CN117568301A - Method for improving erythromycin yield through rhodosporidium saccharum SACE-1646 gene - Google Patents
Method for improving erythromycin yield through rhodosporidium saccharum SACE-1646 gene Download PDFInfo
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- 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
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- C12Y206/01—Transaminases (2.6.1)
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Abstract
The invention relates to the technical field of genetic engineering, and provides a method for improving erythromycin yield by using a Sace_1646 gene of rhodosporidium, wherein the Sace_1646 gene is over-expressed in rhodosporidium by using a genetic engineering method to obtain a high-yield strain of erythromyces saccharopolyspora, and the obtained high-yield strain of erythromyces saccharopolyspora is used for fermenting and producing erythromycin; the nucleotide sequence of the SACE_1646 gene is shown as SEQ ID NO.1, and the corresponding coded amino acid sequence is shown as SEQ ID NO. 2. The invention has the advantages that: the erythromycin is produced by fermenting the obtained erythromycins-polysaccharide high-yield strain, so that the yield can be greatly improved, and technical support is provided for improving the fermentation yield of erythromycin in industrial production.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a method for improving erythromycin yield through rhodosporidium saccharum SACE-1646 genes.
Background
Actinomycetes are an important microbial resource capable of producing secondary metabolites, and are closely related to human beings. About 75% of the antibiotics currently in wide use are produced by actinomycetes, and different types of secondary metabolites such as vancomycin, erythromycin, lincomycin, tetracycline and the like are produced by actinomycetes. The synthesis of antibiotics in actinomycetes requires small molecules from primary metabolism as precursor substances, and the raw material supply of the precursor small molecules is improved by modifying and reforming specific genes in primary metabolic pathways, so that the high yield of antibiotics is realized. Traditional high-yield industrial strains are obtained through random physical or chemical mutagenesis, and random mutagenesis technology has uncertainty and can not carry out rational guidance on breeding. The invention aims to obtain erythromycin high-yield strain by directionally changing genes through a genetic engineering approach, which is used for producing erythromycin or intermediate products.
Erythromycin is currently the most widely used macrolide antibiotic clinically, and was isolated from the secondary metabolite of rhodosporidium saccharopolyspora in 1952 at the earliest. At present, the erythromycin chemical derivative has important application value in the field of medicines, and the world sales of erythromycin and the erythromycin derivative each year reach nearly billions of dollars. Accordingly, the problem of how to increase the yield of erythromycin is also increasingly attracting attention from scientists. The biosynthesis of erythromycin requires propionyl-coA (proyl-coA) as a precursor, which is ultimately produced by the catalysis of polyketide synthase (polyketide synthase, PKS) and a series of post-modification processes. Metabolic conversion of branched-chain amino acids (isoleucine, valine and leucine) in rhodomyces saccharopolyspora is one of the important sources of propionyl-coa. Until now, few studies have been conducted on improvement of erythromycin yield by engineering of branched-chain amino acid metabolic processes in rhodosporidium saccharopolyspora, and only the university of eastern university torch-storage subject group has reported improvement of erythromycin yield by inhibiting the conversion of propionic acid into TCA cycle by inactivating methylmalonyl-coa mutase gene mutB. Based on sequence analysis of rhodosporidium genome, the rhodosporidium SACE_1646 gene codes a branched-chain amino acid aminotransferase IlvE and is responsible for catalyzing the first reaction of metabolic conversion of branched-chain amino acids, and the laboratory research of the invention shows that the rhodosporidium SACE_1646 gene has positive correlation with erythromycin biosynthesis.
Accordingly, there is an urgent need to devise a method for improving erythromycin yield through rhodosporidium saccharopolyspora SACE_1646 gene.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for improving the yield of erythromycin through the rhodosporidium saccharum SACE-1646 gene.
The invention adopts the following technical scheme to solve the technical problems:
a method for improving erythromycin yield through Sace_1646 gene of rhodosporidium includes such steps as over-expressing SACE_1646 gene in rhodosporidium to obtain high-yield strain of erythromyces saccharopolyspora, and fermenting to obtain erythromycin; the nucleotide sequence of the SACE_1646 gene is shown as SEQ ID NO.1, and the corresponding coded amino acid sequence is shown as SEQ ID NO. 2.
As one of the preferred modes of the invention, in the step of over-expressing the SACE_1646 gene in rhodosporidium by a genetic engineering method, rhodosporidium is adopted, in particular to rhodosporidium saccharopolyspora A226 strain.
As one of the preferred embodiments of the present invention, the expression product of the SACE_1646 gene is branched-chain amino acid aminotransferase IlvE.
As one of the preferred modes of the invention, the expression product of the SACE_1646 gene is positively correlated with erythromycin biosynthesis.
Compared with the prior art, the invention has the advantages that:
the invention screens the branched chain amino acid aminotransferase gene SACE-1646 which has positive correlation with erythromycin biosynthesis, and over-expresses the SACE-1646 gene in rhodosporidium saccharopolyspora through a genetic engineering way, so that an erythromycin high-yield strain can be obtained, and technical support is provided for improving the erythromycin fermentation yield in industrial production. Wherein, when SACE_1646 gene is knocked out in rhodosporidium saccharopolyspora A226, erythromycin yield is remarkably reduced by 48.7%; when the SACE-1646 gene is complemented in the delta SACE-1646 deletion mutant, the erythromycin yield is recovered; thus, it was shown that the SACE_1646 gene has a positive correlation with erythromycin biosynthesis. When the copy number of the SACE_1646 gene is increased in the rhodosporidium saccharopolyspora A226, and the SACE_1646 gene is over-expressed in the rhodosporidium saccharopolyspora, the erythromycin yield is greatly improved by 105.3%, which shows that the SACE_1646 gene is over-expressed in the rhodosporidium saccharopolyspora, and a high-yield strain of erythromycin can be constructed.
Drawings
FIG. 1 is a schematic diagram showing the construction process of SACE_1646 gene deletion mutant ΔSACE_1646 of example 2 (using pKC1139 plasmid to make a traceless deletion of 600bp inside SACE_1646 gene on Rhodomycotina saccharopolyspora chromosome);
FIG. 2 is a diagram showing PCR verification of the ΔSACE_1646 mutant strain of example 2 (in the figure, 1 represents positive control 504bp,2 represents negative control 1104bp,3 represents positive clone, the PCR amplified band is identical to 1, and M represents 5000bp DNA Marker);
FIG. 3 is a graph of the analysis of erythromycin production in starting strain A226, mutant ΔSACE_1646, and complementation strain ΔSACE_1646/pIB-1646 of example 7 (specifically including HPLC analysis of erythromycin production in starting strain A226, mutant ΔSACE_1646, complementation strain ΔSACE_1646/pIB-1646, and empty control strain ΔSACE_1646/pIB139 fermentation broth thereof);
FIG. 4 is a chart of biomass analysis of starting strain A226 and mutant strain ΔSACE_1646 of example 7 (specifically, analysis of dry weight of cells during fermentation of starting strain A226 and mutant strain ΔSACE_1646);
FIG. 5 is a chart showing spore growth analyses of the starting strain A226, the mutant strain ΔSACE_1646 and the complementation strain ΔSACE_1646/pIB-1646 in example 7;
FIG. 6 is a graph of transcriptional analysis of genes involved in erythromycin biosynthesis in the starting strain A226 and the ΔSACE_1646 mutant strain of example 7 (specifically, the synthetic gene eryAI, eryBI, eryBIII, eryBIV, eryBVI, eryCI, eryK and the resistance gene ermE in the erythromycin biosynthesis gene cluster);
FIG. 7 is a graph of the analysis of erythromycin yield from over-expressed strain A226/pIB-1646 of example 7 (specifically including HPLC analysis of erythromycin yield in fermentation broths of starting strain A226, over-expressed strain A226/pIB-1646, and empty control strain A226/pIB139 thereof).
Detailed Description
The following describes in detail the examples of the present invention, which are implemented on the premise of the technical solution of the present invention, and detailed embodiments and specific operation procedures are given, but the scope of protection of the present invention is not limited to the following examples.
The strains and plasmids used in the examples below are shown in Table 1, and the primer sequences synthesized are shown in Table 2. Wherein the original strain is rhodosporidium saccharopolyspora A226 which is disclosed at present and can be directly purchased and obtained, and the strain is preserved in China general microbiological culture collection center (CGMCC) 8279.
Meanwhile, E.coli used in the following examples was cultured at 37℃in a liquid LB medium or on an LB solid plate added with 2.0% agar. Rhodosporidium saccharopolyspora A226 is cultured on TSB culture medium or R3M solid plate containing 2.2% agar at 30 ℃, and the culture medium used for fermenting and producing erythromycin by using Rhodosporidium saccharopolyspora is R5 liquid fermentation culture medium, and is cultured at 30 ℃. General procedures for E.coli and Rhodotorula saccharopolyspora are standard procedures. Primer synthesis and DNA sequencing were accomplished by general biosystems (Anhui).
TABLE 1 strains and plasmids used in the invention
TABLE 2 primers used in the present invention
Example 1
Information about sace_1646 gene:
the SACE_1646 gene is positioned on the chromosome of rhodosporidium saccharopolyspora, the nucleotide sequence is shown as SEQ ID NO.1, the corresponding amino acid sequence is shown as SEQ ID NO.2, and the coded branched chain amino acid aminotransferase IlvE.
Based on sequence analysis of rhodosporidium genome, the rhodosporidium SACE_1646 gene codes a branched-chain amino acid aminotransferase IlvE, is responsible for catalyzing the first reaction of metabolic conversion of branched-chain amino acids, and is found by laboratory researches to have positive correlation with erythromycin biosynthesis. Thus, attempts were made to regulate erythromycin yield by engineering the rhodosporidium saccharopolyspora SACE_1646 gene.
Example 2
Construction of SACE_1646 Gene deletion mutant (see FIG. 1):
in order to construct a deletion mutant of the rhodosporidium saccharum SACE_1646 gene, the pKC1139 plasmid was used to perform traceless knockout on the internal sequence of the SACE_1646 gene, and about 1.5kb homology arm DNA fragments were PCR amplified on the upstream and downstream of the SACE_1646 gene using 1646-UF/1646-UR and 1646-DF/1646-DR as primers and the rhodosporidium saccharum genome as templates, respectively. Specific primer sequences are shown in Table 2, wherein the underlined "AAGCTT", "GGATCC" and "GAATTC" sequences are restriction endonuclease HindIII, bamHI and EcoRI cleavage sites, respectively.
Respectively connecting the two DNA fragments to plasmid pKC1139 through enzyme digestion treatment to construct recombinant plasmid pKC-delta SACE_1646; introducing pKC-delta SACE_1646 into rhodosporidium saccharopolyspora protoplast by using PEG3350 as inducer, and screening transformant with apramycin resistance; secondly, carrying out non-resistance relaxation culture on the resistant transformant at 37 ℃ for 1 round or 2 rounds according to the characteristics of pKC1139 replication temperature-sensitive type and chromosome fragment homologous recombination technology; finally, the gene engineering strain of which the SACE_1646 gene is knocked out in a traceless way is obtained by utilizing a non-resistance plate screening, and PCR verification is carried out by using 1646-TF/1646-TR as a primer, and referring to FIG. 2, the correct mutant strain is identified as delta SACE_1646. The specific primer sequences are shown in Table 2.
Example 3
Construction of SACE_1646 Gene-complemented Strain:
to make up the SACE_1646 gene in the ΔSACE_1646 mutant strain, the complete DNA fragment of the SACE_1646 gene was PCR amplified using 1646-CF/1646-CR as primer and the Rhodosporidium saccharopolyspora genome as template. Specific primer sequences are shown in Table 2, wherein the underlined "CATATG" and "TCTAGA" sequences are the restriction enzyme NdeI and XbaI cleavage sites, respectively.
The DNA fragment of the SACE_1646 gene is connected to a plasmid pIB139 through enzyme digestion treatment to construct a recombinant plasmid pIB-1646; introducing pIB-1646 into the mutant strain delta SACE_1646 by a PEG3350 mediated protoplast transformation method, and screening transformants with apramycin resistance; and (3) performing PCR verification by using Apr-F/Apr-R as a primer, wherein the correct verification is the anaplerotic strain delta SACE_1646/pIB-1646. By the same method, an empty vector pIB139 was introduced into the mutant strain ΔSACE_1646 to construct an empty control strain ΔSACE_1646/pIB139.
Example 4
Erythromycin yield detection in rhodosporidium saccharopolyspora series strains:
the rhodosporidium saccharopolyspora a226 and Δsace_1646 series strains were first inoculated into TSB culture for 2 days based on 30 ℃/220rpm shaking culture, and then transferred into R5 liquid fermentation culture for 6 days based on 30 ℃/220rpm shaking culture. And (3) extracting erythromycin in the fermentation liquid after the fermentation is finished and detecting by HPLC.
Example 5
Transcriptional analysis of related genes in Δsace_1646:
and (3) performing RNA extraction on the rhodosporidium saccharopolyspora original strain A226 and the mutant strain delta SACE_1646 in the fermentation culture process by using a Saubause RNA extraction kit, reversing the materials into cDNA, and analyzing the transcription level of genes related to erythromycin biosynthesis by using real-time fluorescence quantitative PCR.
Example 6
Construction of the overexpressing Strain A226/pIB-1646:
the pIB-1646 and the pIB139 are respectively introduced into rhodosporidium saccharopolyspora A226 by a PEG3350 mediated protoplast transformation technology, and the over-expression strain A226/pIB-1646 of the SACE_1646 gene and the empty-load control strain A226/pIB139 thereof are obtained by the same method of screening and PCR verification of apramycin.
Example 7
Specific experimental results of the above examples:
1. erythromycin yield significantly decreased after SACE_1646 gene deletion
ΔSACE_1646 was fermented in R5 liquid medium for 6 days, and after extraction and concentration, the erythromycin yield was reduced by 48.7% compared with that of the starting strain A226 (FIG. 3), indicating that SACE_1646 gene has a positive correlation with erythromycin biosynthesis yield. Meanwhile, the cells cultured by fermentation were sampled and biomass was measured every day, and the results showed that the cell amounts of Δsace_1646 and a226 were not greatly different (fig. 4), suggesting that the deletion of sace_1646 did not affect the cell growth of the strain.
2. SACE_1646 gene complement
In order to confirm that the phenotype of the mutant strain ΔSACE_1646 is entirely due to mutation of the SACE_1646 gene, the invention designs a SACE_1646 gene complementation experiment for verification. pIB-1646 initiates transcriptional expression of the SACE_1646 gene using the strong promoter PermE of the erythromycin resistance gene for complementation of the mutant ΔSACE_1646. The erythromycin production by the anaplerotic strain ΔSACE_1646/pIB-1646 was restored to a level consistent with that of the starting strain A226 (FIG. 3) by fermentation culture and HPLC detection.
3. The SACE-1646 gene is deleted and does not influence the morphological differentiation of the strain
To determine whether the SACE_1646 gene affected sporulation of the cells, mutant ΔSACE_1646, starting strain A226 and complementing strain ΔSACE_1646/pIB-1646 were simultaneously plated on R3M plates and cultured at 30℃to observe spore growth of the strains. The results showed no significant difference in spore morphology of mutant Δsace_1646 compared to a226 (fig. 5), indicating that the deletion of the sace_1646 gene did not affect the formation of rhodosporidium saccharopolyspora spores.
4. Transcription level of erythromycin synthesis gene is obviously reduced after SACE_1646 gene is deleted
qRT-PCR results prove that in the delta SACE-1646 mutant strain, the expression quantity of the structural gene eryAI, eryBI, eryBIII, eryBIV, eryBVI, eryCI, eryK on the erythromycin biosynthesis gene cluster is obviously reduced by 33% -67% compared with that of the original strain A226, and the resistance gene ermE is obviously reduced by 29% (FIG. 6), so that the deletion of SACE-1646 can cause the obvious reduction of the transcription level of the structural gene and the resistance gene in the erythromycin biosynthesis gene cluster.
5. The overexpression of SACE-1646 gene causes the erythromycin yield to be greatly improved
According to the experimental result that the SACE_1646 gene is deleted to cause the obvious reduction of the erythromycin yield in the erythromyces saccharopolyspora, the positive correlation between the SACE_1646 gene and the erythromycin yield is confirmed, so that the over-expression of the SACE_1646 gene in the erythromyces saccharopolyspora is presumed to be likely to improve the erythromycin yield. The invention introduces the expression vector pIB-1646 constructed in the above into the original strain A226 to improve the copy number of SACE_1646 gene, and constructs the over-expression strain A226/pIB-1646. Through fermentation culture and HPLC detection, the yield of erythromycin is greatly improved by 105.3% compared with the control strain A226/pIB139 by the over-expression strain A226/pIB-1646 (figure 7), and the fact that the yield of erythromycin can be improved through the over-expression of SACE_1646 gene in rhodosporidium through a genetic engineering way is proved.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (4)
1. A method for improving the yield of erythromycin through the Sace_1646 gene of rhodosporidium is characterized in that the Sace_1646 gene is over-expressed in rhodosporidium by a genetic engineering method to obtain a high-yield strain of erythromyces saccharopolyspora, and the obtained high-yield strain of erythromyces saccharopolyspora is used for fermenting and producing erythromycin; the nucleotide sequence of the SACE_1646 gene is shown as SEQ ID NO.1, and the corresponding coded amino acid sequence is shown as SEQ ID NO. 2.
2. The method for increasing erythromycin yield by means of the sace_1646 gene of rhodosporidium of claim 1, characterized in that said step of over-expressing the sace_1646 gene in rhodosporidium by means of genetic engineering method uses rhodosporidium, in particular rhodosporidium saccharopolyspora a226 strain.
3. The method for increasing erythromycin yield by means of the sace_1646 gene of rhodosporidium saccharopolyspora according to claim 1, wherein the expression product of the sace_1646 gene is branched-chain amino acid aminotransferase IlvE.
4. A method for increasing erythromycin yield by means of the sace_1646 gene of rhodosporidium saccharopolyspora according to any one of claims 1 to 3, characterized in that the expression product of said sace_1646 gene is positively correlated with erythromycin biosynthesis.
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