An engineered strain of Saccharopolyspora bicolor with high production of butenyl spinosad and its construction method and application

Abstract

The present invention discloses an engineered strain of Saccharopolyspora cerevisiae with high yield of butenyl spinosad, and a construction method and application thereof. The present invention first discloses an engineered strain of Saccharopolyspora cerevisiae, which uses Saccharopolyspora cerevisiae as a starting bacterium to overexpress the TDP‑L‑rhamnosyltransferase gene. The present invention further discloses a construction method of the above-mentioned engineered strain of Saccharopolyspora cerevisiae and its application in the production of butenyl spinosad. The present invention has discovered that the target gene related to the efficient synthesis of butenyl spinosad is the TDP‑L‑rhamnosyltransferase gene, and utilizes genetic engineering technology to overexpress the TDP‑L‑rhamnosyltransferase gene in wild-type Saccharopolyspora cerevisiae to obtain an engineered strain of Saccharopolyspora cerevisiae with high yield of butenyl spinosad. Compared with the wild-type Saccharopolyspora cerevisiae strain, the yield of butenyl spinosad is increased by 76%, providing an excellent strain for the industrial production of butenyl spinosad.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and more specifically to an engineering strain of Saccharopolyspora bicolor with high production of butenyl spinosad, a construction method and application thereof. Background Art

[0002] During grain storage, pest infestation is one of the main causes of grain loss, resulting in a serious reduction in grain quality. At present, the use of chemical synthetic drugs to control stored grain pests is the first choice, but due to the long-term use of chemical agents, a variety of pests have developed serious resistance to chemical agents, and chemical pesticides pose a serious threat to the health of humans and other animals. Butenyl-spinosyn is a new type of green biological insecticide with contact and feeding toxins produced by fermentation of Saccharopolyspora pogona. It has a wider insecticide spectrum than spinosad, a shorter half-life and stronger activity. Wild-type Saccharopolyspora pogona strains have problems such as poor genetic stability, low biomass, difficult growth regulation, and low butenyl-spinosyn production. Therefore, researchers tried to use metabolic engineering to regulate the synthesis of butenyl-spinosyn. The synthesis pathway of butenyl spinosad is long and involves 23 genes, but the regulatory mechanism and target of this synthesis pathway are not clear. In addition, the genome has the characteristics of high GC content and multiple repeating units in the sequence, which makes genetic manipulation relatively difficult, seriously restricting the selection and application of high-yield butenyl spinosad strains.

[0003] Therefore, it is of great significance for the industrial production of butenyl spinosad to find the key rate-limiting steps in the synthesis pathway of butenyl spinosad and genetically modify the wild-type strain to obtain a strain with excellent performance. Summary of the invention

[0004] The object of the present invention is to provide an engineered strain of Saccharopolyspora bicolor with high yield of butenyl spinosad, and a construction method and application thereof.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The invention firstly provides an engineering strain of Saccharopolyspora bicolor, which takes Saccharopolyspora bicolor as a starting bacterium and over-expresses a TDP-L-rhamnosyltransferase gene (busG).

[0007] Furthermore, the Saccharopolyspora pogona is a wild-type Saccharopolyspora pogona strain S. pogona ASAGF58.

[0008] Further, the TDP-L-rhamnosyltransferase gene is a nucleotide sequence of any of the following:

[0009] a1) the nucleotide sequence shown in SEQ ID NO.1;

[0010] b1) the complementary sequence of a1) above;

[0011] c1) A polynucleotide derived from the nucleotide sequence of SEQ ID NO. 1 due to the degeneracy of the genetic code.

[0012] Furthermore, the amino acid sequence encoded by the TDP-L-rhamnosyltransferase gene is shown in SEQ ID NO.2.

[0013] Furthermore, the overexpression of the TDP-L-rhamnosyltransferase gene is to drive the expression of the TDP-L-rhamnosyltransferase gene using a strong promoter Kasop* or other strong promoters.

[0014] Furthermore, the overexpression of the TDP-L-rhamnosyltransferase gene using Saccharopolyspora as a starting bacterium is to introduce a plasmid containing the TDP-L-rhamnosyltransferase gene into Saccharopolyspora.

[0015] Furthermore, the plasmid containing the TDP-L-rhamnosyltransferase gene is a pSET159-busG plasmid, and the pSET159-busG plasmid is obtained by assembling the TDP-L-rhamnosyltransferase gene with the plasmid pSET159.

[0016] In a specific embodiment of the present invention, the construction method of the pSET159-busG plasmid is as follows: PCR amplification of the TDP-L-rhamnosyltransferase gene fragment from the genome of Saccharopolyspora cerebri, PCR amplification of the pSET159 backbone fragment from the plasmid pSET159, and Gibson The TDP-L-rhamnosyltransferase gene fragment and the pSET159 backbone fragment were assembled using an assembly kit (NEB, catalog number E2611L) to obtain the pSET159-busG plasmid.

[0017] In the present invention, the plasmid pSET159 contains a strong promoter Kasop*, which can drive the expression of TDP-L-rhamnosyltransferase gene.

[0018] In a specific embodiment of the present invention, the engineered strain of Saccharopolyspora pogona is Saccharopolyspora pogona ASAG SW-1, whose preservation number is CCTCC NO: M 20221151, and the preservation date is July 22, 2022, and it is preserved in the China Center for Type Culture Collection (address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province).

[0019] The present invention further provides a method for constructing an engineered strain of Saccharopolyspora, comprising the following steps:

[0020] (1) Construction of pSET159-busG plasmid: A TDP-L-rhamnosyltransferase gene fragment was obtained by PCR amplification from the genome of the Saccharopolyspora strain, and a pSET159 backbone fragment was obtained by PCR amplification from the plasmid pSET159. The TDP-L-rhamnosyltransferase gene fragment and the pSET159 backbone fragment were assembled to obtain the pSET159-busG plasmid;

[0021] (2) Transformation of pSET159-busG plasmid: The pSET159-busG plasmid was transformed into the Saccharopolyspora strain using the protoplast transformation method and integrated into its genome to obtain an engineered Saccharopolyspora strain.

[0022] Furthermore, the primers used for PCR amplification to obtain the TDP-L-rhamnosyltransferase gene fragment shown in SEQ ID NO.1 consist of the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4; the primers used for PCR amplification to obtain the pSET159 backbone fragment consist of the nucleotide sequences shown in SEQ ID NO.5 and SEQ ID NO.6.

[0023] Furthermore, the Saccharopolyspora strain is a wild-type Saccharopolyspora strain S. pogona ASAGF58.

[0024] The present invention further provides the use of the above-mentioned Saccharopolyspora engineered strain in the production of butenyl spinosad.

[0025] The method for producing butenyl spinosad by utilizing an engineered strain of Saccharopolyspora bicolor comprises the following steps: fermenting and culturing the engineered strain of Saccharopolyspora bicolor to obtain the butenyl spinosad.

[0026] In a specific embodiment of the present invention, the fermentation culture conditions are 30°C, 200 rpm shaking, or 30°C incubator culture for 7 days.

[0027] The beneficial effects of the present invention are as follows:

[0028] The present invention compares the differences in the transcription levels of the target product spinosad and butenyl spinosad synthesis gene cluster structural genes in Saccharopolyspora spinosa and Saccharopolyspora bicolor, finds that the target gene related to the efficient synthesis of butenyl spinosad is the TDP-L-rhamnosyltransferase gene (busG), and uses genetic engineering technology to overexpress the TDP-L-rhamnosyltransferase gene (busG) in the wild-type Saccharopolyspora bicolor to obtain a Saccharopolyspora bicolor engineered strain with high butenyl spinosad production. Compared with the wild-type Saccharopolyspora bicolor strain, the butenyl spinosad production is increased by 76%, thereby providing an excellent strain for the industrial production of butenyl spinosad. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0030] Figure 1 It is the relative amount of spinosad and butenyl spinosad production.

[0031] Figure 2 Transcription levels of 23 structural genes in S. spinosa ASAGF60 and S. pogona ASAGF19 on days 3 and 5 of fermentation.

[0032] Figure 3 It is constructed from pSET159-busG; wherein, (A) busG fragment; (B) pSET159 backbone; (C) pSET159-busG plasmid map.

[0033] Figure 4 Transformation plate (A) and expansion plate (B) for S. pogona ASAG SW-1.

[0034] Figure 5 The fermentation performance diagram of S.pogonaASAGF58 and S.pogona ASAG SW-1; wherein, (A) relative yield; (B) glucose consumption; (C) biomass. DETAILED DESCRIPTION

[0035] In order to more clearly illustrate the present invention, the present invention is further described below in conjunction with preferred embodiments and accompanying drawings. Similar components in the accompanying drawings are represented by the same reference numerals. It should be understood by those skilled in the art that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0036] Example 1 Construction and performance determination of the engineered strain of Saccharopolyspora pogona ASAG SW-1

[0037] (I) Culture medium and culture conditions

[0038] Culture medium:

[0039] (1) Seed culture medium (g / L): glucose 10.0, MgSO4·7H2O 2.0, KH2PO4 0.5, FM888 30.0, peptone milk 5.0, solid culture medium supplemented with 15%-20% agar.

[0040] (2) Fermentation medium (g / L): glucose 80.0, dextrin 20.0, corn steep liquor powder 10.0 g, cottonseed protein 20.0, CaCO3 20.0, MgSO4·7H2O 1.0, peptone milk 40.0, NaCl 2.0, pH of the fermentation medium 7.2.

[0041] (3) LB medium (g / L): 10.0% peptone, 5.0% yeast extract powder, 10.0% sodium chloride, and 15%-20% agar added to the solid medium.

[0042] (4) R5 medium (g / L): sucrose 103.0, glucose 10.0, acid hydrolyzed casein 0.1, yeast extract powder 5.0, TES 5.73, K2SO4 0.25, MgCl2·6H2O 10.12, trace elements 2.0 mL, agar 15.0-20.0.

[0043] Culture conditions:

[0044] Incubate at 30°C, 200 rpm in a shaker, or in a 30°C incubator.

[0045] (II) Saccharopolyspora fermentation culture and yield detection

[0046] After inoculating S. spinosa ASAGF60 and S. pogona ASAGF19 into seed culture medium and culturing for 48 hours, the inoculation volume of 10% was transferred to fermentation culture medium for fermentation culture to obtain fermentation samples, and fixed-point sampling and yield detection were performed every day. Fermentation sample processing: a certain volume of fermentation broth was taken, 2 volumes of methanol were added, and after sufficient shaking, it was allowed to stand at 4°C overnight. The obtained extract was centrifuged at 4 000r / min for 10 minutes, and the supernatant was filtered with a 0.22μm filter membrane; the yield analysis conditions were: C18 reverse phase column (ZORBAX Ecllipe XDB-C18, 4.6mm×100mm, 3.5um); the mobile phase was V acetonitrile: V methanol: V water = 45:45:10, and the water contained 0.05% ammonium acetate; the injection volume was 10uL.

[0047] The yield of spinosad relative to the yield of butenyl spinosad is as follows Figure 1As shown, when the fermentation reached the 3rd day, the yield of spinosad relative to butenyl spinosad was 140%, and when the culture reached the 6th day, it reached 472%.

[0048] Comparison of the 16SrRNA sequence similarity between S. spinosa ASAGF60 and S. pogona ASAGF58 is 98.88%, and the similarity between the spinosad synthesis gene cluster in S. spinosa and the butenyl spinosad synthesis gene cluster in S. pogona is as high as 94%. However, the production of butenyl spinosad is much lower than that of spinosad. Therefore, it is necessary to compare the differences in the transcriptional levels of the target product spinosad and the structural genes of the butenyl spinosad synthesis gene cluster in S. spinosa and S. pogona to determine the target genes related to the efficient synthesis of butenyl spinosad.

[0049] (III) Analysis of transcriptional levels of the spinosyn and butenyl spinosyn gene clusters

[0050] The transcription level of the structural genes of the spinosad synthesis gene cluster in Saccharopolyspora spinosa and the butenyl spinosad synthesis gene cluster in Saccharopolyspora bicolor was analyzed. The primers used for Saccharopolyspora spinosa are shown in Table 1, and the primers used for Saccharopolyspora bicolor are shown in Table 2. The RNA extraction method for Saccharopolyspora bicolor and Saccharopolyspora spinosa was referred to the instructions of Tiangen RNA extraction kit (Tiangen Biochemical Technology Co., Ltd., catalog number DP430). TM SYBR TM Green Kit Instructions (Applied Biosystems TM , catalog number A25742), the calculation method uses the relative quantitative algorithm: -ΔΔC t This method is based on the C t Adjust the C of the target gene (GOI) in the same two samples t The resulting ΔΔC t The value can be used to determine the fold difference in expression, and the specific formula is as follows:

[0051] Multiple difference = 2 -ΔΔCt (1)

[0052] ΔΔC t =ΔC t样本 -ΔC t标准品 (2)

[0053] ΔC t样本 =C tGOI s –C t正常 s (3)

[0054] ΔC t校准品 =C tGOI c –C t正常 c (4)

[0055] Table 1 Primers used for Saccharopolyspora

[0056]

[0057]

[0058]

[0059] Table 2 Primers used for Saccharopolyspora spinosa

[0060]

[0061]

[0062] RNA was extracted from the fermentation samples of S.pogona ASAGF19 and S.spinosa ASAGF60 on the 3rd, 5th and 7th days, and the transcription levels of 23 structural genes in the spinosad and butenyl spinosad synthetic gene clusters were measured and compared. On the 7th day of culture, it was determined that the bacteria had entered the late stable or decay stage, and the transcription level of the gene was low, so the analysis results on the 7th day were of no reference significance. The transcription levels of the 23 structural genes in the two strains on the 3rd and 5th days were compared and analyzed, and the results were as follows Figure 2 As shown, the relative differences in the transcription levels of TDP-L-rhamnosyltransferase gene (spnG) in S.spinosa ASAGF60 and TDP-L-rhamnosyltransferase gene (busG) in S.pogona ASAGF19 on the 3rd and 5th days reached 93 times and 133 times, respectively. Although the busM and busO genes differed significantly on the 3rd day, their difference levels were significantly lower than busG on the 5th day. Sequence comparison revealed that a total of 29 amino acids in the BusG sequence mutated relative to SpnG. Therefore, busG (nucleotide sequence as shown in SEQ ID NO.1, encoded amino acid sequence as shown in SEQ ID NO.2) may be a potential rate-limiting step in the synthesis of butenyl spinosad, that is, busG may be a target gene related to the efficient synthesis of butenyl spinosad.

[0063] (IV) Construction of the engineered strain S. pogona ASAG SW-1 and verification of its busG transcription level

[0064] 1. Construction of pSET159-busG plasmid

[0065] The wild-type Saccharopolyspora pogona ASAGF58 genome was used as a template and PCR amplification was performed using primers busG-F / R to obtain a busG fragment shown in SEQ ID NO.1. The results of agarose electrophoresis detection were as follows: Figure 3 As shown in A (indicated by "busG" in the figure), the primers used are shown in Table 3, and the PCR program is shown in Table 4;

[0066] Using plasmid pSET159 as a template, PCR amplification was performed with primers pSET159-F / R to obtain the pSET159 backbone fragment (indicated by "pSET159" in the figure), such as Figure 3 As shown in B, the primers used are shown in Table 3, and the PCR program is shown in Table 4;

[0067] Using Gibson The busG fragment containing SEQ ID NO.1 and the pSET159 backbone fragment were assembled using an assembly kit (NEB, catalog number E2611L) to obtain the busG gene driven by the strong promoter KasOp*, namely, the pSET159-busG plasmid (map as shown in Figure 3 (as shown in C).

[0068] Table 3 Plasmid construction primers

[0069]

[0070] Table 4 PCR program

[0071]

[0072] 2. Plasmid Transformation

[0073] (1) The wild-type Saccharopolyspora pogona strain ASAGF58 was cultured in TSB medium, glycine was added at a final concentration of 0.2%, cultured for 48 h, centrifuged at 5000 rpm for 5 min, and at 4° C. to obtain mycelium;

[0074] (2) Wash the mycelium twice with 10.3% sucrose solution and resuspend it in 10 mL of P buffer;

[0075] (3) Prepare a lysozyme solution with a final concentration of 2 mg / mL using P buffer and keep it at 30°C for about 30 min. During this time, gently invert the centrifuge tube every 5 min to promote the release of protoplasts.

[0076] (4) Filter the bacterial solution using a homemade filter tube filled with absorbent cotton, centrifuge at 3000 rpm for 5 min, and collect the protoplasts;

[0077] (5) The protoplasts were spread on R5 medium to detect whether the protoplasts were contaminated and their activity;

[0078] (6) The protoplasts were suspended in 1 mL of P buffer to obtain a protoplast suspension, which was immediately divided into small portions (200 μL of protoplasts were added to each portion) and stored at -80°C or transformed.

[0079] (7) Add 20 μL of pSET159-busG plasmid and 50 μL of 60% PEG 4000 to 200 μL of the prepared protoplast suspension, gently tap the tube wall with your fingers to mix, apply it to the dried R5 plate, and culture it at the regeneration culture temperature of 30°C. When the regenerated protoplasts appear foggy on the plate (24 h), apply an aqueous solution containing an appropriate concentration of antibiotics to the plate.

[0080] After 7 days of cultivation, the transformants obtained on the plate were as follows Figure 4 As shown in A, the transformants were transferred to apramycin resistance plate for expansion. Figure 4 As shown in B, the strain is in good growth condition, indicating that the pSET159-busG plasmid was transformed into Saccharopolyspora pogona and integrated into its genome, that is, the Saccharopolyspora pogona engineering strain was successfully constructed and named Saccharopolyspora pogona ASAG SW-1 (Saccharopolyspora pogona ASAG SW-1 or S.pogona ASAG SW-1). Saccharopolyspora pogona ASAG SW-1 was sent to the China Center for Type Culture Collection (address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province) for preservation, the preservation date is July 22, 2022, and the preservation number is CCTCCNO: M 20221151.

[0081] 3. Verification of busG transcription levels in S. pogona ASAG SW-1

[0082] The wild-type Saccharopolyspora strain S.pogonaASAGF58 and the Saccharopolyspora strain S.pogona ASAGSW-1 were fermented and cultured in the same step (ii) for 7 days, and samples were taken to extract RNA to verify the difference in the transcription level of busG. The results showed that the transcription level of busG in the Saccharopolyspora strain S.pogona ASAG SW-1 was 6.15 times that of busG in the wild-type Saccharopolyspora strain S.pogonaASAGF58, indicating that overexpression of busG in the wild-type Saccharopolyspora strain S.pogonaASAGF58 achieved strong expression of TDP-L-rhamnosyltransferase at the transcription level.

[0083] (V) Fermentation performance verification of strain S.pogona ASAG SW-1

[0084] The wild-type Saccharopolyspora strain S.pogonaASAGF58 and the Saccharopolyspora strain S.pogona ASAGSW-1 were fermented for 7 days and then sampled and tested for yield. Figure 5 As shown in A, the results show that the production of butenyl spinosad by the Saccharopolyspora strain S.pogona ASAG SW-1 was 76% higher than that of S.pogona ASAGF58, and the production of butenyl spinosad was further improved. The above results also confirmed that the strong expression of TDP-L-rhamnosyl transferase can achieve the effect of increasing the fermentation yield of butenyl spinosad, and also reflected that TDP-L-rhamnosyl transferase is one of the key rate-limiting steps in the synthesis of butenyl spinosad. In addition, the glucose consumption and biomass of the Saccharopolyspora strain S.pogona ASAG SW-1 and the wild-type Saccharopolyspora strain S.pogona ASAGF58 were detected, and the results are shown in Figure 5 As shown in B and C, the Saccharopolyspora strain S.pogona ASAG SW-1 and the wild-type Saccharopolyspora strain S.pogona ASAGF58 show glucose consumption ( Figure 5 B) and biomass ( Figure 5 There was no significant difference in C).

[0085] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. An engineered strain of Saccharopolyspora spp., characterized in that: The Saccharopolyspora cellulosa engineered strain is Saccharopolyspora cellulosa ASAG SW-1, and its preservation number is CCTCC NO: M 20221151.

2. The engineered strain of Saccharopolyspora according to claim 1, characterized in that The engineered strain of Saccharopolyspora is derived from Saccharopolyspora as a starting strain and overexpresses the TDP-L-rhamnosyltransferase gene. The Saccharopolyspora is a wild-type Saccharopolyspora strain S. pogona ASAGF58, and the TDP-L-rhamnosyltransferase gene is the nucleotide sequence shown in SEQ ID NO.

1.

3. The engineered strain of Saccharopolyspora according to claim 2, characterized in that The overexpression of the TDP-L-rhamnosyltransferase gene utilizes the strong promoter Kasop* to drive the expression of the TDP-L-rhamnosyltransferase gene.

4. The engineered strain of Saccharopolyspora according to claim 2, characterized in that The method of overexpressing the TDP-L-rhamnosyl transferase gene by using Saccharopolyspora as the starting bacteria is to introduce a plasmid containing the TDP-L-rhamnosyl transferase gene into Saccharopolyspora.

5. Use of the engineered strain of Saccharopolyspora according to any one of claims 1 to 4 in the production of butenyl spinosad.

6. A method for producing butenyl spinosad using an engineered strain of Saccharopolyspora bicolor, characterized in that: The method comprises the following steps: fermenting and culturing the engineered strain of Saccharopolyspora according to any one of claims 1 to 4 to obtain the butenyl spinosad.

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