Method and application of increasing erythromycin production by modifying SACE_5312 gene of Saccharopolyspora erythromycin

Abstract

The present invention provides a method for increasing the yield of erythromycin by modifying the SACE_5312 gene of Saccharopolyspora erythromycin, wherein the SACE_5312 gene of the TetR family in Saccharopolyspora erythromycin is deleted by a genetic engineering method, and then an erythromycin high-yield engineering strain is obtained, and the erythromycin high-yield engineering strain is used to ferment and produce erythromycin; wherein the nucleotide sequence of the SACE_5312 gene is shown in SEQ ID NO.1. The present invention also provides an application of the above method. The present invention has the advantages that the yield can be greatly increased by fermenting and producing erythromycin with the obtained erythromycin high-yield engineering strain, and new technical support is provided for increasing the yield of erythromycin in industrial production.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and in particular to a method and application of increasing erythromycin yield by modifying the gene of Saccharopolyspora erythromyces SACE_5312. Background Art

[0002] Erythromycin, produced by the secondary metabolism of Saccharopolyspora erythromycin, belongs to the macrolide antibiotics and mainly inhibits bacterial growth by preventing the biosynthesis of bacterial proteins. At present, erythromycin derivatives such as clarithromycin, azithromycin, and roxithromycin are widely used in clinical medicine and other fields. As a raw material drug, erythromycin has very important market economic value.

[0003] The molecular structure of erythromycin is very complex. Due to the high cost of chemical synthesis of erythromycin, erythromycin is currently mainly obtained by microbial fermentation. In the erythromycin biosynthesis pathway: first, propionyl-CoA and methylmalonyl-CoA are used as direct precursors, and under the action of a series of enzymes such as polyketide synthase, 6-deoxyerythromycin lactone B, the parent nucleus of erythromycin, is formed; then, it is modified by hydroxylation and glycosylation to generate erythromycin D; finally, erythromycin D is modified by hydroxylation and methylation to generate the final product erythromycin A.

[0004] Studies have shown that the high yield of erythromycin mainly depends on its gene regulation. There are no regulatory genes in the erythromycin biosynthesis gene cluster of Saccharopolyspora erythromycin, indicating that the regulatory factors affecting erythromycin biosynthesis are all located outside the cluster. There are many regulatory genes in the chromosomes of Saccharopolyspora erythromycin, which are divided into 16 families. Among them, BldD (SACE_2077) is the first regulatory factor reported. BldD can not only affect the morphological differentiation of Saccharopolyspora erythromycin, but also positively regulate the biosynthesis of erythromycin. TetR family transcriptional regulatory factors have also been found to be involved in the biosynthesis of erythromycin; among them, SACE_3396 can regulate the biosynthesis of erythromycin by controlling the surrounding genes and affecting the supply of precursors for erythromycin synthesis; SACE_3986 controls the production of erythromycin by directly regulating the gene eryAI in the erythromycin synthesis cluster; and SACE_5754 can regulate the flow of other metabolites, thereby controlling the biosynthesis of erythromycin. All these indicate that the TetR family transcriptional regulatory factors play a very important regulatory role in the biosynthesis of erythromycin. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method and application for increasing the yield of erythromycin by modifying the SACE_5312 gene of Saccharopolyspora erythromycin.

[0006] The present invention adopts the following technical solutions to solve the above technical problems:

[0007] A method for improving erythromycin production by modifying the SACE_5312 gene of Saccharopolyspora erythromycin, wherein the TetR family SACE_5312 gene in Saccharopolyspora erythromycin is deleted by a genetic engineering method, thereby obtaining an erythromycin high-yield engineered strain, and using the erythromycin high-yield engineered strain to ferment and produce erythromycin; wherein the nucleotide sequence of the SACE_5312 gene is as shown in SEQ ID NO.1.

[0008] As one of the preferred embodiments of the present invention, the amino acid sequence encoded by the SACE_5312 gene is shown in SEQ ID NO.2.

[0009] As one of the preferred methods of the present invention, the yield of the fermentation product erythromycin was detected by biological and chemical methods, and it was found that the SACE_5312 gene product can negatively regulate the biosynthesis of erythromycin.

[0010] An application of the above method for increasing erythromycin yield is to knock out the SACE_5312 gene in an industrial high-yield strain to obtain a high-yield mutant strain for erythromycin production.

[0011] As one of the preferred embodiments of the present invention, the industrial high-yield strain is specifically selected from the industrial strain of Saccharopolyspora erythraea WB, and correspondingly, the high-yield mutant is the strain WB-△SACE_5312.

[0012] The advantages of the present invention compared to the prior art are:

[0013] In the present invention, the negative regulator SACE_5312 of erythromycin biosynthesis was screened, and the SACE_5312 gene was deleted in the wild strain A226 of Saccharopolyspora erythromycin and the high-yield strain WB by genetic engineering, and finally the erythromycin high-yield engineered strain WB-△SACE_5312 was obtained, which provided technical support for industrial production to increase the erythromycin fermentation yield. Among them, the SACE_5312 gene was deleted in Saccharopolyspora erythromycin A226, and the erythromycin yield of the obtained mutant strain △SACE_5312 was increased by 60% compared with A226; the SACE_5312 gene was supplemented in △SACE_5312, and the erythromycin fermentation yield was restored; the copy number of SACE_5312 was increased in A226, and the erythromycin yield was reduced by 19%. The SACE_5312 gene was deleted in WB, and the erythromycin yield of the obtained mutant strain WB-△SACE_5312 was increased by 42% compared with WB. These results indicate that SACE_5312 is a negative regulator involved in erythromycin biosynthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the location information map of the SACE_5312 gene and its neighboring genes on the chromosome;

[0015] Figure 2 Schematic diagram of the large chromosome fragment homologous recombination technology of the present invention and PCR verification diagram of the SACE_5312 deletion mutant ( Figure 2 Figure A is a schematic diagram of the construction of the △SACE_5312 mutant; Figure B is a PCR identification diagram of the △SACE_5312 mutant: the length of the tsr resistance gene "360bp" replaced the SACE_5312 gene "740bp" to become "1760bp"; M: 5000bp DNA Maeker);

[0016] Figure 3 This is the fermentation liquid antibacterial experiment and erythromycin production detection diagram ( Figure 3 Figure A is a comparison of the inhibition zone sizes of the fermentation broths of the original strain A226 and the mutant strain △SACE_5312; Figure B is an analysis of the erythromycin production of the original strain A226, the mutant strain △SACE_5312, the complemented control strain △SACE_5312 / pIB139, the complemented strain △SACE_5312 / pIB139-SACE_5312, the overexpression control strain A226 / pIB139 and the overexpression strain A226 / pIB139-SACE_5312; "ns": no significant difference; "***": P < 0.001);

[0017] Figure 4 The spore morphology analysis and mycelium dry weight determination of related strains ( Figure 4 In the figure, Figure A is a spore morphology observation diagram of the original strain A226 and the mutant strain △SACE_5312; Figure B is a mycelium dry weight measurement result diagram of the original strain A226 and the mutant strain △SACE_5312);

[0018] Figure 5 Related gene transcription analysis and EMSA analysis ( Figure 5 Figure A shows the transcription analysis of related genes in the original strain A226 and the mutant strain △SACE_5312. In the mutant strain △SACE_5312, the transcription levels of eryBIV (SACE_0720), eryAI (SACE_0721), eryBI (SACE_0732) and eryCI (SACE_0734) in the erythromycin biosynthesis gene cluster increased; the transcription levels of eryK (SACE_0713), eryBVI (SACE_0717) and eryBIII (SACE_0731) remained unchanged; the transcription level of ermE (SACE_0733) decreased. "ns": no significant difference; "*": P < 0.05; "**": P < 0.01; Figure B shows the EMSA analysis of the promoter region of SACE_5312 and the erythromycin biosynthesis gene cluster):

[0019] Figure 6 This is the analysis of erythromycin production of the industrial high-yield strain WB and the WB-△SACE_5312 mutant (in the figure, "***": P < 0.001). DETAILED DESCRIPTION

[0020] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0021] The strains and plasmids used in the following examples are shown in Table 1, and the synthesized primer sequences are shown in Table 2. The starting strain is Saccharopolyspora erythromyces A226 (CGMCC 8279), which is a strain that can be directly purchased.

[0022] Meanwhile, the Escherichia coli used in the following examples were cultured in liquid LB medium at 37°C or on solid LB plates supplemented with 1.25% agar. Erythromycin-producing bacteria Saccharopolyspora erythraea were cultured in tryptone soy broth (TSB) medium at 30°C or on R3M plates containing 2.2% agar.

[0023] PEG3350, lysozyme, TES, casamino acids, thiostrepton, and apramycin were purchased from Sigma. TSB, yeast extract, and peptone were purchased from Oxoid. Glycine, agar powder, sodium chloride, and other biological reagents were purchased from a reagent company. General techniques for Escherichia coli and Saccharopolyspora erythromyces were performed in accordance with standard procedures. Primer synthesis and DNA sequencing were performed by Sangon Biotech Co., Ltd.

[0024] Table 1 The strains, plasmids and their main properties involved in the present invention

[0025]

[0026]

[0027] Table 2 The present invention relates to primers

[0028]

[0029]

[0030]

[0031]

[0032] Example 1

[0033] Analysis of SACE_5312 gene and construction of SACE_5312 gene deletion mutant strain:

[0034] Based on the gene annotation information of KEGG database (https: / / www.kegg.jp / ), the location information of SACE_5312 and its surrounding genes in the genome of Saccharopolyspora erythraea and their potential gene functions are as follows: Figure 1 The nucleotide sequence of SACE_5312 gene is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2.

[0035] The process of SACE_5312 gene knockout is as follows Figure 2 As shown in A. Using primers 5312-P1, 5312-P2, 5312-P3 and 5312-P4 in Table 2, the A226 genome was used as a template to amplify the upstream and downstream homologous arms of the SACE_5312 gene with a length of 1.5kb respectively. The upstream and downstream homologous arms were inserted into the two ends of the tsr gene in the pUCTSR plasmid to construct pUCTSR-△5312. Using the PEG-mediated protoplast transformation method, the upstream and downstream homologous arm fragments containing the tsr gene were introduced into the A226 protoplasts. After homologous recombination, thiostrepton was used to screen for positive mutant strains. Using primers 5312-P5 and 5312-P6, the pUCSTR-△5312 plasmid was used as the positive template and the A226 genome was used as the negative template for PCR identification, as shown Figure 2 As shown in B, the correctly identified mutant was named △SACE_5312.

[0036] Example 2

[0037] Construction of SACE_5312 gene complementation and overexpression strains:

[0038] Primers 5312-P5 and 5312-P6 were used to amplify the SACE_5312 gene using the A226 genome as a template. The SACE_5312 gene fragment and the pIB139 plasmid were double-digested using NdeI and XbaI endonucleases, and the gene fragment and plasmid after digestion were connected using T4 ligase to obtain the pIB139-SACE_5312 plasmid. The pIB139 and pIB139-SACE_5312 plasmids were introduced into A226 and △SACE_5312 protoplasts using the PEG-mediated protoplast transformation method. Apramycin resistance was used for screening, and then primer pair aac(3)IV-P1 / P2 was used for PCR identification. The obtained complemented control strain, complemented strain, overexpression control strain and overexpression strain were named △SACE_5312 / pIB139, △SACE_5312 / pIB139-SACE_5312, A226 / pIB139 and A226 / pIB139-SACE_5312, respectively.

[0039] Example 3

[0040] Comparison of inhibition zones of fermentation broth and detection of erythromycin production:

[0041] A226 and △SACE_5312 were inoculated into TSB seed medium and cultured at 30℃ for 2 days. The seed solution was then transferred to R5 fermentation medium and cultured at 30℃ for 7 days. The fermentation solution was centrifuged and the supernatant was dropped on a dry plate coated with Bacillus subtilis. After overnight culture, the size of the inhibition zone was observed. Compared with A226, the inhibition zone of △SACE_5312 was larger, as shown in Figure 2. Figure 3 As shown in A.

[0042] A226 and △SACE_5312 strains were inoculated into TSB seed medium and cultured at 30°C for 2 days. The seed solution was then transferred to R5 fermentation medium and cultured at 30°C for 7 days. Chloroform was added to the fermentation solution for extraction. After the extract was dried, methanol was added to dissolve it, and impurities were filtered out. Finally, HPLC was used to detect the erythromycin content. Figure 3 As shown in B, after knocking out SACE_5312, the erythromycin production increased significantly. HPLC results showed that SACE_5312 was negatively correlated with erythromycin production.

[0043] Example 4

[0044] Spore morphology observation and mycelium dry weight determination:

[0045] A226 and △SACE_5312 were inoculated into TSB seed medium for cultivation, and the seed solution was spread on R3M plates. The spore morphology was observed and recorded every 12 hours. Figure 4 As shown in A, there is no difference in the spore morphology of △SACE_5312 compared with A226, indicating that SACE_5312 does not affect the spore morphological differentiation of the strain.

[0046] A226 and △SACE_5312 strains were inoculated into TSB seed medium and cultured at 30°C for 2 days. The seed solution was then transferred to R5 fermentation medium and cultured at 30°C for 7 days. During this period, the fermentation liquid was collected every 24 hours, and the mycelium was collected by centrifugation, and the weight of the mycelium after drying was recorded. Compared with A226, the mycelium weight of △SACE_5312 was also not significantly different, such as Figure 4 As shown in B. This shows that SACE_5312 does not affect bacterial growth.

[0047] Example 5

[0048] Related gene transcription analysis and EMSA analysis:

[0049] A226 and △SACE_5312 strains were inoculated into TSB seed medium and cultured at 30°C for 2 days. The seed solution was then transferred to R5 fermentation medium and cultured at 30°C for another day. The fermentation solution was aspirated and the bacteria were collected by centrifugation. RNA was extracted and reversed into cDNA. The reversed cDNA was used as a template to detect changes in the transcription level of the corresponding gene by qRT-PCR. Figure 5 As shown in A, in the deletion mutant △SACE_5312, the transcription levels of eryBIV (SACE_0720), eryAI (SACE_0721), eryBI (SACE_0732) and eryCI (SACE_0734) in the erythromycin biosynthetic gene cluster increased; the transcription levels of eryK (SACE_0713), eryBVI (SACE_0717) and eryBIII (SACE_0731) remained unchanged; the transcription level of ermE (SACE_0733) decreased. The results show that SACE_5312 can affect the biosynthesis of erythromycin by regulating the transcription of genes in the erythromycin biosynthetic gene cluster.

[0050] Using 5312-28a-P1 and 5312-28a-P2 as primers, using A226 genome as template, amplify SACE_5312 gene. Use NdeI and HindIII endonucleases to double digest SACE_5312 gene fragment and pET28a plasmid, then connect the gene fragment and plasmid after digestion with T4 ligase to obtain pET28a-SACE_5312 plasmid. Using CaCl2-mediated transformation method, pET28a-SACE_5312 plasmid is imported into BL21 (DE3) competence, and coated on a plate containing kanamycin resistance, the positive monoclonal clone obtained is named BL21 (DE3)-SACE_5312. BL21 (DE3)-SACE_5312 is cultivated in LB liquid medium containing kanamycin resistance, and IPTG is added to induce protein expression. Centrifugal collection of thalline, ultrasonic cell disruption, and nickel ion column is used for purification. Finally, high concentration imidazole was used to elute the SACE_5312 protein. Using the A226 genome as a template and different primers, PCR was used to amplify the promoter fragment of the erythromycin biosynthesis gene cluster with FAM fluorescence labeling. The SACE_5312 protein was incubated with the above promoter fragments, and then PAGE electrophoresis analysis was performed. Figure 5 As shown in B, SACE_5312 protein can specifically bind to the promoter regions of the five erythromycin biosynthetic gene clusters. Combined with the transcription results, it can be shown that SACE_5312 can directly control the transcription of some genes in the erythromycin biosynthetic gene cluster and negatively regulate the synthesis of erythromycin.

[0051] Example 6

[0052] Construction of industrial strain WB-△SACE_5312 mutant and analysis of erythromycin production:

[0053] The SACE_5312 gene was knocked out in the high-yield industrial strain WB, and after identification, the mutant was named WB-△SACE_5312. The WB and WB-△SACE_5312 strains were fermented, and the erythromycin fermentation yield of the above strains was detected by HPLC. The mutant strain construction method and HPLC detection method refer to the above Examples 1 and 3. Figure 6 As shown, knockout of SACE_5312 significantly increased erythromycin production.

[0054] In summary, the SACE_5312 gene on the genome of Saccharopolyspora erythromycin can be knocked out by genetic engineering technology to obtain an erythromycin high-yield engineered strain; the present invention provides strong technical support for increasing the yield of erythromycin in industrial production.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for increasing erythromycin production by modifying the SACE_5312 gene of Saccharopolyspora erythromycin, characterized in that: The TetR family SACE_5312 gene in Saccharopolyspora erythromycin is deleted by genetic engineering methods, thereby obtaining an erythromycin high-yield engineered strain, and the erythromycin high-yield engineered strain is used to ferment and produce erythromycin; wherein the nucleotide sequence of the SACE_5312 gene is shown in SEQ ID NO.

1.

2. The method for increasing erythromycin production by modifying the SACE_5312 gene of Saccharopolyspora erythromycin according to claim 1, characterized in that: The amino acid sequence encoded by the SACE_5312 gene is shown in SEQ ID NO.

2.

3. The method for increasing erythromycin production by modifying the SACE_5312 gene of Saccharopolyspora erythromycin according to claim 1, characterized in that: The SACE_5312 gene product negatively regulates erythromycin biosynthesis.

4. An application of the method for increasing erythromycin production by modifying the SACE_5312 gene of Saccharopolyspora erythromycin as claimed in any one of claims 1 to 3, characterized in that: The SACE_5312 gene was knocked out in the industrial high-yield strain to obtain a high-yield mutant strain for erythromycin production.

5. The use of the method for increasing erythromycin production by modifying the SACE_5312 gene of Saccharopolyspora erythromycin according to claim 4, characterized in that: The industrial high-yield strain is specifically selected from the industrial strain of Saccharopolyspora erythrocytes WB, and correspondingly, the high-yield mutant strain is the WB-△SACE_5312 strain.

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