CN106148378A - Improve yield of erythrocin by sugared many spores red mould SACE_Lrp gene approach - Google Patents
Improve yield of erythrocin by sugared many spores red mould SACE_Lrp gene approach Download PDFInfo
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Abstract
The invention discloses a kind of by the red mould of sugared many sporesSACE_ LrpGene approach improves the method for yield of erythrocin, is inactivated in the red mould of sugared many spores by genetic engineering approachSACE_LrpGene, and its target gene of overexpressionSACE_5387‑5386Obtaining sugared many spores red mould erythromycin high-yielding engineering bacterial strain, producing erythromycin with the strain fermentation of gained, the strain fermentation obtaining by described technology can improve yield of erythrocin.
Description
Technical field
The invention mainly relates to a kind of method improving fermenting and producing yield of erythrocin, be specifically related to a kind of red at sugared many spores
The method improving yield of erythrocin by inactivation SACE_Lrp gene and/or overexpression SACE_Lrp target gene in mould.
Background technology
Actinomyces secondary metabolite tool has been widely used, such as antibiotic, anticancer, immunomodulator, pest repellant, elder brother
Worm control agent.In the 23000 kinds of biologically active secondary metabolites having now been found that, kind more than 10000 is had to be that actinomyces produce.So
And, the original yield of these secondary metabolites is very low, needs to obtain industrial production superior strain by screening.Past industry
Produce bacterial strain and mainly pass through random physical or the acquisition of mutagenesis method.Traditional random mutagenesis techniques is not only time-consuming, Er Qiewu
Method instructs and carries out design and rational to breeding.It is red mould to obtain that the object of the invention is through genetic engineering approach gene targeted alteration
Element superior strain, is used for erythromycin or intermediate product produces.
The red mould of the many spores of sugar is nineteen fifty-two thread actinomyces of isolated Gram-positive from soil, and its cometabolism produces
Thing Erythromycin A is important broad-spectrum macrolide class antibiotic, current erythromycin sequence of chemical derivative (CLA, Archie
Mycin, ROX, Ketek etc.) it is widely used for treating infectious diseases.Owing to erythromycin and derivative thereof are annual
World wide sales reaches tens billion of dollar, has attracted many scientists to study and how to have improved its yield.2007, Oliynyk etc.
Report the genome sequence of the red mould NRRL23338 of sugared many spores, but the research of sugared many spores red mould controlling gene is seldom, up till now
Till only bldD (SACE_2077), the controlling gene research report of SACE_7040, SACE_0012 and SACE_5599.
Procaryotic transcription regulator can be divided into LysR, AraC/XylS, TetR, LuxR, LacI, ArsR, IcIR,
16 families such as MerR, Lrp/AsnC, MarR, NtrC (EBP), OmpR, DeoR, Cold shock, GntR and Crp, Lrp/
AsnC family transcription regulatory factor widely exists in bacterium and archeobacteria, and structure has common feature, N end be with
What DNA interacted helix turn helix structure, C end comprises the ligand binding domains that typical α β-sandwich folds,
Be otherwise known as RAM (regulation of amino acid metabolism) domain.The adjusting function of Lrp/AsnC family
Extensively, great majority are involved in the transhipment of amino acid metabolism and little molecule.
Content of the invention
It is an object of the present invention to provide a kind of genetic engineering approach that passes through in the red mould of sugared many spores, inactivate SACE_Lrp gene
And or the red mould yield of erythrocin of the sugared many spores of overexpression SACE_Lrp target gene SACE_5387-5386 raising.
The present invention is achieved by the following technical solutions:
A kind of method building erythromycin superior strain, it is characterised in that described method is: by genetic engineering approach
Inactivate SACE_Lrp gene in the red mould of sugared many spores, and the sugared many spores of overexpression its target gene SACE_5387-5386 acquisition are red
Mould erythromycin high-yielding engineering bacterial strain, produces erythromycin with the strain fermentation of gained.
Build the method for erythromycin superior strain with SACE_Lrp for starting point, it is characterised in that: with the red mould of sugared many spores
SACE_Lrp gene or its expression product are starting point, search out the new gene related to erythromycin biosynthesis or albumen, logical
Cross carry out to all related genes searching out inactivating, increase copy, improving the ways such as expression, to build the red mould of sugared many spores red
Mycin superior strain, is used for fermenting and producing erythromycin or intermediate product.
The invention have the advantage that
Present invention research screens erythromycin biosynthesis negative regulatory factor SACE_Lrp, by genetic engineering approach
Transform SACE_Lrp and target gene SACE_5387-5386 thereof in the red mould of sugared many spores, be obtained in that erythromycin superior strain, be
Industrial production improves abomacetin fermentation yield provides technical support.
When knocking out SACE_Lrp gene in the red mould A226 of the many spores of sugar, yield of erythrocin improves 25%, and at Δ SACE_
Covering SACE_Lrp gene in Lrp deletion mutation strain, yield of erythrocin is restored, and shows that SACE_Lrp is that a participation is red mould
The biosynthetic negative regulatory factor of element.The target gene SACE_5387-to SACE_Lrp in the red mould A226 of sugared many spores further
5386 carry out overexpression, and yield of erythrocin improves 31%.Utilize high industrial production bacterial strain WB as starting strain, disappearance
SACE_Lrp gene, yield of erythrocin improves 19%；Overexpression SACE_5387-on the basis of lacking SACE_Lrp gene
5386, yield of erythrocin improves 41%；Add 10mM valine, the erythromycin of transformation bacterial strain further in industrial fermentation is cultivated
Output increased 48%.
Brief description
Fig. 1 is the PCR checking thiostrepton resistance gene of homology of chromosome recombinant technique schematic diagram and deletion mutation strain
(tsr) substituted for SACE_Lrp gene on the red mould chromosome of sugared many spores, SACE_Lrp gene (441bp) is by tsr (1360bp)
After replacement, length adds 919bp；M,5000bp DNA Marker；
Fig. 2 is yield of erythrocin analysis and the biomass detection of mutant
(A) starting strain A226, deletion mutation strain Δ SACE_Lrp, disappearance covering bacterial strain, process LAN strains A 226/
PIB139-5388 and the unloaded control strain of process LAN 6 days product HPLC of 30 DEG C of fermentations in R5 culture medium analyze
(B) analysis of dry cell weight in starting strain A226 and deletion mutation strain Δ SACE_Lrp sweat；
Fig. 3 is related gene transcription analysis in Δ SACE_Lrp and starting strain A226
In SACE_Lrp deletion mutation strain, erythromycin biosynthesis gene eryAI and resistant gene ermE, neighbouring branch
The transamination enzyme coding gene ilvE in amino acid ABC transporter gene SACE_5387-5386 and amino acid metabolism path
Transcriptional level be all significantly increased；
Fig. 4 is the successful structure of carrier pET28a-Lrp and the expression of SACE_Lrp albumen and purifying thereof,
(A) the double digestion checking of pET28a-Lrp plasmid, M:5000bp DNA Marker
(B) SDS-PAGE detects abduction delivering and the purifying of SACE_3986 albumen: what 7 swimming lanes showed is mesh after purification
Albumen；1-6 swimming lane is shown that the eluent after variable concentrations imidazoles elutes；
Fig. 5 is that the EMSA of SACE_Lrp albumen analyzes
(A) SDS-PAGE analyzes SACE_Lrp albumen and molecular size after purification
(B) EMSA analyze SACE_Lrp albumen respectively with contiguous gene spacer region Plrp-5387, eryAI promoter region and
The combination situation of ilvE promoter region；
Fig. 6 is for representing that SACE_5387-5386 overexpression and branched chain amino acid add the impact (A) on yield of erythrocin
The impact on yield of erythrocin for the SACE_5387-5386 overexpression
(B) branched chain amino acid adds the impact on yield of erythrocin；
Fig. 7 is to lack the red mould of SACE_Lrp and overexpression SACE_5387-5386 in erythromycin high industrial production bacterial strain WB
Element yield HPLC is analyzed.
Detailed description of the invention
Embodiment 1
1 materials and methods:
1.1 bacterial strains, plasmid and growth conditions
The bacterial strain and the plasmid that use in test are shown in Table 1.Escherichia coli at liquid LB (Luria-Bertani) culture medium or
The LB solid plate adding 1.25% agar is cultivated in 37 DEG C.The red mould A226 of the erythromycin producing strain many spores of sugar and
High industrial production bacterium WB enters at pancreas peptone soybean broth (TSB) culture medium or on the R3M flat board containing 2.2% agar in 30 DEG C
Row is cultivated.
1.2 materials, DNA operation and order-checking
PEG3350, lysozyme, TES, casamino acid, thiostrepton, apramycin are bought from Sigma company.TSB、
Yeast extract, peptone are bought in Oxoid company.Glycine, agar powder, sodium chloride and other biological reagents are all purchased from examination
Agent company.The general operation technology of Escherichia coli and the red mould of the many spores of sugar is according to standard operation[11][12].The synthesis of primer and DNA
Order-checking is completed by Sangon Biotech (Shanghai) Co., Ltd..
The bacterial strain using in table 1 test and plasmid
The structure of 1.3SACE_Lrp deletion mutant body
PUCTSR plasmid is to insert 1.36kb thiostrepton between BamH I and the Sma I restriction enzyme site of pUC18 to resist
Property gene (tsr).In order to knock out SACE_Lrp (SACE_5388) gene in the red mould of sugared many spores, respectively with PU1/PU2 and
PD1/PD2 be primer, the red mould genome of sugared many spores be template, each about 1.5kb's of upstream and downstream of PCR amplification SACE_Lrp gene
Homology arm SU and SD DNA fragmentation.
PCR amplification SU DNA fragmentation primer is:
PU1:5 '-AATGGTACCTGGTGTCCTGCTCGGACAGCA-3 ' (sequence of line is the restriction enzyme site of KpnI),
PU2:5 '-TTAGAATTCGATGATTGCGCGATCGAGTTT-3 ' (sequence of line be EcoRI be digested position
Point)；
PCR amplification SD DNA fragmentation primer is:
PD1:5 '-AAAAAGCTTTCACCATGAAAACCCTCAAGC-3 ' (sequence of line be HindIII be digested position
Point),
PD2:5 '-AAATCTAGAGACACGGTGATCTTCTTCCG-3 ' (sequence of line is the restriction enzyme site of XbaI).
Respectively two DNA fragmentations of above-mentioned SU and SD are connected to the tsr resistant gene both sides of pUCTSR, complete to build plasmid
pUC-ΔSACE_Lrp；With PU1 and PD2 as primer, pUC-Δ SACE_Lrp plasmid as template, PCR expands Tsr-Δ SACE_
Lrp DNA fragmentation, utilizes chromosome segment homologous recombination technique to import Tsr-Δ SACE_Lrp fragment in the red mould of sugared many spores,
Screen mutant according to thiostrepton resistance, i.e. obtain SACE_Lrp gene and replaced by thiostrepton resistance gene (tsr)
Engineering strain, use P1/P2 primer PCR verify this mutant by successfully build (see Fig. 1), named Δ SACE_
Lrp.The primer that PCR good authentication obtains deletion mutation strain is:
P1:5 '-AAACATATGATGGACAAACTCGATCGCGC-3 '；
P2:5 '-AAATCTAGATCAGTCGAGCTGCTTGAGGGTTT-3 '
1.4SACE_Lrp gene replys the structure of bacterial strain
In order in Δ SACE_Lrp introduce SACE_Lrp gene, devise PCR amplification SACE_Lrp gene primer:
Upstream: 5 '-AAACATATGATGGACAAACTCGATCGCGC-3 ' (sequence of line is the restriction enzyme site of NdeI),
Downstream: 5 '-AAATCTAGATCAGTCGAGCTGCTTGAGGGTTT-3 ' (sequence of line be XbaI be digested position
Point).
Amplify SACE_Lrp gene from sugared many spores red mould A226 genome, be inserted into Nde I and Xba of pIB139
Between I restriction enzyme site, construction expression plasmid pIB-Lrp, the protoplast transformation method then being mediated by PEG is by pIB-Lrp
Import in Δ SACE_Lrp.It by apramycin Preliminary screening, is that object enters performing PCR mirror with apramycin resistance gene (apr)
Fixed, it is thus achieved that reply Strain Designation be Δ SACE_Lrp/pIB-Lrp.
Overexpression SACE_Lrp gene in the 1.5 red mould A226 of sugared many spores
In the red mould A226 of the many spores of protoplast transformation technological sourcing sugar that pIB-Lrp is mediated by PEG, with apramycin
Resistant gene (apr) is that object enters performing PCR qualification, it is thus achieved that Strain Designation is A226/pIB-Lrp.The 1.6 red mold fermentations of sugared many spores
Product detects
Inoculate the red mould of sugared many spores in TSB culture medium, after 30 DEG C of shaken cultivation 2 days, R5 fluid nutrient medium 30 DEG C of transferring
Shaken cultivation 6 days.Carry out to erythromycin after fermentation ends extracting and HPLC detection.
The transcription analysis of related gene in 1.7 Δ SACE_Lrp
Use SBS Genetech RNA to extract kit to obtain by cultivating the Δ SACE_Lrp of 2 days and starting strain A226 bacterium solution respectively
RNA needed for, after being inverted to cDNA, uses machine testing on real-time fluorescence quantitative PCR instrument.1.8SACE_Lrp protein expression and purification
And EMSA experiment
Amplify SACE_Lrp gene from sugared many spores red mould A226 genome, be inserted into pET28a NdeI and
Between HindIII restriction enzyme site, construct plasmid pET28a-Lrp, be then introduced in BL21 (DE3) competence, obtain
Protein expression host BL21 (DE3)-Lrp.
By protein expression bacterial classification BL21 (the DE3)-Lrp obtaining with 1% inoculum concentration be inoculated into shake in pipe and overnight train
Support, then the inoculum concentration with 2% is inoculated in 50ml liquid LB shaking flask, cultivate for 37 DEG C and add in right amount when reaching 0.6-0.8 to OD
IPTG, and induce 10h in 22 DEG C, cultivate and carry out SDS-PAGE analysis, testing goal egg after taking appropriate bacterium solution thermal denaturation after terminating
White whether successful expression.
Destination protein after successful expression carries out ni-sepharose purification, and the gradient imidazole buffer wash-out first using appropriate concentration is miscellaneous
Destination protein is finally eluted by 500mM imidazole buffer, i.e. obtains destination protein after purification by albumen.
The destination protein taking appropriate above-mentioned purifying (includes SACE_Lrp with SACE_Lrp contiguous gene spacer sequence respectively
With the 30bp within SACE_5387 spacer region and two ends extension gene), eryAI promoter sequence and ilvE promoter sequence
Mix, add a certain amount of combination buffer solution, in 30 DEG C incubate 20min, carry out activity PAGE analyze, observe albumen with
The combination situation of DNA.
Overexpression SACE_5387-5386 gene in the 1.9 red mould A226 of sugared many spores
For overexpression SACE_5387-5386 in A226, devise pcr amplification primer thing:
Upstream: 5 '-AATCATATGGTGCAACAACCCGTACTC-3 ' (sequence of line is the restriction enzyme site of NdeI),
Downstream: 5 '-AAATCTAGATCACAGCACGTTAACGAG-3 ' (sequence of line is the restriction enzyme site of XbaI).
With reference to above-mentioned 1.5 methods, build SACE_5387-5386 gene overexpression bacterial strain in the red mould A226 of sugared many spores, life
Entitled A226/pIB-5387-5386.
1.10 erythromycin high industrial production bacterial strain WB lack SACE_Lrp and overexpression SACE_5387-5386
Lack SACE_Lrp in erythromycin high industrial production bacterial strain WB, verify that correct Strain Designation is WB Δ SACE_Lrp.
Further pIB-5387-5386 is imported in WB Δ SACE_Lrp by the protoplast transformation method that PEG mediates, the transformation obtaining
Strain Designation is WB Δ SACE_Lrp/5387-5386.Mutative symptom process is with reference to above-mentioned 1.3 and 1.9 methods.
2 interpretations of result:
2.1 Δ SACE_Lrp improve than starting strain A226 yield of erythrocin
SACE_Lrp and close on position on the red mould chromosome of sugared many spores for the gene and encoding amino acid sequence sees
NCBI(http://www.ncbi.nlm.nih.gov/gene/？Term=sace_5388 and http: //
www.ncbi.nlm.nih.gov/protein/133914513).The structure of SACE_Lrp gene deletion mutants Δ SACE_Lrp
The process of building is shown in Fig. 1.Screen deletion mutation and confirm (see Fig. 1) by PCR on the R3M flat board containing thiostrepton.Δ
SACE_Lrp ferments 6 days in R5 fluid nutrient medium, and extraction is analyzed through HPLC after concentrating, and its yield of erythrocin compares starting strain
The output increased of A226 25% (see Fig. 2 A), show SACE_Lrp be probably the participation biosynthetic negative regulation of erythromycin because of
Son.The thalline simultaneously taking fermentation every day carries out biomass estimation, and the biomass difference of result display Δ SACE_Lrp and A226 is not
Greatly (see Fig. 2 B), the disappearance that imply SACE_Lrp does not affect the thalli growth of bacterial strain.
2.2SACE_Lrp gene is replied
In order to confirm that the phenotype of mutant strain Δ SACE_Lrp is entirely due to SACE_Lrp gene mutation causes, the present invention
The covering experiment of research and design SACE_Lrp gene.PIB-Lrp include erythromycin resistance gene strong promoter PermE* and
Complete SACE_Lrp gene, is used for covering mutant strain Δ SACE_Lrp.Fermented and HPLC detects, Δ SACE_Lrp/pIB-
The yield of erythrocin of Lrp returns to the consistent level (see Fig. 2 A) with starting strain A226.
In 2.3A226, process LAN SACE_Lrp gene causes yield of erythrocin to reduce
In order to verify function in the red mould of the many spores of host's sugar for the SACE_Lrp further, confirm itself and yield of erythrocin
Relation, the present invention devises the experiment of SACE_Lrp process LAN simultaneously, imports to build in A226 table by expression vector pIB-Lrp
Reach strains A 226/pIB-Lrp.Fermented and HPLC detects, and finds that its yield of erythrocin is compared than comparison A226/pIB139, produces
Amount reduces 23% (see Fig. 2 A), and to sum up result confirms that SACE_Lrp is that in the red mould of sugared many spores, erythromycin is biosynthetic negative
Controlling gene, can improve erythromycin Isoquant by SACE_Lrp gene in the red mould of the sugared many spores of genetic engineering approach disappearance.
The transcription analysis of related gene in 2.4 Δ SACE_Lrp deletion mutation strains
QRT-PCR as a result, it was confirmed that in Δ SACE_Lrp mutant strain, the structural gene on erythromycin biological synthesis gene cluster
The expression of eryAI improves 4 times compared with starting strain A226, and resistant gene ermE improves 2 times, branched chain amino acid metabolic pathway
Middle aminotransferase gene ilvE expression improves 3 times, and the branched chain amino acid ABC transporter gene that downstream is neighbouring
The transcriptional level of SACE_5387-5386 improves 6 times (see Fig. 3), illustrates that the sudden change of SACE_Lrp can cause erythromycin biological
The rising of synthetic gene bunch, branched chain amino acid metabolism and contiguous gene transcriptional level.
The expression and purification of 2.5SACE_Lrp albumen and EMSA analyze
According to method step in 1.8, expression and purification is carried out to destination protein, by 5mM, 40mM, 60mM, 80mM, 160mM,
The protein solution that 300mM, 500mM imidazoles elutes successively carries out SDS-polyacrylamide gel electrophoresis (SDS-PAGE) point
Analysis, result is as shown in figure (see Fig. 4).Result shows, destination protein is purified out under 500mM imidazole concentration, and utilizes egg
SACE_Lrp fusion protein after purification is carried out quantitatively by white quantification kit.
EMSA analyzes display, and SACE_Lrp albumen and Plrp-5387 have a significantly external combination, and with PeryA and
PilvE but there is no fixation phenomenon (see Fig. 5), illustrates SACE_Lrp to neighbouring branched chain amino acid abc transport Protein S ACE_
5387-SACE_5386 gene has direct regulating and controlling effect.Branched chain amino acid as erythromycin biosynthetic premise material,
Have between the effective content and the yield of erythromycin of intracellular and contact closely, imply SACE_Lrp albumen for sugared many spores
The biosynthetic regulation and control of a red mould wherein approach is by regulating and controlling the content of intracellular branched chain amino acid and then affecting erythromycin
Yield.
In 2.6A226, process LAN SACE_5387-5386 gene causes yield of erythrocin to improve
In order to verify the impact on yield of erythrocin for the SACE_5387-5386, expression vector pIB-5387-5386 is imported
Process LAN strains A 226/pIB-5387-5386 is built in A226.Fermented and HPLC detects, and finds its yield of erythrocin ratio
Comparison A226/pIB139 compares, output increased 31% (see Fig. 6 A), and result confirms raising branched chain amino acid abc transport albumen
The copy of SACE_5387-5386 gene can significantly improve the yield of erythromycin.
2.7 branched chain amino acids interpolations cause yield of erythrocin to improve
In order to confirm the impact on yield of erythrocin for the branched chain amino acid, by 3 kinds of branched chain amino acids Leu, Ile and Val respectively
Adding to sugared many spores red mould A226 fermentation medium to final concentration of 10mM, fermented and HPLC detection, discovery is compared not
Interpolation state, after adding Leu, Ile and Val, yield of erythrocin is respectively increased the 16%th, 35% and 47% (see Fig. 6 B), and result is demonstrate,proved
The real effective content increasing intracellular branched chain amino acid can significantly improve the yield of erythromycin.
The high industrial production bacterial strain WB Δ SACE_Lrp/5387-5386 yield of erythrocin of 2.8 transformations significantly improves mutant strain
WB Δ SACE_Lrp, WB Δ SACE_Lrp/5387-5386 activates with control strain WB coated plate, is then respectively connected to industry seed
In the shaking flask of culture medium, after cultivating 2 days under 30 DEG C of rotating speed 220rpm, in switching industrial fermentation culture medium, continue to cultivate 6 days.Send out
Extraction after ferment terminates concentrates through HPLC analysis, and compare starting strain WB, WB Δ SACE_Lrp and WB Δ SACE_Lrp/5387-
The yield of erythrocin of 5386 has been respectively increased 19% and 41% (see Fig. 7).Further WB Δ SACE_Lrp/5387-5386's
Industrial fermentation adds 10mM valine in cultivating, and yield of erythrocin improves further to 48%.
Claims (4)
1.SACE_LrpThe New function of gene:SACE_LrpGene outcome can be with negative regulation erythromycin biosynthesis, SACE_
Lrp gene inactivation can improve yield of erythrocin.
2.SACE_Lrp target geneSACE_5387-5386New function: by genetic engineering approach in the red mould of sugared many spores
OverexpressionSACE_5387-5386Yield of erythrocin can be improved.
3. building a technical method for erythromycin superior strain, described method is: by genetic engineering approach at sugared many spores
Red mould inactivatesSACE_LrpGene, and its target gene of overexpressionSACE_5387-5386Obtain sugared many spores red mould
Bacterium erythromycin high-yielding engineering bacterial strain, produces erythromycin with described strain fermentation.
4. according to claim 1SACE_LrpThe New function of gene, it is characterised in that with the red mould of sugared many sporesSACE_LrpGene or its expression product are starting point, search out the new gene related to erythromycin biosynthesis or albumen,
By carrying out to all related genes searching out inactivating, increasing the red mould of the sugared many spores of way structure such as copy, raising expression
Erythromycin superior strain, is used for fermenting and producing erythromycin or intermediate product.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106520866A (en) * | 2016-12-13 | 2017-03-22 | 安徽大学 | Method for increasing erythromycin yield by transforming saccharopolyspora erythraea SACE_3980 gene |
| CN109321618A (en) * | 2018-11-13 | 2019-02-12 | 安徽农业大学 | A method of yield of erythrocin is improved by the red mould SACE_5717 gene of the more spores of sugar |
| CN117568301A (en) * | 2023-11-16 | 2024-02-20 | 安徽农业大学 | Method for improving erythromycin yield through rhodosporidium saccharum SACE-1646 gene |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106520866A (en) * | 2016-12-13 | 2017-03-22 | 安徽大学 | Method for increasing erythromycin yield by transforming saccharopolyspora erythraea SACE_3980 gene |
| CN106520866B (en) * | 2016-12-13 | 2021-01-01 | 安徽大学 | Method for improving yield of erythromycin by modifying saccharopolyspora erythraea SACE _3980 gene |
| CN109321618A (en) * | 2018-11-13 | 2019-02-12 | 安徽农业大学 | A method of yield of erythrocin is improved by the red mould SACE_5717 gene of the more spores of sugar |
| CN109321618B (en) * | 2018-11-13 | 2021-06-18 | 安徽农业大学 | Method for improving erythromycin yield through saccharopolyspora erythraea SACE _5717 gene |
| CN117568301A (en) * | 2023-11-16 | 2024-02-20 | 安徽农业大学 | Method for improving erythromycin yield through rhodosporidium saccharum SACE-1646 gene |
| CN117568301B (en) * | 2023-11-16 | 2024-05-10 | 安徽农业大学 | Method for improving erythromycin yield through rhodosporidium saccharum SACE-1646 gene |
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