CN109321618A - A method of yield of erythrocin is improved by the red mould SACE_5717 gene of the more spores of sugar - Google Patents
A method of yield of erythrocin is improved by the red mould SACE_5717 gene of the more spores of sugar Download PDFInfo
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Abstract
The present invention provides a kind of methods for improving yield of erythrocin by the red mould SACE_5717 gene of the more spores of sugar, it first passes through genetic engineering approach and inactivates SACE_5717 gene in the more red moulds of spore of sugar, to obtain the more red mould erythromycin superior strains of spore of sugar, then the corresponding red mould erythromycin high-yielding engineering bacterial strain of the more spores of sugar of acquisition is used for fermenting and producing erythromycin；Wherein, the nucleotide sequence of SACE_5717 gene is as shown in SEQ ID NO.1, and nucleotide sequence coded amino acid sequence is as shown in SEQ ID NO.2.The present invention has the advantages that passing through SACE_5717 gene in the more red moulds of spore of genetic engineering approach inactivation sugar, erythromycin superior strain can be obtained, erythromycin is produced with resulting strain fermentation, yield of erythrocin can be improved, yield of erythrocin is improved for industrial production and new technical support is provided.
Description
Technical field
The present invention relates to a kind of method for improving fermenting and producing yield of erythrocin, more particularly to one kind are red mould by the more spores of sugar
The method of bacterium SACE_5717 gene raising yield of erythrocin.
Background technique
Actinomyces are a kind of important microbe resources that can generate secondary metabolite, closely related with the mankind, are answered at present
It in extensive antibiotic about 75% is generated by actinomyces.Common vancomycin, erythromycin, lincomycin, tetracycline etc.
Different type secondary metabolite is generated by actinomyces.Antibiotic synthesis in actinomyces is by synthetic gene cluster control
System, and the transcription of the synthetic gene cluster generally regulation dependent on specific regulatory control albumen or modulin of overall importance, by this
The research of modulin, and regulatory protein gene is modified, to regulate and control to cometabolism, change the production of antibiotic
Amount.Traditional high yield industrial strain is obtained by random physical or chemical mutagenesis, and random mutagenesis techniques not only have uncertainty,
And rationality guidance cannot be carried out to breeding.The object of the invention is exactly obtained by genetic engineering approach gene targeted alteration red
Mycin superior strain, the production for erythromycin or intermediate product.
The more red moulds of spore of sugar are the Gram-positive filiform actinomyces isolated from soil nineteen fifty-two, and Erythromycin A is
Its important macrolide secondary metabolite.Currently, erythromycin chemical derivative has critically important answer in field of medicaments
With value, erythromycin and its annual worldwide sales of derivative reach nearly 10,000,000,000 dollars.Accordingly, on how to improving erythromycin
This problem of yield also gradually causes the concern of more and more scientists.2003, Rodriguez etc. reported erythromycin high yield master
It wants the reason is that due to its controlling gene, rather than synthesizes gene, so that the emphasis of research has turned to controlling gene.2007,
Oliynyk etc. completes the more red mould gene order-checkings of spore of sugar.Up to the present, grinding about sugared more red mould controlling genes of spore
Study carefully still seldom, only only bldD (SACE_2077), SACE_7040, SACE_0012, SACE_5599, SACE_3986,
The research of the controlling genes such as SACE_3986 and SACE_5388 is reported.
The transcription regulator of prokaryotes 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 is widely present in bacterium and archeobacteria, has common feature, nuclear structure in structure
Usually be made of two parts, N-terminal structural domain be by have conservative helix turn helix (helix-turn-helix,
HTH) structure composition, it can specifically bind with target DNA；C-terminal structural domain is ligand binding domains (also referred to as regulation amino acid
Metabolism domain, RAM domain), have typical α β-sandwich foldable structure, C-terminal structural domain also participates in mediating polymer
It is formed.The protein regulation function of Lrp/AsnC family is extensive, most of transhipments for being involved in regulation amino acid metabolism and small molecule.
Speculate that the more red mould SACE_5717 genes of spore of sugar belong to Lrp/AsnC family based on the red mould genome sequence of the more spores of sugar, and
And found through the application laboratory research, the more red mould SACE_5717 genes of spore of sugar are a participation erythromycin biosynthesis
Negative regulatory factor.
Accordingly, it is badly in need of a kind of method that yield of erythrocin is improved by the red mould SACE_5717 gene of the more spores of sugar at present.
Summary of the invention
It is a kind of by the red mould SACE_5717 gene raising of the more spores of sugar technical problem to be solved by the present invention lies in providing
The method of yield of erythrocin.
The present invention solves above-mentioned technical problem using following technical scheme:
A method of yield of erythrocin is improved by the red mould SACE_5717 gene of the more spores of sugar, first passes through genetic engineering
Approach inactivates SACE_5717 gene in the more red moulds of spore of sugar, to obtain the more red mould erythromycin superior strains of spore of sugar, then will obtain
The corresponding red mould erythromycin high-yielding engineering bacterial strain of the more spores of sugar obtained is used for fermenting and producing erythromycin；Wherein, SACE_5717 gene
Nucleotide sequence is as shown in SEQ ID NO.1, and nucleotide sequence coded amino acid sequence is as shown in SEQ ID NO.2.
As one of preferred embodiment of the invention, described inactivate in SACE_5717 gene in the more red moulds of spore of sugar is inactivated
In the step of SACE_5717 gene, the more red mould original strains of spore of sugar of use are specially the more red mould A226 bacterial strains of spore of sugar.
As one of preferred embodiment of the invention, the red mould A226 bacterial strain of the more spores of sugar is to disclose at present and is public
Many resulting bacterial strains, are provided by University of Anhui, and are sent to the medicinal microorganism of China before the application applying date by University of Anhui
Culture presevation administrative center carries out preservation and registration, deposit number CGMCC8279.
As one of preferred embodiment of the invention, the specific method is as follows: with the red mould SACE_5717 gene of the more spores of sugar or its
Expression product is starting point, lacks Lrp/AsnC family transcriptional modulatory gene SACE_5717 in erythromycin superior strain, obtains sugar
More red mould erythromycin superior strains of spore are used for fermenting and producing erythromycin.
As one of preferred embodiment of the invention, the gene product of the red mould SACE_5717 gene of the more spores of sugar is used for
Negative regulation erythromycin biosynthesis.
The present invention compared with prior art the advantages of be:
Erythromycin biosynthesis negative regulator gene SACE_5717 has been screened in present invention research, passes through genetic engineering way
SACE_5717 gene in the more red moulds of spore of diameter inactivation sugar, can obtain erythromycin superior strain, improve erythromycin for industrial production
Fermentation yield provides technical support.Wherein, when knocking out SACE_5717 gene in the more red mould A226 of spore of sugar, erythromycin is produced
Amount improves 27%；And when covering SACE_5717 gene in Δ SACE_5717 Inactivating mutations strain, yield of erythrocin then obtains
Restore；It is indicated above that SACE_5717 is the negative regulatory factor of a participation erythromycin biosynthesis.Utilize superior strain WB
As starting strain, SACE_5717 gene is inactivated, yield of erythrocin improves 25%, and it is red to illustrate that inactivation SACE_5717 gene improves
The technology of mycin yield is equally applicable in high industrial production bacterial strain.
Detailed description of the invention
Fig. 1 is the building process schematic diagram of SACE_5717 gene Inactivating mutations strain Δ SACE_5717 in embodiment 7；
Fig. 2 is that (it is more that sugar is substituted in thiostrepton resistance gene (tsr) for the PCR proof diagram of Inactivating mutations strain in embodiment 7
SACE_5717 gene on the red mould chromosome of spore, length becomes after SACE_5717 gene (501bp) is replaced by tsr (1360bp)
1600bp；In figure, M:5000bp DNA Marker)；
Fig. 3 is yield of erythrocin analysis chart (the specially starting strain A226, Inactivating mutations strain of mutant in embodiment 7
Δ SACE_5717, inactivation covering bacterial strain are overexpressed strains A 226/pIB139-5717 and are overexpressed unloaded control strain and train in R5
Support 30 DEG C of fermentations product HPLC analysis in 6 days in base)；
Fig. 4 is biomass testing result figure (specially starting strain A226 and the Inactivating mutations strain of mutant in embodiment 7
The analysis of dry cell weight in Δ SACE_5717 fermentation process)；
Fig. 5 is the spore growth situation display figure of Δ SACE_5717 mutant strain and wild type A226 bacterial strain in embodiment 7
(in figure, 1:A226 bacterial strain；2: Δ SACE_5717 mutant strain；3: replying bacterial strain Δ SACE_5717/pIB139-5717；4: influencing
The more red mould Morphological Differentiation bacterial strain Δ bldD of spore of sugar)；
Fig. 6 is related gene transcription analysis figure (SACE_5717 in Δ SACE_5717 and starting strain A226 in embodiment 7
In Inactivating mutations strain, erythromycin biosynthetic enzyme genes eryAI (SACE_0721) and erythromycin resistance gene ermE (SACE_
0733), neighbouring lysine transporter gene SACE_5716 transcriptional level is significantly increased)；
Fig. 7 is PCR qualification figure (the SACE_5717 gene of high industrial production WB/ Δ SACE_5717 mutant in embodiment 7
Length becomes 1600bp after (501bp) is replaced by tsr resistant gene (1360bp)；M:5000bp DNA Marker)；
Fig. 8 is the superior strain WB and HPLC of Inactivating mutations strain WB/ Δ SACE_5717 Erythromycin A yield points in embodiment 7
Analysis figure.
The critical strain source explanation that the present invention uses:
The more red mould A226 bacterial strains of spore of sugar that the present invention uses are to disclose at present and be the resulting bacterial strain of the public, by pacifying
Emblem university provides, and be sent in the application applying date before by University of Anhui Chinese medicinal Microbiological Culture Collection administrative center into
Row preservation and registration, identified to show as surviving, deposit number is CGMCC 8279.
The more red mould WB of spore (high industrial production bacterial strain) of sugar that the present invention uses, to disclose at present and being that the public is resulting
Bacterial strain is provided by University of Anhui, and is sent to the medicinal Microbiological Culture Collection of China before the application applying date by University of Anhui
Administrative center carries out preservation and registration, identified to show as surviving, and deposit number is CGMCC 8280.
Specific embodiment
The present invention test in using to bacterial strain and plasmid be shown in Table 1.Escherichia coli are in LB liquid medium or adding
It is cultivated on the LB solid plate of 2.0% agar in 37 DEG C.The red mould A226 of the more spores of erythromycin producing strain sugar and its industry are high
Bacterium WB is produced to be trained in pancreas peptone soybean broth (TSB) culture medium or on the R3M plate containing 2.2% agar in 30 DEG C
It supports.
PEG3350, lysozyme, TES, casamino acid, thiostrepton, the apramycin used in present invention test
It is bought from Sigma company.TSB, yeast extract, peptone purchase are in Oxoid company.Glycine, agar powder, sodium chloride and its
Its biological reagent is all purchased from Reagent Company.The general operation technology of Escherichia coli and the more red moulds of spore of sugar is according to standard operation.
The synthesis of primer and DNA sequencing are completed by general biosystem (Anhui) Co., Ltd.
The bacterial strain and plasmid used in 1 present invention test of table
It elaborates below to the embodiment of the present invention, the present embodiment carries out under the premise of the technical scheme of the present invention
Implement, the detailed implementation method and specific operation process are given, but protection scope of the present invention is not limited to following implementation
Example.
The building of embodiment 1SACE_5717 gene inactivated mutants:
PUCTSR plasmid is to be inserted into 1360bp thiostrepton between BamH I and Sma the I restriction enzyme site of pUC18 to resist
Property gene (tsr).In order to knock out the SACE_5717 gene in the more red moulds of spore of sugar, it is with PUp1/PUp2 and PD1/PD2 respectively
Primer, the more red mould genomes of spore of sugar are template, the homology arm SU of each about 1.5kb of the upstream and downstream of PCR amplification SACE_5717 gene
With SD DNA fragmentation.
The primer of PCR amplification SU DNA fragmentation be PUp1, PUp2, the nucleotide sequence of PUp1 as shown in SEQ ID NO.3,
The nucleotide sequence of Pup2 is as shown in SEQ ID NO.4；Wherein, " AAGCTT " in PUp1 sequence is the digestion position of HindIII
Point, the restriction enzyme site that " TCTAGA " in Pup2 sequence is Xbal.
The primer of PCR amplification SD DNA fragmentation is PD1, PD2, and the nucleotide sequence of PD1 is as shown in SEQ ID NO.5, PD2
Nucleotide sequence as shown in SEQ ID NO.6；Wherein, in PD1 sequence
" GGTACC " is the restriction enzyme site of KpnI, the restriction enzyme site that " GAATTC " in PD2 sequence is EcoRI.
Two DNA fragmentations of above-mentioned SU and SD are connected to the tsr resistant gene two sides of pUCTSR respectively, complete building plasmid
pUC-ΔSACE_5717；It is template, PCR amplification Tsr- Δ by primer, pUC- Δ SACE_5717 plasmid of PUp1 and PD2
SACE_5717DNA segment, it is using chromosome segment homologous recombination technique that the more spores of Tsr- Δ SACE_5717 segment importing sugar are red
In mould protoplast, mutant is screened according to thiostrepton resistance, i.e. acquisition SACE_5717 gene is resisted by thiostrepton
Property gene (tsr) replace engineering strain, verifying the mutant using P1/P2 primer PCR is successfully constructed, and Δ is named as
SACE_5717.Wherein, the nucleotide sequence of P1 primer is as shown in SEQ ID NO.7, the nucleotide sequence of P2 primer such as SEQ ID
Shown in NO.8.
The building of embodiment 2SACE_5717 gene reply bacterial strain:
For the introducing SACE_5717 gene in Δ SACE_5717, PCR amplification SACE_5717 gene primer is devised,
That is P3, P4；The nucleotide sequence of P3 is as shown in SEQ ID NO.9, and the nucleotide sequence of P4 is as shown in SEQ ID NO.10；Its
In, " CATATG " in P3 sequence is the restriction enzyme site of NdeI, the restriction enzyme site that " TCTAGA " in P4 sequence is XbaI.
Amplify SACE_5717 gene from the more red mould A226 genomes of spore of sugar, be inserted into pIB139 Nde I and
Between Xba I restriction enzyme site, expression plasmid pIB139-5717 is constructed, it then will by the protoplast transformation method that PEG is mediated
PIB139-5717 is imported in Δ SACE_5717.It is pair with apramycin resistance gene (apr) by apramycin preliminary screening
As carrying out PCR identification, the reply Strain Designation of acquisition is Δ SACE_5717/pIB139-5717.
Overexpression SACE_5717 gene in the 3 more red mould A226 of spore of sugar of embodiment:
In the more red mould A226 of spore of protoplast transformation technological sourcing sugar that pIB139-5717 is mediated by PEG, Yi Anpu
Mycin resistant gene (apr) is that object carries out PCR identification, and acquisition Strain Designation is A226/pIB139-5717.
The 4 more red mold fermentation product detections of spore of sugar of embodiment:
The more red moulds of spore of inoculation sugar are in TSB culture medium, after 30 DEG C/220rpm shaken cultivation 2 days, R5 Liquid Culture of transferring
30 DEG C/220rpm of base shaken cultivation 6 days.Extraction is carried out to erythromycin after fermentation and HPLC is detected.
The transcription analysis of related gene in 5 Δ SACE_5717 of embodiment:
SBS Genetech RNA extracts kit is used to obtain the Δ SACE_5717 and starting strain A226 bacterium solution that cultivate 2 days respectively
RNA needed for obtaining, after being inverted to cDNA, uses machine testing on real-time fluorescence quantitative PCR instrument.
SACE_5717 is inactivated in 6 erythromycin high industrial production bacterial strain WB of embodiment:
PUCTSR plasmid is to be inserted into 1360bp thiostrepton between BamH I and Sma the I restriction enzyme site of pUC18 to resist
Property gene (tsr).In order to knock out the SACE_5717 gene in erythromycin high industrial production bacterium, respectively with above-mentioned identical PUp1/
PUp2 (with PUp1/PUp2 in embodiment 1) and PD1/PD2 (with PD1/PD2 in embodiment 1) is primer, the more red mould bases of spore of sugar
Because group is template, homology arm SU and the SD DNA fragmentation of each about 1.5kb of the upstream and downstream of PCR amplification SACE_5717 gene.
Two DNA fragmentations of above-mentioned SU and SD are connected to the tsr resistant gene two sides of pUCTSR respectively, complete building plasmid
pUC-ΔSACE_5717；It is template, PCR amplification Tsr- Δ by primer, pUC- Δ SACE_5717 plasmid of PUp1 and PD2
SACE_5717DNA segment, it is using chromosome segment homologous recombination technique that the more spores of Tsr- Δ SACE_5717 segment importing sugar are red
In mould protoplast, mutant is screened according to thiostrepton resistance, i.e. acquisition SACE_5717 gene is resisted by thiostrepton
Property gene (tsr) replace erythromycin high-yield genetic engineering bacterium strain, use P1/P2 primer (with P1/P2 primer in embodiment 1)
PCR verifies the mutant and is successfully constructed, and is named as WB/ Δ SACE_5717.
Embodiment 7
The present embodiment is to analysis and summary the various embodiments described above experimental result:
1, the essential information of SACE_5717 gene
Position of the SACE_5717 gene on the more red mould chromosomes of spore of sugar is referring to NCBI, the nucleosides of SACE_5717 gene
Acid sequence is as shown in SEQ ID NO.1, and nucleotide sequence coded amino acid sequence is as shown in SEQ ID NO.2.
2, Δ SACE_5717 is improved than starting strain A226 yield of erythrocin
The building process of SACE_5717 gene Inactivating mutations strain Δ SACE_5717 is shown in Fig. 1.Containing thiostrepton
Inactivating mutations is screened on R3M plate and (electrophoresis result is shown in Fig. 2) is confirmed by PCR.Δ SACE_5717 is in R5 fluid nutrient medium
Middle fermentation 6 days is analyzed after extraction concentration through HPLC, output increased of the yield of erythrocin than starting strain A226 26.5%
(see Fig. 3) shows that SACE_5717 may be the negative regulator for participating in regulation erythromycin biosynthesis.Meanwhile it taking and daily fermenting
Thallus carries out biomass estimation, and the biomass difference of Δ SACE_5717 and A226 less (see Fig. 4), implies as the result is shown
The inactivation of SACE_5717 does not influence the thalli growth of bacterial strain.
3, SACE_5717 gene is replied
In order to confirm that the phenotype of mutant strain Δ SACE_5717 is this hair entirely due to caused by SACE_5717 gene mutation
The SACE_5717 gene covering experiment of bright researching and designing.PIB139-5717 includes erythromycin resistance gene strong promoter
PermE* and complete SACE_5717 gene, for covering mutant strain Δ SACE_5717.Fermented and HPLC detection, Δ
The yield of erythrocin of SACE_5717/pIB139-5717 is restored to horizontal (see Fig. 3) consistent with starting strain A226's.
4, SACE_5717 gene is overexpressed in A226 causes yield of erythrocin to reduce
In order to further verify function of the SACE_5717 in the more red moulds of spore of host's sugar, itself and yield of erythrocin are confirmed
Relationship, the present invention devise simultaneously SACE_5717 be overexpressed experiment, expression vector pIB139-5717 is imported into A226
Building is overexpressed strains A 226/pIB139-5717.Fermented and HPLC detection finds its yield of erythrocin than control A226/
PIB139 is compared, and yield reduces 19.6% (see Fig. 3), and to sum up result confirms that SACE_5717 is red in the more red moulds of spore of sugar
The negative regulator gene of mycin biosynthesis, can be with by SACE_5717 gene in the more red moulds of spore of genetic engineering approach inactivation sugar
Improve erythromycin Isoquant.
5, the influence of inactivation SACE_5717 gene pairs thallus spore shape differentiation.
In order to determine whether SACE_5717 gene regulates and controls the sporogenesis of thallus, by mutant strain Δ SACE_5717, reply
Bacterial strain Δ SACE_5717/pIB139-5717, be overexpressed strains A 226/pIB139-5717, wild type control strain A226 and
It influences spore shape differentiation bacterial strain Δ bldD to be applied on R3M plate simultaneously, 30 DEG C are cultivated 84 hours, and bacterial strain spore growth feelings are observed
Condition.As the result is shown compared to A226, the spore shape no significant difference (see Fig. 5) of Δ SACE_5717 mutant illustrates SACE_
The inactivation of 5717 genes does not influence the formation of spore.
6, in the strain of Δ SACE_5717 Inactivating mutations related gene transcription analysis
QRT-PCR is as a result, it was confirmed that structure base in Δ SACE_5717 mutant strain, on erythromycin biological synthesis gene cluster
Because the expression quantity of eryAI improves 5 times compared with starting strain A226, resistant gene ermE improves 1.2 times, and upstream is neighbouring relies
The transcriptional level of propylhomoserin transporter gene SACE_5716 improves 4 times (see Fig. 6), illustrates that the mutation of SACE_5717 can make
At the rising of erythromycin biological synthesis gene cluster and contiguous gene transcriptional level.
7, the high industrial production bacterial strain WB/ Δ SACE_5717 yield of erythrocin being transformed significantly improves
Inactivating mutations is screened on the R3M plate containing thiostrepton and (see Fig. 7) is identified by PCR.It will mutation
Strain WB/ Δ SACE_5717 and control strain WB coated plate activate, and are then respectively connected in the shaking flask of industrial seed culture medium, 30 DEG C
Under revolving speed 220rpm after constant-temperature shaking culture 2 days, in industrial fermentation culture medium of transferring, continue culture 6 days.It extracts after fermentation
Concentration is analyzed through HPLC, and compare starting strain WB, and the yield of erythrocin of WB/ Δ SACE_5717 has been respectively increased 25% (see figure
8)。
Embodiment 8
Based on embodiment 1-7 experimental result, we are known: being inactivated in the more red moulds of spore of sugar by genetic engineering approach
SACE_5717 gene, yield of erythrocin are improved；SACE_5717 gene is covered in mutant strain, yield of erythrocin restores
To original level；It is thus determined that SACE_5717 gene is the negative regulation gene for controlling erythromycin biosynthesis.
Accordingly, it can get a kind of method that yield of erythrocin is improved by the red mould SACE_5717 gene of the more spores of sugar: with sugar
More red mould SACE_5717 genes of spore or its expression product are starting point, lack Lrp/AsnC family in erythromycin superior strain
Transcriptional modulatory gene SACE_5717 (is specifically shown in the various embodiments described above), to obtain the more red mould erythromycin superior strains of spore of sugar, then
The corresponding red mould erythromycin high-yielding engineering bacterial strain of the more spores of sugar of acquisition is used for fermenting and producing erythromycin.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
SEQUENCE LISTING
<110>Agricultural University Of Anhui, University of Anhui
<120>a kind of method that yield of erythrocin is improved by the red mould SACE_5717 gene of the more spores of sugar
<130> 2018
<160> 10
<170> PatentIn version 3.3
<210> 1
<211> 474
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<213>the more red moulds of spore of sugar
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cagggcgtga tcagggggtc gcacatcgac gtcgacccgg cggccaccgg gctggcgctg 180
gaggcgatcg tgtcgatcaa cgtccggccg cacacccgcg aagtcgtcga ccagttccgc 240
aagttcgtgc tggcgcagcc cgagacgcgg tcgctgctgc acgtcagcgg gcaggccgac 300
ttcctcctgc acgtcgcggt ggccgacacc gcgcacctgc agggcttcct ggtcgacaag 360
ctggcgtcgc gcccggaggt ccgccacttc accagctcga tcgtgctgga gcaggtgcac 420
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Val Asp Trp Gln Ile Leu Glu Glu Leu Gln Asn Asp Ala Thr Leu Pro
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Asn Arg Thr Leu Ala Asp Arg Val Gly Leu Ala Ala Ser Ser Cys Leu
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Gln Arg Val Arg Arg Leu Arg Glu Gln Gly Val Ile Arg Gly Ser His
35 40 45
Ile Asp Val Asp Pro Ala Ala Thr Gly Leu Ala Leu Glu Ala Ile Val
50 55 60
Ser Ile Asn Val Arg Pro His Thr Arg Glu Val Val Asp Gln Phe Arg
65 70 75 80
Lys Phe Val Leu Ala Gln Pro Glu Thr Arg Ser Leu Leu His Val Ser
85 90 95
Gly Gln Ala Asp Phe Leu Leu His Val Ala Val Ala Asp Thr Ala His
100 105 110
Leu Gln Gly Phe Leu Val Asp Lys Leu Ala Ser Arg Pro Glu Val Arg
115 120 125
His Phe Thr Ser Ser Ile Val Leu Glu Gln Val His Thr Arg Ala Leu
130 135 140
Thr Ala Pro Ala His Leu Arg Pro Lys Arg Arg Arg Lys
145 150 155
<210> 3
<211> 29
<212> DNA
<213>artificial sequence
<400> 3
aaaaagctta ttctgcgcgg agccccggt 29
<210> 4
<211> 29
<212> DNA
<213>artificial sequence
<400> 4
aaatctagat tgcaggcagc tcgacgcgg 29
<210> 5
<211> 29
<212> DNA
<213>artificial sequence
<400> 5
aaaggtaccc acctgcggcc gaagcggcg 29
<210> 6
<211> 29
<212> DNA
<213>artificial sequence
<400> 6
aaagaattcc gaccccgaga cgctgccga 29
<210> 7
<211> 29
<212> DNA
<213>artificial sequence
<400> 7
aaacatatga tgcgtgaatc cgtagaact 29
<210> 8
<211> 29
<212> DNA
<213>artificial sequence
<400> 8
aaatctagac tacttgcgtc gccgcttcg 29
<210> 9
<211> 29
<212> DNA
<213>artificial sequence
<400> 9
aaacatatga tgcgtgaatc cgtagaact 29
<210> 10
<211> 29
<212> DNA
<213>artificial sequence
<400> 10
aaatctagac tacttgcgtc gccgcttcg 29
Claims (4)
1. a kind of method for improving yield of erythrocin by the red mould SACE_5717 gene of the more spores of sugar, which is characterized in that first pass through
Genetic engineering approach inactivates SACE_5717 gene in the more red moulds of spore of sugar, to obtain the more red mould erythromycin Producing Strains of spore of sugar
Strain, then the corresponding red mould erythromycin high-yielding engineering bacterial strain of the more spores of sugar of acquisition is used for fermenting and producing erythromycin；Wherein, SACE_
The nucleotide sequence of 5717 genes is as shown in SEQ ID NO.1, nucleotide sequence coded amino acid sequence such as SEQ ID NO.2
It is shown.
2. the method according to claim 1 that yield of erythrocin is improved by the red mould SACE_5717 gene of the more spores of sugar,
It is characterized in that, it is described to be inactivated in the step of inactivating SACE_5717 gene in SACE_5717 gene in the more red moulds of spore of sugar, it adopts
The more red moulds of spore of sugar are specially the more red mould A226 bacterial strains of spore of sugar.
3. the method according to claim 1 that yield of erythrocin is improved by the red mould SACE_5717 gene of the more spores of sugar,
It is characterized in that, it is specific as follows: using sugared more red mould SACE_5717 genes of spore or its expression product as starting point, in erythromycin height
It produces bacterial strain and lacks Lrp/AsnC family transcriptional modulatory gene SACE_5717, obtain the more red mould erythromycin superior strains of spore of sugar, use
In fermenting and producing erythromycin.
4. according to claim 1 to 3 improve yield of erythrocin by the red mould SACE_5717 gene of the more spores of sugar
Method, which is characterized in that the gene product of the red mould SACE_5717 gene of the more spores of sugar is for negative regulation erythromycin biology
Synthesis.
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Cited By (2)
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| CN111363710A (en) * | 2020-03-04 | 2020-07-03 | 安徽大学 | Method for improving yield of erythromycin through saccharopolyspora erythraea SACE _4839 gene pathway |
| CN115433685A (en) * | 2022-06-08 | 2022-12-06 | 安徽大学 | Method for improving yield of erythromycin through modification of saccharopolyspora erythraea SACE _5812 gene |
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| CN103205451A (en) * | 2013-03-15 | 2013-07-17 | 安徽大学 | Method for improving erythromycin yield by saccharopolyspora erythraea SACE_7301 gene pathway |
| CN106148378A (en) * | 2016-07-19 | 2016-11-23 | 安徽大学 | Improve yield of erythrocin by sugared many spores red mould SACE_Lrp gene approach |
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| CN103205451A (en) * | 2013-03-15 | 2013-07-17 | 安徽大学 | Method for improving erythromycin yield by saccharopolyspora erythraea SACE_7301 gene pathway |
| WO2016196319A1 (en) * | 2015-05-29 | 2016-12-08 | Radiant Genomics, Inc. | Compositions and methods for activating silent gene clusters |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111363710A (en) * | 2020-03-04 | 2020-07-03 | 安徽大学 | Method for improving yield of erythromycin through saccharopolyspora erythraea SACE _4839 gene pathway |
| CN111363710B (en) * | 2020-03-04 | 2022-02-18 | 安徽大学 | Method for improving yield of erythromycin through saccharopolyspora erythraea SACE _4839 gene pathway |
| CN115433685A (en) * | 2022-06-08 | 2022-12-06 | 安徽大学 | Method for improving yield of erythromycin through modification of saccharopolyspora erythraea SACE _5812 gene |
| CN115433685B (en) * | 2022-06-08 | 2023-03-03 | 安徽大学 | Method for improving yield of erythromycin by modifying saccharopolyspora erythraea SACE _5812 gene |
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| CN109321618B (en) | 2021-06-18 |
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