CN117904162A - Method for improving erythromycin yield by modifying rhodosporidium saccharatum SACE_5680 gene and application - Google Patents

Abstract

The invention relates to the technical field of genetic engineering, and provides a method for improving erythromycin yield by modifying a Sace_5680 gene of rhodosporidium, wherein the GntR family SACE_5680 gene in rhodosporidium is deleted by a genetic engineering method, so that a high-yield engineering strain of erythromyces saccharopolyspora erythromycin is obtained, and the obtained high-yield engineering strain of erythromyces saccharopolyspora is used for fermenting and producing erythromycin; wherein the nucleotide sequence of the SACE_5680 gene is shown as SEQ ID NO. 1. The invention also provides an application of the method. The invention screens the erythromycin biosynthesis negative regulator SACE_5680, and the SACE_5680 gene copy on the chromosome of the rhodosporidium is deleted by a genetic engineering way, so that the erythromycin high-yield strain can be obtained, and the technical support is provided for improving the erythromycin fermentation yield in industrial production.

Description

Method for improving erythromycin yield by modifying rhodosporidium saccharatum SACE_5680 gene and application

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a method for improving erythromycin yield by modifying rhodosporidium saccharum SACE_5680 gene and application thereof.

Background

Erythromycin is produced by the secondary metabolism of erythromyces saccharopolyspora, belongs to typical polyketide antibiotics, and comprises the components of erythromycin A (Er-A), erythromycin B (Er-B), erythromycin C (Er-C), erythromycin D (Er-D), erythromycin E (Er-E), erythromycin F (Er-F) and the like. The antibiotics have broad-spectrum antibacterial effect, the antibacterial spectrum of the antibiotics is similar to that of penicillin, and the antibiotics have strong inhibition effect on gram-positive bacteria. Erythromycin A is widely used clinically in the components of erythromycin, and has the highest antibacterial activity, and erythromycin series chemical derivatives (clarithromycin, azithromycin, roxithromycin, telithromycin and the like) are widely used for treating infectious diseases, and the selling amount of erythromycin and derivatives thereof is hundreds of billions of dollars each year.

Erythromycin has an important role in the pharmaceutical field, but its yield is still to be improved. The traditional method for improving the yield of erythromycin A by optimizing the fermentation conditions is time-consuming and uneconomical, and is not suitable for wide application. The erythromycin high-yield strain is obtained by increasing the copy number of the synthetic gene in the rhodosporidium saccharopolyspora chromosome by a genetic engineering method or modifying the regulatory gene by a gene knockout method, and has good prospect.

Ramos et al split the prokaryotic transcriptional regulator into 16 families of LysR, araC/XylS, tetR, luxR, lacI, arsR, icIR, merR, asnC, marR, ntrC (EBP), ompR, deoR, cold stock, gntR and crp based on sequence similarity, structure and function. GntR family transcription regulators are the most widely distributed class of helix-turn-helix (HTH) transcription regulators in bacteria, which contain two functional domains, an N-terminal DNA binding domain and a C-terminal effector binding/oligomerization domain, respectively. The amino acid sequence of the DNA domain is very conserved among GntR family members, but there is a great deal of variability in the amino acid sequence of the effector binding domain/oligomerization domain. Many transcription regulators of the GntR family have been identified, which regulate many different cellular processes of bacteria, such as motility, glucose metabolism, bacterial resistance, pathogenicity of pathogenic bacteria, etc. However, there is little research on the GntR family regulatory genes for the secondary metabolism of rhodosporidium saccharopolyspora.

Disclosure of Invention

The invention aims to provide a method for improving erythromycin yield by modifying rhodosporidium saccharopolyspora SACE_5680 gene and application thereof.

The invention adopts the following technical scheme to solve the technical problems:

A method for improving erythromycin yield by modifying Sace_5680 gene of rhodosporidium, deleting GntR family Sace_5680 gene in rhodosporidium by a genetic engineering method, obtaining a rhodosporidium erythromycin high-yield engineering strain, and fermenting and producing erythromycin by utilizing the obtained rhodosporidium erythromycin high-yield engineering strain; wherein the nucleotide sequence of the SACE_5680 gene is shown as SEQ ID NO. 1.

As one of the preferable modes of the invention, the amino acid sequence coded by the SACE_5680 gene is shown as SEQ ID NO. 2.

As one of the preferred modes of the invention, the SACE 5680 gene product negatively regulates erythromycin biosynthesis.

The application of the method for improving the erythromycin yield by modifying the rhodosporidium saccharum SACE_5680 gene is that the SACE_5680 gene is knocked out in an industrial high-yield strain to obtain a high-yield mutant strain which is used for erythromycin production.

As one of the preferred modes of the invention, the industrial high-yield strain specifically selects the rhodosporidium saccharopolyspora industrial strain WB, and correspondingly, the high-yield mutant strain is a WB-delta SACE_5680 strain.

Compared with the prior art, the invention has the advantages that:

The invention screens the erythromycin biosynthesis negative regulator SACE_5680, and the SACE_5680 gene copy on the chromosome of the rhodosporidium is deleted by a genetic engineering way, so that the erythromycin high-yield strain can be obtained, and the technical support is provided for improving the erythromycin fermentation yield in industrial production. Wherein, when SACE_5680 gene is knocked out in rhodosporidium saccharum A226, erythromycin yield is increased by 22%, and SACE_5680 gene is supplemented back in delta SACE_5680 deletion mutant, erythromycin yield is recovered, which indicates that SACE_5680 is a negative regulatory factor involved in erythromycin biosynthesis. In addition, by using industrial high-yield strain WB as the starting strain, the SACE_5680 gene was deleted on its chromosome to increase erythromycin yield by 53%, and the technique of deleting SACE_5680 gene to increase erythromycin yield was also applicable to industrial high-yield strain.

Drawings

FIG. 1 is a diagram showing the positional information of SACE_5680 gene and peripheral adjacent genes on a chromosome;

FIG. 2 is a schematic diagram of the construction of a ΔSACE_5680 mutant;

FIG. 3 is a PCR identification chart of a ΔSACE_5680 mutant strain (in the figure, SACE_5680 gene is not knocked out, 666bp; SACE_5680 gene is knocked out, 360bp;M,5000bp DNA Marker);

FIG. 4 is a diagram of PCR identification of ΔSACE_5680/pIB139-5680 complementation strain (in the figure, PCR product is apramycin resistance gene, 776bp;M,5000bp DNAMarker);

fig. 5 is an analysis of erythromycin a production by starting strain a226 and deletion mutant Δsace_5680 (in the figure, "x" indicates that there is a significant difference, p < 0.001);

fig. 6 is an HPLC analysis of erythromycin a for the starting strain a226, deletion mutant Δsace_5680, deletion complement strain Δsace_5680/pIB139-5680, and complement empty control strain Δsace_5680/pIB139 (in the figure, "x" indicates that there is a significant difference, p < 0.001).

FIG. 7 is a graph of the biological measurement results of mycelia of the ΔSACE_5680 mutant strain and the wild type A226 strain;

Fig. 8 shows the transcription level of erythromycin synthesis gene cluster-related gene in Δsace_5680 (in the figure, "+" indicates p <0.01, "+" indicates p < 0.001);

fig. 9 is an HPLC analysis of the yield of erythromycin a from high yielding strain WB/Δsace_5680 (in the figure, "×" indicates that there is a significant difference, p < 0.01).

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 (CGMCC 8279) which can be directly purchased.

Meanwhile, E.coli used in the following examples was cultured on a liquid LB medium at 37℃or on a solid LB plate with 1.25% agar added. Erythromyces and its engineering strains were cultured in Tryptone Soy Broth (TSB) medium at 30 ℃ or on R3M plates containing 2.2% agar.

Among the materials used in the examples below, PEG3350, lysozyme, TES, casamino acid, thiostrepton, and apramycin were purchased from Sigma. TSB, yeast extract, peptone were purchased from Oxoid corporation. Glycine, agar powder, sodium chloride and other biological agents are all purchased from reagent companies. General procedures for E.coli and Rhodotorula saccharopolyspora are standard procedures. Primer synthesis and DNA sequencing were performed by biological engineering (Shanghai) Inc.

TABLE 1 bacterial species, plasmids and their main properties according to the invention

TABLE 2 primers according to the invention

Example 1

Analysis of SACE_5680 gene:

The locations of SACE_5680 and adjacent genes on the chromosome of Rhodosporidium saccharopolyspora are shown in FIG. 1.

The nucleotide sequence of the SACE_5680 gene is shown as SEQ ID NO.1, the gene length is 666bp, the encoded amino acid sequence is shown as SEQ ID NO.2, and the protein monomer size is 24.4kDa.

Example 2

Construction of SACE_5680 Gene deletion mutant (see FIG. 2):

The genome DNA of rhodosporidium saccharopolyspora A226 is used as a template, primers 5680UF/5680UR and 5680DF/5680DR are used for PCR amplification to obtain upstream and downstream homology arms of 1.5kb, and HindIII, xba I, ecoR I and Xba I are used for digestion and recovery; simultaneously, the vector pKC1139 is cut out by HindIII and EcoRI enzymes and recovered; the recovered upstream and downstream homology arms and pKC1139 plasmid are connected and transformed and then transferred into DH5 alpha, and the pKC 1139-delta 5680 is obtained through PCR and enzyme digestion identification.

Transferring the constructed plasmid pKC 1139-delta 5680 into the prepared rhodosporidium saccharopolyspora A226 protoplast, coating the protoplast on an R3M solid plate without resistance (placing the plate in a baking oven at 37 ℃ for 48 hours after drying the plate), placing the plate in a constant temperature incubator at 30 ℃ for culturing for about 20 hours, adding aseptic water of apramycin (Apr) to cover the plate (the final concentration is 50 mug/mL apramycin), and culturing for 4-5 days at 30 ℃ continuously until monoclonal grows. The grown monoclonal is transferred to an R3M solid plate containing 50 mug/mL apramycin for enrichment, and spores grow out after culturing for 4-5 days at 30 ℃. And (3) a small amount of spores are taken and scratched in an R3M solid flat plate without resistance, and then the spores are placed in a constant temperature incubator at 37 ℃ for 2-3 d to finish the relaxation and loss of plasmids. When the monoclonal grows out from the plate, half of the monospore is selected in the R3M solid plate without the antibody, and the other half of the monospore is selected in the R3M solid plate containing 50 mug/mL apramycin, and is cultured for 4-5 d at 30 ℃. Bacterial strains growing in R3M solid plates without apramycin are scraped with a small amount of spores for bacterial liquid PCR, the identified primers are 5680JDF and 5680JDR, the positive control template is pKC 1139-delta 5680 plasmid, the negative control template is A226 genome, and the delta SACE_5680 deletion mutant is obtained according to the size of PCR products (see figure 3).

Example 3

Construction of SACE_5680 Gene complementation and overexpression Strain:

The SACE_5680 gene was amplified using the designed primers 5680-P1 and 5680-P2, and electrophoretically recovered, and the recovered SACE_5680 gene fragment and pIB139 were digested with Nde I and Xba I endonucleases, respectively, and the SACE_5680 gene fragment was ligated to pIB139 by T4 DNA ligase to successfully obtain the integrative plasmid pIB139-5680. The pIB139-5680 was then introduced into ΔSACE_5680 protoplasts by PEG-mediated protoplast transformation. By preliminary screening of apramycin, PCR identification was performed with the apramycin resistance gene as a target, and as shown in FIG. 4, the recovered strain was named ΔSACE_5680/pIB139-5680.

Example 4

HPLC detection of rhodosporidium saccharopolyspora fermentation product:

Inoculating rhodosporidium to TSB culture medium, shake culturing at 30deg.C for 48 hr, transferring to R5 liquid culture medium, shake culturing at 30deg.C for 168 hr, extracting fermentation liquid with organic solvent, evaporating to dryness in water bath, adding 1mL methanol for dissolving, treating with 0.22 μm organic filter membrane, and detecting erythromycin A content in sample.

Example 5

Detecting the biomass of rhodosporidium saccharopolyspora mycelium:

The delta SACE_5680 mutant and A226 are respectively inoculated into 30mL of liquid TSB with the same inoculum size, shake-cultured for 48 hours at 30 ℃, then transferred into an R5 culture medium, shake-cultured for 168 hours at the rotational speed of 220rpm at 30 ℃, sampled in different time periods, washed by absolute ethyl alcohol, dried and weighed to obtain the dry weight of the thalli, and after measurement, a biomass curve of the thalli is drawn according to experimental data.

Example 6

Transcriptional analysis of genes within the erythromycin biosynthesis gene cluster in Δsace_5680:

To investigate the level of transcription of genes associated with the erythromycin synthesis gene cluster in the ΔSACE_5680 strain, an RT-qRCR experiment was performed (see Table 2 for relevant primers). And (3) fermenting the delta SACE_5680 and the A226 in an R5 culture medium for 24 hours, taking 1mL of fermentation liquid in a homogenizing tube, centrifuging at 4 ℃ and 12000rpm for 15 minutes, removing the supernatant, adding a 1mL Transzol reagent, homogenizing, vibrating and crushing, extracting total RNA by using an RNA extraction kit, quantifying the Nanodrop, and selecting RNA with the OD260/280 of 1.9-2.2 for subsequent experimental operation. Digesting genomic DNA in RNA, then reversely transcribing the genomic DNA into cDNA in time, finally preparing a reaction system by using the reverse transcribed cDNA as a template and using an RT-qPCR kit, and detecting the transcription level of genes related to erythromycin synthesis gene clusters on a qPCR instrument.

Example 7

Construction of erythromycin industrial high-yield strain WB/delta SACE_5680 strain and HPLC detection:

SACE_5680 was deleted in the erythromycin industrial high-producing strain WB, and the correct strain was verified to be named WB/ΔSACE_5680. And HPLC detection is carried out on fermentation products of the WB high-yield strain and the deletion mutant strain WB/deltaSACE_5680. Mutant construction procedures and HPLC detection are referred to above in the examples.

Example 8

Specific experimental results of the above examples:

1. Deletion mutant ΔSACE_5680 showed increased erythromycin production compared with starting strain A226.

After ΔSACE_5680 was fermented in the fermentation medium for 7d, the yield of erythromycin A was checked by HPLC, and ΔSACE_5680 was found to be 22% higher than that of starting strain A226 (see FIG. 5), and HPLC results indicated that SACE_5680 was a negative regulator involved in erythromycin biosynthesis.

2. The SACE_5680 gene reverts to and overexpresses.

To verify that the increase in erythromycin production in mutant ΔSACE_5680 was due to a SACE_5680 gene deletion, SACE_5680 gene expression vectors pIB139-5680 and pIB139 vectors (as controls) were introduced into protoplasts of the ΔSACE_5680 mutant strain to obtain revertant and empty ΔSACE_5680/pIB139-5680, ΔSACE_5680/pIB139. After PCR identification, A226 and delta SACE_5680 series mutant strains are subjected to shake flask fermentation, and HPLC detection results show that: erythromycin A yield of ΔSACE_5680 was 22% higher than A226; the erythromycin A production of the revertant strain ΔSACE_5680/pIB139-5680 was substantially recovered compared to A226 (see FIG. 6); HPLC detection further indicates that SACE_5680 gene can down regulate erythromycin A biosynthesis.

3. Influence of deletion of SACE_5680 gene on cell growth.

The Δsace_5680 mutant and the strain a226 strain were subjected to fermentation for 7 days, and the dry weight of the cells was plotted to give a corresponding change curve, which showed that Δsace_5680 was not significantly different from the biomass of strain a226 (see fig. 7), suggesting that the deletion of the sace_5680 gene did not affect the primary metabolism of the cells.

4. The Δsace_5680 mutant has increased transcription of genes within the erythromycin biosynthesis gene cluster.

Transcript levels of ermE, eryAI, eryCI, eryK, eryBI, eryBIII, eryBIV and eryBVI were both significantly increased in the Δsace_5680 mutant compared to the starting strain A226. It was demonstrated that SACE_5680 had a direct regulatory effect on transcription of genes within the erythromycin biosynthesis gene cluster (see FIG. 8).

5. The engineered industrial high-yielding strain WB/ΔSACE_5680 resulted in increased erythromycin production.

After constructing the WB/delta SACE_5680 mutant, activating the WB/delta SACE_5680 mutant and the industrial high-yield strain WB by coating plates, respectively inoculating into shake flasks of an industrial seed culture medium, culturing for 2 days at the rotating speed of 220rpm at 30 ℃, transferring into an industrial fermentation culture medium, and continuously culturing for 7 days. After fermentation, the extraction concentration is analyzed by HPLC, and compared with the starting strain WB, the erythromycin yield of WB/delta SACE_5680 is improved by 53 percent (see figure 9). This suggests that the SACE_5680 gene in the high producing strain WB is also involved in regulating erythromycin production.

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 (5)

1. A method for improving the yield of erythromycin by modifying the SACE_5680 gene of rhodosporidium is characterized in that the GntR family SACE_5680 gene in rhodosporidium is deleted by a genetic engineering method, so that a high-yield engineering strain of rhodosporidium erythromycin is obtained, and the obtained high-yield engineering strain of rhodosporidium erythromycin is used for fermenting and producing erythromycin; wherein the nucleotide sequence of the SACE_5680 gene is shown as SEQ ID NO. 1.

2. The method for improving erythromycin yield by modifying the SACE 5680 gene of rhodosporidium saccharopolyspora according to claim 1, wherein the amino acid sequence encoded by the SACE 5680 gene is shown in SEQ ID No. 2.

3. The method for increasing erythromycin yield by engineering a rhodosporidium saccharum SACE 5680 gene according to claim 1 wherein said SACE 5680 gene product negatively regulates erythromycin biosynthesis.

4. Use of a method for increasing the yield of erythromycin by engineering the SACE 5680 gene of rhodosporidium saccharopolyspora as claimed in any of claims 1 to 3, characterized in that the SACE 5680 gene is knocked out in an industrial high-yielding strain, obtaining a high-yielding mutant strain for the production of erythromycin.

5. The use of the method for increasing erythromycin yield by engineering a rhodosporidium SACE 5680 gene according to claim 4, characterized in that said industrial high-yielding strain is specifically selected from rhodosporidium industrial strain WB, correspondingly said high-yielding mutant strain is WB- Δsace 5680 strain.