CN110157756B - Method for improving erythromycin yield by modifying saccharopolyspora erythraea SACE _0303 gene - Google Patents
Method for improving erythromycin yield by modifying saccharopolyspora erythraea SACE _0303 gene Download PDFInfo
- Publication number
- CN110157756B CN110157756B CN201910178832.5A CN201910178832A CN110157756B CN 110157756 B CN110157756 B CN 110157756B CN 201910178832 A CN201910178832 A CN 201910178832A CN 110157756 B CN110157756 B CN 110157756B
- Authority
- CN
- China
- Prior art keywords
- sace
- gene
- erythromycin
- strain
- yield
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 title claims abstract description 103
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 65
- 229960003276 erythromycin Drugs 0.000 title claims abstract description 55
- 241000187559 Saccharopolyspora erythraea Species 0.000 title claims abstract description 29
- 238000000034 method Methods 0.000 title claims abstract description 15
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 16
- 238000010353 genetic engineering Methods 0.000 claims abstract description 8
- 238000013459 approach Methods 0.000 claims abstract description 5
- 238000013518 transcription Methods 0.000 claims abstract description 5
- 230000035897 transcription Effects 0.000 claims abstract description 5
- 238000004519 manufacturing process Methods 0.000 claims description 7
- 230000002103 transcriptional effect Effects 0.000 claims description 6
- 239000002773 nucleotide Substances 0.000 claims 1
- 125000003729 nucleotide group Chemical group 0.000 claims 1
- 230000002018 overexpression Effects 0.000 abstract description 4
- 238000009776 industrial production Methods 0.000 abstract description 3
- 239000013612 plasmid Substances 0.000 description 13
- 102000004169 proteins and genes Human genes 0.000 description 12
- 238000010276 construction Methods 0.000 description 11
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 9
- 229930006677 Erythromycin A Natural products 0.000 description 7
- 239000012634 fragment Substances 0.000 description 7
- 230000014509 gene expression Effects 0.000 description 7
- 101100445522 Saccharopolyspora erythraea (strain ATCC 11635 / DSM 40517 / JCM 4748 / NBRC 13426 / NCIMB 8594 / NRRL 2338) ermE gene Proteins 0.000 description 6
- 238000000855 fermentation Methods 0.000 description 6
- 230000004151 fermentation Effects 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 210000001938 protoplast Anatomy 0.000 description 6
- 238000012224 gene deletion Methods 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 239000002028 Biomass Substances 0.000 description 4
- 108700005075 Regulator Genes Proteins 0.000 description 4
- NSFFHOGKXHRQEW-UHFFFAOYSA-N Thiostrepton B Natural products N1C(=O)C(C)NC(=O)C(=C)NC(=O)C(C)NC(=O)C(C(C)CC)NC(C(C2=N3)O)C=CC2=C(C(C)O)C=C3C(=O)OC(C)C(C=2SC=C(N=2)C2N=3)NC(=O)C(N=4)=CSC=4C(C(C)(O)C(C)O)NC(=O)C(N=4)CSC=4C(=CC)NC(=O)C(C(C)O)NC(=O)C(N=4)=CSC=4C21CCC=3C1=NC(C(=O)NC(=C)C(=O)NC(=C)C(N)=O)=CS1 NSFFHOGKXHRQEW-UHFFFAOYSA-N 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 230000003115 biocidal effect Effects 0.000 description 4
- 239000013604 expression vector Substances 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 229930188070 thiostrepton Natural products 0.000 description 4
- NSFFHOGKXHRQEW-AIHSUZKVSA-N thiostrepton Chemical compound C([C@]12C=3SC=C(N=3)C(=O)N[C@H](C(=O)NC(/C=3SC[C@@H](N=3)C(=O)N[C@H](C=3SC=C(N=3)C(=O)N[C@H](C=3SC=C(N=3)[C@H]1N=1)[C@@H](C)OC(=O)C3=CC(=C4C=C[C@H]([C@@H](C4=N3)O)N[C@H](C(N[C@@H](C)C(=O)NC(=C)C(=O)N[C@@H](C)C(=O)N2)=O)[C@@H](C)CC)[C@H](C)O)[C@](C)(O)[C@@H](C)O)=C\C)[C@@H](C)O)CC=1C1=NC(C(=O)NC(=C)C(=O)NC(=C)C(N)=O)=CS1 NSFFHOGKXHRQEW-AIHSUZKVSA-N 0.000 description 4
- 229940063214 thiostrepton Drugs 0.000 description 4
- NSFFHOGKXHRQEW-OFMUQYBVSA-N thiostrepton A Natural products CC[C@H](C)[C@@H]1N[C@@H]2C=Cc3c(cc(nc3[C@H]2O)C(=O)O[C@H](C)[C@@H]4NC(=O)c5csc(n5)[C@@H](NC(=O)[C@H]6CSC(=N6)C(=CC)NC(=O)[C@@H](NC(=O)c7csc(n7)[C@]8(CCC(=N[C@@H]8c9csc4n9)c%10nc(cs%10)C(=O)NC(=C)C(=O)NC(=C)C(=O)N)NC(=O)[C@H](C)NC(=O)C(=C)NC(=O)[C@H](C)NC1=O)[C@@H](C)O)[C@](C)(O)[C@@H](C)O)[C@H](C)O NSFFHOGKXHRQEW-OFMUQYBVSA-N 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 241000187747 Streptomyces Species 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 210000000349 chromosome Anatomy 0.000 description 3
- 238000012217 deletion Methods 0.000 description 3
- 230000037430 deletion Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 230000004069 differentiation Effects 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 230000000877 morphologic effect Effects 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 240000000220 Panda oleosa Species 0.000 description 2
- 235000016496 Panda oleosa Nutrition 0.000 description 2
- 241000223252 Rhodotorula Species 0.000 description 2
- 241000187560 Saccharopolyspora Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- XZNUGFQTQHRASN-XQENGBIVSA-N apramycin Chemical compound O([C@H]1O[C@@H]2[C@H](O)[C@@H]([C@H](O[C@H]2C[C@H]1N)O[C@@H]1[C@@H]([C@@H](O)[C@H](N)[C@@H](CO)O1)O)NC)[C@@H]1[C@@H](N)C[C@@H](N)[C@H](O)[C@H]1O XZNUGFQTQHRASN-XQENGBIVSA-N 0.000 description 2
- 229950006334 apramycin Drugs 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 108091008053 gene clusters Proteins 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 238000002703 mutagenesis Methods 0.000 description 2
- 231100000350 mutagenesis Toxicity 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 229930000044 secondary metabolite Natural products 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 229920002562 Polyethylene Glycol 3350 Polymers 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 241000221523 Rhodotorula toruloides Species 0.000 description 1
- 241000187399 Streptomyces lincolnensis Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000001851 biosynthetic effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000012407 engineering method Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 229960003444 immunosuppressant agent Drugs 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 239000003120 macrolide antibiotic agent Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000036457 multidrug resistance Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 102000037983 regulatory factors Human genes 0.000 description 1
- 108091008025 regulatory factors Proteins 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000028160 response to osmotic stress Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000024053 secondary metabolic process Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000028070 sporulation Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 108091008023 transcriptional regulators Proteins 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 239000001974 tryptic soy broth Substances 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 101150093896 xylS gene Proteins 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
- C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for improving the yield of erythromycin by modifying a saccharopolyspora erythraea SACE _0303 gene, which comprises the steps of utilizing a screened erythromycin biosynthesis positive regulator SACE _0303, carrying out overexpression on a TetR family transcription gene SACE _0303 gene in the saccharopolyspora erythraea through a genetic engineering approach, obtaining a saccharopolyspora erythraea erythromycin high-yield engineering strain, and fermenting and producing erythromycin by using the strain to provide technical support for improving the yield of erythromycin in industrial production.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to a method for improving the yield of erythromycin by modifying saccharopolyspora erythraea SACE _0303 gene.
Background
Actinomycetes can produce abundant secondary metabolites, and the secondary metabolites and derivatives thereof have wide application, such as antibiotics, anticancer drugs, immunosuppressants and the like. Erythromycin is a macrolide antibiotic with broad-spectrum antibacterial action produced by saccharopolyspora erythraea. Since the discovery in 1952, erythromycin and its derivatives have been widely used clinically, and have been developed to the third generation. It follows that increasing the production of erythromycin is a problem that is urgently needed to be solved.
In the past, industrial production strains have been obtained mainly by physical or chemical mutagenesis methods. The traditional mutagenesis technology is time-consuming and has high randomness, and theoretical guidance cannot be provided for breeding. Through the gene engineering method, the modified antibiotic high-yield strain is obtained by inactivating certain regulatory genes or increasing the copy number of certain genes in the strain, and has good prospect. The invention aims to obtain the erythromycin high-producing strain by directionally changing genes through a genetic engineering way.
In the saccharopolyspora erythraea, no regulatory gene exists in an erythromycin biosynthesis gene cluster, and a high-yield strain obtained by modifying the gene of the saccharopolyspora erythraea through a genetic engineering approach can only modify other regulatory genes from the whole genome.
In 2005, Ramos et al, based on sequence similarity, structure and function, classified prokaryotic transcriptional regulators into 16 families, LysR, AraC/xylS, TetR, LuxR, LacI, ArsR, IcIR, MerR, AsnC, MarR, NtrC (EBP), OmpR, DeoR, Cold shock, GntR and Crp. Among them, the TetR family regulatory factors (TFRs) have high conservation in DNA binding domains and are widely involved in regulating biological activities such as multidrug resistance, antibiotic synthesis, osmotic stress response, and the like. In recent years, various TFRs involved in antibiotic production and morphological differentiation of Streptomyces have been reported, such as SAV _576, SAV _3619, SCO1712, AtrA, SLCG _2919, etc., suggesting the importance of TetR family regulatory genes in Streptomyces secondary metabolism and antibiotic biosynthesis. Among the Saccharopolyspora erythraea, there are 101 TFRs, and currently reported TFRs involved in spore morphological differentiation of Saccharopolyspora erythraea are SACE _0012 and SACE _7040, and TFRs involved in regulation of erythromycin biosynthesis are SACE _5599, SACE _3986, SACE _7301, SACE _3446, BldD (SACE _2077), PccD (SACE _3396), SACE _ Lrp (SACE _5388), SACE _5754 and the like. Indicating the importance of TFRs in erythromycin biosynthesis.
Disclosure of Invention
The invention aims to make up the defects of the prior art and provides a method for improving the yield of erythromycin by modifying a saccharopolyspora erythraea SACE _0303 gene.
The invention is realized by the following technical scheme:
a method for improving the yield of erythromycin by modifying a saccharopolyspora erythraea SACE _0303 gene comprises the steps of over-expressing the SACE _0303 gene in the saccharopolyspora erythraea through a genetic engineering approach to obtain a saccharopolyspora erythraea erythromycin high-yield engineering strain, and fermenting and producing erythromycin by using the strain.
The SACE _0303 gene product can positively regulate erythromycin biosynthesis.
The invention also provides application of the SACE _0303 gene in an industrial strain WB of saccharopolyspora erythraea, wherein the high-yield strain is obtained by over-expressing the TetR family transcriptional gene SACE _0303 gene in the industrial high-yield strain WB and can be used for producing erythromycin.
The invention has the advantages that:
in the research of the invention, an erythromycin biosynthesis positive regulator SACE _0303 is screened, and the saccharopolyspora erythraea SACE _0303 gene is overexpressed through a genetic engineering approach, so that a saccharopolyspora erythraea high-yield strain can be obtained, and a technical support is provided for improving the yield of erythromycin in industrial production.
Drawings
FIG. 1 shows the positional information of SACE _0303 gene and neighboring genes on chromosome.
FIG. 2 shows the construction of a Δ SACE _0303 mutant, wherein (A) is a schematic diagram of the construction of a Δ SACE _0303 mutant; (B) for PCR identification of Δ SACE _0303 mutants, M: 5000bp DNA Marker; 1: Δ SACE _0303 strain.
FIG. 3 shows the erythromycin production analysis of wild strain A226 and deletion mutant strain Δ SACE _ 0303.
FIG. 4 shows SACE _0303 gene complementation, construction of over-expressed strain and erythromycin yield analysis, wherein (A) is Δ SACE _0303/pIB139-0303 complementation, Δ SACE _0303/pIB139 empty load, A226/pIB139-0303 over-expression, PCR identification of A226/pIB139 empty load strain, and PCR product is apr resistance gene (776 bp); m: 5000bp DNA Marker; +: pIB139 plasmid; lane 1: Δ SACE _0303/pIB139 back-supplemented no-load control strains; 2: Δ SACE _0303/pIB139-0303 anaplerotic strains; 3: A226/pIB139 over-expressed empty control strain; 4: A226/pIB139-0303 overexpression strain; (B) erythromycin production analyses for strains A226,. DELTA.SACE _0303/pIB139-0303,. DELTA.SACE _0303/pIB139, A226/pIB 139-0303.
FIG. 5 shows the effect of SACE _0303 gene on biological and morphological differentiation, where (A) is a biomass assay of Δ SACE _0303 mutant and wild strain A226 mycelium; (B) is the spore growth of the mutant strain Δ SACE _0303 and wild strain A226.
FIG. 6 shows gene transcript level analysis, wherein (A) is the transcript levels of erythromycin biosynthetic enzyme gene eryAI (SACE _0721) and erythromycin resistance gene ermE (SACE _0733) in Δ SACE _ 0303; (B) is the self SACE _0303 transcript level in Δ SACE _ 0303.
FIG. 7 shows the purified expression of SACE _0303 protein and EMSA analysis of promoter region of related gene, in which (A) is the construction of SACE _0303 protein expression vector pET28 a-0303; (B) purifying SACE _0303 protein; (C) the EMSA analysis of SACE _0303 protein and eryAI promoter region, ermE promoter region, SACE _0303 promoter region.
FIG. 8 shows the modification of an industrial high-yielding strain WB of erythromycin using overexpression of SACE _0303 gene, wherein (A) is the PCR identification of WB/pIB139-0303 strain; SACE _0303 gene (420bp) was replaced by tsr resistance gene (1360 bp); m: 5000bp DNA Marker; 1: WB/pIB139-0303 strain; (B) the method is used for analyzing the yield of the erythromycin in the industrial high-yield strains WB and WB/pIB139-0303 of the erythromycin.
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.
The strains and plasmids used in the examples are shown in Table 1. Coli were cultured on liquid LB medium at 37 ℃ or on solid LB plates supplemented with 1.25% agar. Erythromycin producing bacteria Rhodosporidium toruloides wild strain A226 and its engineered strain WB were cultured in Tryptone Soy Broth (TSBY) medium at 30 ℃ or on MGM plates containing 2.2% agar.
TABLE 1 strains and plasmids used in this example
Examples PEG3350, lysozyme, TES, casamino acids, thiostrepton, apramycin were purchased from Sigma. TSB, yeast extract, peptone were purchased from Oxoid. Glycine, agar powder, sodium chloride and other biological reagents were purchased from reagent companies. General procedures for E.coli and S.lincolnensis were performed according to standard protocols. The synthesis of primers and DNA sequencing were performed by Biotechnology engineering (Shanghai) Inc.
The sequences of the primers synthesized in the experiment are shown in Table 2:
TABLE 2 primer List
(1) Construction of SACE _0303 Gene deletion mutants:
the pUCTSR plasmid is obtained by inserting 1360bp thiostrepton resistance gene (tsr) between BamHI and SmaI cleavage sites of pUC 18. Using the genome of wild strain A226 as template, primers SACE _0303up-F/R and SACE _0303down-F/R were used to amplify homologous recombination upstream and downstream homologous fragments 0303-up and 0303-down of about 1500 bp.
Connecting the two segments 0303-up and 0303-down to two sides of tsr resistance gene sequence of pUCTSR in sequence to complete construction of plasmid pUCTS-SACE _0303 LR; the tsr-0303LR large fragment is transformed into saccharopolyspora erythraea A226 by utilizing chromosome homologous recombination technology, and a positive mutant strain is screened by thiostrepton, so that a genetic engineering strain with the SACE _0303 gene replaced by the tsr resistance gene is obtained. And c0303-F/R is used as an identification primer, plasmid pUCTS-SACE _0303LR is used as a positive template, A226 genome is used as a negative template for PCR identification, and the positive deletion mutant strain is named as delta SACE _ 0303.
(2) Construction of SACE _0303 Gene reverted and overexpressed strains:
the genome of the wild strain A226 is used as a template, and a complete SACE _0303 gene fragment is amplified by using a back-filling primer c 0303-F/R. The amplification product was electrophoresed and recovered using a kit. The recovered SACE _0303 fragment and plasmid pIB139 are subjected to NdeI and XbaI double enzyme digestion, and the double-enzyme digested SACE _0303 fragment is cloned onto the digested plasmid pIB 139. And (3) carrying out PCR verification on the selected monoclonal antibody, and screening the integrative plasmid pIB 139-0303.
The recombinant plasmid pIB139-0303 is transferred into wild strain A226 protoplast and mutant strain delta SACE _0303 protoplast, and the pIB139 no-load plasmid is transferred into A226 protoplast and mutant strain delta SACE _0303 protoplast in the same way. Approximately 24h after transformation, the medium was covered with an aqueous apramycin solution and cultured until transformants grew. And carrying out amplification culture on the picked transformant, and then carrying out bacteria liquid PCR identification and screening on strains delta SACE _0303/pIB139, delta SACE _0303/pIB139-0303, A226/pIB139 and A226/pIB139-0303 by using a pair of Apr primers Apr-p1/p2 designed on the pIB139 no-load plasmid.
(3) Construction of WB/pIB-0303 erythromycin high-producing strains in industrial high-producing strains WB:
the constructed pIB139-0303 plasmid is transferred into a saccharopolyspora erythraea industrial strain WB protoplast to construct a WB/pIB-0303 strain, and the steps are referred to for the construction and screening method.
(4) HPLC detection of the fermentation product of saccharopolyspora erythraea:
inoculating saccharopolyspora erythraea series strains into a TSB culture medium, carrying out shake culture at 30 ℃ for 48 hours, transferring to an R5 liquid culture medium, carrying out shake culture at 30 ℃ for 6 days, extracting fermentation liquor by using an organic solvent, evaporating to dryness by using a water bath kettle, adding 1mL of methanol for dissolving, treating by using a 0.22 mu m organic filter membrane, and then loading on a computer to detect the content of erythromycin A in a sample.
(5) And (3) detecting the biomass of saccharopolyspora erythraea mycelium:
inoculating the delta SACE _0303 mutant strain and A226 in 50mL of liquid TSR, carrying out shake cultivation at 30 ℃ for 48 hours, then transferring the mutant strain to R5 culture medium, carrying out shake cultivation at 30 ℃ and 240rpm for 6d, setting sampling at different time periods, washing with absolute ethyl alcohol, drying, weighing the dry weight of the thallus, repeatedly sampling twice each time, obtaining an average value, and drawing a thallus biomass curve according to experimental data after the measurement is finished.
(6) And (3) observing the spore morphology of saccharopolyspora erythraea:
marking on an R3 culture medium, respectively coating the spore glycerol bacterial liquids of the delta SACE _0303, the delta SACE _0303/pIB139-0303, the delta SACE _0303/pIB139 and the wild strain A226 on the R3M culture medium by the same inoculation amount, airing in a super clean bench, and then placing in a 30 ℃ constant temperature incubator for inverted growth. Spore growth was observed and recorded every 24 h.
(7) Transcriptional analysis of the relevant genes in Δ SACE _ 0303:
collecting the culture liquid of the delta SACE _0303 and the wild strain A226 which are cultured for 24h, obtaining the required RNA by adopting a full-type gold RNA extraction kit, inverting the RNA into cDNA, and detecting on a computer by using a real-time fluorescence quantitative PCR instrument.
(8) Protein expression of SACE _0303 and EMSA analysis of promoter regions of related genes:
according to the information noted on NCBI, a protein expression primer p28a-0303-F/R with NdeI and HindIII enzyme cutting sites is designed, and the SACE _0303 complete gene fragment is amplified and connected with pET28 a. And carrying out PCR verification on the selected monoclonal antibody.
The successfully constructed protein expression vector pET28a-0303 is transferred into a competent cell of escherichia coli BL21(DE3), a single colony is selected on a Kana resistant LB solid culture medium, after amplification culture, PCR identification of bacterial liquid is carried out by using pET28a-0303-F/R primers, and the screened bacterial strain is named as BL21/pET28 a-0303.
BL21/pET28a-0303 is inoculated in Kana resistance LB liquid culture medium and cultured overnight at 37 ℃; inoculating according to 2% of inoculum size the next day, and culturing at 37 deg.C to obtain thallus OD600Adding IPTG (final concentration 0.5mM) between 0.5 and 1, and inducing expression at 30 ℃ and 180rpm for 5 h; collecting thallus, ultrasonic crushing, centrifuging, taking supernatant, performing Ni-affinity chromatography, eluting with imidazole of different concentrations, and detecting by SDS-PAGE. The predicted protein size is 20.57kDa, plus a sequence on the vector of about 22 kDa.
Respectively carrying out PCR amplification on promoter regions of eryAI, ermE and SACE _0303 genes in an erythromycin cluster by using an eryAI-eryBIV-F/R primer and an ermE-eryCI-F/R, SACE _0303-SACE _0304-F/R primer, and carrying out electrophoresis and recovery on amplification products. The amplified fragment was named Probe PeryAI-eryBIV、PermE-eryCI、PSACE_0303-SACE_0304. And respectively incubating the probes with the purified SACE _0303 protein at the temperature of 30 ℃, wherein the incubation system is 20 mu L, and performing active PAGE detection after 10 min.
And (4) analyzing results:
the location of SACE _0303 and adjacent genes on the chromosome of Rhodosporidium saccharopolyspora is shown in FIG. 1. The construction process of SACE _0303 gene deletion mutant A226/delta SACE _0303 is shown in FIG. 2A. SACE _0303 gene deletion mutants were screened on R3M plates containing 30. mu.g/ml thiostrepton and confirmed by PCR (FIG. 2B).
After A226/delta SACE _0303 is fermented in R5 fermentation medium for 6d, the yield of erythromycin is detected by HPLC, and the yield of A226/delta SACE _0303 is reduced by 40% compared with the yield of wild strain A226 (FIG. 3), and the HPLC result shows that SACE _0303 is a positive regulator involved in erythromycin biosynthesis.
To further verify that the improvement of erythromycin production in mutant Δ SACE _0303 was due to SACE _0303 gene deletion, SACE _0303 gene expression vectors pIB139-0303 and pIB139 vector (control) were introduced into protoplasts of the Δ SACE _0303 mutant strain and wild strain A226, respectively, to obtain a revertant strain Δ SACE _0303/pIB139-0303 and an empty Δ SACE _0303/pIB139, and an over-expressed strain A226/pIB139-0303 and an empty A226/pIB 139. A226 and delta SACE _0303 series strains are subjected to shake flask fermentation, and HPLC detection results show that: the yield of the erythromycin A in the delta SACE _0303 is reduced by 37 percent compared with the yield of the A226; the yield of erythromycin A in the revertant strain Δ SACE _0303/pIB139-0303 was restored to the level of erythromycin A in A226; the yield of erythromycin A in A226/pIB139-0303 is improved by 30% compared with A226 (FIG. 4). These results indicate that SACE _0303 is positively regulating erythromycin biosynthesis.
The dry weight of cells of the mutant strain Δ SACE _0303 and the strain A226 were measured for 6 days of fermentation, and the results of plotting the corresponding change curves showed that the biomass of Δ SACE _0303 was not much different from that of A226 (FIG. 5A), suggesting that the deletion of SACE _0303 gene did not affect the growth of cells.
To determine whether SACE _0303 gene regulates the sporulation of the thallus, mutant strain Δ SACE _0303, anaplerotic strain Δ SACE _0303/pIB139-0303, empty load control strain Δ SACE _0303/pIB139 and wild type strain A226 were simultaneously smeared on an R3M plate, cultured at 30 ℃ for 72 hours, and the spore growth of the strain was observed. The results show no significant difference in spore morphology for the Δ SACE _0303 mutant compared to a226 (fig. 5B), indicating that the SACE _0303 gene deletion does not affect spore formation.
qRT-PCR data showed that the transcription levels of eryAI and ermE genes in the erythromycin gene cluster in the mutant strain Δ SACE _0303 were respectively down-regulated by 3.2-fold and 3.3-fold (FIG. 6A) compared to wild strain A226, indicating that SACE _0303 regulates the transcription level of genes in the cluster to control erythromycin biosynthesis; the SACE _0303 transcriptional level was down-regulated (FIG. 6B), indicating that SACE _0303 protein can promote the transcriptional expression of self-genes.
SACE _0303 protein expression vector construction is shown in FIG. 7A, protein purification is shown in FIG. 7B, and binding experiment with eryAI, ermE, SACE _0303 promoter region is shown in FIG. 7C; EMSA analysis shows that SACE _0303 protein does not bind to eryAI and ermE promoter regions and specifically binds to SACE _0303 promoter regions, which indicates that SACE _0303 indirectly regulates the transcriptional expression of genes in erythromycin biosynthesis clusters; directly promote the self gene transcription and then regulate the biosynthesis of erythromycin.
The constructed WB/pIB139-0303 strain was identified as shown in FIG. 8A. And carrying out shake flask parallel fermentation on the constructed WB/pIB139-0303 strain and the WB strain, and detecting the yield of the erythromycin A by HPLC. The results show that: the production of erythromycin A in WB/pIB139-0303 strain was improved by 27% compared to WB (FIG. 8B).
Sequence listing
<110> university of Anhui
<120> a method for improving the yield of erythromycin by modifying saccharopolyspora erythraea SACE _0303 gene
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 564
<212> DNA
<213> Rhodotorula saccharopolyspora (Streptomyces erythraea)
<400> 1
atgacccgca ccgacgaaag ccgccgtcgc cgagaactgc tgtccaagct gctggagtac 60
tcggccgacc acggcctctc cgaggtgtcg ctgcgcccgc tggccgcagc cgtcgggtcg 120
agcccgcgca tgctgctgta cttcttcggc tccaaggagg gcctcgtccg cgaggtgcac 180
cggcacgccc gcgaggccca gctcgccctc ctggccgacg cgctcggcga caggctcgcg 240
accggctccc gcgccgacgc gctccgcgcg ctgtgggagt ggctgagcga tcccgcgcac 300
cacaacgtcg tgcggttctt cttcgagagc tacgcccgct ccctccacgc acgcgaggac 360
gcctgggagg gcttcggcga ggccagcgtc cgggaatggc tgccgcacat ccggcgggcc 420
ctcgggtcgt cggacgcgga ggcgacgctc gtgctggccg cgctacgcgg cctgatgctc 480
gacctgctcg ccaccggcga caccgaacgg gtgcgcgcgg gcttcgagtc actggtcgcc 540
ggactgccgg tcgagccggc gtag 564
Claims (3)
1. A method for improving the yield of erythromycin through modifying a saccharopolyspora erythraea SACE _0303 gene is characterized in that a TetR family transcription gene SACE _0303 gene in the saccharopolyspora erythraea is overexpressed through a genetic engineering approach to obtain an engineering strain with improved saccharopolyspora erythraea erythromycin yield, and erythromycin is produced by fermenting the engineering strain, wherein the nucleotide sequence of the SACE _0303 gene is shown as a sequence 1.
2. The method of claim 1, wherein said SACE _0303 gene positively regulates erythromycin biosynthesis.
3. The method according to claim 1, characterized in that the use of SACE _0303 gene in the saccharopolyspora erythraea industrial strain WB overexpresses the TetR family transcriptional gene SACE _0303 gene in said industrial strain WB, obtaining an engineered strain for erythromycin production.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910178832.5A CN110157756B (en) | 2019-03-11 | 2019-03-11 | Method for improving erythromycin yield by modifying saccharopolyspora erythraea SACE _0303 gene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910178832.5A CN110157756B (en) | 2019-03-11 | 2019-03-11 | Method for improving erythromycin yield by modifying saccharopolyspora erythraea SACE _0303 gene |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110157756A CN110157756A (en) | 2019-08-23 |
| CN110157756B true CN110157756B (en) | 2022-03-29 |
Family
ID=67638426
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910178832.5A Active CN110157756B (en) | 2019-03-11 | 2019-03-11 | Method for improving erythromycin yield by modifying saccharopolyspora erythraea SACE _0303 gene |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110157756B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111139192B (en) * | 2019-12-16 | 2022-04-15 | 安徽大学 | Method for improving erythromycin yield by modifying saccharopolyspora erythraea SACE _4682 gene |
| CN111197019B (en) * | 2020-01-10 | 2022-04-15 | 安徽大学 | A kind of method of improving erythromycin yield through Saccharopolyspora SACE_1906 gene pathway |
| CN112458136A (en) * | 2020-12-11 | 2021-03-09 | 安徽大学 | Saccharopolyspora erythraea with modified SACE-1409 gene and application thereof |
| CN114457101B (en) * | 2022-01-12 | 2024-01-12 | 安徽大学 | Method for improving erythromycin yield by modifying rhodosporidium saccharatum SACE_1558 gene and application |
| CN116121163A (en) * | 2022-12-29 | 2023-05-16 | 安徽农业大学 | Alteration of thaxtomin A biosynthesis by the transcription factor gene bldD in Streptomyces scabies |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001027306A2 (en) * | 1999-10-13 | 2001-04-19 | The Board Of Trustees Of The Leland Stanford Junior University | Biosynthesis of polyketide synthase substrates |
| CN103849642A (en) * | 2014-01-13 | 2014-06-11 | 安徽大学 | Method for improving yield of erythromycin through saccharopolyspora erythraea SACE _3986 gene |
| CN106520866A (en) * | 2016-12-13 | 2017-03-22 | 安徽大学 | Method for improving yield of erythromycin by modifying saccharopolyspora erythraea SACE _3980 gene |
-
2019
- 2019-03-11 CN CN201910178832.5A patent/CN110157756B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001027306A2 (en) * | 1999-10-13 | 2001-04-19 | The Board Of Trustees Of The Leland Stanford Junior University | Biosynthesis of polyketide synthase substrates |
| CN103849642A (en) * | 2014-01-13 | 2014-06-11 | 安徽大学 | Method for improving yield of erythromycin through saccharopolyspora erythraea SACE _3986 gene |
| CN106520866A (en) * | 2016-12-13 | 2017-03-22 | 安徽大学 | Method for improving yield of erythromycin by modifying saccharopolyspora erythraea SACE _3980 gene |
Non-Patent Citations (5)
| Title |
|---|
| "Complete genome sequence of the erythromycinproducing bacterium Saccharopolyspora erythraea producing bacterium Saccharopolyspora erythraea NRRL23338";Markiyan Oliynyk et al.;《Nature Biotechnology》;20070318;第25卷(第4期);第447-453页 * |
| "Saccharopolyspora erythraea NRRL2338 complete genome"，Accession Number：AM420293.1;Oliynyk,M.et al.;《Genbank》;20150227;第1-2页 * |
| "Uncovering and Engineering a Mini-Regulatory Network of the TetR-Family Regulator SACE_0303 for Yield Improvement of Erythromycin in Saccharopolyspora erythraea";Ying Liu et al.;《Frontiers in Bioengineering and Biotechnology》;20210914;第9卷;第1-13页 * |
| "放线菌中与抗生素合成相关TetR家族转录因子的研究进展";倪静姝 等;《微生物学通报》;20190125;第1-11页 * |
| "糖多孢红霉菌红霉素生物合成的级联调控初探";刘英 等;《中国生物工程学会第十三届学术年会暨2019年全国生物技术大会论文集》;20191130;第586页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110157756A (en) | 2019-08-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110157756B (en) | Method for improving erythromycin yield by modifying saccharopolyspora erythraea SACE _0303 gene | |
| Liang et al. | Biosensor-assisted transcriptional regulator engineering for Methylobacterium extorquens AM1 to improve mevalonate synthesis by increasing the acetyl-CoA supply | |
| CN110484481B (en) | A method for improving the yield of lincomycin by modifying the gene of Streptomyces lincosides SLCG_3128 | |
| CN106520866B (en) | A method for improving erythromycin yield by modifying the gene of Saccharopolysporidium fungus SACE_3980 | |
| CN105200072B (en) | The biological synthesis gene cluster of aromatic polyketones class atypia square ring element fluostatins a kind of and its application | |
| CN107881190B (en) | Method for improving lincomycin yield by modifying streptomyces lincolnensis SLCG _2919 gene | |
| CN111621458B (en) | BVG90_11450 gene-deleted serratia marcescens engineering bacterium | |
| WO2012119484A1 (en) | Plasmid and method for using plasmid to increase yield of streptomyces antibiotic | |
| CN109321618B (en) | A kind of method for improving erythromycin yield by Saccharopolyspora SACE_5717 gene | |
| CN117106816A (en) | A method and application for improving lincomycin production by modifying the Streptomyces lincomis SLCG_1608 gene | |
| WO2020048125A1 (en) | Construction and application of efficient streptomyces genome simplification system | |
| CN117230100A (en) | Method for improving yield of lincomycin by modifying streptomyces forestation SLCG_1979 gene and application thereof | |
| CN109136253B (en) | Method for improving yield of erythromycin through saccharopolyspora erythraea SACE _5754 gene approach | |
| CN111139192B (en) | Method for improving erythromycin yield by modifying saccharopolyspora erythraea SACE _4682 gene | |
| CN112251456B (en) | A method for improving the yield of lincomycin by Streptomyces lincolis regulatory gene combination modification | |
| CN112899210B (en) | Method for improving validamycin fermentation level by enhancing positive regulatory protein gene expression | |
| CN119736354A (en) | A method for efficiently and greenly synthesizing L-carnosine and its application | |
| CN104073507B (en) | A kind of dovemycin biosynthetic gene cluster and its application | |
| CN113755517A (en) | Construction method and application of SLCG _5407 gene modified streptomyces lincolnensis | |
| CN111363710B (en) | A kind of method for improving erythromycin yield through Saccharopolysporidium mold SACE_4839 gene pathway | |
| CN110174514B (en) | Mining method and application of efficient promoters for secondary metabolism of Streptomyces based on proteomics | |
| CN111197019B (en) | A kind of method of improving erythromycin yield through Saccharopolyspora SACE_1906 gene pathway | |
| CN115927151B (en) | Method and application of increasing erythromycin production by modifying SACE_5312 gene of Saccharopolyspora erythromycin | |
| CN111718884B (en) | BVG90_08615 gene-deleted serratia marcescens engineering bacterium | |
| CN114457101B (en) | Method for improving erythromycin yield by modifying rhodosporidium saccharatum SACE_1558 gene and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |