A method for improving the fermentation yield of spinosad heterologous strains by modifying Streptomyces albus B4 chassis

Abstract

The present invention discloses a method for improving the fermentation yield of a heterologous strain of spinosad by modifying a Streptomyces albus B4 chassis. The method comprises the following steps: overexpressing a TetR family transcriptional regulatory factor XNR_0706 involved in regulating fatty acid β-oxidation from S. albus B4 through a genetic engineering approach, obtaining a chassis strain with increased polyketide compound precursor acyl-CoA, and heterologously expressing a spinosad gene cluster in the modified chassis strain to achieve increased fermentation yield of spinosad. Furthermore, when exogenous fatty acids are added, the yield of spinosad is further increased, thereby providing technical support for increasing the yield of spinosad in industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to a method for improving the fermentation yield of a heterologous strain of spinosad by transforming a Streptomyces albus B4 chassis. Background Art

[0002] Spinosad is a polyketide-derived macrolide secondary metabolite produced by aerobic fermentation of Saccharopolyspora spinosa. Due to its readily biodegradable, low-residue, low-pollution, and high safety profile, it has promising applications and broad market prospects in controlling agricultural pests, stored-grain pests, and animal parasites. Spinosad is composed of one propionyl-CoA molecule, nine malonyl-CoA molecules, and one methylmalonyl-CoA molecule, forming a polyketide backbone catalyzed by type I polyketide synthase. This is then modified by 14 other genes in the spinosad biosynthesis gene cluster to form a mixture of spinosads. Spinosad A and D exhibit the highest activity.

[0003] S. spinosa faces challenges both in laboratory and industrial production, such as difficulty in cultivation, slow growth, and long fermentation cycles. Furthermore, genetic manipulation of S. spinosa is challenging due to its stringent restriction and modification system, which strongly restricts and modifies foreign DNA. Heterologous expression of the spinosad biosynthetic gene cluster in a heterologous host with a clear genetic background, ease of cultivation, a short growth cycle, and amenability to transformation and genetic manipulation is expected to increase spinosad production. Although some complex polyketides can currently be synthesized through heterologous expression, their yields are often low. The key limiting factor for this is an insufficient supply of intracellular precursors or biosynthetic enzymes. Therefore, metabolic engineering and genetic manipulation of the heterologous host are aimed at increasing the supply of intracellular precursors and thereby improving spinosad production.

[0004] Microbial secondary metabolic pathways are the intrinsic foundation of microbial drug synthesis. Stimulated by specific external signals, the relevant drug synthesis pathways are activated, and the synthesis of specific drug molecules is achieved through the combination of multiple catalytic and regulatory elements. In addition to the structural genes encoding secondary metabolite production, the Streptomyces secondary metabolite biosynthesis gene cluster also contains regulatory genes that sense developmental status, nutrient availability, and respond to various stresses. Existing research indicates that approximately 12% of Streptomyces chromosomes contain regulatory genes. These regulatory genes form a vast and complex regulatory network that systematically regulates growth, differentiation, and secondary metabolite production. Transcriptional regulatory factors in Streptomyces are generally classified into families such as LysR, AraC / XylS, TetR, LuxR, and ArsR based on sequence similarity, structure, and function. The TetR family of transcriptional regulatory factors can act both globally and through pathway-specific regulation of antibiotic biosynthesis. Their abundance in the Streptomyces genome suggests a crucial role in the regulation of antibiotic biosynthesis. Therefore, deepening the understanding of the regulatory molecular basis of microbial drugs, discovering and determining the mechanism of action of key regulatory elements in the synthesis pathway, rationally designing and modifying the regulatory elements, and controlling the activation level and continuity of the corresponding drug synthesis pathway are of great significance for breeding high-yield strains and significantly increasing drug production. Summary of the Invention

[0005] In view of the above problems, the present invention aims to provide a method for improving the fermentation yield of polymyxin B by modifying the chassis of Streptomyces albus B4.

[0006] The specific technical solutions are as follows:

[0007] A method for improving the fermentation yield of a strain heterologously expressing a spinosad gene cluster by metabolically engineering an S.albus B4 strain based on a regulatory protein XNR_0706. The method comprises overexpressing the TetR family transcriptional regulatory factor XNR_0706, which regulates fatty acid β-oxidation in S.albus B4, through a genetic engineering method to obtain an engineered strain with increased polyketide compound precursor acyl-CoA. The engineered strain is used to heterologously express the spinosad gene cluster to achieve increased spinosad fermentation yield. Exogenous oleic acid and linoleic acid are added to further increase the spinosad fermentation yield. The nucleotide sequence of the XNR_0706 gene is shown in SEQ ID NO.1.

[0008] Furthermore, the amino acid sequence encoded by the XNR_0706 gene is shown in SEQ ID NO.2.

[0009] Furthermore, the genes involved in fatty acid β-oxidation regulated by the XNR_0706 gene include XNR_0345, XNR_0454, XNR_0513, and XNR_1438, whose nucleotide sequences are shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively, and their amino acid sequences are shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQID NO.10, respectively.

[0010] Furthermore, the XNR_0706 gene positively regulates the fatty acid β-oxidation process.

[0011] The beneficial effects of the present invention are:

[0012] The present invention screens the TetR family transcriptional regulatory factor XNR_0706 that positively regulates fatty acid β-oxidation in the chassis strain S.albus B4, expresses the XNR_0706 gene through a genetic engineering approach, increases the transcriptional expression levels of genes XNR_0345, XNR_0454, XNR_0513, and XNR_1438 involved in fatty acid β-oxidation, obtains a chassis strain with increased intracellular acyl-CoA content, and on this basis obtains an engineered strain for producing spinosad by heterologously expressing a spinosad gene cluster, thereby providing technical support for increasing the yield of spinosad in industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The location information of XNR_0706 gene and its neighboring genes on chromosomes;

[0014] Figure 2 The location information of genes involved in regulating fatty acid β-oxidation and adjacent genes on chromosomes;

[0015] Figure 3 Figure 1 is the purification of XNR_0706 protein and EMSA analysis of its target;

[0016] Figure 4 Schematic diagram of the fragment homologous recombination technology of the present invention and the construction of the knockout strain ΔXNR_0706 and the complemented strain ΔXNR_0706 / pIB139-0706;

[0017] Figure 5 Transcriptional analysis of genes XNR_0345, XNR_0454, XNR_0513, and XNR_1438 involved in fatty acid β-oxidation in S. albus B4, knockout strain ΔXNR_0706, and complemented strain ΔXNR_0706 / pIB139-0706, as well as growth analysis;

[0018] Figure 6 Analysis of intracellular Acetyl-CoA and Malonyl-CoA contents in S. albusB4, knockout strain ΔXNR_0706, and complemented strain ΔXNR_0706 / pIB139-0706;

[0019] Figure 7 To construct the S.albusB4 spnNEW p15A plus-0706 overexpression strain and to analyze the spinosad production of S.albusB4spnNEW and S.albusB4 spnNEW p15A plus-0706 strains in SFM fermentation medium; DETAILED DESCRIPTION

[0020] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process. However, 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. Streptomyces albus B4 and B4 spnNEW strains were collected in the laboratory.

[0022] Meanwhile, E. coli used in the following examples was cultured in liquid LB medium at 37°C or on solid LB plates supplemented with 1.5% agar. S. albus B4 was cultured in tryptic soy broth (TSB), liquid CSM, SFM, or solid SM medium supplemented with 3% agar at 30°C.

[0023] HindIII, KpnI, XbaI, and EcoRI used in the following examples were purchased from Thermo Fisher Scientific. TSB, casamino acids, yeast extract, and peptone were purchased from Oxoid. Agar, apramycin, kanamycin, thiostrepton, apramycin, and chloramphenicol were purchased from Sangon Biotech (Shanghai) Co., Ltd. Oleic acid and linoleic acid were purchased from Beijing Solaibao Biotechnology Co., Ltd. TES, acetonitrile, and other chemicals were purchased from reagent companies. Routine operations for E. coli and S. albus B4 were performed according to standard operating techniques. DNA synthesis and sequencing were commissioned to Sangon Biotech (Shanghai) Co., Ltd.

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

[0025]

[0026] Table 2 Primers involved in the present invention

[0027]

[0028]

[0029]

[0030] Example 1

[0031] Mining regulatory factor XNR_0706

[0032] It has been reported that a TetR family transcriptional regulator SAV7471 exists in Streptomyces Avermitilis. It can directly inhibit the expression of genes SAV7472-SAV7473 encoding those involved in CoA metabolism, regulate the intracellular acyl-CoA content, and affect the synthesis of avermectin. We searched the KEGG database for interspecies homologous genes of SAV7471 in the chassis strain S. albus B4 and obtained the TetR family transcriptional regulator XNR_0706, as shown in Figure 2. Figure 1 shown.

[0033] Example 2

[0034] EMSA experiments on XNR_0706 protein expression and promoters of its regulatory genes XNR_0345, XNR_0454, XNR_0513, and XNR_1438

[0035] The full-length XNR_0706 gene fragment was amplified using the primer pair 0706-28a-F / R in Table 2 and the S. albus B4 genome as a template. The fragment was then ligated with the NdeI and EcoRI-digested vector pET28a(+) by homologous recombination to construct the protein expression vector pET28a-0706.

[0036] Transform an appropriate amount of the target plasmid pET28a-0706 into the competent BL21(DE3) strain for inducible expression. Select a single clone and culture it in LB medium containing the appropriate antibiotics. Culture overnight at 37°C, 220 rpm. Add 2% of the overnight culture to 200 mL of LB medium. Add the appropriate antibiotic at 1 / 1000th of the medium volume. Cultivate at 37°C, 220 rpm, until the OD600 reaches 0.4-0.6. Add an appropriate amount of IPTG solution to a final concentration of 0.6 mM. Induce at 16°C, 220 rpm, for 24 hours.

[0037] Remove the induced bacterial suspension and place on ice for approximately 10 minutes. Aliquot into centrifuge tubes and centrifuge at 8000 rpm at 4°C for 15 minutes to collect the cells. Resuspend the cells in PBS buffer and centrifuge at 8000 rpm at 4°C for 15 minutes. Repeat two to three times. Add 25 mL of buffer A to the cells and resuspend them. Ultrasonicate the suspension over ice water until clear. Ultrasonicate for 3 seconds on, 7 seconds off, for a total of 40 minutes. Centrifuge at 13000 rpm at 4°C for 40 minutes. Collect the supernatant and set aside. Remove the pre-packed nickel column from the refrigerator, discard the 20% ethanol added during storage, and wash with 20 mL of deionized water. Elute with 20 mL of charge buffer. Equilibrate the column with 20 mL of buffer A containing 10 mM imidazole. Add the collected supernatant to the equilibrated column and allow the liquid to drain by gravity. Repeated loading can be performed to increase binding efficiency. Subsequently, elution was performed using buffer A and buffer B containing 80mM, 100mM, and 200mM imidazole, respectively. The eluates containing different concentrations of imidazole were collected and concentrated using ultrafiltration tubes. When the final volume was 0.5-1mL, Buffer C was added to the ultrafiltration tube to replace the original buffer in the system. This was replaced twice. The liquid in the ultrafiltration tube was removed and stored at -80°C until further use. The protein concentration was determined according to the instructions of the BCA protein concentration assay kit.

[0038] Take 10 μL of purified protein and an appropriate amount of 6×SDS Loading Buffer and mix them, heat them in a metal bath for 10 to 15 minutes, and centrifuge them at 12000rpm for 1 minute to obtain the sample. Prepare protein gel of corresponding concentration according to the molecular weight of the target protein. In this project, the size of the target protein is 23.0kDa, so a 12% protein gel is selected. After the prepared protein gel solidifies, add the sample to the corresponding lane, set the electrophoresis instrument parameters to 90V for 30min, and then adjust the parameters to 120V for 80min. Take out the protein gel, add the staining solution and boil it for 10 minutes to stain, then add the destaining solution for decolorization after washing, and judge according to the position of the band and the size of the marker (such as Figure 3 (as shown in A in the figure).

[0039] The location of genes involved in regulating fatty acid β-oxidation and adjacent genes on chromosomes is as follows: Figure 2As shown in Table 2, the putative promoter regions of the target genes were amplified by PCR using primer pairs 0706-EMSA-F / R, 0345-EMSA-F / R, 0454-EMSA-F / R, 0513-EMSA-F / R, and 1438-EMSA-F / R. Biotin-labeled PCR products were then purified and recovered using agarose gel electrophoresis and a PCR product purification kit to obtain labeled probes. The biotin-labeled probes were incubated with varying concentrations of protein at 25°C for 20 minutes. Unlabeled specific probes and nonspecific competitor (salmon sperm DNA) were used as controls. After incubation, samples were separated on 8% native polyacrylamide gels in pre-chilled 0.5× TBE buffer at 100 V. The gel bands were transferred to a positively charged nylon membrane using a wet electrotransfer device. Place the nylon membrane on dry filter paper and irradiate it under UV light for 10-15 minutes to crosslink the DNA. Then use chemiluminescence to detect the biotin-labeled probe. The results are as follows Figure 3 As shown in B, C, D, E, and F, among which B is the EMSA analysis of XNR_0706 and target gene XNR_0705-0706-int, C is the EMSA analysis of XNR_0706 and target gene XNR_0344-0345-int, D is the EMSA analysis of XNR_0706 and target gene XNR_0453-0454-int; E is the EMSA analysis of XNR_0706 and target gene XNR_0512-0513-int; F is the EMSA analysis of XNR_0706 and target gene XNR_1438-1439-int.

[0040] The XNR-0706 protein can bind to the promoter regions of genes involved in fatty acid β-oxidation, including XNR_0345, XNR_0454, XNR_0513, and XNR_1438. This suggests that the regulatory factor XNR_0706 can directly regulate the expression of genes XNR_0345, XNR_0454, XNR_0513, and XNR_1438 in S. albus B4.

[0041] XNR_0345, XNR_0454, XNR_0513, and XNR_1438, their nucleotide sequences are shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively, and their amino acid sequences are shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively.

[0042] Example 3

[0043] Construction of XNR_0706 gene knockout mutant

[0044] Based on the annotation information of the KEGG database (https: / / www.kegg.jp / ), the location information of the XNR_0706 gene and its surrounding genes in the S. albus B4 genome and the functions of genes involved in fatty acid β-oxidation were analyzed. Figure 1 As shown, the full length of the XNR_0706 gene is 576 bp, consisting of 191 amino acids, its nucleotide sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2.

[0045] The process of XNR-0706 gene knockout is as follows Figure 4 Figure 2 shows the schematic diagram of the construction of ΔXNR_0706. Using the primer pairs 0706-up-F / R and 0706-down-F / R in Table 2, the upstream and downstream homology arms of the XNR_0706 gene were amplified approximately 1.5 kb, respectively, using the S. albus B4 genome as template. These homology arms were then ligated into the pJTU1278 vector digested with EcoRI and HindIII by homologous recombination to construct the knockout plasmid pJTU1278-0706. Plasmid pJTU1278-0706 was transformed into Escherichia coli ET12567 (PUZ8002) and then transferred into S. albus B4 by conjugation. Conjugates were selected in TSB medium containing apramycin and nalidixic acid to obtain single-crossover strains. Subsequently, after two rounds of relaxation, thiostrepton- and apramycin-resistant mutants were obtained. PCR verification was performed using primers 0706-CF / R and 0706-C-F2 / R2. Figure 4 As shown in B, in the figure, lanes 1 and 2 are the corresponding fragments of S.albusB4 amplified using primer pairs 0706-CF / R and 0706-C-F2 / R2, wherein 0706-CF / R corresponds to lane 1, the fragment size is 1026bp, and primer pair 0706-C-F2 / R2 corresponds to lane 2, the fragment size is 211bp; M: 8000bp DNA Marker; the corresponding fragment of the knockout strain ΔXNR_0706 was amplified using primer pairs 0706-CF / R and 0706-C-F2 / R2, among which 0706-CF / R corresponded to lane 3, and the fragment size was 815 bp. The primer pair 0706-C-F2 / R2 corresponded to lane 4, and no corresponding band appeared in this lane, proving that the XNR_0706 gene had been knocked out. Sequencing further verified that the XNR_0706 gene was knocked out, thus obtaining the XNR_0706 gene-deficient strain ΔXNR_0706.

[0046] Example 4

[0047] Construction of XNR_0706 gene complementation strain

[0048] Using the primer pair 0706-C-F3 / R3 in Table 2 and the S. albus B4 genome as a template, a 576bp DNA fragment containing the XNR_0706 promoter region and coding region was amplified. By homologous recombination, it was ligated with the vector pIB139 digested with EcoRI and HindIII to construct the complementing plasmid pIB139-0706. The plasmid was transformed into Escherichia coli ET12567 (PUZ8002) and transferred into the knockout strain XNR_0706 through conjugation. After apramycin selection, the complementing strain ΔXNR_0706 / pIB139-0706 was obtained and PCR verification was performed using primers Apr-F and Apr-R. Figure 4 As shown in C in FIG, C is PCR identification of the complemented strain ΔXNR_0706 / pIB139-0706. In the figure, M: 8000 bp DNA marker; lanes 1, 2, and 3 are PCR amplifications using primers Apr-F / R, and the target band is the 773 bp apramycin resistance gene.

[0049] Example 5

[0050] In order to explore the regulatory function of the regulatory factor XNR_0706 in the S.albus B4 strain, we measured the growth curves of S.albus B4, XNR_0706 and XNR_0706 / pIB139-0706 in TSB medium. Figure 5 As shown in Figure B, the growth of these three strains in TSB medium was basically consistent, without showing obvious growth differences, indicating that the loss of the regulatory factor XNR_0706 did not affect the bacterial growth of S.albus B4.

[0051] In S.albus B4, we found four genes that were regulated by the regulatory factor XNR_0706 and involved in fatty acid metabolism. However, it is not clear how the regulatory factor regulates the expression of these genes. In order to explore the role of the regulatory factor XNR_0706 in fatty acid metabolism, we analyzed their transcription levels by RT-qPCR. Total RNA was extracted from the three strains of S.albus B4, XNR_0706 and XNR_0706 / pIB139-0706 grown in TSB medium to the logarithmic growth phase, and the RNA was analyzed by RT-qPCR. Relative quantitative analysis of XNR_0454, XNR_0513, and XNR_1438 genes was performed to compare the differences in the transcription levels of these four genes in the three strains, with the hrdB gene as an internal reference gene. Figure 5 As shown in A.

[0052] After knockout of the XNR_0706 gene, the transcription of genes involved in fatty acid β-oxidation, XNR_0345, XNR_0454, XNR_0513, and XNR_1438, decreased in the XNR_0706 strain compared to S. albus B4. The transcript levels of XNR_0513 and XNR_1438 decreased by 54% and 49% respectively. In contrast, in the XNR_0706 / pIB139-0706 strain, the transcript levels of these four genes were upregulated, with XNR_0513 and XNR_1438 increasing by 3-fold and 1.3-fold respectively. This suggests that the regulatory factor XNR_0706 plays a positive role in regulating the expression of the fatty acid β-oxidation genes XNR_0345, XNR_0454, XNR_0513, and XNR_1438 in S. albus B4.

[0053] To further verify the hypothesis, S. albus B4, XNR_0706 and XNR_0706 / pIB139-0706 were inoculated into TSB medium supplemented with glucose and fatty acids. The OD600 of the bacteria in the medium was measured every 12 hours after inoculation, and a growth curve was drawn. Figure 5 As shown in Figure D, in the medium supplemented with exogenous fatty acids, the three strains showed obvious growth differences, with the XNR_0706 strain growing the slowest, while the growth of S.albus B4 and XNR_0706::pIB-0706 strains remained basically the same. In the medium supplemented with exogenous glucose, the three strains did not show obvious growth differences, as shown in Figure 4. Figure 5 As shown in C.

[0054] Example 6

[0055] Analysis of intracellular acyl-CoA content in knockout strain ΔXNR_0706, complemented strain ΔXNR_0706 / pIB139-0706 and S. albus B4

[0056] The activated strain was obtained according to the method in Example 4, and then the activated bacterial solution was adjusted to the initial OD 600Inoculate 50 mL of TSB medium with 0.05 μg of the culture medium and incubate at 30°C, 220 rpm, for 48 hours. Centrifuge 1 mL of the bacterial suspension at 12,000 × g for 2 minutes, then wash twice with phosphate-buffered saline (PBS, pH 8.0). Add 800 μL of lysis buffer (10% trichloroacetic acid, 90% 2 mM dithiothreitol) and mix thoroughly. Freeze and thaw the cell lysate in liquid nitrogen and ice water two to three times, centrifuge at 15,000 × g for 10 minutes at 4°C, and transfer the supernatant to an activated and equilibrated Sep-Pak column (1 mL, 50 mg tC18; Waters, Milford, MA). Allow to stand for 3 to 5 minutes. Once the sample is fully adsorbed, slowly drain the column, wash the column with 1 mL of ddH2O, and elute with 400 μL of 40% acetonitrile. Collect the eluate. Freeze-drying was performed using a vacuum freeze dryer, and the freeze-dried sample was dissolved in 100 μL of acetonitrile. Acyl-CoA was separated and determined using HPLC. The device used a reverse phase C18 column chromatographic separation C18, 5 μm, 4.6×250 mm. The analysis conditions were: mobile phase A was 50 mM KH2PO4, pH 5.5 and mobile phase B was acetonitrile. The sample was detected at 254 nm with a flow rate of 1 mL / min. The mobile phase was set to: 0-8 min 98%-95% A; 8-12 min 95%-90% A; 12-15 min 90%-85% A; 15-19 min 85%-70% A; 19-22 min 70%-98% A. Figure 6 As shown in Figures A and B ("*" indicates P < 0.05, "**" indicates P < 0.01, and "***" indicates P < 0.001), in the deletion strain ΔXNR_0706, the intracellular acyl-CoA content was lower than that in S. albus B4, while in the complemented strain ΔXNR_0706 / pIB139-0706, the intracellular acyl-CoA content was restored to a level similar to that of S. albus B4, as shown in Figures B and C. Figure 6 The experiment showed that the regulatory factor XNR_0706 positively regulates fatty acid β-oxidation and promotes the production of intracellular acyl-CoA.

[0057] Example 7

[0058] Construction of XNR_0706 overexpression strain

[0059] Using the primer pair 0706-C-F4 / R4 in Table 2 and the S. albus B4 genome as a template, a 576bp DNA fragment containing the XNR_0706 promoter region and coding region was amplified. By homologous recombination, it was ligated with the NdeⅠ and HindⅠⅠⅠ digested vector p15A plus to construct the overexpression plasmid p15Aplus-0706. This plasmid was transformed into Escherichia coli ET12567 (PUZ8002) and then transferred into the strain B4spnNEW containing the spinosad gene cluster by conjugation to obtain the overexpression strain B4spnNEW / p15A plus-0706. The primers 0706-C-test-F / R were used for verification, as shown in Figure 2. Figure 7 As shown in A, A is the PCR identification of the XNR_0706 overexpression strain B4 spnNEW p15A plus-0706, where M: 8000bp DNA Marker; Lane 1 is the PCR amplification using primer pair 0706-C-test-F / R, and the target fragment size is 699bp.

[0060] Example 8

[0061] Spinosad production assay

[0062] The polymyxin B4 spnNEW strain and the overexpression strain B4 spnNEW / p15Aplus-0706 were inoculated into 50 mL TSB medium and cultured at 220 rpm and 30 °C for 48 h. 600 The mixture was transferred to a new 50 mL TSB shake flask at a concentration of 0.05 and cultured at 30°C and 220 rpm. At the designated time, the fermentation broth of the strain was collected and the pH of the fermentation broth was adjusted to 5.0 with glacial acetic acid. 2 mL of acetonitrile was added to 1 mL of the bacterial broth, and the mixture was ultrasonically shaken for 10-15 minutes and centrifuged at 10,700 × g for 10 minutes. The supernatant was filtered through a 0.22 μm organic membrane to obtain the sample to be tested. Exogenous fatty acids were added during the transfer.

[0063] To analyze and determine the production of spinosad, the prepared sample was injected onto a C18 column (C18, 2.2 μm, 4.0 × 200 mm) and analyzed by a 6530C Q-TOF LC / MS. The analytical conditions were: mobile phase A: ultrapure water containing 0.1% (v / v) formic acid, mobile phase B: acetonitrile (LC / MS), flow rate 0.8 mL / min, column temperature 30°C, and a standard ESI source. The elution profile was: 10% to 54% mobile phase B (0–10 min); 54% to 62% mobile phase B (10–16 min); 62% to 90% mobile phase B (16–20 min); and 90% to 10% mobile phase B (20–25 min). The peak times of spinosad were determined by comparison with standard curves prepared with spinosad A and spinosad D standards, and the contents of spinosad A and spinosad D in the fermentation broth were calculated. The standard deviation was calculated from three independent experiments.

[0064] When B4 spnNEW and B4 spnNEW / pBC422-0706 were fermented in SFM medium, the production of spinosad reached the highest on the 4th day, with the production of spinosad being 1524.135 μg / L and 1870.86 μg / L, respectively. Figure 7 As shown in Figure A. When exogenous fatty acids were added to the SFM medium, the production of spinosad in the B4 / spnNEW strain and the B4 / spnNEW pBC422-0706 strain reached 855.741 μg / L and 1108.182 μg / L, respectively. Figure 7 As shown in B.

[0065] 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 scope of protection of the present invention.

Claims

1. A method for increasing the fermentation yield of a heterologous strain of spinosad by modifying the chassis of Streptomyces albus B4, characterized in that: By genetically engineering the overexpression of the TetR family transcriptional regulator XNR_0706, which regulates fatty acid β-oxidation, in Streptomyces albus B4, an engineered strain with increased acyl-CoA, a precursor of polyketide compounds, was obtained. The modified engineered strain was used to heterologously express the spinosad gene cluster to increase the spinosad fermentation yield. The addition of exogenous oleic acid and linoleic acid further increased the spinosad fermentation yield. XNR_0706 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The method according to claim 1, wherein XNR_0706 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

2.

3. The method according to claim 1, wherein quilt XNR_0706 Genes regulated by genes involved in fatty acid β-oxidation include XNR_0345、XNR_0454、XNR_0513、XNR_1438 , their nucleotide sequences are shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, respectively, and their amino acid sequences are shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively.

4. The method according to claim 1, wherein XNR_0706 The gene positively regulates the fatty acid β-oxidation process.

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