CN111454873B

CN111454873B - Streptomyces albus genetic engineering bacterium and application thereof in production of epsilon-polylysine

Info

Publication number: CN111454873B
Application number: CN202010283789.1A
Authority: CN
Inventors: 秦加阳; 王爱霞; 薛宇斌; 王秀文; 于波
Original assignee: Institute of Microbiology of CAS; Binzhou Medical College
Current assignee: Institute of Microbiology of CAS; Binzhou Medical College
Priority date: 2019-11-29
Filing date: 2020-04-13
Publication date: 2023-01-17
Anticipated expiration: 2040-04-13
Also published as: CN111454873A

Abstract

The invention discloses a gene engineering strain Streptomyces albugus (Streptomyces albugus) Q-PL2, which is obtained by overexpression of an epsilon-polylysine synthetase gene in a Streptomyces albugus wild strain Q-PL by using a gene engineering method. The invention also discloses the application of the genetic engineering bacteria in the fermentation production of the epsilon-polylysine, namely, the epsilon-polylysine is obtained by culturing and fermenting the streptomyces albidoflauvs, and the yield of the epsilon-polylysine reaches up to 42.9 g/l, the production rate reaches 11.7 g/l/day, and the production capacity of the epsilon-polylysine is 2 to 5 times of that of the wild streptomyces albidoflauvs Q-PL.

Description

Streptomyces albus genetic engineering bacterium and application thereof in production of epsilon-polylysine

Technical Field

The invention relates to a genetic engineering bacterium and application thereof, in particular to a streptomyces albidoflavus genetic engineering bacterium, a construction method thereof and a method for producing epsilon-polylysine by utilizing the strain through fermentation.

Background

Epsilon-polylysine (. Epsilon. -PL) is a polymer in which the alpha-amino group and epsilon-carboxyl group of L-lysine are linked by an amide bond. Epsilon-PL has strong antibacterial activity, wide antibacterial spectrum, high safety, no toxic or side effect, biodegradability and edibility. In the last 80 s epsilon-PL was approved for food preservatives in japan, usa, korea and other countries. In 2014, epsilon-PL and hydrochloride thereof are approved by relevant departments in China as food preservatives. At present, epsilon-PL has been applied to industries such as food, cosmetics, medical care and health as a preservative. Besides being used as food preservative, the epsilon-PL can also be used as gene carrier, weight-losing health care product, drug carrier, novel water-absorbing material, chip, biological electron coating agent and the like.

Currently, the main production method of epsilon-PL is a microbial fermentation method, the microorganism commonly used for fermentation is Streptomyces albus, and the Streptomyces albus mutant strain is utilized by the Japan Shikonin Corporation (Chisso Corporation) to realize the industrial production of epsilon-PL on a kiloton-scale annually. The breeding work of the high yield epsilon-PL streptomyces albidoides strain in China is continuously promoted and has achieved certain success.

The search of the prior art shows that Chinese patent document No. CN201610665007.4, granted publication No. 2019-06-11, discloses an epsilon-PL high-yield strain and a method for producing epsilon-PL, wherein the strain is obtained by mutagenesis screening, the yield of epsilon-PL is up to 7.5 g/l after fermentation for 42 hours, and the production speed is 4.3 g/l/day.

Chinese patent document No. CN201110274326.X, application date 2011-09-16 discloses a method for producing epsilon-PL by fermenting glucose and glycerol mixed carbon source, and the production speed of producing epsilon-PL by using the method is 3-5 g/L/day.

Chinese patent document No. CN201510886138.0, granted publication No. 2019-01-18, discloses a Streptomyces albus genetic engineering strain, a construction method and application thereof, the strain is used for over-expressing an ammonium transporter gene amtB in Streptomyces albus, the utilization rate of the strain on a nitrogen source in a fermentation liquid is improved, the yield of epsilon-PL is improved, 35.7 g/l of epsilon-PL is produced in 168 hours by using the strain, and the production speed is 5.1 g/l/day.

Chinese patent document No. CN201610551417.6, granted publication No. 2019-07-02, discloses a method for increasing yield of epsilon-PL, which is to add exogenous substances such as calcium gluconate, lysine, aspartic acid and the like into a fermentation culture medium, so that the yield of the epsilon-PL of streptomycete in a total synthesis culture medium for 46 hours is increased to 5.12 g/L, and the production speed is 2.7 g/L/day.

Disclosure of Invention

Aiming at the defect of low production speed of epsilon-PL in the prior art, the invention aims to provide a streptomyces albus genetic engineering strain and realize high-efficiency production of epsilon-PL by utilizing the strain.

According to one aspect of the invention, the invention provides a streptomyces albus genetically engineered bacterium Q-PL2, wherein the streptomyces albus genetically engineered bacterium Q-PL2 comprises one or more epsilon-PL synthetase genes introduced by genetic engineering and expression elements thereof.

According to some embodiments of the invention, the expression elements are in particular a promoter and a ribosome binding site.

According to some embodiments of the invention, the nucleotide sequence of the epsilon-PL synthetase gene comprises the sequence of SEQ ID No.2 or a homologous sequence thereof.

According to some embodiments of the invention, the homologous sequence refers to a nucleotide sequence that is at least 70% identical to the sequence.

According to certain embodiments of the invention, the homologous sequence has substantially the same activity as the sequence disclosed herein.

According to some embodiments of the invention, the Streptomyces albus genetically engineered bacterium Q-PL2 has 2 to 25 times higher ability to produce epsilon-PL by fermentation than a Streptomyces albus wild strain.

According to one aspect of the invention, the invention provides a method for producing epsilon-PL by using streptomyces albus genetic engineering bacteria Q-PL2.

According to certain embodiments of the invention, the method uses a medium comprising citrate to ferment the S.albilineans genetically engineered bacterium Q-PL2 to produce epsilon-PL.

According to certain embodiments of the invention, the medium is a fermentation medium.

According to certain embodiments of the invention, the fermentation medium comprises 1 to 20 grams per liter citrate.

According to some embodiments of the invention, the method comprises:

plate culture: inoculating streptomyces albidoflavus Q-PL2 to an MS solid culture medium containing 60-120 micrograms/ml apramycin, culturing for 4-7 days at 25-35 ℃, and waiting until the surface of the culture medium is full of black spores for later use;

seed culture: collecting spores from the flat plate, inoculating the spores into a seed culture medium containing 60-120 micrograms/ml apramycin, and culturing for 36-72 hours at the temperature of 25-35 ℃;

fermentation culture: inoculating the cultured seed culture solution into a fermentation tank filled with a fermentation culture medium for culture, wherein the culture temperature is 25-35 ℃, dissolved oxygen is controlled to be 10% -100%, the aeration ratio is 1-5 vvm, when the pH value of the fermentation solution is reduced to 3.8-4.2, the pH value of the fermentation solution is maintained to be constant by using alkali, the concentration of a carbon source in the fermentation solution is monitored in the culture process, a feed supplement culture medium is fed in a flowing manner when the concentration of the carbon source is 5-15 g/l, the feeding speed is adjusted by monitoring the concentration of the carbon source in the fermentation solution in a feedback manner, the yield of epsilon-PL in the fermentation solution is measured at intervals, and the total time of the fermentation culture is 36-144 hours.

According to some embodiments of the invention, the fermentation medium comprises a carbon source in an amount of 40 to 100 g/l, a nitrogen source in an amount of 2 to 20 g/l, citrate in an amount of 1 to 20 g/l, dipotassium hydrogen phosphate in an amount of 0.2 to 2 g/l, potassium dihydrogen phosphate in an amount of 0.5 to 5 g/l, zinc sulfate heptahydrate in an amount of 0.01 to 0.1 g/l, magnesium sulfate heptahydrate in an amount of 0.1 to 2 g/l, ferrous sulfate heptahydrate in an amount of 0.01 to 0.1 g/l, and a pH in an amount of 5.5 to 7.0.

According to some embodiments of the invention, the feed medium comprises carbon source 0-600 g/l, nitrogen source 20-200 g/l, citrate 10-100 g/l.

According to certain embodiments of the invention, the carbon source, nitrogen source and citrate in the feed medium are the same as the carbon source, nitrogen source and citrate in the fermentation medium.

According to certain embodiments of the invention, the carbon source is one or more combinations of glucose, glycerol, xylose, fructose, mannitol.

According to some embodiments of the invention, the nitrogen source is one or more combinations of ammonium sulfate, ammonium chloride, urea, ammonium nitrate, yeast powder, soy peptone, corn steep liquor, peanut cake powder, peptone.

According to certain embodiments of the invention, the citrate salt is one or more combinations of sodium citrate, ammonium citrate, potassium citrate, zinc citrate, calcium citrate, magnesium citrate, sodium dihydrogen citrate, potassium dihydrogen citrate, sodium potassium citrate, ferric citrate, diammonium hydrogen citrate.

Genetically engineered bacterium Streptomyces albus (Streptomyces albulus) Q-PL2

The genetic engineering strain is named as Streptomyces albulus Q-PL2 in a laboratory, and is obtained by over-expressing epsilon-PL synthetase in Streptomyces albulus wild strain. The strain is preserved in China general microbiological culture Collection center (CGMCC for short, the address is microorganism research institute of Zhongkoyao academy of sciences in Xilu No.1, beijing, chaoyang, north Cheng) within 30 months and 10 months in 2019, and the preservation registration number is CGMCC NO.18772.

The Streptomyces albidoflavus wild strain is selected from soil samples in the school of Binzhou medical college in Laishu, taiwan, shandong province, and the laboratory of the strain is named as Streptomyces albulus Q-PL.

The genetic engineering bacterium Streptomyces albus (Streptomyces albulus) Q-PL2 contains a genetic element capable of over-expressing polylysine synthetase; wherein, the length of the strong promoter kasOp and the ribosome binding site sequence from the phage phi C31 capsid protein is 96 bases, and the nucleotide sequence is shown as SEQ ID NO. 1; the length of the gene sequence of the epsilon-PL synthetase gene PLs is 3960 bases, and the nucleotide sequence is shown in SEQ ID NO. 2.

In SEQ ID NO.1, the sequence of the strong promoter kasOp is "tgttcacattcgaacggtctctgctttgacaacatgctgtgcggtgttgtaaagtcgtggcca". The sequence of the ribosome binding site from the phage φ C31 capsid protein is "tctaagtaaggagtgtccat".

The genetic engineering bacterium Streptomyces albugus (Streptomyces albugus) Q-PL2 can be used for producing epsilon-PL by fermentation with glucose, glycerol, xylose and the like as substrates.

The preferred culture temperature of the genetically engineered bacterium Streptomyces albus (Streptomyces albulus) Q-PL2 is 25-35 ℃, and the genetically engineered bacterium Streptomyces albus can grow on a culture medium containing 60-120 micrograms/ml of apramycin.

The invention relates to application of a genetic engineering bacterium Streptomyces albugus (Streptomyces albulus) Q-PL2 in epsilon-PL production.

The application relates to the following implementation steps:

(1) Plate culture

Inoculating streptomyces albidoflavus Q-PL2 to an MS solid culture medium containing 60-120 micrograms/ml apramycin, culturing for 4-7 days at 25-35 ℃, and waiting until the surface of the culture medium is full of black spores for later use.

(2) Seed culture

Collecting spores from the plate, inoculating the spores into a seed culture medium containing 60-120 micrograms/ml apramycin, and culturing at 25-35 ℃ for 36-72 hours.

(3) Fermentation culture

The strain is fermented in a shake flask during fermentation comparison and culture medium optimization, and is fermented in a fermentation tank during production.

And (3) shaking flask fermentation: inoculating the cultured seed culture solution into a conical flask filled with a fermentation culture medium according to the inoculation amount of 5-20% (volume ratio), performing shake culture on a shaking table at 25-35 ℃ at 200-300 r/min for 48-84 hours, and measuring the yield of epsilon-PL in the fermentation liquid.

Fermentation in a fermentation tank: inoculating the cultured seed culture solution to a fermentation tank filled with a fermentation culture medium according to the inoculation amount of 5-20% (volume ratio) for culturing, wherein the culture temperature is 25-35 ℃, the dissolved oxygen is controlled to be more than 5-30% by adjusting the stirring speed of the fermentation tank to be 200-1200 r/min, the aeration ratio is 1-5 vvm, when the pH value of the fermentation solution is naturally reduced to 3.8-4.2, ammonia water is used for maintaining the pH value of the fermentation solution unchanged, the concentration of a carbon source in the fermentation solution is monitored in the culture process, the carbon source, a nitrogen source and citrate are supplemented when the concentration of the carbon source is lower than 10 g/l, the yield of epsilon-PL in the fermentation solution is measured every 4-12 hours, and the total time of the fermentation culture is 36-144 hours.

Wherein the formula of the MS solid culture medium in the step (1) comprises 10-30 g/L of mannitol, 10-30 g/L of soybean meal and 15-20 g/L of agar powder.

The formula of the MS solid culture medium in the step (1) is as follows: 10 to 30 g/L of mannitol, 10 to 30 g/L of soybean meal and 15 to 20 g/L of agar powder.

The formula of the seed culture medium in the step (2) comprises 10-100 g/L of glucose, 2-20 g/L of yeast powder, 2-20 g/L of ammonium sulfate, 1-20 g/L of sodium citrate, 0.1-2 g/L of dipotassium phosphate, 0.5-5 g/L of monopotassium phosphate, 0.01-0.1 g/L of zinc sulfate heptahydrate, 0.1-2 g/L of magnesium sulfate heptahydrate and 0.01-0.1 g/L of ferrous sulfate heptahydrate.

The formula of the seed culture medium in the step (2) is as follows: 10 to 100 grams/liter of glucose, 2 to 20 grams/liter of yeast powder, 2 to 20 grams/liter of ammonium sulfate, 1 to 20 grams/liter of sodium citrate, 0.1 to 2 grams/liter of dipotassium phosphate, 0.5 to 5 grams/liter of monopotassium phosphate, 0.01 to 0.1 gram/liter of zinc sulfate heptahydrate, 0.1 to 2 grams/liter of magnesium sulfate heptahydrate and 0.01 to 0.1 gram/liter of ferrous sulfate heptahydrate.

The formula of the fermentation medium in the step (3) comprises 40-100 g/L of carbon source, 2-20 g/L of nitrogen source, 1-20 g/L of citrate, 0.2-2 g/L of dipotassium phosphate, 0.5-5 g/L of potassium dihydrogen phosphate, 0.01-0.1 g/L of zinc sulfate heptahydrate, 0.1-2 g/L of magnesium sulfate heptahydrate and 0.01-0.1 g/L of ferrous sulfate heptahydrate.

The formula of the fermentation medium in the step (3) is as follows: 40-100 g/L of carbon source, 2-20 g/L of nitrogen source, 1-20 g/L of citrate, 0.2-2 g/L of dipotassium hydrogen phosphate, 0.5-5 g/L of potassium dihydrogen phosphate, 0.01-0.1 g/L of zinc sulfate heptahydrate, 0.1-2 g/L of magnesium sulfate heptahydrate and 0.01-0.1 g/L of ferrous sulfate heptahydrate.

The carbon source in the fermentation medium formula is one or more of glucose, glycerol, xylose, fructose and mannitol. The nitrogen source is an organic nitrogen source, an inorganic nitrogen source or a combination of the two. According to certain embodiments of the present invention, the nitrogen source, organic nitrogen source or inorganic nitrogen source, may be combined in any proportion in the fermentation medium. The organic nitrogen source is one or more of yeast powder, soybean peptone, corn steep liquor, peanut cake powder and peptone. The inorganic nitrogen source is one or more of ammonium sulfate, ammonium chloride, urea and ammonium nitrate. The citrate is one or more of sodium citrate, ammonium citrate, potassium citrate, zinc citrate, calcium citrate, magnesium citrate, sodium dihydrogen citrate, potassium sodium citrate, ferric citrate, and diammonium hydrogen citrate.

The genetic engineering bacterium Streptomyces albus (Streptomyces albulus) Q-PL2 takes the carbon sources such as glucose, glycerol and the like as substrates, the yield of the epsilon-PL is up to 42.9 g/l, the production rate is 11.7 g/l/day, and the production capacity of the epsilon-PL is 2-5 times of that of the wild strain Streptomyces albulus Q-PL under the condition of 28-32 ℃ and high-efficiency fermentation production.

Detailed Description

Streptomyces albus

According to certain embodiments of the present invention, the present invention provides a strain of Streptomyces albus genetically engineered bacterium Q-PL2, wherein the Streptomyces albus genetically engineered bacterium Q-PL2 comprises one or more epsilon-PL synthetase genes introduced by genetic engineering and expression elements thereof.

According to some embodiments of the invention, the Streptomyces albus genetically engineered bacterium Q-PL2 is genetically engineered such that the genome comprises one or more genetically engineered epsilon-PL synthetase genes and expression elements thereof.

According to some embodiments of the invention, the Streptomyces albus genetically engineered bacterium Q-PL2 comprises an epsilon-PL synthetase gene and expression elements thereof introduced by genetic engineering.

According to some embodiments of the invention, the Streptomyces albus genetically engineered bacterium Q-PL2 comprises two genetically engineered epsilon-PL synthetase genes and expression elements thereof. According to some embodiments of the invention, the Streptomyces albus genetically engineered bacterium Q-PL2 comprises three genetically engineered epsilon-PL synthetase genes and expression elements thereof. According to some embodiments of the invention, the Streptomyces albus genetically engineered bacterium Q-PL2 comprises four genetically engineered introduced epsilon-PL synthetase genes and expression elements thereof. According to some embodiments of the invention, the S.albidoides genetically engineered bacterium Q-PL2 comprises five genetically engineered introduced epsilon-PL synthetase genes and expression elements thereof. According to some embodiments of the invention, the genetically engineered strain of S.albus Q-PL2 comprises more genetically engineered epsilon-PL synthetase genes and expression elements thereof.

According to some embodiments of the invention, the epsilon-PL synthetase gene and its expression elements are integrated into the genome of S.albus genetically engineered bacterium Q-PL2 by means of the pSET152-pro-PLs plasmid. According to certain embodiments of the invention, the epsilon-PL synthetase gene may also be integrated into the genome of S.albus genetically engineered bacterium Q-PL2 by other plasmids known in the art.

According to some embodiments of the invention, the pSET152-pro-PLs plasmid is constructed to contain an epsilon-PL synthetase gene and its expression elements. According to some embodiments of the invention, the pSET152-pro-PLs plasmid is constructed to contain two epsilon-PL synthetase genes and their expression elements. According to some embodiments of the invention, the pSET152-pro-PLs plasmid is constructed to contain three epsilon-PL synthetase genes and their expression elements. According to some embodiments of the invention, the pSET152-pro-PLs plasmid is constructed to contain four epsilon-PL synthetase genes and their expression elements. According to certain embodiments of the invention, the pSET152-pro-PLs plasmid is constructed to contain five epsilon-PL synthetase genes and their expression elements. According to some embodiments of the invention, the pSET152-pro-PLs plasmid is constructed to contain more epsilon-PL synthetase genes and their expression elements.

According to some embodiments of the invention, the epsilon-PL synthetase gene is 3960 bases in length.

According to some embodiments of the invention, the nucleotide sequence of the epsilon-PL synthetase gene is set forth in SEQ ID No. 2.

According to some embodiments of the invention, the epsilon-PL synthetase gene expression elements are specifically a promoter and a ribosome binding site.

According to some embodiments of the invention, the promoter is a constitutive promoter. According to some embodiments of the invention, the promoter is a strong promoter. According to some embodiments of the invention, the promoter is a strong promoter, kasOp. According to certain embodiments of the invention, the promoter is another promoter that can be used for expression in S.albus.

According to certain embodiments of the invention, the ribosome binding site is the ribosome binding site from the phage Φ C31 capsid protein. According to certain embodiments of the invention, the ribosome binding site is another ribosome binding site that can be used in S.albidus.

According to some embodiments of the invention, the epsilon-PL synthetase gene expression elements are specifically strong promoter and ribosome binding site sequences.

According to some embodiments of the invention, the epsilon-PL synthetase gene expression elements are in particular the strong promoter kasOp and the ribosome binding site sequence, 96 bases in length.

According to some embodiments of the invention, the epsilon-PL synthetase gene expression elements are specifically a strong promoter and a ribosome binding site sequence from the phage phi C31 capsid protein. According to some embodiments of the invention, the nucleotide sequence of the epsilon-PL synthetase gene expression element is set forth in SEQ ID No. 1.

According to certain embodiments of the invention, the epsilon-PL synthetase gene is that of Streptomyces albus. According to certain embodiments of the invention, the epsilon-PL synthetase gene is an epsilon-PL synthetase gene of other species known in the art.

According to some embodiments of the invention, the nucleotide sequence of the epsilon-PL synthetase gene is the sequence of SEQ ID No.2 or a homologous sequence thereof.

According to certain embodiments of the invention, the nucleotide sequence of the epsilon-PL synthetase gene comprises the sequence of SEQ ID No. 2. According to some embodiments of the invention, the nucleotide sequence of the epsilon-PL synthetase gene is the sequence of SEQ ID No. 2.

According to some embodiments of the invention, the homologous sequence refers to a nucleotide sequence that is at least 70% identical to the sequence. According to certain embodiments of the invention, the homologous sequences have a nucleotide sequence that is at least 70% identical, such as a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, or at least 99% identical.

According to certain embodiments of the invention, the homologous sequences have substantially the same activity as the sequences disclosed herein. According to certain embodiments of the invention, the homologous sequence has an activity that is at least 70% identical, e.g., at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, or at least 99% identical to a sequence disclosed herein.

According to some embodiments of the present invention, the Streptomyces albus genetically engineered bacterium Q-PL2, when integrated with an epsilon-PL synthetase gene and its expression elements, has a fermentation production capacity of 2 to 5 times, such as 2 to 4 times, 2 to 3 times, 3 to 5 times, 3 to 4 times, or 4 to 5 times that of a wild strain of Streptomyces albus. According to some embodiments of the invention, the Streptomyces albus genetically engineered bacterium Q-PL2 has a fermentation production capacity of 2 times, 2.2 times, 2.5 times, 2.8 times, 3 times, 3.2 times, 3.5 times, 3.8 times, 4 times, 4.2 times, 4.5 times, 4.8 times or 5 times that of a wild strain of Streptomyces albus.

According to some embodiments of the present invention, the Streptomyces albus genetically engineered bacterium Q-PL2, when two epsilon-PL synthetase genes and expression elements thereof are integrated, has a fermentation production capacity of epsilon-PL 4-10 times, such as 4-8 times, 4-6 times, 6-10 times, 6-8 times or 8-10 times that of a wild strain of Streptomyces albus. According to some embodiments of the invention, the Streptomyces albus genetically engineered bacterium Q-PL2, when integrated with two epsilon-PL synthetase genes and expression elements thereof, has a fermentation production capacity of 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times or 10 times that of a wild strain of Streptomyces albus.

According to some embodiments of the present invention, the Streptomyces albus genetically engineered bacterium Q-PL2, when integrated with three epsilon-PL synthetase genes and expression elements thereof, has a fermentation production capacity of 6 to 15 times, such as 6 to 12 times, 6 to 10 times, 6 to 8 times, 8 to 15 times, 8 to 12 times, 8 to 10 times, 10 to 15 times, 10 to 12 times, or 12 to 15 times that of a wild strain of Streptomyces albus. According to some embodiments of the present invention, the Streptomyces albus genetically engineered bacterium Q-PL2, when integrated with three epsilon-PL synthetase genes and expression elements thereof, has a fermentation production capacity of 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 10.5-fold, 11-fold, 11.5-fold, 12-fold, 12.5-fold, 13-fold, 13.5-fold, 14-fold, 14.5-fold, or 15-fold that of a wild strain of Streptomyces albus.

According to some embodiments of the present invention, the Streptomyces albus genetically engineered bacterium Q-PL2, when integrated with four epsilon-PL synthetase genes and expression elements thereof, has a fermentation production capacity of epsilon-PL 8 to 20 times, such as 8 to 18 times, 8 to 15 times, 8 to 12 times, 8 to 10 times, 10 to 20 times, 10 to 18 times, 10 to 15 times, 10 to 12 times, 12 to 20 times, 12 to 18 times, 12 to 15 times, 15 to 20 times, 15 to 18 times or 18 to 20 times, that of a wild strain of Streptomyces albus. According to some embodiments of the invention, the Streptomyces albus genetically engineered bacterium Q-PL2, when integrated with four epsilon-PL synthetase genes and expression elements thereof, has an ability to produce epsilon-PL by fermentation that is 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times or 20 times that of a wild strain of Streptomyces albus.

According to some embodiments of the invention, the Streptomyces albus genetically engineered bacterium Q-PL2, when integrated with five epsilon-PL synthetase genes, has a fermentation production capacity of epsilon-PL 10 to 25 times, such as 10 to 20 times, 10 to 15 times, 15 to 25 times, 15 to 20 times or 20 to 25 times that of a wild strain of Streptomyces albus. According to some embodiments of the invention, the Streptomyces albus genetically engineered bacterium Q-PL2, when integrated with five epsilon-PL synthetase genes, has a fermentation production capacity of 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 21 times, 22 times, 23 times, 24 times or 25 times that of a wild strain of Streptomyces albus.

According to certain embodiments of the invention, when multiple epsilon-PL synthetase genes are integrated into the Streptomyces albus genetically engineered bacterium Q-PL2, the fold between the ability to ferment and produce epsilon-PL and the wild strain of Streptomyces albus is increased. According to some embodiments of the invention, when multiple epsilon-PL synthetase genes are integrated into the Streptomyces albus genetically engineered bacterium Q-PL2, the ability to ferment and produce epsilon-PL is doubled compared with the wild strain of Streptomyces albus.

According to some embodiments of the invention, the Streptomyces albus genetically engineered bacterium Q-PL2 has a fermentation production capacity of 2 to 25 times, e.g., 2 to 20 times, 2 to 15 times, 2 to 10 times, 2 to 5 times, 5 to 25 times, 5 to 20 times, 5 to 15 times, 5 to 10 times, 10 to 25 times, 10 to 20 times, 10 to 15 times, 15 to 25 times, 15 to 20 times, or 20 to 25 times, that of a wild strain of Streptomyces albus. According to certain embodiments of the invention, the Streptomyces albus genetically engineered bacterium Q-PL2 has a fermentation production ability of 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, or 25-fold that of a wild strain of Streptomyces albus.

Method for producing epsilon-PL

According to certain embodiments of the invention, the invention provides methods for producing epsilon-PL using the Streptomyces albus genetically engineered bacterium Q-PL2.

According to certain embodiments of the invention, the method of producing epsilon-PL is a fermentation method.

According to certain embodiments of the invention, the method for producing epsilon-PL uses a medium comprising citrate to produce the genetically engineered strain of S.albidoides Q-PL2 by fermentation.

According to certain embodiments of the invention, the medium of the method of producing epsilon-PL comprises a fermentation medium and a feed medium. According to some embodiments of the invention, the fermentation medium comprises 1 to 20 g/l citrate.

According to certain embodiments of the invention, the fermentation medium comprises citrate 1 to 20 g/l, such as 1 to 15 g/l, 1 to 10 g/l, 1 to 5 g/l, 5 to 20 g/l, 5 to 15 g/l, 5 to 10 g/l, 10 to 20 g/l, 10 to 15 g/l or 15 to 20 g/l. According to certain embodiments of the invention, the fermentation medium comprises citrate 1 g/l, 2 g/l, 3 g/l, 4 g/l, 5 g/l, 6 g/l, 7 g/l, 8 g/l, 9 g/l, 10 g/l, 11 g/l, 12 g/l, 13 g/l, 14 g/l, 15 g/l, 16 g/l, 17 g/l, 18 g/l, 19 g/l or 20 g/l.

According to some embodiments of the invention, the feed medium comprises citrate 10-100 g/l.

According to certain embodiments of the invention, the feed medium comprises citrate 10-100 g/l, such as 10-80 g/l, 10-50 g/l, 10-30 g/l, 30-100 g/l, 30-80 g/l, 30-50 g/l, 50-100 g/l, 50-80 g/l or 80-100 g/l. According to certain embodiments of the invention, the feed medium comprises citrate 10 g/l, 20 g/l, 30 g/l, 40 g/l, 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l or 100 g/l.

According to certain embodiments of the invention, the citrate salt in the fermentation medium is one or more combinations of sodium citrate, ammonium citrate, potassium citrate, zinc citrate, calcium citrate, magnesium citrate, sodium dihydrogen citrate, potassium dihydrogen citrate, sodium potassium citrate, ferric citrate, diammonium hydrogen citrate. According to certain embodiments of the invention, the citrate may be combined in any ratio in the fermentation medium. According to certain embodiments of the invention, the citrate in the fermentation medium is sodium citrate. According to some embodiments of the invention, the citrate in the fermentation medium is 5 g/l sodium citrate.

(1) Plate culture

According to some embodiments of the invention, the seeding comprises streaking, coating, or the like.

According to some embodiments of the invention, the MS solid medium comprises apramycin in the range of 60 to 120 micrograms/ml, such as 60 to 100 micrograms/ml, 60 to 80 micrograms/ml, 80 to 120 micrograms/ml, 80 to 100 micrograms/ml, or 100 to 120 micrograms/ml. According to certain embodiments of the invention, the MS solid medium comprises apramycin at 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 micrograms/ml.

According to certain embodiments of the invention, streptomyces parvus Q-PL2 is cultured at 25 to 35 ℃, e.g., 25 to 32 ℃,25 to 30 ℃,25 to 28 ℃, 28 to 35 ℃, 28 to 32 ℃, 28 to 30 ℃,30 to 35 ℃,30 to 32 ℃ or 32 to 35 ℃. According to some embodiments of the invention, S.parvulus Q-PL2 is cultured at 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃,30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃ or 35 ℃.

According to certain embodiments of the invention, S.parvulus Q-PL2 is cultured for 4 to 7 days, e.g., 4 days, 5 days, 6 days, or 7 days.

According to some embodiments of the invention, streptomyces albus Q-PL2 is inoculated on MS solid culture medium containing 80 microgram/ml apramycin, and is cultured for 5 days at 30 ℃, and black spores are grown on the surface of the culture medium for standby.

MS solid culture medium

According to some embodiments of the invention, the formulation of the MS solid medium comprises 10 to 30 g/l mannitol, 10 to 30 g/l soybean meal, and 15 to 20 g/l agar powder.

According to some embodiments of the invention, the formulation of the MS solid medium is: 10 to 30 g/L of mannitol, 10 to 30 g/L of soybean meal and 15 to 20 g/L of agar powder.

According to certain embodiments of the invention, the formulation of the MS solid medium comprises 10 to 30 g/l, such as 10 to 20 g/l or 20 to 30 g/l, of mannitol. According to certain embodiments of the invention, the formulation of the MS solid medium comprises mannitol 10 g/l, 11 g/l, 12 g/l, 13 g/l, 14 g/l, 15 g/l, 16 g/l, 17 g/l, 18 g/l, 19 g/l, 20 g/l, 21 g/l, 22 g/l, 23 g/l, 24 g/l, 25 g/l, 26 g/l, 27 g/l, 28 g/l, 29 g/l or 30 g/l.

According to some embodiments of the invention, the formulation of the MS solid medium comprises 10 to 30 g/l of soy flour, such as 10 to 20 g/l or 20 to 30 g/l. According to some embodiments of the invention, the formulation of the MS solid medium comprises soybean meal 10 g/l, 11 g/l, 12 g/l, 13 g/l, 14 g/l, 15 g/l, 16 g/l, 17 g/l, 18 g/l, 19 g/l, 20 g/l, 21 g/l, 22 g/l, 23 g/l, 24 g/l, 25 g/l, 26 g/l, 27 g/l, 28 g/l, 29 g/l or 30 g/l.

According to some embodiments of the invention, the formulation of the MS solid medium comprises 15 to 20 g/l, such as 15 g/l, 16 g/l, 17 g/l, 18 g/l, 19 g/l or 20 g/l, of agar powder.

According to some embodiments of the invention, the formulation of the MS solid medium comprises 20 g/l mannitol, 20 g/l soybean meal and 20 g/l agar powder.

According to some embodiments of the invention, the formulation of the MS solid medium is mannitol 20 g/l, soy flour 20 g/l, agar powder 20 g/l.

(2) Seed culture

According to certain embodiments of the invention, the inoculation includes direct inoculation by scraping or cutting off spores from a plate, liquid inoculation after washing spores from a plate, and the like.

According to some embodiments of the invention, the seed medium comprises apramycin in the range of 60 to 120 micrograms/ml, such as 60 to 100 micrograms/ml, 60 to 80 micrograms/ml, 80 to 120 micrograms/ml, 80 to 100 micrograms/ml, or 100 to 120 micrograms/ml. According to certain embodiments of the invention, the seed medium comprises apramycin at 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 micrograms/ml.

According to some embodiments of the invention, the spores are cultured at 25-35 ℃, e.g., 25-32 ℃, 25-30 ℃, 25-28 ℃, 28-35 ℃, 28-32 ℃, 28-30 ℃, 30-35 ℃, 30-32 ℃ or 32-35 ℃. According to some embodiments of the invention, the spores are cultured at 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃,30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃ or 35 ℃.

According to some embodiments of the invention, the spores are cultured for 36 to 72 hours, such as 36 to 60 hours, 36 to 48 hours, 48 to 72 hours, 48 to 60 hours, or 60 to 72 hours. According to certain embodiments of the invention, the spores are cultured for 36 hours, 40 hours, 44 hours, 48 hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, or 72 hours.

According to some embodiments of the invention, the culturing is shaking culturing.

According to some embodiments of the invention, the rotational speed of the rocking platforms is 200 to 300 revolutions per minute, such as 200 to 280 revolutions per minute, 200 to 260 revolutions per minute, 200 to 240 revolutions per minute, 200 to 220 revolutions per minute, 220 to 300 revolutions per minute, 220 to 280 revolutions per minute, 220 to 260 revolutions per minute, 220 to 240 revolutions per minute, 240 to 300 revolutions per minute, 240 to 280 revolutions per minute, 240 to 260 revolutions per minute, 260 to 300 revolutions per minute, 260 to 280 revolutions per minute or 280 to 300 revolutions per minute. According to some embodiments of the invention, the rotational speed of the rocking platforms is 200 rpm, 210 rpm, 220 rpm, 230 rpm, 240 rpm, 250 rpm, 260 rpm, 270 rpm, 280 rpm, 290 rpm or 300 rpm. According to some embodiments of the invention, the rotational speed of the rocking platforms is 220 rpm.

According to some embodiments of the invention, spores are collected from the plate, inoculated into seed medium containing 80. Mu.g/ml apramycin, and incubated at 30 ℃ for 72 hours.

Seed culture medium

According to some embodiments of the present invention, the seed medium formulation comprises glucose 10-100 g/l, yeast powder 2-20 g/l, ammonium sulfate 2-20 g/l, sodium citrate 1-20 g/l, dipotassium hydrogen phosphate 0.1-2 g/l, potassium dihydrogen phosphate 0.5-5 g/l, zinc sulfate heptahydrate 0.01-0.1 g/l, magnesium sulfate heptahydrate 0.1-2 g/l, ferrous sulfate heptahydrate 0.01-0.1 g/l, and pH 5.5-7.0.

According to some embodiments of the invention, the formulation of the seed culture medium is: 10 to 100 grams/liter of glucose, 2 to 20 grams/liter of yeast powder, 2 to 20 grams/liter of ammonium sulfate, 1 to 20 grams/liter of sodium citrate, 0.1 to 2 grams/liter of dipotassium phosphate, 0.5 to 5 grams/liter of monopotassium phosphate, 0.01 to 0.1 gram/liter of zinc sulfate heptahydrate, 0.1 to 2 grams/liter of magnesium sulfate heptahydrate, 0.01 to 0.1 gram/liter of ferrous sulfate heptahydrate, and the pH value of 5.5 to 7.0.

According to some embodiments of the invention, the seed medium comprises glucose in the range of 10 to 100 g/l, such as 10 to 80 g/l, 10 to 50 g/l, 10 to 30 g/l, 30 to 100 g/l, 30 to 80 g/l, 30 to 50 g/l, 50 to 100 g/l, 50 to 80 g/l or 80 to 100 g/l. According to some embodiments of the invention, the seed medium comprises glucose 10 g/l, 20 g/l, 30 g/l, 40 g/l, 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l or 100 g/l.

According to certain embodiments of the invention, the seed medium comprises 2 to 20 g/l yeast powder, such as 2 to 15 g/l, 2 to 10 g/l, 2 to 5 g/l, 5 to 20 g/l, 5 to 15 g/l, 5 to 10 g/l, 10 to 20 g/l, 10 to 15 g/l or 15 to 20 g/l. According to some embodiments of the invention, the seed culture medium comprises yeast powder 2 g/l, 3 g/l, 4 g/l, 5 g/l, 6 g/l, 7 g/l, 8 g/l, 9 g/l, 10 g/l, 11 g/l, 12 g/l, 13 g/l, 14 g/l, 15 g/l, 16 g/l, 17 g/l, 18 g/l, 19 g/l or 20 g/l.

According to certain embodiments of the invention, the seed medium comprises 2 to 20 g/l ammonium sulfate, such as 2 to 15 g/l, 2 to 10 g/l, 2 to 5 g/l, 5 to 20 g/l, 5 to 15 g/l, 5 to 10 g/l, 10 to 20 g/l, 10 to 15 g/l or 15 to 20 g/l. According to certain embodiments of the invention, the seed medium comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 grams/liter of ammonium sulfate.

According to certain embodiments of the invention, the seed medium comprises sodium citrate 1 to 20 g/l, such as 1 to 15 g/l, 1 to 10 g/l, 1 to 5 g/l, 5 to 20 g/l, 5 to 15 g/l, 5 to 10 g/l, 10 to 20 g/l, 10 to 15 g/l or 15 to 20 g/l. According to certain embodiments of the invention, the seed medium comprises sodium citrate 1 g/l, 2 g/l, 3 g/l, 4 g/l, 5 g/l, 6 g/l, 7 g/l, 8 g/l, 9 g/l, 10 g/l, 11 g/l, 12 g/l, 13 g/l, 14 g/l, 15 g/l, 16 g/l, 17 g/l, 18 g/l, 19 g/l or 20 g/l.

According to certain embodiments of the invention, the seed medium comprises dipotassium hydrogen phosphate 0.1 to 2 grams per liter, such as 0.1 to 1.5 grams per liter, 0.1 to 1 gram per liter, 0.1 to 0.5 grams per liter, 0.5 to 2 grams per liter, 0.5 to 1.5 grams per liter, 0.5 to 1 gram per liter, 1 to 2 grams per liter, 1 to 1.5 grams per liter, or 1.5 to 2 grams per liter. According to certain embodiments of the invention, the seed medium comprises dipotassium hydrogen phosphate 0.1 g/l, 0.2 g/l, 0.3 g/l, 0.4 g/l, 0.5 g/l, 0.6 g/l, 0.7 g/l, 0.8 g/l, 0.9 g/l, 1 g/l, 1.1 g/l, 1.2 g/l, 1.3 g/l, 1.4 g/l, 1.5 g/l, 1.6 g/l, 1.7 g/l, 1.8 g/l, 1.9 g/l or 2 g/l.

According to some embodiments of the invention, the seed medium comprises potassium dihydrogen phosphate in an amount of 0.5 to 5 g/l, such as 0.5 to 4 g/l, 0.5 to 3 g/l, 0.5 to 2 g/l, 0.5 to 1 g/l, 1 to 5 g/l, 1 to 4 g/l, 1 to 3 g/l, 1 to 2 g/l, 2 to 5 g/l, 2 to 4 g/l, 2 to 3 g/l, 3 to 5 g/l, 3 to 4 g/l or 4 to 5 g/l. According to certain embodiments of the invention, the seed medium comprises monopotassium phosphate 0.5 g/l, 1 g/l, 1.5 g/l, 2 g/l, 2.5 g/l, 3 g/l, 3.5 g/l, 4 g/l, 4.5 g/l or 5 g/l.

According to some embodiments of the invention, the seed medium comprises zinc sulphate heptahydrate between 0.01 and 0.1 g/l, such as between 0.01 and 0.08 g/l, between 0.01 and 0.05 g/l, between 0.05 and 0.1 g/l, between 0.05 and 0.08 g/l or between 0.08 and 0.1 g/l. According to certain embodiments of the invention, the seed medium comprises zinc sulfate heptahydrate 0.01 g/l, 0.02 g/l, 0.03 g/l, 0.04 g/l, 0.05 g/l, 0.06 g/l, 0.07 g/l, 0.08 g/l, 0.09 g/l, or 0.1 g/l.

According to some embodiments of the invention, the seed medium comprises magnesium sulfate heptahydrate 0.1 to 2 g/l, such as 0.1 to 1.5 g/l, 0.1 to 1 g/l, 0.1 to 0.5 g/l, 0.5 to 2 g/l, 0.5 to 1.5 g/l, 0.5 to 1 g/l, 1 to 2 g/l, 1 to 1.5 g/l, or 1.5 to 2 g/l. According to certain embodiments of the invention, the seed medium comprises magnesium sulfate heptahydrate 0.1 g/l, 0.2 g/l, 0.3 g/l, 0.4 g/l, 0.5 g/l, 0.6 g/l, 0.7 g/l, 0.8 g/l, 0.9 g/l, 1 g/l, 1.1 g/l, 1.2 g/l, 1.3 g/l, 1.4 g/l, 1.5 g/l, 1.6 g/l, 1.7 g/l, 1.8 g/l, 1.9 g/l or 2 g/l.

According to certain embodiments of the invention, the seed medium comprises ferrous sulfate heptahydrate 0.01 to 0.1 g/l, such as 0.01 to 0.08 g/l, 0.01 to 0.05 g/l, 0.05 to 0.1 g/l, 0.05 to 0.08 g/l, or 0.08 to 0.1 g/l. According to certain embodiments of the invention, the seed medium comprises 0.01 g/l, 0.02 g/l, 0.03 g/l, 0.04 g/l, 0.05 g/l, 0.06 g/l, 0.07 g/l, 0.08 g/l, 0.09 g/l, or 0.1 g/l ferrous sulfate heptahydrate.

According to some embodiments of the invention, the seed medium is at pH 5.5-7.0, such as pH 5.5-6.5, pH 5.5-6.0, pH 6.0-7.0, pH 6.0-6.5, or pH 6.5-7.0. According to certain embodiments of the invention, the seed medium is pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9 or pH 7.0. According to certain embodiments of the invention, the seed medium is pH 6.0.

According to some embodiments of the invention, the seed medium formulation comprises glucose 50 g/l, yeast powder 5 g/l, ammonium sulfate 10 g/l, sodium citrate 5 g/l, dipotassium hydrogen phosphate 0.8 g/l, potassium dihydrogen phosphate 1.36 g/l, zinc sulfate heptahydrate 0.04 g/l, magnesium sulfate heptahydrate 0.5 g/l, ferrous sulfate heptahydrate 0.03 g/l, pH 6.0.

According to some embodiments of the invention, the seed medium is formulated with glucose 50 g/l, yeast powder 5 g/l, ammonium sulfate 10 g/l, sodium citrate 5 g/l, dipotassium hydrogen phosphate 0.8 g/l, potassium dihydrogen phosphate 1.36 g/l, zinc sulfate heptahydrate 0.04 g/l, magnesium sulfate heptahydrate 0.5 g/l, ferrous sulfate heptahydrate 0.03 g/l, pH 6.0.

(3) Fermentation culture

(3.1) Shake flask fermentation

According to some embodiments of the present invention, the shake flask fermentation is performed by inoculating the cultured seed culture solution into a conical flask containing a fermentation medium, culturing at 25-35 ℃ for 48-84 hours, and determining the yield of epsilon-PL in the fermentation broth.

According to some embodiments of the invention, the inoculation is at an amount of 5% to 20% (by volume).

According to certain embodiments of the invention, the inoculum size is 5% to 20% (by volume), such as 5% to 15% (by volume), 5% to 10% (by volume), 10% to 15% (by volume), 10% to 20% (by volume), or 15% to 20% (by volume). According to some embodiments of the invention, the inoculum is 5% (vol), 6% (vol), 7% (vol), 8% (vol), 9% (vol), 10% (vol), 11% (vol), 12% (vol), 13% (vol), 14% (vol), 15% (vol), 16% (vol), 17% (vol), 18% (vol), 19% (vol) or 20% (vol).

According to certain embodiments of the invention, the temperature is 25 to 35 ℃, such as 25 to 32 ℃,25 to 30 ℃,25 to 28 ℃, 28 to 35 ℃, 28 to 32 ℃, 28 to 30 ℃,30 to 35 ℃,30 to 32 ℃ or 32 to 35 ℃. According to certain embodiments of the invention, the temperature is 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃,30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃ or 35 ℃.

According to some embodiments of the invention, the culturing is shaking culturing at 200-300 rpm of the shaker, for example 200-250 rpm or 250-300 rpm. For example, 200 to 280 rpm, 200 to 260 rpm, 200 to 240 rpm, 200 to 220 rpm, 220 to 300 rpm, 220 to 280 rpm, 220 to 260 rpm, 220 to 240 rpm, 240 to 300 rpm, 240 to 280 rpm, 240 to 260 rpm, 260 to 300 rpm, 260 to 280 rpm or 280 to 300 rpm. According to some embodiments of the invention, the culturing is shaking culturing at 200 rpm, 210 rpm, 220 rpm, 230 rpm, 240 rpm, 250 rpm, 260 rpm, 270 rpm, 280 rpm, 290 rpm or 300 rpm of the shaker. According to some embodiments of the invention, the culturing is shaking culturing at 220 rpm of the shaker.

According to certain embodiments of the invention, the culturing is for 48 to 84 hours, such as 48 to 72 hours, 48 to 60 hours, 60 to 84 hours, 60 to 72 hours, or 72 to 84 hours. According to certain embodiments of the invention, the culturing is for 48 hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours, 76 hours, 80 hours, or 84 hours.

According to some embodiments of the present invention, the shake flask fermentation is performed by inoculating the cultured seed culture fluid into a conical flask containing a fermentation medium, culturing at 30 ℃ for 72 hours, and determining the yield of epsilon-PL in the fermentation fluid.

According to some embodiments of the invention, the inoculation is a 10% (volume by volume) inoculum.

According to some embodiments of the invention, the culturing is shaking culturing at 220 rpm of the shaker.

(3.2) fermentation in a fermenter

According to some embodiments of the present invention, the fermentation in the fermentation tank is to inoculate the cultured seed culture solution to the fermentation tank filled with the fermentation culture medium for culturing, the culture temperature is 25-35 ℃, the dissolved oxygen is controlled at 10% -100%, the aeration ratio is 1-5 vvm, when the pH value of the fermentation solution is reduced to 3.8-4.2, the pH value of the fermentation solution is maintained unchanged by using alkali, the concentration of the carbon source in the fermentation solution is monitored during the culture process, the fed-batch culture medium is fed when the concentration of the carbon source is 5-15 g/l, the feeding speed is adjusted by monitoring the concentration of the carbon source in the fermentation solution, the yield of epsilon-PL in the fermentation solution is measured at intervals, and the total time of the fermentation culture is 36-144 hours.

According to certain embodiments of the invention, the inoculum size of the inoculation is 5% to 20% (by volume), such as 5% to 15% (by volume), 5% to 10% (by volume), 10% to 15% (by volume), 10% to 20% (by volume) or 15% to 20% (by volume). According to some embodiments of the invention, the inoculum is 5% (vol), 6% (vol), 7% (vol), 8% (vol), 9% (vol), 10% (vol), 11% (vol), 12% (vol), 13% (vol), 14% (vol), 15% (vol), 16% (vol), 17% (vol), 18% (vol), 19% (vol) or 20% (vol).

According to some embodiments of the invention, the method of controlling dissolved oxygen is by adjusting the fermentor agitation speed. According to some embodiments of the invention, the dissolved oxygen is controlled by adjusting the rotation speed of the fermenter to 200-1200 rpm.

According to some embodiments of the invention, the fermenter is stirred at a speed of 200 to 1200 rpm, such as 200 to 1000 rpm, 200 to 800 rpm, 200 to 500 rpm, 500 to 1200 rpm, 500 to 1000 rpm, 500 to 800 rpm, 800 to 1200 rpm, 800 to 1000 rpm or 1000 to 1200 rpm. According to some embodiments of the invention, the fermenter stirring speed is 200 rpm, 300 rpm, 400 rpm, 500 rpm, 600 rpm, 700 rpm, 800 rpm, 900 rpm, 1000 rpm, 1100 rpm or 1200 rpm.

According to certain embodiments of the invention, the dissolved oxygen is dissolved oxygen 10% to 100%, such as 10% to 80%, 10% to 50%, 10% to 30%, 30% to 100%, 30% to 80%, 30% to 50%, 50% to 100%, 50% to 80%, or 80% to 100%. According to certain embodiments of the invention, the dissolved oxygen is dissolved oxygen 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.

According to certain embodiments of the invention, the aeration ratio is an aeration ratio of 1 to 5vvm, such as 1 to 3vvm or 3 to 5vvm. According to some embodiments of the invention, the aeration ratio is 1vvm, 2vvm, 3vvm, 4vvm or 5vvm.

According to certain embodiments of the invention, the pH of the fermentation broth is not controlled until the pH of the fermentation broth is reduced to a value between 3.8 and 4.2.

According to some embodiments of the invention, the reduction of the pH of the fermentation broth to 3.8-4.2 is a natural reduction to 3.8-4.2.

According to certain embodiments of the invention, the pH is reduced to 3.8 to 4.2, such as 3.8 to 4.0 or 4.0 to 4.2. According to certain embodiments of the invention, the pH is lowered to 3.8, 3.9, 4.0, 4.1 or 4.2.

According to certain embodiments of the invention, the base is ammonia or sodium hydroxide.

According to certain embodiments of the invention, the carbon source is fed-batch medium at a concentration of 5 to 15 g/l, for example 5 to 12 g/l, 5 to 10 g/l, 5 to 8 g/l, 8 to 15 g/l, 8 to 12 g/l, 8 to 10 g/l, 10 to 15 g/l, 10 to 12 g/l or 12 to 15 g/l. According to certain embodiments of the invention, the carbon source is fed-batch medium at a concentration of 5 to 15 g/l, for example at a concentration of 5 g/l, 6 g/l, 7 g/l, 8 g/l, 9 g/l, 10 g/l, 11 g/l, 12 g/l, 13 g/l, 14 g/l or 15 g/l.

According to some embodiments of the invention, the production of ε -PL in the fermentation broth is measured at intervals of 4 to 12 hours, such as 4 to 10 hours, 4 to 8 hours, 4 to 6 hours, 6 to 12 hours, 6 to 10 hours, 6 to 8 hours, 8 to 12 hours, 8 to 10 hours, or 10 to 12 hours. According to some embodiments of the invention, the production of e-PL in the fermentation broth is measured at intervals of 4 to 12 hours, such as 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours.

According to some embodiments of the invention, the total time of the fermentation culture is 36 to 144 hours, such as 36 to 120 hours, 36 to 96 hours, 36 to 72 hours, 36 to 48 hours, 48 to 144 hours, 48 to 120 hours, 48 to 96 hours, 48 to 72 hours, 72 to 144 hours, 72 to 120 hours, 72 to 96 hours, 96 to 144 hours, 96 to 120 hours, or 120 to 144 hours. According to certain embodiments of the invention, the total time of the fermentation culture is 36 hours, 38 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, 120 hours, 132 hours, or 144 hours.

According to some embodiments of the present invention, the fermentation in the fermentation tank is performed by inoculating the cultured seed culture solution into the fermentation tank filled with the fermentation culture medium, controlling the fermentation temperature in the fermentation tank to be 30 ℃, controlling the dissolved oxygen to be 30%, controlling the air introduction amount to be 3vvm, feeding ammonia water when the pH value of the fermentation solution is reduced to 4.0, controlling the pH value to be 4.0, and feeding the supplement culture medium to the fermentation solution when the glucose concentration in the fermentation solution is lower than 10 g/l. The yield of epsilon-PL in the fermentation broth was measured at intervals, and the total time of fermentation was 96 hours.

According to some embodiments of the invention, the amount of inoculation is 10% (volume by volume).

According to some embodiments of the invention, the production of ε -PL in the fermentation broth is measured at intervals of 6 hours.

Fermentation medium

According to some embodiments of the invention, the fermentation medium is formulated as: 40 to 100 grams/liter of carbon source, 2 to 20 grams/liter of nitrogen source, 1 to 20 grams/liter of citrate, 0.2 to 2 grams/liter of dipotassium hydrogen phosphate, 0.5 to 5 grams/liter of potassium dihydrogen phosphate, 0.01 to 0.1 gram/liter of zinc sulfate heptahydrate, 0.1 to 2 grams/liter of magnesium sulfate heptahydrate, 0.01 to 0.1 gram/liter of ferrous sulfate heptahydrate and pH of 5.5 to 7.0.

According to certain embodiments of the invention, the fermentation medium comprises a carbon source in the range of 40 to 100 g/l, such as 40 to 80 g/l, 40 to 60 g/l, 60 to 100 g/l, 60 to 80 g/l or 80 to 100 g/l. According to certain embodiments of the invention, the fermentation medium comprises a carbon source of 40 g/l, 45 g/l, 50 g/l, 55 g/l, 60 g/l, 65 g/l, 70 g/l, 75 g/l, 80 g/l, 85 g/l, 90 g/l, 95 g/l or 100 g/l.

According to certain embodiments of the invention, the fermentation medium comprises 2 to 20 g/l of nitrogen source, such as 2 to 15 g/l, 2 to 10 g/l, 2 to 5 g/l, 5 to 20 g/l, 5 to 15 g/l, 5 to 10 g/l, 10 to 20 g/l, 10 to 15 g/l or 15 to 20 g/l. According to certain embodiments of the invention, the fermentation medium comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 g/l of an organic nitrogen source.

According to certain embodiments of the invention, the fermentation medium comprises dipotassium hydrogen phosphate 0.2 to 2 g/l, such as 0.2 to 1.5 g/l, 0.2 to 1 g/l, 0.2 to 0.5 g/l, 0.5 to 2 g/l, 0.5 to 1.5 g/l, 0.5 to 1 g/l, 1 to 2 g/l, 1 to 1.5 g/l, or 1.5 to 2 g/l. According to certain embodiments of the invention, the fermentation medium comprises dipotassium hydrogen phosphate 0.2 g/l, 0.3 g/l, 0.4 g/l, 0.5 g/l, 0.6 g/l, 0.7 g/l, 0.8 g/l, 0.9 g/l, 1 g/l, 1.1 g/l, 1.2 g/l, 1.3 g/l, 1.4 g/l, 1.5 g/l, 1.6 g/l, 1.7 g/l, 1.8 g/l, 1.9 g/l or 2 g/l.

According to certain embodiments of the invention, the fermentation medium comprises potassium dihydrogen phosphate 0.5 to 5 g/l, such as 0.5 to 3 g/l, 0.5 to 1 g/l, 1 to 5 g/l, 1 to 3 g/l, or 3 to 5 g/l. According to certain embodiments of the invention, the fermentation medium comprises monopotassium phosphate 0.5 g/l, 1 g/l, 1.5 g/l, 2 g/l, 2.5 g/l, 3 g/l, 3.5 g/l, 4 g/l, 4.5 g/l or 5 g/l.

According to certain embodiments of the invention, the fermentation medium comprises zinc sulfate heptahydrate 0.01 to 0.1 g/l, such as 0.01 to 0.08 g/l, 0.01 to 0.05 g/l, 0.05 to 0.1 g/l, 0.05 to 0.08 g/l, or 0.08 to 0.1 g/l. According to certain embodiments of the invention, the fermentation medium comprises zinc sulfate heptahydrate 0.01 g/l, 0.02 g/l, 0.03 g/l, 0.04 g/l, 0.05 g/l, 0.06 g/l, 0.07 g/l, 0.08 g/l, 0.09 g/l, or 0.1 g/l.

According to some embodiments of the invention, the fermentation medium comprises magnesium sulfate heptahydrate from 0.1 to 2 grams per liter, e.g., from 0.1 to 1.5 grams per liter, from 0.1 to 1 gram per liter, from 0.1 to 0.5 grams per liter, from 0.5 to 2 grams per liter, from 0.5 to 1.5 grams per liter, from 0.5 to 1 gram per liter, from 1 to 2 grams per liter, from 1 to 1.5 grams per liter, or from 1.5 to 2 grams per liter. According to certain embodiments of the invention, the fermentation medium comprises magnesium sulfate heptahydrate 0.1 g/l, 0.2 g/l, 0.3 g/l, 0.4 g/l, 0.5 g/l, 0.6 g/l, 0.7 g/l, 0.8 g/l, 0.9 g/l, 1 g/l, 1.1 g/l, 1.2 g/l, 1.3 g/l, 1.4 g/l, 1.5 g/l, 1.6 g/l, 1.7 g/l, 1.8 g/l, 1.9 g/l or 2 g/l.

According to certain embodiments of the invention, the fermentation medium comprises 0.01 to 0.1 g/l, such as 0.01 to 0.08 g/l, 0.01 to 0.05 g/l, 0.05 to 0.1 g/l, 0.05 to 0.08 g/l, or 0.08 to 0.1 g/l, of ferrous sulfate heptahydrate. According to certain embodiments of the invention, the fermentation medium comprises 0.01 g/l, 0.02 g/l, 0.03 g/l, 0.04 g/l, 0.05 g/l, 0.06 g/l, 0.07 g/l, 0.08 g/l, 0.09 g/l, or 0.1 g/l of ferrous sulfate heptahydrate.

According to certain embodiments of the invention, the seed medium is at a pH of 5.5 to 7.0, such as pH 5.5 to 6.5, pH 5.5 to 6.0, pH6.0 to 7.0, pH6.0 to 6.5 or pH 6.5 to 7.0. According to certain embodiments of the invention, the seed medium is pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9 or pH 7.0. According to certain embodiments of the invention, the seed medium is pH 6.0.

According to certain embodiments of the invention, the fermentation medium formulation comprises glucose 25 g/l, glycerol 25 g/l, soy peptide 5 g/l, ammonium sulfate 10 g/l, sodium citrate 5 g/l, dipotassium phosphate 0.8 g/l, potassium dihydrogen phosphate 1.36 g/l, zinc sulfate heptahydrate 0.04 g/l, magnesium sulfate heptahydrate 0.5 g/l, ferrous sulfate heptahydrate 0.03 g/l, pH 6.0.

According to certain embodiments of the invention, the fermentation medium is formulated with glucose 25 g/l, glycerol 25 g/l, soy peptide 5 g/l, ammonium sulfate 10 g/l, sodium citrate 5 g/l, dipotassium phosphate 0.8 g/l, potassium dihydrogen phosphate 1.36 g/l, zinc sulfate heptahydrate 0.04 g/l, magnesium sulfate heptahydrate 0.5 g/l, ferrous sulfate heptahydrate 0.03 g/l, pH 6.0.

According to certain embodiments of the invention, the carbon source in the fermentation medium is one or more combinations of glucose, glycerol, xylose, fructose, mannitol. According to certain embodiments of the invention, the carbon sources may be combined in any ratio in the fermentation medium.

According to certain embodiments of the invention, the nitrogen source in the fermentation medium is an organic nitrogen source, an inorganic nitrogen source, or a combination of both. According to certain embodiments of the present invention, the organic nitrogen source or the inorganic nitrogen source may be combined in any ratio in the fermentation medium.

According to some embodiments of the invention, the organic nitrogen source in the fermentation medium is one or more of yeast powder, soy peptone, corn steep liquor, peanut meal, and peptone. According to certain embodiments of the invention, the organic nitrogen source may be combined in any proportion in the fermentation medium.

According to some embodiments of the invention, the inorganic nitrogen source is in particular one or more combinations of ammonium sulfate, ammonium chloride, urea, ammonium nitrate in the fermentation medium. According to certain embodiments of the invention, the nitrogen sources may be combined in any proportion in the fermentation medium.

According to some embodiments of the invention, the nitrogen source in the fermentation medium is one or more of ammonium sulfate, ammonium chloride, urea, ammonium nitrate, yeast powder, soy peptone, corn steep liquor, peanut meal, peptone, and combinations thereof. According to certain embodiments of the invention, the nitrogen sources may be combined in any proportion in the fermentation medium.

According to certain embodiments of the invention, the citrate salt is one or more of sodium citrate, ammonium citrate, potassium citrate, zinc citrate, calcium citrate, magnesium citrate, sodium dihydrogen citrate, potassium dihydrogen citrate, sodium potassium citrate, ferric citrate, diammonium hydrogen citrate in the fermentation medium. According to certain embodiments of the invention, the citrate may be combined in any ratio in the fermentation medium.

Supplementary culture medium

The components of the feed medium comprise 0-600 g/L of carbon source, 20-200 g/L of nitrogen source and 10-100 g/L of citrate.

According to certain embodiments of the invention, the feed medium comprises a carbon source in the range of 0 to 600 g/l, such as 0 to 500 g/l, 0 to 400 g/l, 0 to 300 g/l, 0 to 200 g/l, 0 to 100 g/l, 100 to 600 g/l, 100 to 500 g/l, 100 to 400 g/l, 100 to 300 g/l, 100 to 200 g/l, 200 to 600 g/l, 200 to 500 g/l, 200 to 400 g/l, 200 to 300 g/l, 300 to 600 g/l, 300 to 500 g/l, 300 to 400 g/l, 400 to 600 g/l, 400 to 500 g/l or 500 to 600 g/l. According to certain embodiments of the invention, the feed medium comprises a carbon source of 10 g/l, 50 g/l, 100 g/l, 150 g/l, 200 g/l, 250 g/l, 300 g/l, 350 g/l, 400 g/l, 450 g/l, 500 g/l, 550 g/l or 600 g/l.

According to certain embodiments of the invention, the feed medium comprises a nitrogen source in the range of 20 to 200 g/l, such as 20 to 150 g/l, 20 to 100 g/l, 20 to 50 g/l, 50 to 200 g/l, 50 to 150 g/l, 50 to 100 g/l, 100 to 200 g/l, 100 to 150 g/l or 150 to 200 g/l. According to some embodiments of the invention, the feed medium comprises a nitrogen source of 20 g/l, 30 g/l, 40 g/l, 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, 100 g/l, 110 g/l, 120 g/l, 130 g/l, 140 g/l, 150 g/l, 160 g/l, 170 g/l, 180 g/l, 190 g/l or 200 g/l.

According to some embodiments of the invention, the feed medium comprises glucose 0-600 g/l, glycerol 0-600 g/l, ammonium sulfate 20-200 g/l, sodium citrate 10-100 g/l.

According to some embodiments of the invention, the feed medium has the composition glucose 250 g/l, glycerol 250 g/l, ammonium sulphate 100 g/l, sodium citrate 50 g/l.

According to certain embodiments of the invention, the carbon source in the feed medium is one or more combinations of glucose, glycerol, xylose, fructose, mannitol. According to certain embodiments of the invention, the carbon sources may be combined in any ratio in the feed medium.

According to some embodiments of the invention, the nitrogen source in the feed medium is an organic nitrogen source, an inorganic nitrogen source, or a combination of both. According to certain embodiments of the present invention, the organic nitrogen source or the inorganic nitrogen source may be combined in any ratio in the fermentation medium. According to some embodiments of the invention, the organic nitrogen source in the feed medium is one or more of yeast powder, soy peptone, corn steep liquor, peanut meal, peptone, and combinations thereof. According to certain embodiments of the invention, the organic nitrogen sources may be combined in any ratio in the feed medium.

According to some embodiments of the invention, the inorganic nitrogen source in the feed medium is in particular one or more combinations of ammonium sulfate, ammonium chloride, urea, ammonium nitrate. According to certain embodiments of the invention, the inorganic nitrogen sources may be combined in any ratio in the feed medium.

According to some embodiments of the invention, the nitrogen source in the feed medium is one or more combinations of ammonium sulfate, ammonium chloride, urea, ammonium nitrate, yeast powder, soy peptone, corn steep liquor, peanut meal, peptone. According to certain embodiments of the invention, the nitrogen sources may be combined in any ratio in the feed medium.

According to certain embodiments of the invention, the citrate salt in the feed medium is one or more combinations of sodium citrate, ammonium citrate, potassium citrate, zinc citrate, calcium citrate, magnesium citrate, sodium dihydrogen citrate, potassium dihydrogen citrate, sodium potassium citrate, ferric citrate, diammonium hydrogen citrate. According to certain embodiments of the invention, the citrate may be combined in any ratio in the feed medium.

According to certain embodiments of the invention, the carbon source in the feed medium is the same as the carbon source in the fermentation medium. According to some embodiments of the invention, the nitrogen source in the feed medium is the same as the nitrogen source in the fermentation medium. According to some embodiments of the invention, the citrate in the feed medium is the same as the citrate in the fermentation medium.

Determination of ε -PL

According to certain embodiments of the invention, the concentration of ε -PL in the fermentation broth is measured during fermentation.

According to certain embodiments of the invention, the concentration of ε -PL in the fermentation broth is determined using the Dragendorff's method, the Itzhaki methyl orange colorimetry, or liquid chromatography.

According to certain embodiments of the invention, the concentration of ε -PL in the fermentation broth is determined using the Dragendorff's method. According to certain embodiments of the invention, the concentration of ε -PL in the fermentation broth is determined using the Itzhaki methyl orange colorimetry. According to certain embodiments of the invention, the concentration of ε -PL in the fermentation broth is determined using liquid chromatography. According to certain embodiments of the invention, other useful methods are used to determine the concentration of ε -PL in a fermentation broth.

According to certain embodiments of the invention, the Dragendorff's method comprises the steps of: accurately sucking 500 microliters of an epsilon-PL sample, placing the sample into a 2 milliliter centrifuge tube, adding 500 microliters of DR reagent, shaking up to form a precipitate, centrifuging the sample at 7000 rpm for 10 minutes, discarding the supernatant, washing the precipitate with 1 milliliter of absolute ethyl alcohol, centrifuging the precipitate at 7000 rpm for 10 minutes, discarding the supernatant, adding 1 milliliter of 1% Na ₂ S solution, generating black precipitate, centrifuging at 12000 r/min for 10 min, discarding the supernatant, adding 1 ml of concentrated nitric acid into the precipitate, after 20 min, after the precipitate is dissolved, diluting to 5 ml with deionized water, taking out 100. Mu.l, adding 500. Mu.l of 3% thiourea solution, adding thiourea into 20% nitric acid solution as blank, and measuring the absorbance value at 435 nm. The preparation method of the DR reagent comprises the following steps: 0.8 g of bismuth nitrate pentahydrate was dissolved in 50 ml of 20% glacial acetic acid and mixed with 20 ml of 40% aqueous potassium iodide solution. ε -PL Standard solution: concentration gradients of 0, 0.2, 0.4, 0.6, 1.2 and 1.5 grams/liter were made up with deionized water. The standard curve equation is: y =9.0826x-0.3491, where y is the epsilon-PL concentration (grams/liter) and x is the absorbance at 435 nm.

According to certain embodiments of the present invention, the Itzhaki methyl orange colorimetric method comprises the steps of: after the fermentation supernatant was diluted appropriately with 0.1 mmol/l phosphate buffer, 2 ml of the diluted solution was mixed with 2 ml of 1 mmol/l methyl orange solution, and the mixture was shaken at 30 ℃ for 30 minutes in a shaker, centrifuged at 5000 rpm for 15 minutes to remove the precipitate, 0.5 ml of the supernatant was diluted to 10 ml, and the absorbance value was measured at 465nm, and the content of ε -PL was calculated based on the standard curve. ε -PL Standard solution: concentration gradients of 0, 0.02, 0.04, 0.06, 0.08 and 0.1 g/l were made up with 0.1 mmol/l phosphate buffer. The standard curve equation is: y =0.1956-0.3125x, where y is the epsilon-PL concentration (grams/liter) and x is the absorbance value of 465 nm.

According to certain embodiments of the invention, the liquid chromatography comprises the steps of: the fermentation supernatant was treated and then measured by a Thermo Ultimate 3000 liquid chromatograph equipped with a C18 reverse phase column Tskgel ODS-120T (4.6 mm. Times.250mm, tosho Co., ltd., japan), the mobile phase was 0.1% phosphoric acid, the flow rate was 0.4 ml/min, the column temperature was 30 ℃ and the detection wavelength was 215nm, and the concentration of ε -PL in the fermentation sample was calculated from the ratio of the ε -PL peak area in the standard and the fermentation sample.

Determination of the concentration of carbon sources

According to certain embodiments of the invention, the concentration of the carbon source in the fermentation broth is determined during fermentation.

According to certain embodiments of the invention, the concentration of the carbon source in the fermentation broth is determined using liquid chromatography.

According to certain embodiments of the present invention, the liquid chromatography method for determining the concentration of a carbon source in a fermentation broth comprises the steps of: the fermentation supernatant was treated and measured by a Thermo Ultimate 3000 liquid chromatograph equipped with an ion exchange column Aminex HPX-87H (7.8 mm. Times.300mm, bio-rad, USA), the mobile phase was 5 mmol/l sulfuric acid, the flow rate was 0.6 ml/min, the column temperature was 60 ℃, the detector used was a differential refractometer, and the concentration of the carbon source in the fermentation sample was calculated from the ratio of the peak area of the carbon source in the standard and the fermentation sample.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

When "about" is used in this application to modify a numerical value, it is meant that the numerical value can fluctuate within a range of ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1%.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the application (including the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" as used herein are to be construed as open-ended terms (i.e., "including, but not limited to,") unless otherwise indicated herein or clearly contradicted by context. All methods described herein can be performed in any suitable order, as understood by those skilled in the art, unless otherwise indicated herein or otherwise clearly contradicted by context.

All patents, patent applications, and references cited in this application are incorporated by reference into this application in their entirety to the same extent as if each individual reference were individually incorporated by reference. In the event of a conflict between the present application and the references provided herein, the present application shall control.

Drawings

FIG. 1 is a schematic representation of a recombinant vector pSET152-pro-PLs for overexpression of the epsilon-PL synthetase gene integrated into the S.parvulus Q-PL genome;

FIG. 2 shows the differential expression multiple of epsilon-PL synthetase genes of Streptomyces albus gene engineering bacteria Q-PL2 and wild bacteria Q-PL in different fermentation periods;

FIG. 3 is a graph comparing the enhancement of sodium citrate on the production capacity of epsilon-PL by Streptomyces albus genetically engineered bacterium Q-PL2 and wild bacterium Q-PL;

FIG. 4 is the effect of sodium citrate concentration on the ability of Streptomyces albus genetically engineered bacterium Q-PL2 to produce epsilon-PL;

FIG. 5 is a diagram comparing production of epsilon-PL by shaking flask fermentation of Streptomyces albus genetically engineered bacterium Q-PL2 and wild bacterium Q-PL;

FIG. 6 is a concentration change curve of the Streptomyces albus genetically engineered bacterium Q-PL2 produced by the fermentation tank.

Detailed Description

The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.

Example 1

Bacterial strains and plasmids used and involved in the experiments of the invention

1. Streptomyces albus (Streptomyces albulus) Q-PL (wild strain, selected from soil sample collected from Binzhou medical college in Laiyshan district, tai city, shandong province)

2. Escherichia coli (Escherichia coli) ET12567/pUZ8002 (conjugal transfer donor bacteria, paget ET al, J Bacteriol, 1999,181

3. Escherichia coli Top10 (from Invitrogen, cat # C404006)

4. pSET152 (Streptomyces genomic integration plasmid, apramycin resistance, flett et al, FEMS Microbiol Lett, 1997, 155

5. pSET152-pro-PLs (apramycin resistance, epsilon-PL synthetase overexpression plasmid constructed according to the invention)

6. Streptomyces albus (Streptomyces albulus) Q-PL2 (obtained by integrating plasmid pSET152-pro-PLs into Streptomyces albus Q-PL genome and capable of over-expressing epsilon-PL synthetase, and the high yield epsilon-PL genetic engineering strain constructed by the invention)

Example 2

Gene engineering strain streptomyces albidoides (Stre)ptomyces albulus) Q-PL2 construction

1. Cloning and vector construction of streptomyces albidovis strong promoter, ribosome binding site and epsilon-PL synthetase gene

Annealing with primers pro1, pro2 and pro3 resulted in strong promoter kasOp and ribosome binding site DNA fragments, the sequences of the primers are as follows:

pro1:gccaagcttgggctgcaggtcgactctagatgttcacattcgaacggtctctgctttg

pro2:tgttcacattcgaacggtctctgctttgacaacatgctgtgcggtgttgtaaagtcgtggccaggagaata

pro3:atggacactccttacttagatctagtattctcctggccacgactttacaacaccgcacagcatgtt

the genome of Streptomyces albugineus (Streptomyces albulus) Q-PL is taken as a template, primers PLs-F and PLs-R are used for amplifying the epsilon-PL synthetase gene, and the PCR reaction conditions are as follows: 30 cycles were repeated at 94 ℃ 30s,55 ℃ 30s,72 ℃ for 2 min. The primer sequences are as follows:

pls-F:tactagatctaagtaaggagtgtccatatgtcgtcgccccttctcgaatcgtccttc

pls-R:caggaaacagctatgacatgattacgaattctcacgcggccgcacctccctccgcgcg

the resulting strong promoter kasOp, the ribosome binding site DNA fragment and the ε -PL synthetase gene fragment were mixed as a template, and an overlap PCR reaction was performed using primers pro1 and PLs-R to obtain a complete expression element comprising the strong promoter, the ribosome binding site and the ε -PL synthetase gene. The PCR reaction conditions are as follows: 30 cycles were repeated at 94 ℃ 30s,55 ℃ 30s,72 ℃ for 2 min. The DNA fragment of the expression element and a vector pSET152 subjected to double enzyme digestion by XbaI and EcoRI are subjected to Gibson ligation reaction and then transformed into Escherichia coli (Escherichia coli) Top10, a correctly ligated transformant is screened and sequenced for verification, and a recombinant over-expression plasmid is obtained and named as pSET152-pro-pls, and the plasmid map of the plasmid is shown in the attached figure 1.

2. Transformation of streptomyces albus by recombinant plasmid

The recombinant plasmid pSET152-pro-PLs is firstly transferred into Escherichia coli (Escherichia coli) ET12567/pUZ8002, and then the pSET152-pro-PLs is introduced into Streptomyces albulus Q-PL by utilizing a conjugative transfer method.

The specific steps of the bonding transfer are as follows: donor Escherichia coli (Escherichia coli) ET12567/pUZ8002 single colonies were picked up and cultured in LB medium containing 50. Mu.g/ml kanamycin, 50. Mu.g/ml chloramphenicol and 50. Mu.g/ml apramycin at 37 ℃ until OD600 became 0.6, and the cells were collected, washed 3 times with fresh LB medium, and finally resuspended with 200. Mu.l LB medium for use. Suspending Streptomyces albus spores in 400 microliter 2 XYT culture medium (tryptone 16 g/L, yeast powder 10 g/L, sodium chloride 5 g/L), thermally shocking in 50 ℃ water bath for 10 minutes, cooling to room temperature, mixing with prepared donor bacteria, oscillating at 30 ℃ for 1 hour (100 r/min), centrifuging, discarding part of supernatant, spreading on MS solid culture medium (mannitol 20 g/L, soybean powder 20 g/L, agar powder 20 g/L), after 14 hours, covering with 1 milliliter sterile water containing 80 microgram/milliliter of apramycin and 25 microgram/milliliter of nalidixic acid, culturing at 30 ℃ for 2 days to see resistant zygospores, and culturing the spores to obtain the genetic engineering strain Streptomyces albus Q-PL2.

3. Verification of expression level of Epsilon-PL synthetase Gene

The real-time fluorescent quantitative PCR method is adopted to verify the difference of the expression levels of epsilon-PL synthetase genes in a gene engineering strain streptomyces albidoides Q-PL2 and a wild strain streptomyces albidoides Q-PL, and the method comprises the following specific steps: taking out two strains from a refrigerator at minus 80 ℃, respectively inoculating the two strains to an MS solid culture medium for activation, collecting spores after 5-6 days, respectively inoculating 200 microliters of the two strains into conical flasks filled with 50 milliliters of seed culture medium, respectively taking 1 milliliter of bacteria when the culture is carried out for 12 hours, 24 hours, 36 hours, 48 hours and 60 hours, and collecting the bacteria by low-temperature centrifugation for RNA extraction.

The components of the seed culture medium are 50 g/L of glucose, 5 g/L of yeast powder, 10 g/L of ammonium sulfate, 5 g/L of sodium citrate, 0.8 g/L of dipotassium hydrogen phosphate, 1.36 g/L of potassium dihydrogen phosphate, 0.04 g/L of zinc sulfate heptahydrate, 0.5 g/L of magnesium sulfate heptahydrate, 0.03 g/L of ferrous sulfate heptahydrate and pH 6.0.

Total RNA of two strains is extracted by using a bacterial total RNA extraction kit (Tiangen company, a cargo number DP 430), cDNA is obtained by reverse transcription of EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix (a whole gold company, a cargo number AE 311-02), real-time fluorescence quantitative PCR reaction is carried out by using QuantiNova SYBR Green PCR kit (a Qiagen company, a cargo number 208054), and the PCR reaction conditions are as follows: 95 ℃ for 5s,60 ℃ for 10s,40 cycles. RNA polymerase sigma factor gene (hrdB) is used as an internal reference gene according to the proportion of 2 ^-ΔΔCt Relatively quantitatively calculating the difference of the expression levels of the epsilon-PL synthetase genes in the two strainsThe difference multiple.

Wherein, the primer sequences of the epsilon-PL synthetase gene for amplifying are as follows:

RT-LS1：GCGAGATGTGGAACACCTACGG

RT-LS2：GCGAGCTGCCAGCCCTTCA

the primer sequences for amplifying the reference gene hrdB are as follows:

RT-HrdB1：CTGACCAGATTCCGCCAACCC

RT-HrdB2：GCCTCTGCGGCACTGACCAT

the results of real-time fluorescent quantitative PCR are shown in FIG. 2: the expression levels of the epsilon-PL synthetase genes in the genetic engineering strain streptomyces albidoflauvs Q-PL2 in 12 hours, 24 hours, 36 hours, 48 hours and 60 hours are about 1320.5, 28.0, 14.6, 17.9 and 4.6 times of the expression levels in the wild strain streptomyces albidoflauvs Q-PL at the same time point.

Therefore, the gene engineering strain Streptomyces albus Q-PL2 adopts a constitutive promoter, so that the gene engineering strain expresses the epsilon-PL synthetase gene at high level from the beginning. Therefore, the gene engineering strain streptomyces albidoflavus Q-PL2 can express the epsilon-PL synthetase gene in a short time and high efficiency.

Example 3

Influence of sodium citrate on capability of producing epsilon-PL by fermenting streptomyces albidoflauvs genetic engineering strain Q-PL2 and wild strain Q-PL Sound box

Streaking streptomyces albidoidis Q-PL2 and Q-PL to an MS solid culture medium, culturing for 5-6 days at 30 ℃, collecting spores from a flat plate after black spores grow on the surface of the culture medium, inoculating the spores to a conical flask filled with 50 ml of seed culture medium, and carrying out shaking culture on a shaking table at 30 ℃ for 220 r/min for 48 hours to obtain seed liquid; inoculating the cultured seed culture solution into a conical flask filled with a fermentation culture medium according to the inoculation amount of 10 percent (volume ratio). The yield of ε -PL was measured by fermentation of both media after shaking culture at 30 ℃ for 72 hours at 220 rpm, as shown in FIG. 3.

The components of the fermentation medium are 50 g/L glucose, 5 g/L yeast powder, 10 g/L ammonium sulfate, 0 g/L or 2 g/L sodium citrate, 0.8 g/L dipotassium hydrogen phosphate, 1.36 g/L potassium dihydrogen phosphate, 0.04 g/L zinc sulfate heptahydrate, 0.5 g/L magnesium sulfate heptahydrate, 0.03 g/L ferrous sulfate heptahydrate and pH 6.7.

The results in FIG. 3 show that the addition of sodium citrate increases the yield of epsilon-PL produced by the genetically engineered strain Q-PL2 from 0.85 g/L to 1.81 g/L, with an increase rate of 113.5%; the yield of the wild strain Q-PL for producing the epsilon-PL is improved from 0.45 g/L to 0.58 g/L, and the growth rate is only 28.8 percent. Under the condition of not adding sodium citrate and adding sodium citrate, the yield of the epsilon-PL produced by the genetic engineering strain Q-PL2 is 88.9 percent and 212.1 percent higher than that of the wild strain Q-PL respectively.

Therefore, the capacity improvement of sodium citrate on the fermentation production of the epsilon-PL by the streptomyces albidoflauvs genetic engineering strain Q-PL2 is obviously higher than that of a wild strain Q-PL, and the sodium citrate can form a synergistic effect with the overexpression of an epsilon-PL synthetase gene.

Example 4

Influence of sodium citrate concentration on capability of streptomyces albidoflauvs genetic engineering bacteria Q-PL2 in producing epsilon-PL

Streptomyces albus Q-PL2 was cultured as described in example 3, except that the fermentation medium contained sodium citrate at various concentrations and the other components were the same. The concentrations of sodium citrate used in the examples were 0 g/l, 2.5 g/l, 5 g/l, 10 g/l and 15 g/l, respectively. The production of ε -PL was measured after 72 hours of fermentation, and the results are shown in FIG. 4.

The results in FIG. 4 show that the yield of ε -PL increased with increasing sodium citrate concentration when the sodium citrate concentration was less than 5 g/l; however, when the concentration of sodium citrate exceeds 5 g/L, the yield of ε -PL decreases. Therefore, the optimum amount of sodium citrate is 5 g/l.

Example 5

Comparison of capability of Streptomyces albus gene engineering strain Q-PL2 in shake flask fermentation production of epsilon-PL with wild strain Q-PL

Streptomyces parvus Q-PL2 and Q-PL were cultured separately as described in example 3, except that the fermentation medium contained 2 g/l ammonium citrate and the other components were the same. Shaking culture was carried out at 30 ℃ for 84 hours at 220 rpm, and the yield of ε -PL in the fermentation broth was measured every 12 hours, as shown in FIG. 5.

The results in FIG. 5 show that the ability of the genetically engineered strain Q-PL2 to produce epsilon-PL by fermentation is obviously stronger than that of the wild strain Q-PL. The yield of the former reaches 2.04 g/L and the yield of the latter is only 0.57 g/L after fermentation for 72 hours, and the capability of producing epsilon-PL by fermentation of the genetic engineering strain Q-PL2 is 3.58 times of that of the wild strain Q-PL.

Example 6

Streptomyces albus gene engineering strain Q-PL2 fermentation tank for producing epsilon-PL

Marking out Streptomyces albus Q-PL2 to an MS solid culture medium, culturing for 5-6 days at 30 ℃, collecting spores from a flat plate after black spores grow on the surface of the culture medium, inoculating the spores into a conical flask filled with 50 ml of seed culture medium, and carrying out shaking culture on a shaking table at 30 ℃ for 220 r/min for 48 hours to obtain a seed solution; inoculating the cultured seed culture solution into a fermentation tank filled with a fermentation medium according to the inoculation amount of 10% (volume ratio).

The fermentation medium comprises 25 g/L of glucose, 25 g/L of glycerol, 5 g/L of soybean peptide, 10 g/L of ammonium sulfate, 5 g/L of sodium citrate, 0.8 g/L of dipotassium phosphate, 1.36 g/L of potassium dihydrogen phosphate, 0.04 g/L of zinc sulfate heptahydrate, 0.5 g/L of magnesium sulfate heptahydrate, 0.03 g/L of ferrous sulfate heptahydrate and pH 6.0.

Controlling the fermentation temperature of the fermentation tank to be 30 ℃, controlling the air introduction amount to be 3vvm, controlling the dissolved oxygen to be more than 30% by adjusting the stirring speed to be 200-1200 r/min, not controlling the pH in the early stage of fermentation, feeding ammonia water when the pH value of the fermentation solution is reduced to 4.0 to control the pH to be 4.0, and feeding a supplemented medium into the fermentation solution when the concentration of glucose in the fermentation solution is lower than 10 g/l. The components of the feed medium are 250 g/L of glucose, 250 g/L of glycerol, 100 g/L of ammonium sulfate and 50 g/L of sodium citrate. The yield of epsilon-PL in the fermentation broth was measured every several hours, and the fermentation results are shown in FIG. 6.

The results in FIG. 6 show that the Streptomyces albus genetically engineered bacterium Q-PL2 can produce 42.9 g/L of epsilon-PL within 88 hours, the production rate reaches 11.7 g/L/day, and the production rate is obviously higher than that of the prior art by 3-5 g/L/day.

The invention discloses a genetically engineered Streptomyces albus (Streptomyces albulus) Q-PL2, the capability of producing epsilon-PL by fermentation is 2-5 times of that of a wild strain of the Streptomyces albus, and by applying the Streptomyces albus and a special fermentation method thereof, the yield of the epsilon-PL in 88 hours reaches 42.9 g/l, and the production rate reaches 11.7 g/l/day.

The embodiments of the present application are exemplarily described above with reference to the drawings. Those skilled in the art can easily conceive of the disclosure of the present specification that various embodiments can be appropriately modified and recombined according to actual needs without departing from the spirit of the present application. The protection scope of this application is subject to the claims of this application.

Sequence listing

<110> Binzhou medical college

<120> streptomyces albidoflauvs genetic engineering bacterium and application thereof in epsilon-polylysine production

<160>2

<210>1

<211>96

<212>DNA

<213> Streptomyces albus (Streptomyces albulus)

<221> promoter kasOp and ribosome binding site sequence

<400>1

tgttcacattcgaacggtctctgctttgacaacatgctgtgcggtgttgtaaagtcgtggccaggagaataCTAGAtctaagtaagg agtgtccat

<210>2

<211>3960

<212>DNA

<213> Streptomyces albus (Streptomyces albulus)

<221> < pls Gene

<400>2

Atgtcgtcgccccttctcgaatcgtccttcgagccgtccgagccagcgccccaacaggccctgtaccgcaccgccggcaacccgg ccccgcggaccctgctcgacgtgctcgatgccaccgccgccgcacatccccaggcgatcgccctggacacgggctccgaggcgct cacctaccgcgacctgtgtatcgagatcgaacgccgcgcacggcagctcagggaccgcggcatcggtcccggcgaccgggtcgga gtccgcgtcccctccgggaccgccgagctgtacctgtccatcctcgccgtcctgcgcagcggagcggcctacgtgccggtcgacg ccgacgaccccgacgagcgggccgccaccgtcttccgcgaggccgccgtctgcgccgtcctcggccccgacggcccgctgcccgg cccggcccggcccctcggcgacccgcgttccgcgggcccccaggacgacgcctggatcatcttcacctcgggttcgaccggcgcg cccaagggcgtggcggtcagccaccgctccgccgccgccttcgtcgacgccgaggccgacctgttctgccaggaccagccgttgg gccccggcgaccgggtgctggccgggctgtccgtcgccttcgacgcctcctgcgaggagatgtggctcgcctggcggtacggcgc ctgcctggtgcccgcaccccgcgcgctggtccgggccggccacgaactcggcccctggctcgtcgagcgcggcatcaccgtcgtc tccaccgtgcccaccctcgccgcgctctggccggacgaggcgatgcgccgggtccgcctgctgatcgtcggcggcgaatcctgcc cggccgggctcgtcgaccgcttcgccggacccggccgcgagatgtggaacacctacggcccgaccgagaccaccgtcgtcgcctg cgccgcccgcctgctgccgggcgagccggtccgcatcggcctgcccctgaagggctggcagctcgccgtcgtcgaccgcaccggg cagccggtgcccttcggcgccgagggcgaactgctgatcagcggcgtcggcacggcccgctacctcgaccccgccaaggacgccg aacggttccggcccgacgacgccctgggggccgcccgcgtctaccgcaccggcgacctggtccgggccgaacccgagggcctgct cttcgtcggccgcgccgacgaccagatcaaactcggcggccgccgcatcgagctgggcgagatcgacgccgccctggccgccctg cccggcgtccgcggggccgccgcggccgtccagacgacgccggccggcacccaggtgctggtcggctacgtcgttcccgagcagc gcaccgccgacggttccagcttccagcaggacaaggcccgcgcactgctccaggaacgcctgcccgcgcagttggtcccggtcct cgcggaggtcgagtccctgcccacccggacctccggcaaggtcgaccgcaaggcgctgccctggccgctgccgtccgccccggtc gactccgccaccggcgatccggccacggcgctggacggcaccgccgcccggctcgccgggatctgggaggaactcctcggcgtcc ggcccggcccggacagcgacttcgtctccctcggcggcaccagcctggtcgccgcccgcatggcgtcccagctccgcatccacca ccccggcgtctcggtcgccgacctctaccgccacccggtgctgcgcgacatggccgagcacctcgactcgctgggcggcccggtg gacgaggtccgcccggtccgccccgtcccgcgccgcaccggattcgtccaactcctcgtccagaccggcctgtacggcatcgccg gcctgcgcggactggtcgggctcgcgctcgcggacaacgtcctcggcctgctcgccccgcaggtctgggccccgcacaccgcgtg gtggctgatcatcgtcggctgggtggtgctctacagcgccccgatgcgttgcgccctcggcgcactggccgcccgcgcgctcgcc ggcaccatcaagcccggcgcctacccgcgcggcggcgccacccacctgcgcctgtggaccgccgaacgcgtcgtcgccgccttcg gcgtcccctccctgctcggcaccccctgggcgcggctctacgcccggagcctgggctgcgccacagggcggaacgtggcgctgca caccatgccgccggtcaccggcctcgccgaactcggcgacggctgcagcgtcgaacccgaggccgacatctccggctggtggctc gacggcgacaccctgcacatcggcgcggtccggatcggcgccggcgcccgggtcgcccaccgcagcatgctgatgcccggcgccg tcgtcggccagggcgccgaactcgcctccggcgcctgcctggacggagagatccccgacggcgcctcgtggtccggctccccggc ccgcccggccggcgccgccgagcggatggccggcgccgcctggcccgcccccgcctggcagcgctcgcgccgctggagcgccgcc tacggactgaccctgctgggcctgccgctgctggccctgctgtccaccgcgcccgccctggtcggcgcgtacttcctgctccgcg acagcggcaccctcgccacagccgggcttcgcctgctgctggccgtcccggtcttcacgctcctgaccactggctgctccctcct cgtcaccgccgccgtggtgcgcctcctcggccgcggcatcacgccgggactgcaccccgcgagcggtggcgtcgcctggcgcgcc tggctggtcacccgcctcctggacggcgcccgcggcagcctcttcccgctctacgccagcctcggcaccccgcactggctgcggc tgctcggcgccaaggtcggccggcacgcggagatctccaccgtgctgccgctgccctccctgctgcacgtcgaggacggcgcgtt cctcgccgacgacaccctggtggcgcccttcgaactccgcggcggctggctgcggttggggaccgtccggatcggtcgccgggcc ttcgtcggcaactccggcatcgtcgaccccggccacgacgtgcccgatcacagcctggtcggcgtgctctccaacgcccccgccg acggcgagcccggctcgtcctggctgggccggcccgccatgccgctgccccgggtggcgacccaggccgacccggcgcgcacctt cgcaccgccgcgcaggctggtccgggcccgcgccgccgtcgagctgtgccgggtgctgccgctgatgtgcggcctggcgctcgcc gagggcgtgttcctcaccgagcaggacgccttcgcccagggcggcctcggtctcgccgcactggtcggcgccccgctgctgctgg cctcgggcctcgtggcgctgctcgtcaccaccctcgcgaagtggctgctggtcggccgcttcacggtgagcgagcaccccctgtg gtcgtcgttcgtgtggcgcaacgagctctacgacaccttcgtcgaatcgctcgccgtgccgtcgatggccggcgcgttcaccggc accccggtcctgaactggtggctgcgcaccctcggcgccaagatcgggcgcggggtctggttggagagctactggctgccggaga ccgacctgatcaccgtcgccgacggcgtcagcgtcaaccgcggctgcgtcctgcagacccacctcttccacgaccggatcatgcg gctggacaccgtccgcctcgccgaaggctcctcgctcggcccgcacggcatcgtgctccccggcaccgaggtcggggcgcgcgcc tcgatcgcgccgtcgtccctggtcatgcgcggcgagagcgtcccggcccacacccggtgggccggcaacccgatcgccggcgaac gccccgcccgccccgtcccggcacgcgcggagggaggtgcggccgcgtga。

<110> Bizhou medical college

<160>2

<210>1

<211>96

<212>DNA

<213>Streptomyces albus (Streptomyces albulus）

<221>PromoterskasOp and ribosome binding site sequence

<400>1

tgttcacattcgaacggtctctgctttgacaacatgctgtgcggtgttgtaaagtcgtggccaggagaataCTAGAtctaagtaaggagtgtccat

<210>2

<211>3960

<212>DNA

<213>Streptomyces albus (Streptomyces albulus）

<221> pls Gene

<400>2

Atgtcgtcgccccttctcgaatcgtccttcgagccgtccgagccagcgccccaacaggccctgtaccgcaccgccggcaacccggccccgcggaccctgctcgacgtgctcgatgccaccgccgccgcacatccccaggcgatcgccctggacacgggctccgaggcgctcacctaccgcgacctgtgtatcgagatcgaacgccgcgcacggcagctcagggaccgcggcatcggtcccggcgaccgggtcggagtccgcgtcccctccgggaccgccgagctgtacctgtccatcctcgccgtcctgcgcagcggagcggcctacgtgccggtcgacgccgacgaccccgacgagcgggccgccaccgtcttccgcgaggccgccgtctgcgccgtcctcggccccgacggcccgctgcccggcccggcccggcccctcggcgacccgcgttccgcgggcccccaggacgacgcctggatcatcttcacctcgggttcgaccggcgcgcccaagggcgtggcggtcagccaccgctccgccgccgccttcgtcgacgccgaggccgacctgttctgccaggaccagccgttgggccccggcgaccgggtgctggccgggctgtccgtcgccttcgacgcctcctgcgaggagatgtggctcgcctggcggtacggcgcctgcctggtgcccgcaccccgcgcgctggtccgggccggccacgaactcggcccctggctcgtcgagcgcggcatcaccgtcgtctccaccgtgcccaccctcgccgcgctctggccggacgaggcgatgcgccgggtccgcctgctgatcgtcggcggcgaatcctgcccggccgggctcgtcgaccgcttcgccggacccggccgcgagatgtggaacacctacggcccgaccgagaccaccgtcgtcgcctgcgccgcccgcctgctgccgggcgagccggtccgcatcggcctgcccctgaagggctggcagctcgccgtcgtcgaccgcaccgggcagccggtgcccttcggcgccgagggcgaactgctgatcagcggcgtcggcacggcccgctacctcgaccccgccaaggacgccgaacggttccggcccgacgacgccctgggggccgcccgcgtctaccgcaccggcgacctggtccgggccgaacccgagggcctgctcttcgtcggccgcgccgacgaccagatcaaactcggcggccgccgcatcgagctgggcgagatcgacgccgccctggccgccctgcccggcgtccgcggggccgccgcggccgtccagacgacgccggccggcacccaggtgctggtcggctacgtcgttcccgagcagcgcaccgccgacggttccagcttccagcaggacaaggcccgcgcactgctccaggaacgcctgcccgcgcagttggtcccggtcctcgcggaggtcgagtccctgcccacccggacctccggcaaggtcgaccgcaaggcgctgccctggccgctgccgtccgccccggtcgactccgccaccggcgatccggccacggcgctggacggcaccgccgcccggctcgccgggatctgggaggaactcctcggcgtccggcccggcccggacagcgacttcgtctccctcggcggcaccagcctggtcgccgcccgcatggcgtcccagctccgcatccaccaccccggcgtctcggtcgccgacctctaccgccacccggtgctgcgcgacatggccgagcacctcgactcgctgggcggcccggtggacgaggtccgcccggtccgccccgtcccgcgccgcaccggattcgtccaactcctcgtccagaccggcctgtacggcatcgccggcctgcgcggactggtcgggctcgcgctcgcggacaacgtcctcggcctgctcgccccgcaggtctgggccccgcacaccgcgtggtggctgatcatcgtcggctgggtggtgctctacagcgccccgatgcgttgcgccctcggcgcactggccgcccgcgcgctcgccggcaccatcaagcccggcgcctacccgcgcggcggcgccacccacctgcgcctgtggaccgccgaacgcgtcgtcgccgccttcggcgtcccctccctgctcggcaccccctgggcgcggctctacgcccggagcctgggctgcgccacagggcggaacgtggcgctgcacaccatgccgccggtcaccggcctcgccgaactcggcgacggctgcagcgtcgaacccgaggccgacatctccggctggtggctcgacggcgacaccctgcacatcggcgcggtccggatcggcgccggcgcccgggtcgcccaccgcagcatgctgatgcccggcgccgtcgtcggccagggcgccgaactcgcctccggcgcctgcctggacggagagatccccgacggcgcctcgtggtccggctccccggcccgcccggccggcgccgccgagcggatggccggcgccgcctggcccgcccccgcctggcagcgctcgcgccgctggagcgccgcctacggactgaccctgctgggcctgccgctgctggccctgctgtccaccgcgcccgccctggtcggcgcgtacttcctgctccgcgacagcggcaccctcgccacagccgggcttcgcctgctgctggccgtcccggtcttcacgctcctgaccactggctgctccctcctcgtcaccgccgccgtggtgcgcctcctcggccgcggcatcacgccgggactgcaccccgcgagcggtggcgtcgcctggcgcgcctggctggtcacccgcctcctggacggcgcccgcggcagcctcttcccgctctacgccagcctcggcaccccgcactggctgcggctgctcggcgccaaggtcggccggcacgcggagatctccaccgtgctgccgctgccctccctgctgcacgtcgaggacggcgcgttcctcgccgacgacaccctggtggcgcccttcgaactccgcggcggctggctgcggttggggaccgtccggatcggtcgccgggccttcgtcggcaactccggcatcgtcgaccccggccacgacgtgcccgatcacagcctggtcggcgtgctctccaacgcccccgccgacggcgagcccggctcgtcctggctgggccggcccgccatgccgctgccccgggtggcgacccaggccgacccggcgcgcaccttcgcaccgccgcgcaggctggtccgggcccgcgccgccgtcgagctgtgccgggtgctgccgctgatgtgcggcctggcgctcgccgagggcgtgttcctcaccgagcaggacgccttcgcccagggcggcctcggtctcgccgcactggtcggcgccccgctgctgctggcctcgggcctcgtggcgctgctcgtcaccaccctcgcgaagtggctgctggtcggccgcttcacggtgagcgagcaccccctgtggtcgtcgttcgtgtggcgcaacgagctctacgacaccttcgtcgaatcgctcgccgtgccgtcgatggccggcgcgttcaccggcaccccggtcctgaactggtggctgcgcaccctcggcgccaagatcgggcgcggggtctggttggagagctactggctgccggagaccgacctgatcaccgtcgccgacggcgtcagcgtcaaccgcggctgcgtcctgcagacccacctcttccacgaccggatcatgcggctggacaccgtccgcctcgccgaaggctcctcgctcggcccgcacggcatcgtgctccccggcaccgaggtcggggcgcgcgcctcgatcgcgccgtcgtccctggtcatgcgcggcgagagcgtcccggcccacacccggtgggccggcaacccgatcgccggcgaacgccccgcccgccccgtcccggcacgcgcggagggaggtgcggccgcgtga

Claims

1. The streptomyces albidoflauvs genetically engineered bacterium Q-PL2 is preserved in China general microbiological culture collection management center in 2019, 10 months and 30 days, and the preservation registration number is CGMCC NO.18772, wherein the streptomyces albidoflauvs genetically engineered bacterium Q-PL2 comprises one or more epsilon-polylysine synthetase genes introduced by genetic engineering and expression elements thereof.

2. The Streptomyces albus genetically engineered bacterium Q-PL2 according to claim 1, wherein the expression elements are specifically a promoter and a ribosome binding site.

3. The streptomyces parvulus genetically engineered bacterium Q-PL2 of claim 1, wherein the nucleotide sequence of the epsilon-polylysine synthase gene comprises SEQ ID No.2 or a homologous sequence thereof.

4. The streptomyces parvulus genetically engineered bacterium Q-PL2 of claim 1, wherein the streptomyces parvulus genetically engineered bacterium Q-PL2 has 2-25 times of the capability of producing epsilon-polylysine by fermentation compared with a wild strain of streptomyces parvulus.

5. A method for producing epsilon-polylysine by using the streptomyces albidoflauvs genetic engineering strain Q-PL2 as claimed in claim 1.

6. The method of claim 5, wherein the method uses a culture medium containing citrate to ferment the genetically engineered strain of streptomyces albidoflauvs Q-PL2 to produce epsilon-polylysine.

7. The method of claim 6, wherein the medium is a fermentation medium.

8. The method of claim 6, wherein the fermentation medium comprises 1 to 20 grams per liter citrate.

9. The method of claim 6, wherein the method comprises:

plate culture: inoculating streptomyces albidoflavus Q-PL2 to an MS solid culture medium containing 60-120 microgram/ml apramycin, culturing for 4-7 days at 25-35 ℃, and waiting until black spores are grown on the surface of the culture medium for later use;

seed culture: collecting spores from the flat plate, inoculating the spores into a seed culture medium containing 60-120 micrograms/ml apramycin, and culturing for 36-72 hours at 25-35 ℃;

fermentation culture: inoculating the cultured seed culture solution to a fermentation tank filled with a fermentation culture medium for culture, wherein the culture temperature is 25-35 ℃, dissolved oxygen is controlled to be 10-100%, the aeration ratio is 1-5 vvm, when the pH value of the fermentation solution is reduced to 3.8-4.2, the pH value of the fermentation solution is kept unchanged by using alkali, the concentration of a carbon source in the fermentation solution is monitored in the culture process, a fed-batch culture medium is fed when the concentration of the carbon source is 5-15 g/l, the feeding speed is adjusted by monitoring the concentration of the carbon source in the fermentation solution in a feedback mode, the yield of epsilon-polylysine in the fermentation solution is measured at intervals, and the total time of the fermentation culture is 36-144 hours.

10. The method of claim 9, wherein the fermentation medium has a formulation comprising 40 to 100 g/l of a carbon source, 2 to 20 g/l of a nitrogen source, 1 to 20 g/l of citrate, 0.2 to 2 g/l of dipotassium hydrogen phosphate, 0.5 to 5 g/l of potassium dihydrogen phosphate, 0.01 to 0.1 g/l of zinc sulfate heptahydrate, 0.1 to 2 g/l of magnesium sulfate heptahydrate, 0.01 to 0.1 g/l of ferrous sulfate heptahydrate, and a pH of 5.5 to 7.0; the components of the feed medium comprise 0-600 g/L of carbon source, 20-200 g/L of nitrogen source and 10-100 g/L of citrate.

11. The method of claim 10, wherein the carbon source, nitrogen source, and citrate in the feed medium are the same as the carbon source, nitrogen source, and citrate in the fermentation medium.

12. The method of claim 10 or 11, wherein the carbon source is one or more combinations of glucose, glycerol, xylose, fructose, mannitol.

13. The method of claim 10 or 11, wherein the nitrogen source is one or more of ammonium sulfate, ammonium chloride, urea, ammonium nitrate, yeast powder, soy peptone, corn steep liquor, peanut meal, peptone, or a combination thereof.

14. The method of claim 10 or 11, wherein the citrate salt is one or more combinations of sodium citrate, ammonium citrate, potassium citrate, zinc citrate, calcium citrate, magnesium citrate, sodium dihydrogen citrate, potassium dihydrogen citrate, sodium potassium citrate, ferric citrate, diammonium hydrogen citrate.