of method of utilizing engineering bacterial strain M 18 G to carry plasmid pME 6032 Phz to produce phenazine-1-carboxylic acid is provided, in order to improve the output of phenazine-1-carboxylic acid, reduce the cost of preparation sterilant Shen piperazine mycin, effectively prevent and treat rice disease.
For achieving the above object, the present invention amplifies the biosynthetic encoding gene fragment of phenazine-1-carboxylic acid phzA1-G1 from growth-promoting antagonistic bacterium M18 genome, and this gene fragment is inserted among the expression plasmid pME6032, places strong promoter P
TacControl under, be built into recombinant plasmid pME6032Phz; Then, this recombinant plasmid pME6032Phz is imported among the derivative strain M18G of growth-promoting antagonistic bacterium M18, be built into engineering strain M18G/pME6032Phz.This project bacterial strain is realized the high efficiency stable expression of encoding gene in the copy number of the biosynthetic encoding gene of amplification phenazine-1-carboxylic acid.At last, engineering strain M18G/pME6032Phz cultivates in the analysis for soybean powder nutrient solution, produces phenazine-1-carboxylic acid efficiently and stably, makes the output of phenazine-1-carboxylic acid reach 5700～6600 milligrams every liter level.
Method of the present invention specifically may further comprise the steps:
1, the amplification biosynthetic encoding gene fragment of phenazine-1-carboxylic acid (phzA1-G1)
Design a pair of primer, the nucleotide sequence of primer is as follows:
Forward: 5 '-ATATAT
GGTACCGCCAGCGAATAACCGATGCCGCGAGGGAA-3 '
Oppositely: 5 '-TGCGTA
AGATCTCGATGGGTTCGCTCATGGGTGCTTCCTTTT-3 '
Underscore is the restriction enzyme site of restriction enzyme KpnI, BglII in the sequence; Be template with growth-promoting antagonistic bacterium M18 genomic dna then, utilize the primer of archaeal dna polymerase LA Taq and design, the biosynthetic encoding gene of amplification phenazine-1-carboxylic acid, amplified production detects by agarose electrophoresis, and reclaiming length is the gene fragment phzA1-G1 of 6.8kb.
2, construction recombination plasmid pME6032Phz
With the gene amplification fragment phzA1-G1 that reclaims, cut through restriction enzyme KpnI, BglII enzyme, ligase enzyme connects, and inserts intestinal bacteria/pseudomonas shuttle expression plasmid pME6032, and the expression of phenazine-1-carboxylic acid biosynthesizing encoding gene is placed strong promoter P
TacUnder the control, formation recombinant plasmid pME6032Phz and conversion enter intestinal bacteria; From intestinal bacteria, extract the gene recombination plasmid pME6032Phz that makes up.
3, make up engineering strain M18G/pME6032Phz
The competent cell of preparation antagonizing bacteria M 18 strain M18G, and said gene recombinant plasmid pME6032Phz is transformed in the competent cell of M18G, under 28～37 ℃ of conditions, cultivated 1～2 day, therefrom filter out engineering strain M18G/pME6032Phz.
4, the cultivation of engineering strain M18G/pME6032Phz
Engineering strain M18G/pME6032Phz is seeded on the flat board of glycerin medium, under 26～30 ℃, activation growth 20～24 hours, on the flat board of glycerin medium, draw the bacterium piece once more, 26～30 ℃ activate 10～12 hours down, then activatory M18G/pME6032Phz bacterium piece is transferred to that to contain 25 milliliters of glycerine nutrient solutions, volume be that shaking culture is 9～11 hours in 26～30 ℃ shaking table in 250 milliliters the triangular flask, shaking speed is 160～180 rev/mins; Be transferred at last that to contain 65 milliliters of analysis for soybean powder nutrient solutions, volume be in 250 milliliters the triangular flask, to amplify fermentation culture, temperature and rotating speed are constant, and fermentation time is 60～72 hours, and obtaining phenazine-1-carboxylic acid content is 5700～6600 milligrams every liter.
Among the present invention, the weight percentages of components that contains in described glycerin medium and the glycerine nutrient solution is: peptone 1.8～2.2%, glycerine 1.3～1.7%, sal epsom 0.05～0.1%, potassium primary phosphate 0.01～0.05%, surplus are water, and pH 6.8～7.2.
The weight percentages of components of described analysis for soybean powder nutrient solution is: analysis for soybean powder 4.5～5.5%, corn steep liquor 1.4～1.7%, glucose 1.0～1.4%, surplus are water, and pH 6.5～7.0.
The present invention utilizes engineering strain M18G to carry plasmid pME6032Phz, and the advantage of producing the method for phenazine-1-carboxylic acid is:
1, improves the copy number of phenazine-1-carboxylic acid synthetic gene.The biosynthetic encoding gene of phenazine-1-carboxylic acid is inserted plasmid pME6032, import among the bacterial strain M18G, improved the copy number of the biosynthetic encoding gene of phenazine-1-carboxylic acid.Because plasmid can be stably independently duplicated outside karyomit(e), originally the karyomit(e) of each cell only carried the encoding gene of 2 copies, and after being carried by plasmid now, each intracellular plasmid number can reach 8 to 10 copies; Thereby the copy number of phenazine-1-carboxylic acid biosynthesizing encoding gene has also increased by 8 to 10 copies.
2, the expression amount of enhancing and stable phenazine-1-carboxylic acid synthetic gene.In plasmid, the biosynthetic encoding gene of phenazine-1-carboxylic acid places strong promoter P
TacControl under, the expression activity of this promotor is not subjected to that various suppressors have improved the expression amount of encoding gene greatly to the control of phenazine-1-carboxylic acid composite coding genetic expression in the M18G cell, and makes encoding gene obtain stable expression.
3, efficient and stably synthetic phenazine-1-carboxylic acid.Under the dual function of polygene copy and high efficiency stable expression, engineering strain M18G/pME6032Phz cultivates in the analysis for soybean powder nutrient solution, the resultant quantity of phenazine-1-carboxylic acid reaches 5700～6600 milligrams every liter, compare with original derivative strain M18G, fermentation titer on average improves 2.6～3.1 times.
4, reduce the production cost of phenazine-1-carboxylic acid.Produce the glucose culture solution of phenazine-1-carboxylic acid compares with derivative strain M18G, when utilizing engineering strain M18G/pME6032Phz to produce phenazine-1-carboxylic acid, in the analysis for soybean powder nutrient solution composition that is adopted, corn steep liquor has replaced about 2/3rds glucose consumption, and, the consumption of corn steep liquor be about replace 50% of glucose amount, simultaneously, the corn steep liquor price only is 90% of a glucose.Like this, utilize engineering strain M18G/pME6032Phz to produce phenazine-1-carboxylic acid, under the prerequisite that improves fermentation titer, also further reduced production cost.
5, phenazine-1-carboxylic acid is produced in the high-yield genetic engineering bacterium strain that utilizes the present invention to make up, preparation sterilant Shen piperazine mycin, be used for plant is sprayed or irritates root and use, can prevent and treat the mould and mould Plant diseases that causes of epidemic disease of rice sheath blight disease, bacterial leaf-blight, cucumber fusarium axysporum, watermelon blight, gummy stem blight of melon, cotton wilt, anthrax, damping-off and various corruption.Simultaneously, because of greatly reducing production cost, reach this requirement of farming that is applied to controlling plant diseases, be beneficial to Shen piperazine mycin and in agriculture production, obtain large-scale promotion application.
Description of drawings
Fig. 1 is the structure synoptic diagram of plasmid pME6032Phz among the present invention.
Embodiment
Below, by accompanying drawing and specific embodiment technical scheme of the present invention and technique effect are further described.Following examples do not constitute limitation of the invention.
Embodiment 1
1, the amplification biosynthetic encoding gene fragment of phenazine-1-carboxylic acid (phzA1-G1)
Design a pair of primer, in order to the amplification biosynthetic encoding gene fragment of phenazine-1-carboxylic acid (phzA1-G1), the nucleotide sequence of primer is as follows:
Forward: 5 '-ATATAT
GGTACCGCCAGCGAATAACCGATGCCGCGAGGGAA-3 '
Oppositely: 5 '-TGCGTA
AGATCTCGATGGGTTCGCTCATGGGTGCTTCCTTTT-3 '
Underscore in the sequence is the restriction enzyme site of restriction enzyme KpnI, BglII.This primer entrusts Shanghai Sangon Biological Engineering Technology And Service Co., Ltd synthetic.
Then, with growth-promoting antagonistic bacterium M18 genomic dna is template, utilize the primer of archaeal dna polymerase LA Taq and design, the biosynthetic encoding gene of amplification phenazine-1-carboxylic acid, product detects by 0.7% agarose electrophoresis, reclaim the gene fragment (phzA1-G1) that length is about 6.8kb, the accuracy of gene fragment is verified through nucleotide sequencing.Wherein, the preparation of described M18 genomic dna utilizes AxyPrep bacterial genomes DNA test kit to carry out, the recycling AxyPrep dna gel of gene fragment reclaims test kit to carry out, provide by biotechnology (Hangzhou) company limited of liking to pursue progress, catalog number is respectively AP-MN-BT-GDNA-4 and AP-GX-50; The condition of described gene amplification reaction and agarose electrophoresis, write according to J. Sa nurse Brooker, D.W. Russell respectively, method described in " molecular cloning experiment guide (third edition) " that Science Press in 2002 publishes, the 387th～400 page in the 611st～618 page in the 8th chapter and the 5th chapter is carried out.Wherein, employed archaeal dna polymerase LA Taq and cdna amplification kit are available from Shanghai shipping agency of TAKARA company, catalog number: DRR002AG.Agarose is available from Shanghai shipping agency of GENE TECH company.The nucleotide sequencing checking of gene fragment (phzA1-G1) entrusts Shanghai Ying Jun Bioisystech Co., Ltd to finish.
2, construction recombination plasmid pME6032Phz
With the gene amplification fragment (phzA1-G1) that reclaims in the step 1, cut through restriction enzyme KpnI, BglII enzyme, ligase enzyme connects, and inserts intestinal bacteria/pseudomonas shuttle expression plasmid pME6032, and the expression of phenazine-1-carboxylic acid biosynthesizing encoding gene is placed strong promoter P
TacUnder the control, formation recombinant plasmid pME6032Phz and conversion enter intestinal bacteria.At last, from intestinal bacteria, extract gene recombination plasmid pME6032Phz and the checking that makes up.
The gene recombination plasmid pME6032Phz that makes up as shown in Figure 1, the fragment of biosynthesizing encoding gene that contains phenazine-1-carboxylic acid is inserted among the plasmid pME6032, and is placed strong promoter P after Restriction Enzyme KpnI, BglII enzyme are cut
TacControl under, be built into recombinant plasmid pME6032Phz.Among the figure: 9816bp represents the length of plasmid pME6032, is 9816 base pairs; PhzA1-G1 is the biosynthesizing encoding gene fragment of phenazine-1-carboxylic acid; PhzM is the upstream modifying factor; // shorten segmental symbol for gene; KpnI, BglII are restriction endonuclease sites; Numeral among the figure is represented the Nucleotide sequence number in the gene respectively, and+1 is the transcription initiation site of phzA1-G1 biosynthesizing encoding gene.TetA and TetR are the selectable marker gene among the plasmid pME6032; P
TacBe promotor; T4_32term is a terminator; P15A origin is the replication origin of plasmid; The gene that LacIQ repressor is indicated is the aporepressor encoding gene.
Directed extraction and the checking of inserting plasmid, the colibacillary preparation of competence, conversion and recombinant plasmid of said gene fragment write according to J. Sa nurse Brooker, D.W. Russell respectively, method described in " molecular cloning experiment guide (third edition) " that Science Press in 2002 publishes, the 663rd～666 page in the 68th～71 page in the 1st chapter and the 96th～99 page and the 8th chapter is carried out.Wherein: plasmid pME6032 is provided by department of microbiology Dieter doctor Haas of Lausanne, SUI university.The ligase enzyme of restriction enzyme and insertion usefulness is all available from brilliant Bioisystech Co., Ltd in Shenzhen.The a small amount of plasmid rapid extraction of Type B test kit is adopted in the extraction of recombinant plasmid in the intestinal bacteria, and biological gene technology limited liability company provides catalog number by vast Tyke, Beijing: MK014-2.The employed Restriction Enzyme of the checking of recombinant plasmid, archaeal dna polymerase LA Taq and cdna amplification kit are all available from Shanghai shipping agency of TAKARA company, catalog number: DRR002AG.Agarose is available from Shanghai shipping agency of GENE TECH company.
3, make up engineering strain M18G/pME6032Phz
The competent cell of preparation antagonizing bacteria M 18 strain M18G, and above-mentioned recombinant plasmid pME6032Phz is transformed in the competent cell of M18G, under 28 ℃ of conditions, cultivated 2 days, therefrom filter out engineering strain M18G/pME6032Phz.
The preparation method of the competent cell of antagonizing bacteria M 18 strain M18G, recombinant plasmid pME6032Phz are transformed into the screening method of the engineering strain M18G/pME6032Phz of the competent cell of M18G and High-efficient Production phenazine-1-carboxylic acid, all write according to J. Sa nurse Brooker, D.W. Russell, method described in " molecular cloning experiment guide (third edition) " that Science Press in 2002 publishes, the 96th～99 page in the 1st chapter is carried out.
4, the cultivation of engineering strain M18G/pME6032Phz
Engineering strain M18G/pME6032Phz is seeded on the glycerin medium flat board, activate growth 24 hours down at 26 ℃, and then on the flat board of glycerin medium, draw the bacterium piece, 26 ℃ activate 10 hours down, then, activatory M18G/pME6032Phz bacterium piece is transferred to contain 25 milliliters of glycerine nutrient solutions, volume be that shaking culture is 9 hours in 26 ℃ shaking table in 250 milliliters the triangular flask, shaking speed is 160 rev/mins; Be transferred at last that to contain 65 milliliters of analysis for soybean powder nutrient solutions, volume be in 250 milliliters the special triangular flask, to amplify fermentation culture, temperature and rotating speed are constant, and fermentation time is 60 hours, and in fermented liquid, obtaining phenazine-1-carboxylic acid content is 6200 milligrams every liter.
Wherein, the weight percentages of components that contains in described glycerin medium and the glycerine nutrient solution is: peptone 1.8%, glycerine 1.3%, sal epsom 0.07%, potassium primary phosphate 0.03%, surplus are water, pH7.0.
The weight percentages of components of described analysis for soybean powder nutrient solution is: analysis for soybean powder 4.5%, corn steep liquor 1.4%, glucose 1.0%, surplus are water, and pH 6.5.
Phenazine-1-carboxylic acid output under this prescription is compared with the output of the derivative strain M18G of growth-promoting antagonistic bacterium, has improved about 2.9 times.
Embodiment 2
1, the identical biosynthetic encoding gene of method amplification phenazine-1-carboxylic acid of employing and embodiment 1, product detects by 1.0% agarose electrophoresis, reclaims to obtain the gene fragment (phzA1-G1) that length is about 6.8kb.
2, with the gene amplification fragment (phzA1-G1) that reclaims, cut through restriction enzyme KpnI, BglII enzyme, ligase enzyme connects, and inserts intestinal bacteria/pseudomonas shuttle expression plasmid pME6032, and the expression of phenazine-1-carboxylic acid biosynthesizing encoding gene is placed strong promoter P
TacUnder the control, formation recombinant plasmid pME6032Phz and conversion enter intestinal bacteria.At last, from intestinal bacteria, extract recombinant plasmid and checking.
3, the competent cell of preparation antagonizing bacteria M 18 strain M18G, and above-mentioned recombinant plasmid pME6032Phz is transformed in the competent cell of M18G, under 30 ℃ of conditions, cultivated 1 day, therefrom filter out engineering strain M18G/pME6032Phz.
4, engineering strain M18G/pME6032Phz is seeded on the flat board of glycerin medium, activate growth 22 hours down at 28 ℃, and then on the flat board of glycerin medium, draw the bacterium piece, 28 ℃ activate 11 hours down, then activatory M18G/pME6032Phz bacterium piece is transferred to that to contain 25 milliliters of glycerine nutrient solutions, volume be in 250 milliliters the triangular flask, shaking culture is 10 hours in 28 ℃ shaking table, and shaking speed is 170 rev/mins; Be transferred at last that to contain 65 milliliters of analysis for soybean powder nutrient solutions, volume be in 250 milliliters the triangular flask, to amplify fermentation culture, temperature and rotating speed are constant, and fermentation time is 66 hours, in fermented liquid, obtains phenazine-1-carboxylic acid content for rising per 6600 milligrams.
Wherein, the weight percentages of components that contains in the described glycerin medium is: peptone 2.2%, glycerine 1.7%, sal epsom 0.05%, potassium primary phosphate 0.01%, surplus are water, pH7.2.
The weight percentages of components of described analysis for soybean powder nutrient solution is: analysis for soybean powder 5.0%, corn steep liquor 1.6%, glucose 1.2%, surplus are water, and pH 6.8.
Phenazine-1-carboxylic acid output under this prescription is compared the derivative strain M18G of growth-promoting antagonistic bacterium, has improved about 3.1 times.
Embodiment 3
1, the identical biosynthetic encoding gene of method amplification phenazine-1-carboxylic acid of employing and embodiment 1, product detects by 1.0% agarose electrophoresis, reclaims to obtain the gene fragment (phzA1-G1) that length is about 6.8kb.
2, with the gene amplification fragment (phzA1-G1) that reclaims, cut through restriction enzyme KpnI, BglII enzyme, ligase enzyme connects, and inserts intestinal bacteria/pseudomonas shuttle expression plasmid pME6032, and the expression of phenazine-1-carboxylic acid biosynthesizing encoding gene is placed strong promoter P
TacUnder the control, formation recombinant plasmid pME6032Phz and conversion enter intestinal bacteria.At last, from intestinal bacteria, extract recombinant plasmid and checking.
3, the competent cell of preparation antagonizing bacteria M 18 strain M18G, and above-mentioned recombinant plasmid pME6032Phz is transformed in the competent cell of M18G, under 32 ℃ of conditions, cultivated 2 days, therefrom filter out the engineering strain M18G/pME6032Phz of High-efficient Production phenazine-1-carboxylic acid.
4, the engineering strain M18G/pME6032Phz that will utilize genetic engineering technique to make up, be seeded on the flat board of glycerin medium, activate growth 20 hours down at 30 ℃, and then on the flat board of glycerin medium, draw the bacterium piece, 30 ℃ activate 12 hours down, then activatory M18G/pME6032Phz bacterial strain is transferred to that to contain 25 milliliters of glycerine nutrient solutions, volume be to amplify in 250 milliliters the triangular flask, shaking culture is 11 hours in 30 ℃ shaking table, and shaking speed is 180 rev/mins; Be transferred at last that to contain 65 milliliters of analysis for soybean powder nutrient solutions, volume be in 250 milliliters the triangular flask, to amplify fermentation culture, temperature and rotating speed are constant, and fermentation time is 72 hours, and in fermented liquid, obtaining phenazine-1-carboxylic acid output is 5700 milligrams every liter.
Wherein, the weight percentages of components that contains in the described glycerin medium is: peptone 2.0%, glycerine 1.5%, sal epsom 0.1%, potassium primary phosphate 0.05%, surplus are water, pH6.8;
The weight percentages of components of described analysis for soybean powder nutrient solution is: analysis for soybean powder 5.5%, corn steep liquor 1.7%, glucose 1.4%, surplus are water, and pH 7.0.
Under this prescription, utilize engineering strain M18G/pME6032Phz fermentative production phenazine-1-carboxylic acid, compare, improved about 2.6 times with the output of growth-promoting antagonistic bacterium derivative strain M18G.
Embodiment 4
1, the identical biosynthetic encoding gene of method amplification phenazine-1-carboxylic acid of employing and embodiment 1, product detects by 1.0% agarose electrophoresis, reclaims to obtain the gene fragment (phzA1-G1) that length is about 6.8kb.
2, with the gene amplification fragment (phzA1-G1) that reclaims in a step, cut through restriction enzyme KpnI, BglII enzyme, ligase enzyme connects, and inserts intestinal bacteria/pseudomonas shuttle expression plasmid pME6032, and the expression of phenazine-1-carboxylic acid biosynthesizing encoding gene is placed strong promoter P
TacUnder the control, formation recombinant plasmid pME6032Phz and conversion enter intestinal bacteria.At last, from intestinal bacteria, extract recombinant plasmid and checking.
3, the competent cell of preparation antagonizing bacteria M 18 strain M18G, and above-mentioned recombinant plasmid pME6032Phz is transformed in the competent cell of M18G, under 35 ℃ of conditions, cultivated 1 day, therefrom filter out the engineering strain M18G/pME6032Phz of High-efficient Production phenazine-1-carboxylic acid.
4, the engineering strain M18G/pME6032Phz that will utilize genetic engineering technique to make up, be seeded on the flat board of glycerin medium, activate growth 20 hours down at 30 ℃, and then on the flat board of glycerin medium, draw the bacterium piece, 30 ℃ activate 12 hours down, then activatory M18G/pME6032Phz bacterial strain is transferred to that to contain 25 milliliters of glycerine nutrient solutions, volume be to amplify in 250 milliliters the triangular flask, shaking culture is 11 hours in 28 ℃ shaking table, and shaking speed is 180 rev/mins; Be transferred at last that to contain 65 milliliters of analysis for soybean powder nutrient solutions, volume be in 250 milliliters the triangular flask, to amplify fermentation culture, temperature and rotating speed are constant, and fermentation time is 72 hours, and in fermented liquid, obtaining phenazine-1-carboxylic acid output is 6400 milligrams every liter. Wherein, the weight percentages of components that contains in the described glycerin medium is: peptone 2.0%, glycerine 1.5%, sal epsom 0.1%, potassium primary phosphate 0.05%, surplus are water, pH6.8;
The weight percentages of components of described analysis for soybean powder nutrient solution is: analysis for soybean powder 5.0%, corn steep liquor 1.7%, glucose 1.4%, surplus are water, and pH 6.8.Under this prescription, utilize engineering strain M18G/pME6032Phz fermentative production phenazine-1-carboxylic acid, compare, improved about 3.0 times with the output of growth-promoting antagonistic bacterium derivative strain M18G.
Embodiment 5
1, the identical biosynthetic encoding gene of method amplification phenazine-1-carboxylic acid of employing and embodiment 1, product detects by 1.0% agarose electrophoresis, reclaims to obtain the gene fragment (phzA1-G1) that length is about 6.8kb.
2, with the gene amplification fragment (phzA1-G1) that reclaims in a step, cut through restriction enzyme KpnI, BglII enzyme, ligase enzyme connects, and inserts intestinal bacteria/pseudomonas shuttle expression plasmid pME6032, and the expression of phenazine-1-carboxylic acid biosynthesizing encoding gene is placed strong promoter P
TacUnder the control, formation recombinant plasmid pME6032Phz and conversion enter intestinal bacteria.At last, from intestinal bacteria, extract recombinant plasmid and checking.The gene recombination plasmid pME6032Phz that makes up.
3, the competent cell of preparation antagonizing bacteria M 18 strain M18G, and above-mentioned recombinant plasmid pME6032Phz is transformed in the competent cell of M18G, under 37 ℃ of conditions, cultivated 1 day, therefrom filter out the engineering strain M18G/pME6032Phz of High-efficient Production phenazine-1-carboxylic acid.
4, the engineering strain M18G/pME6032Phz that will utilize genetic engineering technique to make up, be seeded on the glycerin medium flat board, activate growth 20 hours down at 28 ℃, and then on the flat board of glycerin medium, draw the bacterium piece, 28 ℃ activate 12 hours down, then activatory M18G/pME6032Phz bacterial strain is transferred to that to contain 25 milliliters of glycerine nutrient solutions, volume be to amplify in 250 milliliters the triangular flask, shaking culture is 11 hours in 30 ℃ shaking table, and shaking speed is 180 rev/mins; Be transferred at last that to contain 65 milliliters of analysis for soybean powder nutrient solutions, volume be in 250 milliliters the triangular flask, to amplify fermentation culture, temperature and rotating speed are constant, and fermentation time is 72 hours, and in fermented liquid, obtaining phenazine-1-carboxylic acid output is 6000 milligrams every liter.
Wherein, the weight percentages of components that contains in the described glycerin medium is: peptone 2.0%, glycerine 1.5%, sal epsom 0.1%, potassium primary phosphate 0.05%, surplus are water, pH6.8;
The weight percentages of components of described analysis for soybean powder nutrient solution is: analysis for soybean powder 4.8%, corn steep liquor 1.5%, glucose 1.4%, surplus are water, and pH 7.0.
Under this prescription, utilize engineering strain M18G/pME6032Phz fermentative production phenazine-1-carboxylic acid, compare, improved about 2.8 times with the output of growth-promoting antagonistic bacterium derivative strain M18G.
Claims (1)
1. a method of utilizing engineering bacterial strain M 18 G to carry plasmid pME 6032 Phz to produce phenazine-1-carboxylic acid is characterized in that comprising the steps:
1) design a pair of primer, the nucleotide sequence of primer is as follows:
Forward: 5 '-ATATAT
GGTACCGCCAGCGAATAACCGATGCCGCGAGGGAA-3 '
Oppositely: 5 '-TGCGTA
AGATCTCGATGGGTTCGCTCATGGGTGCTTCCTTTT-3 '
Underscore is the restriction enzyme site of restriction enzyme Kpn I, Bg1 II in the sequence; Be template with growth-promoting antagonistic bacterium M18 genomic dna then, utilize the primer of archaeal dna polymerase LA Taq and design, the biosynthetic encoding gene of amplification phenazine-1-carboxylic acid, amplified production detects by agarose electrophoresis, and reclaiming length is the gene fragment phzA1-G1 of 6.8kb;
2) with the gene amplification fragment phzA1-G1 that reclaims, cut through restriction enzyme kpn I, Bg1 II enzyme, ligase enzyme connects, and inserts intestinal bacteria/pseudomonas shuttle expression plasmid pME6032, and the expression of phenazine-1-carboxylic acid biosynthesizing encoding gene is placed strong promoter P
TacUnder the control, formation recombinant plasmid pME6032Phz and conversion enter intestinal bacteria; From intestinal bacteria, extract the gene recombination plasmid pME6032Phz that makes up;
3) competent cell of preparation antagonizing bacteria M 18 strain M18G, and said gene recombinant plasmid pME6032Phz is transformed in the competent cell of M18G, under 28～37 ℃ of conditions, cultivated 1～2 day, therefrom filter out engineering strain M18G/pME6032Phz;
4) engineering strain M18G/pME6032Phz is seeded on the flat board of glycerin medium, under 26～30 ℃, activation growth 20～24 hours, on the flat board of glycerin medium, draw the bacterium piece once more, 26～30 ℃ activate 10～12 hours down, then activatory M18G/pME6032Phz bacterium piece is transferred to that to contain 25 milliliters of glycerine nutrient solutions, volume be that shaking culture is 9～11 hours in 26～30 ℃ shaking table in 250 milliliters the triangular flask, shaking speed is 160～180 rev/mins; Be transferred at last that to contain 65 milliliters of analysis for soybean powder nutrient solutions, volume be in 250 milliliters the triangular flask, to amplify fermentation culture, temperature and rotating speed are constant, and fermentation time is 60～72 hours, and obtaining phenazine-1-carboxylic acid content is 5700～6600 milligrams every liter;
Wherein, the weight percentages of components that contains in the described glycerine nutrient solution is: peptone 1.8～2.2%, glycerine 1.3～1.7%, sal epsom 0.05～0.1%, potassium primary phosphate 0.01～0.05%, surplus are water, pH6.8～7.2;
The weight percentages of components of described analysis for soybean powder nutrient solution is: analysis for soybean powder 4.5～5.5%, corn steep liquor 1.4～1.7%, glucose 1.0～1.4%, surplus are water, and pH 6.5～7.0.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101986642A CN101705265B (en) | 2009-11-12 | 2009-11-12 | Method for producing phenazine-1-carboxylic acid by using engineering bacterial strain M18G to carry plasmid pME6032Phz |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101986642A CN101705265B (en) | 2009-11-12 | 2009-11-12 | Method for producing phenazine-1-carboxylic acid by using engineering bacterial strain M18G to carry plasmid pME6032Phz |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101705265A CN101705265A (en) | 2010-05-12 |
| CN101705265B true CN101705265B (en) | 2011-07-27 |
Family
ID=42375520
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009101986642A Active CN101705265B (en) | 2009-11-12 | 2009-11-12 | Method for producing phenazine-1-carboxylic acid by using engineering bacterial strain M18G to carry plasmid pME6032Phz |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101705265B (en) |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399737B (en) * | 2011-11-07 | 2015-04-08 | 上海交通大学 | Biological engineering bacterial strain for producing novel microorganism source fungicide and application of biological engineering bacterial strain |
| CN103271050A (en) * | 2013-06-19 | 2013-09-04 | 北京燕化永乐农药有限公司 | Compound bactericide |
| CN104293692A (en) * | 2014-07-14 | 2015-01-21 | 武汉汉申生物科技有限责任公司 | Bioengineering bacterial strain, preparation method and application thereof |
| CN104263748A (en) * | 2014-07-14 | 2015-01-07 | 武汉汉申生物科技有限责任公司 | Biological engineering strain, and preparation method and application thereof |
| CN104957145B (en) * | 2014-09-30 | 2017-04-05 | 四川利尔作物科学有限公司 | Bactericidal composition and its application |
| CN104957157B (en) * | 2015-01-08 | 2017-04-05 | 四川利尔作物科学有限公司 | Bactericidal composition and its application |
| CN104946552B (en) * | 2015-02-03 | 2018-02-09 | 上海交通大学 | The engineering strain of safe and efficient production shenqinmycin and its application |
| CN105087455A (en) * | 2015-03-27 | 2015-11-25 | 上海交通大学 | Genetic engineering strain for producing phenazine-1-carboxylic acid and application of genetic engineering strain |
| CN109042685B (en) * | 2018-08-22 | 2020-06-30 | 南京林业大学 | Composite antibacterial agent and use method thereof |
| CN111485009B (en) * | 2020-04-10 | 2022-03-18 | 上海农乐生物制品股份有限公司 | Preparation method of fermentation medium of shenqinmycin and fermentation medium |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1539959A (en) * | 2003-10-30 | 2004-10-27 | 上海交通大学 | Method for inserting gene of pseudomonas fluorescens M18 into mutation strain H18G |
-
2009
- 2009-11-12 CN CN2009101986642A patent/CN101705265B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1539959A (en) * | 2003-10-30 | 2004-10-27 | 上海交通大学 | Method for inserting gene of pseudomonas fluorescens M18 into mutation strain H18G |
Non-Patent Citations (2)
| Title |
|---|
| 史鸣奇等.GacA对假单胞菌株M18两个phz基因簇和菌群传感的调控.《微生物学报》.2009,第49卷(第10期),1306-1406. * |
| 朱栋华等.荧光假单胞菌M18 rpoD克隆及其对抗生素合成的影响.《微生物学报》.2003,第43卷(第3期),315-323. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101705265A (en) | 2010-05-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101705265B (en) | Method for producing phenazine-1-carboxylic acid by using engineering bacterial strain M18G to carry plasmid pME6032Phz | |
| CN102399737B (en) | Biological engineering bacterial strain for producing novel microorganism source fungicide and application of biological engineering bacterial strain | |
| Lau et al. | Cyanobacteria: photoautotrophic microbial factories for the sustainable synthesis of industrial products | |
| Vassilev et al. | Potential application of glycerol in the production of plant beneficial microorganisms | |
| Zou et al. | Emerging technologies of algae‐based wastewater remediation for bio‐fertilizer production: a promising pathway to sustainable agriculture | |
| Khairina et al. | Indigenous bacteria, an excellent reservoir of functional plant growth promoters for enhancing duckweed biomass yield on site | |
| CN105670958A (en) | Low-temperature biocontrol bacterial strain and application thereof | |
| Rao et al. | Isolation, screening and optimization of laccase-producing endophytic fungi from Euphorbia milii | |
| CN105087455A (en) | Genetic engineering strain for producing phenazine-1-carboxylic acid and application of genetic engineering strain | |
| Zhu et al. | Evaluation of the plant-growth-promoting abilities of endophytic bacteria from the psammophyte Ammodendron bifolium | |
| Lee et al. | Genome sequence of a native-feather degrading extremely thermophilic Eubacterium, Fervidobacterium islandicum AW-1 | |
| Rettenmaier et al. | Hungateiclostridium mesophilum sp. nov., a mesophilic, cellulolytic and spore-forming bacterium isolated from a biogas fermenter fed with maize silage | |
| Morais et al. | Sugiyamaella xylanicola sp. nov., a xylan-degrading yeast species isolated from rotting wood | |
| CN101338319A (en) | Recombinant vector pM43HF for expressing harpin protein and engineering strain thereof | |
| Xu et al. | Whole-genome analysis of Bacillus velezensis ZF2, a biocontrol agent that protects cucumis sativus against corynespora leaf spot diseases | |
| CN104946552B (en) | The engineering strain of safe and efficient production shenqinmycin and its application | |
| CN100411521C (en) | Method for preparing bactericidal agent using antagonizing bacteria M18 strain | |
| Zhang et al. | Complete genome sequence of the drought resistance-promoting endophyte Klebsiella sp. LTGPAF-6F | |
| Jiang et al. | Methylobacterium planium sp. nov., isolated from a lichen sample | |
| Yuan et al. | Ecofriendly conversion of algal waste into valuable plant growth-promoting rhizobacteria (PGPR) biomass | |
| Chauhan et al. | Exo-polysaccharide producing bacteria can induce maize plant growth and soil health under saline conditions | |
| CN104293692A (en) | Bioengineering bacterial strain, preparation method and application thereof | |
| CN109536390B (en) | Citrus endophytic fungi and application thereof | |
| Luo et al. | Production of 5‐aminolevulinic acid from hydrolysates of cassava residue and fish waste by engineered Bacillus cereus PT1 | |
| CN101348775B (en) | Enterobacteria recombinant strain and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |