Milbemycins high-yield strain as well as construction method and application thereof

Abstract

The invention relates to the technical field of microbial genetic engineering, in particular to a milbemycins high-yield strain as well as a construction method and application thereof, and the strain is constructed by introducing an integrated CRISPRi plasmid capable of dynamically inhibiting target gene transcription on the basis of streptomyces hygroscopicus SIPI-054. The integrated CRISPRi plasmid comprises a nucleotide sequence for expressing dCas9 protein activity, a time sequence type promoter for driving dCas9 protein expression, sgRNA for a target gene and a constitutive promoter for driving sgRNA transcription, the target gene is a primary metabolism key node gene, and the time sequence type promoter drives dCas9 protein dynamic expression. According to the invention, an endogenous quorum sensing system SbbR / SbbA of streptomyces hygroscopicus SIPI-054 is coupled with dCas9-mediated CRISPRi, and a sequential starting element sbbAp is used for driving dCas9 expression, so that an engineering strain autonomously and dynamically inhibits key nodes of a primary metabolic pathway in a cell density-dependent manner to maintain dynamic balance of primary metabolism and secondary metabolism, and the expression of the dCas9 is promoted. Further, the fermentation level of milbemycins is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial genetic engineering, and particularly relates to a milbemycin high-yield strain and a construction method and application thereof, and particularly uses quorum sensing system-driven gene transcription inhibition (CRISPRi) technology to reshape the primary metabolism of milbemycin-producing bacteria, thereby achieving the synergy of primary metabolism and secondary metabolism and significantly improving the fermentation level of milbemycin. Background Art

[0002] Streptomyces hygroscopicus SIPI-054 is an important industrial actinomycete. Its main secondary metabolite, milbemycin, is an efficient, green, and non-toxic biological pesticide raw material. The fermentation yield of milbemycin is low, resulting in high industrialization costs and weak competitiveness. In the construction of high-yield antibiotic strains, random mutagenesis is very common, but its low forward mutation frequency and huge workload have brought great trouble to the high-yield transformation of milbemycin-producing strains. More advanced rational metabolic engineering strategies are urgently needed to effectively transform them and improve their fermentation level. In recent years, in the metabolic engineering of milbemycin, the production of milbemycin A3 / A4 can be increased by overexpressing rate-limiting enzyme encoding genes or positive regulatory genes (such as post-modification genes milC, milE, positive regulatory genes milR, milR2, etc. in its biosynthetic gene cluster), knocking out negative regulatory factors (such as the global negative regulatory factor nsdA), knocking out competitive pathways (such as II polyketide synthase encoding gene kelBCD), and exploring transport proteins (such as TP2 and TP5).

[0003] In the static metabolic engineering strategy, in order to improve the carbon flux of the microbial target product synthesis pathway, a similar "one-size-fits-all" approach is often used to constitutively inhibit (i.e., continuously inhibit during bacterial growth) the competing pathways of the target product, such as the synthesis pathways of other secondary metabolites of the bacteria themselves. It is worth noting that the primary metabolism of cells is necessary for their own growth on the one hand, and provides synthetic precursors for secondary metabolic processes on the other hand. For example, fatty acid synthesis requires acetyl coenzyme A, which is also the basic module unit for the biosynthesis of the secondary metabolite milbemycin. Premature or excessive inhibition of the fatty acid synthesis pathway is likely to cause "stunted development" of the bacteria, that is, growth retardation, and ultimately lead to a significant decrease in fermentation biomass, and a decrease in the synthesis of the target product. This contradiction between primary metabolism and secondary metabolism caused by cell growth and product synthesis is difficult to effectively solve by traditional static metabolic engineering.

[0004] In recent years, under laboratory conditions, inducers have been used to artificially intervene in the metabolic flow distribution during bacterial fermentation. However, bacterial growth is a complex biological process that is difficult to accurately control externally. In addition, as the fermentation flux increases, the cost of inducers also rises, and the chemical separation process becomes complicated, which will cause an environmental burden and is not in line with the principles of green economic production.

[0005] During the fermentation process, actinomycetes undergo a transition from primary metabolism to secondary metabolism. Usually, during primary metabolism, bacterial cells consume external nutrients and grow rapidly. Secondary metabolites are often produced only when external nutrients are limited or when cells age or stop growing. Therefore, the benign synergy between cell growth and product synthesis is a huge challenge in metabolic engineering. Summary of the invention

[0006] In order to solve the potential deficiencies of the prior art, the present invention couples the endogenous quorum sensing system SbbR / SbbA of Streptomyces hygroscopicus SIPI-054 with dCas9-mediated CRISPRi, and uses the temporal element sbbAp (a promoter that drives the expression intensity of downstream genes to increase continuously as the bacterial density increases) as a promoter to drive dCas9 expression. As a result, bacteria will autonomously and dynamically inhibit the key node genes of primary metabolism in a cell density-dependent manner to maintain a dynamic balance between primary metabolism and secondary metabolism, and achieve high yield of milbemycin.

[0007] The present invention adopts the following technical solution:

[0008] In a first aspect of the present invention, a milbemycin high-producing strain is provided, which is constructed by introducing an integrative CRISPRi plasmid capable of dynamically inhibiting target gene expression based on Streptomyces hygroscopicus SIPI-054, wherein the integrative CRISPRi plasmid comprises a nucleotide sequence expressing dCas9 protein activity, a temporal promoter driving dCas9 protein expression, sgRNA targeting the target gene, and a constitutive promoter driving sgRNA transcription, wherein the target gene is a key node gene of primary metabolism, and the temporal promoter drives the dynamic expression of dCas9 protein.

[0009] Preferably, the temporal promoter is the sbbAp promoter, which drives the expression intensity of downstream genes to increase continuously as the cell density increases. Thus, the bacteria can autonomously and dynamically inhibit the primary metabolic pathway of milbemycin-producing bacteria in a cell density-dependent manner, achieving a dynamic balance between primary metabolism and secondary metabolism and high yield of milbemycin.

[0010] Preferably, the target gene is a combination of one or more genes.

[0011] Preferably, the target gene is one or a combination of two of the citrate synthase gene gltA1 and the β-ketoacyl-ACP (ketoacyl-ACP) synthase gene fabH8.

[0012] Preferably, the sgRNA target site corresponding to the citrate synthase gene gltA1 is 419 nt to 438 nt away from the start codon of the gene or 629 nt to 648 nt away from the start codon of the gene.

[0013] Preferably, the sgRNA target site corresponding to the ketoacyl-ACP synthetase gene fabH8 is 376nt to 395nt away from the start codon of the gene or 648nt to 667nt away from the start codon of the gene.

[0014] Preferably, dCas9 is the SpCas9 protein derived from Streptococcus pyogenes (S. pyogenes).

[0015] Preferably, the constitutive promoter is promoter J23119.

[0016] The second aspect of the present invention provides a method for constructing the above-mentioned milbemycin high-producing strain, characterized in that it comprises the following steps:

[0017] S1. Using the integrative plasmid pSET152-ermEp*-dCas9 as the starting skeleton, an integrative CRISPRi plasmid that dynamically inhibits target gene expression is constructed. The integrative CRISPRi plasmid contains a nucleotide sequence that expresses dCas9 protein activity, a temporal promoter that drives dCas9 protein expression, sgRNA for the target gene, and a constitutive promoter that drives sgRNA transcription. The target gene is a key node gene of the primary metabolism of milbemycin-producing bacteria, and the temporal promoter drives the dynamic expression of dCas9 protein.

[0018] S2. Using Streptomyces hygroscopicus SIPI-054 as the starting strain, the integrated CRISPRi plasmid was transferred into Streptomyces hygroscopicus SIPI-054 to obtain a milbemycin high-producing strain.

[0019] Preferably, the temporal promoter is the sbbAp promoter.

[0020] Preferably, the target gene is one or a combination of two of the citrate synthase gene gltA1 and the β-ketoacyl-ACP (ketoacyl-ACP) synthase gene fabH8.

[0021] The third aspect of the present invention provides the use of the above-mentioned milbemycin high-producing strain for synthesizing milbemycin.

[0022] By implementing the above technical solution, the present invention has the following advantages:

[0023] 1. The present invention couples the endogenous quorum sensing system SbbR / SbbA of Streptomyces hygroscopicus SIPI-054 with dCas9-mediated CRISPRi, and uses the temporal element sbbAp (a promoter that drives the expression intensity of downstream genes to increase continuously as the bacterial density increases) as a promoter to drive dCas9 expression. As a result, bacteria can autonomously and dynamically inhibit key nodes of the primary metabolic pathway in a cell density-dependent manner to maintain a balance between primary metabolism and secondary metabolism, and achieve high yield of milbemycin.

[0024] 2. Based on transcriptome sequencing technology (RNA sequencing, RNA-seq), the present invention screened multiple key node genes of primary metabolism related to SIPI-054 growth, inhibited their expression through dynamic regulation, and increased the yield of the target product. Then, the inhibition intensity was adjusted by adjusting the targeting position of sgRNA (single guide RNA), further increasing the yield of milbemycin.

[0025] 3. In Streptomyces hygroscopicus SIPI-054, dynamic CRISPRi gene regulation technology was used for the first time to reshape the primary metabolic network, achieve a benign synergy between primary metabolism and secondary metabolism, and construct a milbemycin high-yield strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The schematic diagram of the dynamic regulation design and the candidate genes of the key nodes of primary metabolism in SIPI-054; Figure A is a schematic diagram of the dynamic CRISPRi principle based on dCas9, GBL: γ-butyrolactone; sbbA: GBL synthase encoding gene; sbbAp: sbbA promoter; SbbR: TetR family repressor protein, which can bind to sbbAp and inhibit the expression of sbbA gene; oriT: intermolecular conjugation start site; acc(3)IV: apramycin resistance gene; Rep: vector replicon; Figure B is the milbemycins biosynthesis precursor unit and its related primary metabolic pathway.

[0027] Figure 2Screening and preliminary test results of candidate genes for key nodes of primary metabolic pathways; A is the transcriptome data analysis of the gene encoding citrate synthase (gltA) in the TCA cycle, FPKM: Fragments Per Kilobase Million; B is the transcriptome data analysis of the gene encoding ketoacyl-ACP synthase (fabH) in the fatty acid (FA) synthesis pathway; C is the fermentation yield of mutant milbemycin containing CRISPRi plasmids with different strengths of dynamic and static (constitutive) inhibition of gltA1, SIPI-054 is the starting strain, and milbemycin A3 / A4 is the main target product; D is the fermentation yield of mutant milbemycin containing CRISPRi plasmids with different strengths of dynamic and static (constitutive) inhibition of fabH8; the data are the mean ± standard error of 3 biological replicates.

[0028] Figure 3 Determination of target gene transcription level under different inhibition intensities; A is the dynamic inhibition of transcription level changes of gltA1 gene mutant strain on the 3rd, 6th and 9th day of fermentation (compared with the starting strain); B is the dynamic inhibition of transcription level changes of fabH8 gene mutant strain on the 3rd, 6th and 9th day of fermentation (compared with the starting strain); C is the static inhibition of transcription level changes of gltA1 gene mutant strain on the 3rd, 6th and 9th day of fermentation (compared with the starting strain); D is the static inhibition of transcription level changes of fabH8 gene mutant strain on the 3rd, 6th and 9th day of fermentation (compared with the starting strain); the internal reference gene of qRT-PCR is hrdB, the control is the starting strain SIPI-054, and the relative fold changes value adopts 2 -ΔΔCT The data were calculated using the Livak method; the data are the mean ± standard error of three biological replicates.

[0029] Figure 4 It is a multi-gene combination dynamic inhibition strategy; A is a schematic diagram of the dynamic combination inhibition plasmid, and sgRNA inhibits the target genes gltA and fabH in combination in series; B is a schematic diagram of the dynamic combination inhibition library, the orange and green genes represent gltA1 and fabH8, and sg1, sg2, and sg3 below the genes represent sgRNAs of three different strengths, strong, medium, and weak, respectively. The specific position of the targeted gene is marked above the gene, and the presentation form is the relative distance between the sgRNA and the start codon.

[0030] Figure 5is the combined dynamic inhibition effect; A is the fermentation yield of milbemycin in the dynamic combined inhibition library; the ordinate is the sum of the titers of the target products milbemycin A3 and A4; B is the change in the transcription level of the two genes inhibited simultaneously; the two mutants with the highest yield in the test library were sampled on the 3rd, 6th and 9th days of fermentation, and the transcription levels of the target genes gltA1 and fabH8 after inhibition were characterized by qRT-PCR. The starting strain was SIPI-054, and the other strains were constructed by introducing the corresponding integrated CRISPRi plasmids based on SIPI-054. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present invention are described clearly and completely below through the accompanying drawings. The described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Example 1

[0033] Establishment of a CRISPRi dynamic regulation system in Streptomyces hygroscopicus SIPI-054

[0034] SIPI-054 contains a quorum sensing sequential promoter sbbAp that responds to γ-butyrolactone (GBL) signals. The strength of this element increases with the increase of cell density, and it can be used as a biosensor to regulate primary metabolic flow. In the early growth stage of this bacterium, the promoter sbbAp is inhibited by the SbbR protein, with low intensity, and almost does not drive dCas9 expression. As the bacterial density increases, the amount of GBL synthesis catalyzed by SbbA reaches a specific threshold, gradually releasing the inhibition of SbbR on the sbbAp promoter, and the intensity of driving dCas9 increases, and the inhibition intensity of the target gene increases ( Figure 1 A in the figure, which drives the metabolic flux to secondary metabolism. dCas9 is a Cas9 endonuclease with DNase inactivation, but its ability to target and bind to DNA is still retained, so it can sterically hinder RNA polymerase and cause transcriptional inhibition. dCas9-mediated CRSPRi has become an effective tool that can simultaneously inhibit the expression of multiple target genes.

[0035] Milbemycin is a 16-membered macrolide compound. The starting units of its synthesis are malonyl-CoA and methylmalonyl-CoA. It can be synthesized from acetyl-CoA as a precursor ( Figure 1B in the figure). However, even after bacterial growth enters the plateau phase, acetyl-CoA will still flow into the two primary metabolic pathways of tricarboxylic acid (TCA) cycle and fatty acid (FA) synthesis, which competes with the synthesis of the target product. The dynamic regulation strategy was used to dynamically inhibit the key nodes of the TCA cycle and FA synthesis of Streptomyces hygroscopicus, citrate synthase (gltA) and ketoacyl-ACP synthase (fabH). Figure 1 B) in the figure, thereby coupling the primary metabolism and secondary metabolism pathways of the cell, tilting the carbon flux toward the biosynthesis of milbemycin at the appropriate time point, and realizing real-time automatic adjustment of the metabolic network of the microorganism.

[0036] Through KEGG (Kyoto Encyclopedia of Genes and Genomes) bioinformatics analysis, we found two citrate synthases (gltA1, gltA2) and 12 ketoacyl-ACP synthase genes (fabH1-12) in the starting strain SIPI-054, and selected these 14 genes as candidate genes. After analyzing the transcriptome data of the starting strain SIPI-054, we found that gltA1 and fabH8 were the most actively expressed, and they were selected as the target genes for CRISPRi ( Figure 2 A).

[0037] Dynamic inhibition was performed using CRISPRi technology based on the temporal promoter sbbAp and dCas9, and static inhibition was performed using CRISPRi mediated by the constitutive strong promoter ermEp* and dCas9. At the same time, because the target genes are important nodes of primary metabolism closely related to bacterial growth, we carefully selected three different strengths of sgRNA, strong (sg1), medium (sg2), and weak (sg3), to inhibit them in a gradient manner to avoid growth retardation or bacterial death caused by imbalance in central metabolism.

[0038] 1. Dynamically inhibit key nodes of primary metabolism (single gene)

[0039] The integrative plasmid pSET152-ermEp*-dCas9 (Zhao Yawei, Lei Li, Zheng Guosong, et al. CRISPR / dCas9-mediated multiplex gene repression in Streptomyces. Biotechnology Journal, 13(9): e1800121. doi: 10.1002 / biot.201800121) was used as the starting skeleton and linearized by double restriction digestion with XbaI and NdeI. This process also removed the constitutive promoter ermEp*. The sbbAp promoter amplified by primers F-sbbAp, R-sbbAp, and SIPI-054 genome (template) was then ligated to the vector ( Figure 1 A in Figure 1 yields pSET152-sbbAp-dCas9. sgRNA is driven by the constitutive strong promoter J23119.

[0040] In this experiment, three sgRNAs with different inhibition strengths were designed for the target gene gltA1 (NLG24_RS33945) (online design software CRISPOR, http: / / crispor.tefor.net / ), targeting nt 120-139 (gltA1-sg1), nt 419-438 (gltA1-sg2), and nt 629-648 (gltA1-sg3) of the non-template chain, respectively. The numerical value indicates the relative position of the sgRNA target site from the gene start codon. The vector was then used as a template to amplify the corresponding sgRNAs using primers F-gltA1-sg1, F-gltA1-sg2, F-gltA1-sg3 and universal primer R-dCas9-sgRNA, and ligated to pSET152-ermEp*-dCas9 and pSET152-sbbAp-dCas9 linearized with SpeI and BsrGI. A total of 6 inhibition plasmids (three static inhibition plasmids were used as controls): pSET-ermEp*-dCas9-gltA1-sg1, pSET-ermEp*-dCas9-gltA1-sg2, pSET-ermEp*-dCas9-gltA1-sg3, pSET-sbbAp-dCas9-gltA1-sg1, pSET-sbbAp-dCas9-gltA1-sg2, and pSET-sbbAp-dCas9-gltA1-sg3. For the target gene fabH8 (NLG24_RS33235), the same principles were used to design nt 199-218 (fabH8-sg1), nt 376-395 (fabH8-sg2), and nt 648-667 (fabH8-sg3) targeting the non-template chain, respectively, to obtain the following vectors: pSET-ermEp*-dCas9-fabH8-sg1, pSET-ermEp*-dCas9-fabH8-sg2, pSET-ermEp*-dCas9-fabH8-sg3, pSET-sbbAp-dCas9-fabH8-sg1, pSET-sbbAp-dCas9-fabH8-sg2, and pSET-sbbAp-dCas9-fabH8-sg3.

[0041] All plasmids were introduced into Streptomyces hygroscopicus SIPI-054 through intergeneric conjugative transfer to construct corresponding mutants, and pSET-ermEp*-dCas9 and pSET-sbbAp-dCas9 were used as negative controls. The obtained engineered strains were fermented separately, and finally samples were taken for high-performance liquid chromatography (HPLC) detection.

[0042] The experimental results showed that the fermentation yield of the mutant with static inhibition of gltA1 did not increase significantly, and even decreased to a certain extent. However, the fermentation yield of the mutant strain constructed by the dynamic inhibition strategy showed an upward trend. Among them, the yield of the mutant SIPI-054 / pSET-sbbAp-dCas9-gltA1-sg2 was 2946±298mg / L, which was about 31.5% higher than that of the starting strain, and the change was the most obvious ( Figure 2 Among the different mutants inhibiting fabH8, although the static inhibition strategy also improved the fermentation yield of the starting strain to a certain extent, the dynamic inhibition showed a better improvement effect, among which the mutant SIPI-054 / pSET-sbbAp-dCas9-fabH8-sg1 had a milbemycin yield of 3111±198 mg / L, which was 38.9% higher than that of the starting strain ( Figure 2 B).

[0043] In order to further confirm the effect of dynamic inhibition in the present invention at the transcriptional level, we further characterized the transcriptional levels of target genes in different time periods by qRT-PCR (real-time fluorescence quantitative PCR), and analyzed the changes in transcriptional levels of gltA1 and fabH8 in mutant strains driven by sbbAp or ermEp* in CRISPRi by comparing with the starting bacterial strain. The experiment was carried out using SYBR Green PCR premix kit (manufacturer: Bio-Rad), and the PCR program was: 95°C pre-denaturation for 2 minutes, 95°C denaturation for 20 seconds, 60°C annealing for 20 seconds, 72°C extension for 15 seconds, and 40 cycles. Using the corresponding cDNA as a template and the corresponding primers, qRT-PCR (manufacturer: Roche) was performed to detect the fluorescent CT value of the target gene. The internal reference gene of qRT-PCR was hrdB, and the control was the starting strain SIPI-054. The changes in transcriptional levels were expressed by relative values ​​(Relative fold changes), which were expressed in 2 -ΔΔCT The results showed that the transcription levels of the static inhibitory strains gltA1 and fabH8 driven by the ermEp* constitutive promoter were significantly downregulated to 1.5-16% and 3.7-25.3% on days 3, 6, and 9, respectively. Figure 3 Although sbbAp-mediated dynamic inhibition had little effect on the transcription of gltA1 and fabH8 in the early growth period (day 3), a significant decrease in gene transcription was detected from day 6, and the decrease in transcription level slowed down as the sgRNA intensity weakened ( Figure 2A and B in the figure indicate that the inhibition of target genes occurs in a cell density-dependent manner, and the inhibition intensity gradually increases with the growth of the bacterial population. Moreover, under the control of sgRNAs of different strengths, this dynamic regulation strategy can achieve deep coordination of metabolic fluxes among the TCA cycle, FA synthesis pathway, and milbemycin biosynthesis, thereby significantly increasing the yield of milbemycin.

[0044] 2. Dynamically inhibiting key nodes of primary metabolism (double genes)

[0045] For gltA and fabH, which are key synthase encoding genes in important pathways of primary metabolism in actinomycetes, we screened the actively expressed parts by combining bioinformatics prediction with transcriptome data and inhibited them at different intensities. From the experimental results, we observed that when the inhibition was constitutive or highly sequential, the antibiotic production of the bacteria did not increase, but decreased instead ( Figure 2 C, D), which is most likely due to the lack of primary metabolites leading to a reduction in total biomass or a retardation in unit cell growth. There is no doubt that bacterial growth is a complex process, and the necessary process of primary metabolism is sensitive to any organism. Even if the dynamic regulatory system shows extremely high autonomy and timing, we still have to carefully adjust the inhibition intensity. Fortunately, in the initial tests of these two key genes, we also received positive feedback, and we found suitable conditions in separate inhibition. Therefore, in order for the dynamic regulatory system to be able to synergistically inhibit these two independent primary metabolic pathways, we tried to combine them for inhibition, thereby increasing the flux upper limit of the secondary metabolic flow.

[0046] In order to avoid excessive inhibition of accumulated primary metabolites in two independent primary pathways, we retained sgRNAs with different strengths of inhibiting gltA1 and fabH8 genes, arranged and combined them, constructed a mutant combination inhibition library, and finally screened high-quality mutant strains by fermentation ( Figure 4 B).

[0047] The specific process is:

[0048] The above-constructed integrated CRISPRi plasmids pSET-sbbAp-dCas9-fabH8-sg1, pSET-sbbAp-dCas9-fabH8-sg2, and pSET-sbbAp-dCas9-fabH8-sg3 were used as the starting backbones and linearized with EcoRV / KpnI ( Figure 4A), using primers F-gltA1-sg1, F-gltA1-sg2, F-gltA1-sg3 and universal primer R-T0, the corresponding sgRNAs were amplified using the vector as a template: fabH8-sg1, fabH8-sg2, and fabH8-sg3, which were inserted into the 5' end of the original sgRNA promoter by recombination and ligation, and then tandemly combined to obtain the plasmid library: pSET-sbbAp-dCas9-gltA1-sg1-fabH8-sg1, pSET-sbbAp-dCas9-gltA1-sg1-fabH8-sg2, pSET-sbbAp-dCas9-gltA1-sg1-fabH8-sg3. pSET-sbbAp-dCas9-gltA1-sg1-fabH8-sg1, pSET-sbbAp-dCas9-gltA1-sg2-fabH8-sg2, pSET-sbbAp-dCas9-gltA1-sg2-fabH8-sg3, pSET-sbbAp-dCas9-gltA1-sg3-fabH8-sg1, pSET-sbbAp-dCas9-gltA1-sg3-fabH8-sg2, pSET-sbbAp-dCas9-gltA1-sg3-fabH8-sg3 Figure 4 B). All plasmids were introduced into Streptomyces hygroscopicus SIPI-054 to construct corresponding mutants, and the obtained strains were fermented respectively, and finally samples were taken for high performance liquid chromatography (HPLC) detection.

[0049] The experimental results showed that the high-intensity inhibition combination had a side effect on the synthesis of antibiotics ( Figure 5 A), but it also shows that the system does play a synergistic role and can achieve the effect of multiple inhibition. Under the condition of appropriate inhibition intensity, the combined dynamic inhibition significantly increases the yield of milbemycin greater than that of single inhibition. The yield of the mutant strain SIPI-054 / gltA1-sg2-fabH8-sg3 is 3772±289mg / L, which is about 79.5% higher than that of the starting strain (2113±151mg / L).

[0050] Example 2

[0051] Verification of the behavior of the dynamic inhibitory pathway

[0052] In order to verify the inhibitory ability of dCas9 when targeting multiple target genes, the two mutants with the highest yield (SIPI-054 / gltA1-sg2-fabH8-sg2 and SIPI-054 / gltA1-sg2-fabH8-sg3) in the mutant strains that inhibited the double genes in Example 1 were sampled on the 3rd, 6th, and 9th days of shake flask fermentation, and the total RNA of the bacteria was extracted. After gel identification, it was reverse transcribed to obtain the cDNA of the starting strain and the corresponding mutant strain (three biological replicates per group). Primers for verifying the transcription level of the target gene were designed, namely F-rt-gltA1, R-rt-gltA1 (testing the transcription level of gtlA1); F-rt-fabH8, R-rt-fabH8 (testing the transcription level of fabH8); F-rt-hrdB, R-rt-hrdB (determining the internal reference gene).

[0053] The results of qRT-PCR analysis showed that in the combined inhibition of dual sgRNA, its inhibitory ability was slightly attenuated, but the trend was similar to that of single inhibition, which indicates that the combined dynamic inhibition strategy has superior ability than single inhibition, achieving better synergistic effects between primary metabolism and secondary metabolism and high production of milbemycin.

[0054] The primer sequences used in the above examples are shown in Table 1.

[0055] Table 1 Primer sequences

[0056]

[0057]

Claims

1. A milbemycin high-producing strain, characterized in that: The milbemycin high-producing strain is constructed by transferring an integrative CRISPRi plasmid capable of dynamically inhibiting target gene transcription into Streptomyces hygroscopicus SIPI-054, wherein the integrative CRISPRi plasmid comprises a nucleotide sequence expressing dCas9 protein activity, a temporal promoter driving dCas9 protein expression, sgRNA targeting the target gene, and a constitutive promoter driving sgRNA transcription, wherein the target gene is a key node gene of primary metabolism, and the temporal promoter drives the dynamic expression of dCas9 protein.

2. A milbemycin high-producing strain according to claim 1, characterized in that: The temporal promoter is the sbbAp promoter.

3. A milbemycin high-producing strain according to claim 1, characterized in that: The target gene is a combination of one or more genes.

4. A milbemycin high-producing strain according to claim 3, characterized in that: The target gene is one or a combination of two of the citrate synthase gene gltA1 and the β-ketoacyl-ACP (ketoacyl-ACP) synthase gene fabH8.

5. A milbemycin high-producing strain according to claim 4, characterized in that: The sgRNA target site corresponding to the citrate synthase gene gltA1 is 419 nt to 438 nt away from the start codon of the gene or 629 nt to 648 nt away from the start codon of the gene.

6. A milbemycin high-producing strain according to claim 4, characterized in that: The sgRNA target site corresponding to the ketoacyl-ACP synthetase gene fabH8 is 376nt to 395nt away from the start codon of the gene or 648nt to 667nt away from the start codon of the gene.

7. A milbemycin high-producing strain according to claim 1, characterized in that: The dCas9 is the SpCas9 protein derived from Streptococcus pyogenes.

8. The milbemycin high-producing strain according to claim 1, characterized in that: The constitutive promoter is promoter J23119.

9. A method for constructing a milbemycin high-yielding strain, characterized in that: The following steps are included: S1. Using the integrative plasmid pSET152-ermEp*-dCas9 as the starting vector, an integrative CRISPRi plasmid capable of dynamically inhibiting the expression of the target gene was constructed. The integrative CRISPRi plasmid contained a nucleotide sequence expressing the activity of the dCas9 protein, a temporal promoter driving the expression of the dCas9 protein, an sgRNA targeting the target gene, and a constitutive promoter driving the transcription of the sgRNA. The target gene was a key node gene of primary metabolism, and the temporal promoter drove the dynamic expression of the dCas9 protein. S2. Using Streptomyces hygroscopicus SIPI-054 as the starting strain, the integrated CRISPRi plasmid was transferred into Streptomyces hygroscopicus SIPI-054 to obtain a milbemycin high-producing strain.

10. Use of the milbemycin high-producing strain according to any one of claims 1 to 9 for biosynthesis of milbemycin.

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