CN104762247B - Improve the engineering strain and construction method of production ascosin yield - Google Patents
Improve the engineering strain and construction method of production ascosin yield Download PDFInfo
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- CN104762247B CN104762247B CN201510128637.3A CN201510128637A CN104762247B CN 104762247 B CN104762247 B CN 104762247B CN 201510128637 A CN201510128637 A CN 201510128637A CN 104762247 B CN104762247 B CN 104762247B
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Abstract
The present invention relates to the engineering strain and construction method for improving production ascosin yield.Genetic engineering bacterium is streptomyces hygroscopicus Streptomyces hygroscopicus var.ascomyceticus TD01, is preserved in China General Microbiological culture presevation administrative center, and deposit number is CGMCC 10615.By cloning fkbN and frr genes, the dual-gene series connection over-express vectors of fkbN and frr are built using Escherichia coli streptomycete shuttle plasmid pIB139, it is transformed into Escherichia coli ET12567 bodies, streptomyces hygroscopicus ascus subspecies are converted by the method for engaging transfer, over-express vector is set to be expressed in starting strain body, the yield of structure ascosin engineering strain TD01 ascosin has reached 500mg/L, and original strain improves 42%;With great potentiality and application value.
Description
Technical field
The invention belongs to gene engineering technology field, and in particular to by building dual-gene series connection over-express vector, and
Expressed in streptomyces hygroscopicus ascus subspecies thalline, build efficient ascosin genetic engineering bacterium and its construction method and engineered strain
Application in ascosin fermenting and producing.More particularly to the engineering strain and structure for improving production ascosin yield
Method.
Background technology
Ascosin (ascomycin, FK-520) is by streptomyces hygroscopicus ascus subspecies (Streptomyces
Hygroscopicus var.ascomyceticus) fermentation caused by a kind of macrolides being made up of two thirteen rings resist
Raw element.Synthesized jointly by polyketide synthase/non-ribosomal synzyme, there are the antimycotic and natural compounds of immunosuppressive activity
Thing.Ascosin is separated from S.hygroscopicus var ascomyceticus tunnings earliest, is considered as
It is antimycotic antibiotic, is found that ascosin has immunosupress feature when studying hypotoxicity FK506 analog.Son
Capsule mycin was once used successfully to prevention of organ transplant rejection, and also had in the treatment of rheumatoid arthritis and psoriasis etc.
Remarkable efficacy.Ascosin in autoimmune disease is treated except having the effect of good, also with anti-spasm, anti-malarial, god
Isoreactivity is regenerated through protection, there is huge medical value and market value.(Okuhara M(1990)Tricyclo
compounds,a process for their production and pharmaceutical composition
containing the same:US,4894366;Sierra P G(2008)pharmacology and therapeutic
potential as anticonvul-sants and neuroprotectants.CNS Neurosci Ther,14:36-
46) at present, the correlative study of the ascosin in China is also in developing stage, obtains the height with China's independent intellectual property right
Engineered strain and the fermentation technique for producing ascosin are extremely urgent.
The expression of the different type albumen of specific regulatory gene code is different to the yield effect of secondary metabolite,
Such as:Production of the overexpression of the homologous protein of the AsnC modulins of allN gene codes to secondary metabolite tacrolimus
Amount thing significantly influences, and the albumen of the LysR-type transcription factors family of fkbR, fkbN coding and LAL transcriptional controls family
Overexpression can significantly improve the yield of tacrolimus.Therefore, it is overexpressed the specific regulatory of secondary metabolites biosynthesis
It is necessary that gene pairs, which improves the yield of secondary metabolites and reduces production cost,.The present invention is to ascosin biosynthesis
Gene cluster sequence carries out bioinformatic analysis, and proposition fkbN is ascosin biosynthesis related genes, its albumen encoded
Belong to the ATP combination transcriptional regulation proteins of LAL families, ATP combinations transcription factor can promote the synthesis of secondary metabolites.
The ribosomes regeneration factor (RRF) that the frr gene codes in streptomyces hygroscopicus genome be present can be catalyzed termination
Preceding compound ribosomes-mRNA-tRNA dissociation and ribosomal recycling, RRF can be effectively facilitated the work of albumen synthesis
Power, the recycle component of compound can improve the efficiency of albumen synthesis before termination, and RRF overexpression can effectively increase
The strong synthesis of albumen and the high yield of antibiotic.The fkbN and coding RRF albumen of present invention selection coding ATP combination transcription factors
Frr genes are the over-express vector that target gene builds dual-gene series connection, jointly high by the overexpression of fkbN and frr genes
Effect promotes the biosynthesis of ascosin, builds efficiently production ascosin engineered strain.
At present, it is gradually improved for the construction method of streptomyces gene engineered strain, and both at home and abroad for streptomyces hygroscopicus
The research of ascus subspecies engineering strain is seldom, and builds ascosin base by being overexpressed fkbN and frr tandem genes
Because the patent research of engineered strain has no report.
The content of the invention
The present invention over-express vector dual-gene by building fkbN and frr, is expressed so that fkbN in starting strain body
Albumen and RRF albumen overexpression obtain efficiently production ascosin engineered strain so as to strengthen the biosynthesis of ascosin
Streptomyces hygroscopicus var.ascomyceticus TD01.Present invention structure is overexpressed fkbN and frr strings
Symbasis because production ascosin engineered strain Streptomyces hygroscopicus var.ascomyceticus
TD01, there is realistic meaning and application value to the yield for improving ascosin.
Technical scheme is as follows:
A kind of engineering strain for improving production ascosin yield;It is characterized in that genetic engineering bacterium is water suction strepto-
Bacterium Streptomyces hygroscopicus var.ascomyceticus TD01, are preserved in China General Microbiological strain
Preservation administrative center, deposit number are CGMCC 10615.
The construction method of gene engineering strain method of the present invention;It is characterized in that:With S.hygroscopicus
Var.ascomyceticus ATCC14891 are starting strain, are overexpressed fkbN and frr tandem genes, and structure obtains efficiently
Produce ascosin engineered strain Streptomyces hygroscopicus var.ascomyceticus TD01.
Streptomyces hygroscopicus var.ascomyceticus TD01 construction step is as follows:
(1) PCR primer of fkbN genes and the degenerate primer of frr genes are designed;
(2) using the genomic DNA of streptomyces hygroscopicus as template, fkbN and frr genetic fragments are expanded respectively;
(3) the cloning vector pUCNR for being connected into frr and fkbN encoding genes is established;
(4) by cloning vector pUCNR, the over-express vector pIBNR for being connected into frr and fkbN encoding genes, heat shock are established
ET12567 competent escherichia coli cells are transformed into, are screened on apramycin, chloramphenicol, kalamycin resistance flat board positive
Transformant, enter performing PCR and double digestion checking, establish the recombinant bacterial strain for importing external source frr and fkbN gene overexpression carrier;
(5) the Escherichia coli ET12567 for importing over-express vector is subjected to engagement transfer with streptomyces hygroscopicus F-strain,
(6) engineering bacteria Streptomyces hygroscopicus of the screening with apramycin resistance
var.ascomyceticus TD01。
Engineering strain Streptomyces hygroscopicus var.ascomyceticus TD01 construction steps
Describe in detail as follows:
(1) the S.hygroscopicus var.ascomyceticus ATCC14891 (Gene reported according to NCBI
Bank:AF235504.1) the DNA sequence dna of the fkbN in gene cluster, the full base of pcr amplification primer thing, at present streptomyces hygroscopicus is designed
Because organizing also not sequencing completely, frr gene orders are not reported, pass through the ammonia of the ribosomes regeneration factor (RRF) to frr gene codes
The related homology analysis of base acid sequence, the degenerate primer of design amplification frr genes;
(2) using the genomic DNA of streptomyces hygroscopicus as template, fkbN and frr genetic fragments are expanded respectively;
(3) the over-express vector pIBNR for being connected into frr and fkbN encoding genes is established, it is heat-shock transformed to arrive ET12567 large intestine bars
Bacterium competence cell, positive transformant is screened on apramycin, chloramphenicol, kalamycin resistance flat board, enter performing PCR and double enzymes
Checking is cut, that is, obtains importing the recombinant bacterial strain of external source frr and fkbN gene overexpression carrier;
(4) the Escherichia coli ET12567 for importing over-express vector is subjected to engagement transfer with streptomyces hygroscopicus bacterial strain, screened
Tool
There are the streptomyces hygroscopicus genetic engineering bacterium Streptomyces hygroscopicus of apramycin resistance
var.ascomyceticus
TD01;
(5) engineered strain Streptomyces hygroscopicus var.ascomyceticus TD01 are shaken
Bottle fermentation examination
Test, detect the yield of cometabolism ascosin.
PCR primer sequence is:
(1) fkbN gene magnifications primer sequence
fkbN-F25’-GGTTGCATATGTAATGTGATGCCTATGGGTTTCC-3 ' (NdeI restriction enzyme sites)
fkbN-R25’-GTATTCTAGATTTAGGATCCGCACGGGCATCCACCAGG-3’
(XbaI, BamHI restriction enzyme site)
(2) frr gene magnifications degenerate primer sequence
frr-F25’GTTGCATATGTAAGAGATCTATCGAAGAGACCCTCCTCGA-3’
(NdeI, BglII restriction enzyme site)
frr-R25’-TCATTCTAGATTTAGGATCCTCAGACYTCGAGCAGCTCSG-3’
(XbaI, BamHI restriction enzyme site)
Clone fkbN and frr genes respectively from streptomyces hygroscopicus, pUC18 is connected with filling-in after BamHI digestions, then uses
It is connected after NdeI-XbaI double digestions with same with the fkbN genes of NdeI-XbaI double digestions, plasmid pUCN, by plasmid pUCN
With BamHI-XbaI double digestions, frr genes BglII-XbaI double digestions, pUCNR is named as after connection.Plasmid pUCNR is used
With the pIB139 connections of identical digestion after NdeI-XbaI double digestions, that is, obtain and be overexpressed plasmid pIBNR, recombinant plasmid pIBNR structures
Build collection of illustrative plates and see accompanying drawing.Over-express vector plasmid is transformed into host strain by engaging transfer, by apramycin resistance screening,
Structure is overexpressed the engineered strain Streptomyces hygroscopicus var.ascomyceticus of fkbN and frr genes
TD01, fermentation test then is carried out to engineering strain, detect ascosin change of production.
The production ascosin engineering bacteria of the present invention, is Streptomyces hygroscopicus
Var.ascomyceticus TD01, on March 12nd, 2015 in the Chinese general of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Logical Microbiological Culture Collection administrative center, deposit number is CGMCC 10615.
The present invention realizes the dual-gene series connection of fkbN and frr using the means of genetic engineering in streptomyces hygroscopicus mycelidium
Overexpression, successfully construct the engineering bacteria Streptomyces hygroscopicus of high yield ascosin
var.ascomyceticus TD01.Ascosin shake flask fermentation experiment, structure of the present invention are carried out using genetic engineering bacterium of the present invention
The yield for building the ascosin of the efficient production ascosin engineering bacteria of acquisition improves 42% than original strain, engineering bacteria
The yield of Streptomyces hygroscopicus var.ascomyceticus TD01 ascosin has reached 500mg/
L.Available for the fermenting and producing of ascosin, yield reduction cost can be improved.The genetic engineering bacterium that the present invention is built
Streptomyces hygroscopicus var.ascomyceticus TD01 are sent out in the production of ascosin industrial fermentation
Great effect is waved, the application value with great potentiality and Guan Kuo.
Brief description of the drawings
Fig. 1:Construction recombination plasmid pIBNR collection of illustrative plates.
Embodiment
With reference to embodiment, the present invention will be described, and following embodiments are illustrative, is not limited, ability
The professional in domain can be made improvements and change according to the spirit of the present invention, and described such modifications and variations all should
It is considered as within the scope of the invention, the scope of the present invention and essence have the right requirement to limit.
The present invention utilizes the research meanses of genetic engineering, with S.hygroscopicus var.ascomyceticus
ATCC14891 bacterial strains clone fkbN and frr genes, utilize Escherichia coli-streptomycete shuttle plasmid as starting strain
PIB139, over-express vector dual-gene structure fkbN and frr, is transformed into Escherichia coli ET12567 bodies, then by connecing
The method conversion streptomyces hygroscopicus of transfer is closed, over-express vector is expressed in starting strain body, strengthens the biology of ascosin
Synthesis obtains the engineered strain of efficiently production ascosin so as to improve ascosin yield on fermentation level
Streptomyces hygroscopicus var.ascomyceticus TD01。
Material:
1.Taq archaeal dna polymerases (Tiangeng biochemical technology Co., Ltd, BeiJing, China).
2. the restriction enzymes such as T4 ligases and NdeI, XbaI (Fermentas Products, Chinese Shanghai).
3. plasmid extraction kit (Tiangeng biochemical technology Co., Ltd, BeiJing, China).
4. DNA purifying QIAquick Gel Extraction Kits (Tiangeng biochemical technology Co., Ltd, BeiJing, China).
5. DNA gel QIAquick Gel Extraction Kit (Tiangeng biochemical technology Co., Ltd, BeiJing, China).
6. ampicillin, chloramphenicol, apramycin, kanamycins (Sigma companies).
7. LB fluid nutrient mediums (g/L):Peptone 10, yeast extract 5, sodium chloride 10, autoclaving.
8. LB solid mediums (g/L):Peptone 10, yeast extract 5, sodium chloride 10, agar 20, autoclaving.
9. YEME fluid nutrient mediums (g/L):Yeast extract 3, peptone 5, malt extract 3, glucose 10, sucrose
2mL MgCl is added after 100, pH 6.8,121 DEG C of sterilizing 20min2·6H2O(2.5M)。
10. 2 × YT culture mediums (g/L):Peptone 16, yeast extract 10, NaCl 5, pH 6.8.
11. 2CMY culture mediums (g/L):Soluble starch 10, tryptone 2, NaCl 1, (NH4)2SO42, K2HPO41,
CaCO32, inorganic salt solution 1ml/L, agar 20, PH 7.2, inorganic salt solution:FeSO47H2O 1, MgCl2·6H2O1,
ZnSO4·7H2O
Embodiment one:Dual-gene series connection over-express vector pIBNR structure
Using the genome extracted in S.hygroscopicus var.ascomyceticus mycelium as template, design is drawn
Thing PCR expands fkbN and frr genetic fragments, and the PCR primer of gained is detected through 0.8% agarose gel electrophoresis, obtained big
The small electrophoretic band for respectively 2700bp, 500bp or so.It is and corresponding after the purifying of frr gene amplification fragments, digestion, recovery
The pUC18 plasmids connection of digestion, converts DH5 α competent escherichia coli cells, is screened on amicillin resistance flat board positive
Transformant, plasmid is extracted, double digestion checking is carried out using restriction enzyme NdeI and XbaI, through Ago-Gel after double digestion
Electrophoresis obtains 500bp or so endonuclease bamhi, shows to obtain the correct recombinant plasmid of insetion sequence, is named as pUCR, is transferred to
The bacterium solution of recombinant plasmid is sequenced, and sequence is SEQ NO.1.
PUC18 is connected with filling-in after BamHI digestions, then uses NdeI-XbaI double with same with after NdeI-XbaI double digestions
The fkbN genes connection of digestion, plasmid pUC18-fkbN, by plasmid pUCN BamHI-XbaI double digestions, frr genes are used
BglII-XbaI double digestions, pUCNR is named as after connection.Plasmid pUCNR is cut through with after NdeI-XbaI double digestions with same enzyme
PIB139 connections, be named as pIBNR, Transformed E T12567 competent escherichia coli cells, sieved in apramycin resistant panel
Select positive transformant to extract plasmid, double digestion checking is carried out using restriction enzyme NdeI and XbaI, through agar after double digestion
Sugared gel electrophoresis obtains 3300bp or so endonuclease bamhi, shows that obtaining the dual-gene series connection for inserting correct sequence is overexpressed load
Body.
Primer sequence:
fkbN-F25’-GGTTGCATATGTAATGTGATGCCTATGGGTTTCC-3 ' (NdeI restriction enzyme sites)
fkbN-R25’-GTATTCTAGATTTAGGATCCGCACGGGCATCCACCAGG-3’
(XbaI, BamHI restriction enzyme site)
frr-F25’-GTTGCATATGTAAGAGATCTATCGAAGAGACCCTCCTCGA-3’
(NdeI, BglII restriction enzyme site)
frr-R25’-TCATTCTAGATTTAGGATCCTCAGACYTCGAGCAGCTCSG-3’
(XbaI, BamHI restriction enzyme site)
Double digestion system:DNA fragmentation 15 μ L, NdeI 1 μ L, XbaI 1 μ L, 10 × buffer 5 μ L, ddH2O 28 μ L, 37
DEG C reaction 2h.
Linked system:The μ L of target gene fragment 12 after digestion, 7.5 μ L, T4 ligase of plasmid vector fragment 0.5 μ L, 22
DEG C reaction 2h.
Embodiment two:Between S.hygroscopicus var.ascomyceticus-E.Coli ET12567 (pIBNR) category
Engage shift experiment
(1) donor bacterium is the E.coli ET12567 (PU28002) of the pIBNR containing over-express vector, and recipient bacterium is water suction chain
The Fresh spores of mould.
(2) take 500 μ L E. coli donor industry to be inoculated into 10mL and contain corresponding antibiotic (the μ g/mL of apramycin 50, card
That mycin 25 μ g/mL, the μ g/mL of chloramphenicol 25) fresh LB in, 37 DEG C are incubated overnight;1mL overnight cultures are taken to connect again
In the fresh LB that kind contains corresponding antibiotic to 50mL, 37 DEG C of cultures to OD600Received for 0.4-0.6,4000r/min centrifugations 3min
Collect thalline,
(3) (wash antibiotic off) twice with isometric fresh LB washing thallines, finally hanged with 2mL LB fluid nutrient mediums
It is floating.
(4) scrape off the fresh spore of streptomyces hygroscopicus to use, monospore suspension is prepared, with 500 μ L 2 × YT fluid nutrient mediums
Washing one time, 500 μ L 2 × YT fluid nutrient mediums, adjustment concentration to 10 are resuspended in after centrifugation8Individual/mL, 50 DEG C of metal bath heat shocks
10min, 37 DEG C of shaking table low speed (150rpm) activation 2h are placed in after being cooled to room temperature.
(5) certain Escherichia coli handled well and acceptor spore is taken to be well mixed, the mixing ratio of recipient bacterium and donor bacterium
Example is 1:1、1:10、1:100, and appropriate bacterium solution is coated on MgCl containing 10mmol/L2Engagement transport medium
In flat board, 14-20h is cultivated on flat board,
(6) with the 1mL aqueous solution uniform folds containing 100mg/mL nalidixic acids and 50mg/mL apramycins in flat board
It is interior, flat board is continued to be placed in 30 DEG C and cultivated 2 days, sub- growing state is shifted in observation engagement.
(7) engagement transformant is selected on streptomyces hygroscopicus solid plate, and flat board contains 50 μ g/m L nalidixic acids and 50 μ
G/mL apramycins.
(8) select the single bacterium grown on flat board and drop down onto the YEME fluid nutrient mediums containing 50 μ g/mL apramycins, 28 DEG C of vibrations
Culture.
(9) primer is designed with carrier pIB139 DNA sequence dna, extracts the genome of the transformant after shaken cultivation, pass through
PCR amplification checking transformants, the PCR primer of gained detect through 0.8% agarose gel electrophoresis, and it is left for 3600bp to obtain size
Right electrophoretic band, is consistent with gross data, i.e., the expression vector of dual-gene series connection successfully has been imported into aimed strain body
It is interior, it have successfully been obtained efficiently production ascosin engineering strain Streptomyces hygroscopicus
Var.ascomyceticus TD01, the genetic engineering bacterium of structure can be used for follow-up fermenting experiment.
Primer sequence:
pIB-F 5’-CGATGCTGTTGTGGGCACA-3’
pIB-R 5’-CGCGTTGGCCGATTCAT-3’。
Embodiment three:Produce ascosin engineered strain Streptomyces hygroscopicus
Var.ascomyceticus TD01 fermenting experiment
(1) culture medium
Seed fluid nutrient mediums of saccharomycete (g/L):Starch 10, glucose 30, peptone 6, dusty yeast 6, CaCO32, pH 7.0
Fermentation medium (g/L):Starch 20, DEXTRIN, peptone 5, dusty yeast 5, corn steep liquor 5, CaCO31, MgSO4·
7H2O 1, MnSO4·H2O 0.5, (NH4)2SO41.5, K2HPO4·3H2O 0.5, soya-bean oil 11.
(2) fermenting experiment:The efficient production ascosin engineering strain that an oese present invention obtains is scraped respectively
Streptomyces hygroscopicus var.ascomyceticus TD01 and the spore for the strain streptomyces hygroscopicus that sets out,
It is inoculated into the 250mL of 40mL seed culture mediums shaking flask, 28 DEG C, 60h is cultivated in 220rpm shaking tables, prepares seed liquor;Press body
Ratio is accumulated as 10% inoculum concentration, in the shaking flask for the 250mL for being inoculated into 40mL fermentation mediums, 28 DEG C, 220rpm shaking table culture,
External source addition 3g/L shikimic acid, fermentation terminate fermentation after 7 days after thalli growth 24h.
(3) measure of ascosin content:2mL zymotic fluids are drawn in 7mL centrifuge tubes, add the mixing of 2mL absolute ethyl alcohols
After uniformly, 50 DEG C of water-bath 2.5h are extracted, and then 5000rpm centrifuges 10min, takes supernatant through the organic filter membrane mistakes of 0.22 μ L
Film, sample analysis is carried out by high performance liquid chromatography.Measurement result shows:The efficient production ascosin work that present invention structure obtains
The yield of the ascosin of journey bacterium improves 42% than original strain, engineering bacteria Streptomyces hygroscopicus
The yield of var.ascomyceticus TD01 ascosin has reached 500mg/L.The engineered strain application that the present invention obtains
In ascosin fermenting and producing, yield reduction production cost can be effectively improved.
Claims (3)
- A kind of 1. engineering strain for improving production ascosin yield;It is characterized in that genetic engineering bacterium is streptomyces hygroscopicus Streptomyces hygroscopicus var.ascomyceticus TD01, it is preserved in China General Microbiological strain guarantor Administrative center is hidden, deposit number is CGMCC 10615.
- 2. the construction method of gene engineering strain method of claim 1;It is characterized in that:With S.hygroscopicus Var.ascomyceticus ATCC14891 are starting strain, are overexpressed fkbN and frr tandem genes, and structure obtains efficiently Produce ascosin engineered strain Streptomyces hygroscopicus var.ascomyceticus TD01.
- 3. method as claimed in claim 2, it is characterized in that Streptomyces hygroscopicus Var.ascomyceticus TD01 construction step is as follows:(1) PCR primer of fkbN genes and the degenerate primer of frr genes are designed;(2) using the genomic DNA of streptomyces hygroscopicus as template, fkbN and frr genetic fragments are expanded respectively;(3) the cloning vector pUCNR for being connected into frr and fkbN encoding genes is established;(4) by cloning vector pUCNR, the over-express vector pIBNR for being connected into frr and fkbN encoding genes is established, it is heat-shock transformed To ET12567 competent escherichia coli cells, positive transformants are screened on apramycin, chloramphenicol, kalamycin resistance flat board Son, enter performing PCR and double digestion checking, establish the recombinant bacterial strain for importing external source frr and fkbN gene overexpression carrier;(5) the Escherichia coli ET12567 for importing over-express vector is subjected to engagement transfer with streptomyces hygroscopicus F-strain,(6) engineering bacteria Streptomyces hygroscopicus of the screening with apramycin resistance var.ascomyceticus TD01。
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CN107955800B (en) * | 2016-10-17 | 2021-05-28 | 上海医药工业研究院 | Genetic engineering bacterium for producing ascomycin FK520 and preparation method thereof |
CN107245460B (en) * | 2017-05-18 | 2020-01-03 | 福建省微生物研究所 | Streptomyces hygroscopicus mutant strain for high yield of ascomycin and application thereof |
CN109468253B (en) * | 2018-11-05 | 2021-08-06 | 山东省药学科学院 | Streptomyces hygroscopicus with high rapamycin yield |
CN112111486B (en) * | 2019-06-19 | 2022-09-13 | 中国科学院寒区旱区环境与工程研究所 | tRNAmu and application thereof in improving yield of streptomycete antibiotics |
CN112410274B (en) * | 2019-08-23 | 2023-01-24 | 上海医药工业研究院 | Genetic engineering bacterium for producing ascomycin and preparation method and application thereof |
CN112301072B (en) * | 2020-11-05 | 2022-08-30 | 天津大学 | Method for improving yield of ascomycin by using polyhydroxybutyrate as intracellular carbon depot |
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