Bacterial multi-target non-specific genome engineering method and application thereof

Abstract

The invention discloses a bacterium multi-target non-specific genome engineering method and application thereof, and relates to the field of gene engineering. According to the invention, the MTI system is successfully expanded from eukaryotic cells to a prokaryotic bacterium system for the first time, the dependence on a long-segment specific attachment site (attB) is thoroughly eliminated in mechanism, and any pre-modified specific sequence does not need to be carried out on a host genome, so that real host-independent editing is realized. On the basis, a set of genome engineering platform which is simple and convenient to operate and universal is constructed, the platform supports stable integration of multi-copy and large-fragment DNA and is successfully applied to various bacteria including gram-positive bacteria and gram-negative bacteria, and the operable host range of genome engineering is greatly expanded.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and more particularly to a method for multi-target non-specific genome engineering of bacteria and its application. Background Technology

[0002] Genome engineering plays a crucial role in metabolic engineering and synthetic biology, and is widely used in the industrial production of biofuels, chemicals, pharmaceuticals, and other high-value-added compounds (Volk et al., 2023; Meng et al., 2020; Lee et al., 2019). Compared with plasmid expression systems, chromosome integration can overcome population heterogeneity and has better genetic stability. It can reliably maintain and express the required metabolic genes or pathways while reducing metabolic load, making it particularly suitable for large-scale, long-term fermentation.

[0003] Compared to traditional homologous recombination or transposon-mediated DNA integration technologies, site-specific integration driven by large serine recombinases offers significant mechanistic advantages: it has no clear upper limit on donor DNA size, making it particularly suitable for genomic insertion into large metabolic pathways such as bacterial natural product biosynthesis gene clusters (approximately 10-150 kb) and nitrogen fixation gene clusters (approximately 10-60 kb) (Merrick et al., 2018; Fogg et al., 2014; Seshadri et al., 2025; Smanski et al., 2014). In fact, large serine recombinases (LSRs) can integrate their own mobile genetic elements, ranging in size from 20 kb to over 500 kb, into recipient chromosomes without relying on host genetic repair mechanisms or cellular cofactors (Olorunniji et al., 2016). These characteristics make LSRs an important tool for constructing microbial cell factories.

[0004] However, the practical application of LSRs in the rapidly developing fields of metabolic engineering and synthetic biology is limited by a number of factors: Existing site-specific integration tools, such as large serine recombinases (LSRs), rely heavily on specific attachment sites pre-existing in the bacterial genome. attB The vast majority of non-model industrial strains lack such sites, which necessitates time-consuming, inefficient, and narrowly applicable genome pre-modification before application. This is the core bottleneck restricting the widespread application of genome engineering technology in industrial microorganisms.

[0005] When using traditional site-specific integrase (such as PhiC31 and PhiBT1) to optimize yield, simply increasing the copy number of biosynthetic gene clusters will face clear limitations: for example, when the copy number of UK-2A increases to three, the yield will decrease instead of increase due to the introduction of excessive metabolic burden, and it will be impossible to achieve a sustained increase in yield.

[0006] Traditional site-specific integration systems face technical bottlenecks in non-model industrial strains such as *Saccharomyces cerevisiae*, including a scarcity of genetic tools and low operational efficiency. For example, when using the conventional Int32 site-specific integration system to introduce the spinosad J / L biosynthetic gene cluster, the yield increase is limited, only 36.3% higher than the original strain, making it difficult to achieve a breakthrough in yield. Traditional site-specific integration systems also face dual technical bottlenecks: a lack of specific integration sites in certain Gram-negative bacteria and the expression toxicity of MNGE driven by strong promoters. For example, traditional specific integration sites are lacking in *Chromobacterium* and *Burkholderia*; while directly driving the expression of MTI1 integrase in these bacteria using strong promoters leads to significant cytotoxicity, severely limiting or even preventing the growth of engineered bacteria in culture media. Furthermore, there are also problems such as low integration efficiency and a limited number of identified LSRs. To overcome these limitations, there is an urgent need to develop universal, host-independent genome engineering tools to achieve stable chromosomal expression of functional genes or large metabolic pathways in different bacteria.

[0007] Regular LSR attP / attB Site recombination has led some LSRs to evolve multi-targeting or transposition capabilities. Multi-target integrase (MTIs) have more relaxed sequence specificity than site-specific LSRs like PhiC31, allowing them to target multiple genomic sites (Figure 1a). Durrant et al. (2023) identified over 60 LSRs from bacterial metagenomics that can effectively integrate 7 kb of exogenous DNA into human cells, discovering MTI clades. Among them, MTI_Cp36 and others can non-specifically integrate into the human genome, and 63% of MTIs (such as MTI_2871) contain the rare DUF4368 domain (present in only 0.73% of site-specific integrase), which may be the reason for their multi-targeting characteristics (Figures 1b, c) (Durrant et al., 2023). MTIs are theoretically applicable to large-scale non-specific DNA integration in various organisms such as plants, fungi, and bacteria, but this has only been validated in human cells so far.

[0008] Therefore, those skilled in the art are dedicated to developing a host-independent, universal MNGE method that extends the MTI system from human cells to bacterial systems, enabling random chromosome integration in both Gram-positive and Gram-negative bacteria. Summary of the Invention

[0009] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is to develop a bacterial multi-target non-specific genome engineering method and its application.

[0010] To achieve the above objectives, the present invention provides a method for bacterial multi-target non-specific genome engineering.

[0011] Furthermore, a method for bacterial multi-target non-specific genome engineering includes the following steps: Step 1: The MTI integrase encoding gene, codon-optimized from the target bacteria, and the native MTI-related phage... attP The site, adaptor promoter, and target functional gene or biosynthetic gene cluster are cloned into the plasmid backbone via in vivo or in vitro recombination to form a recombinant integrative plasmid; the MTI integrase encoding gene is selected from at least one of the MTI_1737, MTI_2871, or MTI_6538 genes; the nucleotide sequences of the MTI_1737, MTI_2871, and MTI_6538 genes are shown in SEQ ID NO:4, SEQ ID NO:1, and SEQ ID NO:7, respectively; wherein, the integrase encoded by the MTI_1737 gene can recognize the GG conserved dinucleotide core sequence of the host genome; the integrase encoded by the MTI_2871 gene can recognize the TT conserved dinucleotide core sequence of the host genome; and the integrase encoded by the MTI_6538 gene can recognize the AA conserved dinucleotide core sequence of the host genome; the adaptor promoter is... ermEp , stnYp , sp44 , tipAp (Guo et al., 2023) or Rhap (Guo et al., 2023); the plasmid backbone is derived from pLC01, pCAP01, pCL01 or pHZ series plasmids; the codon optimization is based on the codon usage frequency table of the target bacteria; Step 2: The recombinant integrative plasmid is introduced into the target bacteria via conjugation transfer. Positive conjugates are cultured and screened on a selection medium containing apramycin to obtain engineered bacteria with multiple copies of the target functional gene or biosynthetic gene cluster randomly integrated into the host genome.

[0012] Further, in step two, the target bacteria include at least one of UK-2 actinomycetes and FK228 chromobacteria; wherein the UK-2 actinomycete is *Streptomyces huiliensis* (…). Streptomyces huiliensis (The accession number is GDMCC 4.215).

[0013] Further, in step two, the engineered bacteria are *Streptomyces albopictus* J1074 / pCAP-UK-C2-MTI1-7 or *Polyspora spicata* 301 / pCL01-spi-MTI1-Δ spnK No. 19.

[0014] Furthermore, in step one, when the target bacteria are Gram-positive bacteria, the appropriate method is to select... ermEp , stnYp or sp44 Strong constitutive promoters; when the target bacteria are Gram-negative bacteria, select [the appropriate promoter]. tipAp or Rhap Inducible promoters; wherein, for Gram-negative bacteria, the strongly constitutive promoters may also be selected in the initial testing phase.

[0015] Furthermore, in step one, if the plasmid backbone originally carries the PhiC31 or Int32 integration system, the integration system is first precisely removed using CRISPR / Cas9 gene editing technology before being used for in vivo recombination.

[0016] Furthermore, in step two, the conditions for the conjugation transfer are: when the target bacterium is a Gram-positive bacterium, the donor bacterium is *Escherichia coli* S17-1 or... E. coli ET12567 / pUZ8002, the recipient bacteria are spore suspensions or mycelia, co-cultured in a solution containing 10 mM MgCl2. MS or 2CMC agar medium was co-cultured at 30°C for 18 hours, and then covered with a selection plate containing 50 μg / mL apramycin and 25 μg / mL nalidixic acid to screen for conjugates. When the target bacteria were Gram-negative bacteria, the donor bacteria were Escherichia coli WM3064, co-cultured on LB solid medium containing 0.5 mM diaminopimelic acid at 37°C for 6 hours, and then transferred to LB medium containing 100 μg / mL apramycin.

[0017] Furthermore, the target functional gene or biosynthetic gene cluster is idgS-sfp Reporter genes, UK-2 biosynthetic gene cluster, spinosad J / L biosynthetic gene cluster, or FK228 biosynthetic gene cluster.

[0018] Furthermore, the application of the bacterial multi-target non-specific genome engineering method in bacterial genome engineering is specifically used to achieve non-specific multi-target integration of exogenous functional genes or biosynthetic gene clusters.

[0019] Further, a recombinant integrative plasmid comprises: 1) a plasmid backbone derived from pLC01, pCAP01, pCL01, or pHZ series plasmids, containing the origin of replication of Streptomyces / Gram-negative bacteria and an apramycin resistance selection marker; if the plasmid backbone originally carried a PhiC31 or Int32 integration system, the integration system has been excised using gene editing technology; 2) a functional expression unit consisting of an aptamer promoter, an MTI integrase encoding gene, and a natural... attP The site and target functional gene / biosynthetic gene cluster are sequentially tandemly arranged; wherein: the MTI integrase encoding gene is selected from one of the MTI_1737 gene, MTI_2871 gene, or MTI_6538 gene; the integrase encoded by the MTI_1737 gene can recognize the GG conserved dinucleotide core sequence of the host genome, and its corresponding natural attP The site is intrinsic to the phage to which MTI_1737 belongs. attP The two sites form a specific recognition pair; the integrase encoded by the MTI_2871 gene can recognize the TT conserved dinucleotide core sequence of the host genome, whose corresponding natural... attP The site is intrinsic to the phage to which MTI_2871 belongs. attP The two sites form a specific recognition pair; the integrase encoded by the MTI_6538 gene can recognize the AA conserved dinucleotide core sequence of the host genome, whose corresponding natural... attP The site is intrinsic to the phage to which MTI_6538 belongs. attP The sites form a specific recognition pair; the aptor promoter is ermEp , stnYp or sp44 , tipAp or Rhap .

[0020] Further, the plasmid is pMTI_2871 (pMTI), pMTI- sp44 pMTI- stnYp pMTI- tipAp pMTI- Rhap pMTI- idgS pMTI- stnYp - idgS , pCAP-UK-MTI1, pCL01-spi-MTI1-Δ spnK pHZ-FK228-MTI1- tipAp pMTI_1737, pMTI_1737- stnYp pMTI_6538 or pMTI_6538- stnYp .

[0021] Technical effects: 1. This invention is the first to successfully extend the MTI system from eukaryotic cells to prokaryotic bacterial systems, fundamentally eliminating the need for long-segment specific attachment sites. attB It does not depend on the host genome, does not require pre-modification of the host genome, is easy to operate, and truly achieves "host-independent" editing; 2. The universal genome engineering platform constructed in this invention is applicable to multiple strains, is easy to operate and does not require pre-installation of any specific attachment sites, and supports stable integration of multiple copies and large fragments of DNA; 3. This invention breaks through the host limitations and sequence dependence of traditional integration technologies, supports efficient editing of Gram-positive and Gram-negative bacteria, and greatly expands the operable host range of genome engineering.

[0022] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0023] Figure 1 illustrates the design principle and tool construction of the MNGE platform based on multi-target integrases (MTIs) of this invention: Figure a shows the site-specific integrase and multi-target integrase in the target gene ( attB Figure 1 compares the site-specific recognition specificity of PhiC31 and MTI_2871; Figure 2 shows the differences in protein domain composition between the representative site-specific integrase (PhiC31) and the multi-target integrase (MTI_2871); Figure 3 compares the three-dimensional protein structure models of PhiC31 and MTI_2871 predicted by AlphaFold2; Figure 4 shows the natural and organic properties of four candidate MTIs in the heterologous host Streptomyces albopictus J1074. attB Sequence identification and integration efficiency evaluation; Figure e is a schematic diagram of the evolution of genome engineering technology from MSGE, aMSGE to MNGE; Figure 2 is a schematic diagram illustrating the principle verification of multi-target non-specific integration of functional genes using MNGE technology in a preferred embodiment 2 of the present invention (within the figure). idgS-sfp For example): Figure a shows the natural form of MTI1, the core tool enzyme of MNGE. attP and attB Site sequence; Figure b shows MTI1 in its natural host. C. innocuum Genomic evidence for VE303-07 mediating double-copy integration of mobile genetic elements; Figure c shows MTI1 mediating integration in three Streptomyces heterologous hosts. idgS-sfp Evaluation of the integration efficiency of reporter gene clusters; Figure d shows the integration efficiency of the MTI1-based system. S. albus Introduced in J1074 idgS-sfp Observation of the growth phenotype of post-conjugators; Figure e shows the analysis of MTI1 in the genome through whole-genome sequencing. S. albus J1074 Genome Mediation idgS-sfp Integration sites and copy numbers; Figure f shows the diversity targeted by MTI1 to achieve genome integration. attB Core sequence feature alignment; Figure 3 illustrates MTI1-mediated transformation in a preferred embodiment 2 of the present invention. idgS-sfp Figure a shows the correlation between multicopy integration and indigo yield: Figure a shows the correlation between multicopy integration and indigo yield among 14 high-yielding engineered strains of indigo. idgS-sfp Genomic site distribution integrated via the MTI1 system; Figure b shows the distribution of 14 selected strains. idgS-sfp Copy number statistics integrated through the MTI1 system; Figure c shows the indigo yield and... idgS-sfp Correlation of integration copy number; Figure d shows the change in indigo yield of engineered bacteria with different copy numbers after supplementation with 5 mM L-glutamine (based on single-copy integration strain S. albusJ1074 / pLC01- idgS -C1 and double-copy integrated strains S. albus J1074 / pLC01- idgS -C2 is the control group); Figure 4 is an analysis of the integration efficiency of three MTI1 series plasmids in three non-model Streptomyces in a preferred embodiment 2 of the present invention: Figure a shows the integration efficiency of the three MTI1 series plasmids in Streptomyces vena cava ATCC10712; Figure b shows the integration efficiency of the three MTI1 series plasmids in Streptomyces ponsey ATCC27952; Figure c shows the integration efficiency of the three MTI1 series plasmids in Streptomyces avermitilis NRRL8165 (with pLC01 plasmid as a control); Figure 5 shows the HPLC analysis of UK-2 production in four heterologous Streptomyces hosts in a preferred embodiment 3 of the present invention: Figure a shows the HPLC analysis of UK-2 production in *Streptomyces whiteus* J1074 when heterologously expressed in five different culture media; Figure b shows the HPLC analysis of UK-2 production in *Streptomyces cerevisiae* SBT5 when heterologously expressed in five different culture media; Figure c shows the HPLC analysis of UK-2 production in *Streptomyces cerevisiae* RedStrep1.7 when heterologously expressed in five different culture media; Figure d shows the UK-2 production in *Streptomyces marineus* ZH16 when heterologously expressed in five different culture media. Figure 6 is an analysis of the in situ and heterologous expression yields of the UK-2 biosynthetic gene cluster in a preferred embodiment 3 of the present invention: Figure a shows the chemical structures of fenpicoxamid and its related natural products UK-2A, B, C, and D; Figure b shows the UK-2 biosynthetic gene cluster from Streptomyces huiligensis GDMCC 4.215; Figure c compares the product yields of UK-2 BGC expressed in the original producing strain and four heterologous Streptomyces hosts; Figure d shows the single-copy UK-2 BGC engineered strain. S. albus The yield-time curve of J1074 / pCAP-UK-C1 during fermentation; Figure e shows the double-copy UK-2 BGC engineered strain. S. albus Yield-time curve of J1074 / pCAP-UK-C2 during fermentation; Figure 7 shows the UK-2 yield enhancement using MNGE technology based on *Streptomyces albopictus* J1074 / pCAP-UK-C1 in a preferred embodiment 3 of the present invention: Figure a is a schematic diagram of non-specific integration of the UK-2 gene cluster mediated by MTI1 (with site-specific integration mediated by PhiBT1 as a control); Figure b is the screening of high-yielding strains of MTI1-mediated UK-2 gene cluster genome integration conjugates (samples were taken on the fifth day of fermentation for HPLC analysis); Figure c is the fermentation verification of the high-yielding UK-2 engineered strain mediated by MTI1 (samples were taken on the fifth day of fermentation for HPLC analysis); Figure d is the optimal engineered strain. S. albus UK-2 production time series analysis of J1074 / pCAP-UK-C1-MTI1-24; Figure 8 illustrates the efficient enhancement of UK-2 production in *Streptomyces albus* J1074 using the MNGE method in a preferred embodiment 3 of the present invention: Figure a is a schematic diagram of the strategy of non-specific and site-specific integration using MTI1 and PhiBT1 based on the double-copy chassis bacterium *Streptomyces albus* J1074 / pCAP-UK-C2; Figure b is a preliminary high-throughput screening of UK-2 production capacity in the engineered strain library constructed using MNGE technology (fermentation samples were detected by HPLC on day 5); Figure c is a repeatability verification of the UK-2 production capacity of the high-yielding engineered strains obtained by MNGE screening (fermentation samples were detected by HPLC on day 5); Figure d is the yield-time curve of the optimal MNGE engineered strain *S. albus* J1074 / pCAP-UK-C2-MTI1-7 during fermentation; Figure 9 shows the application of MNGE in a preferred embodiment 4 of the present invention to increase the yield of spinosad J / L in the natural producer *Polyspora spinosae*: Figure a shows the chemical structures of the target products spinosad J and L; Figure b shows the spinosad J / L biosynthetic gene cluster derived from *Polyspora spinosae* 301; Figure c compares the non-specific integration based on MTI1 with the site-specific integration strategy based on Int32; Figure d shows the integration efficiency of three MTI1 series plasmids in the natural producer of spinosad J / L, *Polyspora spinosae* 301 (pMTI, pMTI-...). sp44 and pMTI- stnYp MTI1 / attP The expression of the system is influenced by promoters. ermEp , sp44 and stnYp (Plasmids pLC01 and pSI01 were used as negative and positive controls, respectively). Figure e shows the screening of conjugates for high spinosad J / L production mediated by MTI1; Figure f shows the replication validation of progeny conjugates for high spinosad J / L production mediated by MTI1 (fermentation samples used for HPLC analysis were collected on day 14). Figure 10 is a growth phenotype observation of Chromobacterium tumefaciens Beijing after the introduction of three strong promoter-controlled MTI1 series plasmids in a preferred embodiment 5 of the present invention: plasmid pMTI, pMTI- sp44 and pMTI- stnYp The expression of MTI1 is influenced by strong promoters. ermEp , sp44 and stnYp Regulation; growth phenotypes were observed using solid (top) and liquid (bottom) LB media; pLC01 plasmid was used as a control; Figure 11 is a phenotypic observation of Burkholderia ATCC10248 after incorporation with MTI1 series plasmids controlled by promoters of different strengths in a preferred embodiment 5 of the present invention: Figure a shows the effect of plasmids controlled by strong promoters on the growth phenotype of Burkholderia ATCC10248 (pMTI- sp44 and pMTI- stnYp Respected sp44 and stnYp Strong promoter regulation, with ermEp (The pMTI plasmid controlled by a strong promoter was used as a control); Figure b shows the effect of the weak promoter-controlled plasmid on the growth phenotype of Burkholderia ATCC10248 (pMTI- tipAp and pMTI- Rhap Respected tipAp and Rhap Weakly inducible promoter regulation (see Figure a for control); Figures a and b both use solid culture medium (top) and liquid CYMG culture medium (bottom) for phenotypic observation. Figure 12 is an analytical diagram illustrating the high-yield production of the anticancer drug FK228 in Gram-negative bacteria using the optimized MNGE method in a preferred embodiment 5 of the present invention: Figure a shows the chemical structure of the antitumor drug FK228; Figure b shows the FK228 biosynthetic gene cluster derived from Chromobacterium Beijing; Figure c shows the growth phenotype of Chromobacterium Beijing when different MTI1 series plasmids are introduced (plastmid pMTI, pMTI- tipAp and pMTI- Rhap MTI1 / attP The expression of the system is influenced by promoters. ermEp , tipAp and Rhap The regulation was observed using solid and liquid LB media, with plasmid pLC01 as a positive control; Figure d shows a schematic diagram of non-specific integration of the FK228 biosynthetic gene cluster mediated by MTI1 (with site-specific integration of the FK228 biosynthetic gene cluster mediated by PhiC31 as a negative control); Figure e shows the screening of high-yielding strains of progeny conjugates of MTI1-mediated FK228 biosynthetic gene cluster genomic integration; Figure f shows the verification of progeny conjugates that efficiently produce FK228 mediated by MTI1 (fermentation samples used for HPLC analysis were collected on the third day). Detailed Implementation

[0024] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0025] The strains and culture conditions used in the embodiments of this invention are as follows: This invention relates to a variety of microbial strains, mainly including actinomycetes (Streptomyces, Polysporum spinosum, etc.) and Gram-negative bacteria (Chlorobacterium, Burkholderia, etc.). All actinomycete strains (including...) S. albus J1074 S. coelicolor M1152 S. lividans SBT5, S. lividans RedStrep 1.7 S. venezuelae ATCC 10712 S. avermitilis NRRL 8165 S. huiliensis GDMCC 4.215 S. atratus SCSIO ZH16NSEP-Δligase (ZH16), S. peucetius ATCC 27952 and S. spinosa All 301 samples were cultured at 30°C. S. spinosa Spore preparation and conjugation transfer of strain 301 and its derivatives were performed using 2CMC agar medium (containing 10 g / L soluble starch, 2 g / L tryptone, 1 g / L NaCl, 2 g / L (NH4)2SO4, 1 g / L K2HPO4, 2 g / L MgSO4·7H2O, 2 g / L casein amino acids, 2 g / L CaCO3, 20 g / L agar, and supplemented with trace element solution including 1.55 mg / L MnCl2·4H2O and 1.75 mg / L ZnSO4·7H2O, pH 7.2). S. atratus ZH16 was cultured on YMS medium (4 g / L yeast extract, 10 g / L malt extract, 4 g / L soluble starch, 7.5 g / L oats, 2 g / L CaCO3, 20 g / L agar, pH 7.4). S. peucetius ATCC 27952 was cultured on MY medium (maltodextrin 20 g / L, yeast extract 15 g / L, casein amino acids 5 g / L, glucose 4 g / L, peptone 2.5 g / L, K₂HPO₄ 1.5 g / L, CaCO₃ 2 g / L, agar 20 g / L, pH 7.2-7.4); other Streptomyces strains were cultured on MS agar medium (soybean flour 20 g / L, mannitol 20 g / L, agar 20 g / L). Among Gram-negative bacteria... Chromobacterium sp. Beijing used LB medium. B. gladioli ATCC 10248 and its derivative strains were cultured on CYMG agar medium containing 50 μg / mL gentamicin (8 g / L tryptone, 4 g / L yeast extract, 4.06 g / L MgCl2·2H2O, 10 g / L glycerol, and 20 g / L agar).

[0026] The culture medium of this invention: MS medium (with 10 mM MgCl2 added) is suitable for... S. albus J1074 S. coelicolor M1152 S. lividans SBT5 and S. avermitilis NRRL 8165 conjugation transfer; M-Isp4 medium (containing soybean meal 5 g / L, mannitol 5 g / L, starch 5 g / L, tryptone 2 g / L, yeast extract 1 g / L, NaCl 1 g / L, (NH4)2SO4 2 g / L, HP 1 g / L, CaCO3 2 g / L, agar 20 g / L, add 1 mL / L trace element solution, pH 7.0-7.2; Trace element solution: ZnSO4·7H2O 1 g / L, FeSO4·7H2O 1 g / L, MnSO4·H2O 1 g / L) with 10 mM MgCl2 added. S. lividans RedStrep 1.7 S. venezuelae ATCC 10712 S. peucetius ATCC 27952 and S. atratus Conjugation transfer of SCSIO ZH16NSEP-Δligase (ZH16); 2CMC medium (with 20 mM MgCl2 added) specifically for S. spinosa 301 bonding transfer; for B. gladioli Conjugation transfer was performed using LB medium containing 0.5 mM DAP and CYMG medium containing 50 μg / mL apramycin. For genomic DNA extraction, S. huiliensis GDMCC 4.215 S. albus J1074 and its derived strains were cultured in TSB liquid medium. Chromobacterium sp. Beijing used LB liquid medium.

[0027] The tool strains used in this invention include: E. coli DH5α and EPI300 are used for routine DNA cloning. E.coli DH10B is used to build the BAC library for FK228 BGC. E. coli BL23 or E. coli DH10B / pCB006 is used for editing large-fragment natural product BGCs (such as UK-2 BGCs). Conjugation transfer donor bacteria. E.coli ET12567 / pUZ8002 or S17-1 is used for conjugation transfer of Streptomyces. E. coli S17-1 is specifically designed for S. spinosa 301 bonding transfer, E. coli WM3064 (DAP auxotrophic type) is used for B. gladioli Conjugation transfer was performed. All *E. coli* strains were cultured in LB medium at 37°C, with appropriate antibiotics added as needed (50 μg / mL ampicillin, apramycin, chloramphenicol, gentamicin, kanamycin, or spectinomycin). *Saccharomyces cerevisiae* V6-48, used to capture UK-2 BGCs, was cultured in YPD medium (containing 20 g / L glucose, 10 g / L yeast extract, 20 g / L peptone, and 0.08 g / L adenine sulfate) at 30°C.

[0028] Example 1: Screening of multi-target integrase (MTI) and identification of core tools for MNGE

[0029] 1. Materials and Methods

[0030] The strains and plasmids used in Example 1 are shown in Table 1-2.

[0031] Table 1. Strains used in Example 1 of the present invention

[0032] Table 2. Plasmids used in Example 1

[0033] 1.1 Potential MTI Bioinformatics Analysis

[0034] First, the integration mechanisms of the four MTI systems were analyzed using a bioinformatics analysis system. It was found that they each possess unique conserved dinucleotide core sequences: MTI_1737 (GG core), MTI_2871 (TT core), MTI_6538 (AA core), and MTI_Cp36 (non-conserved core), and each targets 17, 14, 21, and 33 different natural nucleotides, respectively. attB The integration site exhibits highly degenerate sequence specificity.

[0035] 1.2 Construction of pMTI series plasmids

[0036] Subsequently, bacterial codon optimization was performed on four MTI genes, and their chemical synthesis was completed. These genes were then combined with natural... attP Cloning sites separately into those containing ermEp and stnYp In the expression frameworks of two strong promoters, a series of integration plasmids were constructed: pMTI_1737, pMTI_2871, pMTI_6538, pMTI_Cp36, and pMTI_1737-. stnYp pMTI_2871- stnYp pMTI_6538- stnYp pMTI_Cp36- stnYp .

[0037] Specifically, taking the construction of plasmid pMTI_2871 (pMTI) as an example: by using EcoR I and Hind III. The synthesized plasmid pUC57-MTI_2871 was double-digested to obtain... ermEp -MTI_2871 DNA fragment. Simultaneously, using plasmid pLC01 as a template, the pLC01 backbone was obtained by PCR amplification using primer pair pLC-skeleton-HindIII-fw / pLC-skeleton-EcoRI-rev. The two fragments were then recombined in vitro using DNA Assembly Mix Plus to generate plasmid pMTI_2871. Using pMTI as a template, the pMTI backbone was obtained by PCR using primer pair MTI-fw / pMTI-skeleton-rev. Simultaneously, using pSET- sp44 - indC and pSET- stnYp - indC Using the template, primer pairs skeleton- sp44 -fw / sp44 -MTI-rev and skeleton- stnYp -fw / stnYp -MTI-rev obtained respectively sp44 and stnYp Promoter. Subsequently, the pMTI backbone and promoter fragment were recombined in vitro to generate plasmid pMTI- sp44 and pMTI- stnYp .

[0038] Construction of other MTI system plasmids (pMTI_1737, pMTI_6538, pMTI_Cp36): Using pMTI as a template, primer pair pMTI-skeleton-fw / ermEp -rev Obtain the pMTI backbone via PCR. Simultaneously, via PCR (using primer pairs) ermEp The codon-optimized MTI_1737 gene fragment was obtained using the PCR product (-1737-fw / 1737-rev). These two PCR products were then recombined in vitro to generate plasmid pMTI_1737. Plasmids pMTI_6538 and pMTI_Cp36 were constructed using a similar strategy. stnYp Promoter-derived plasmid pMTI_1737- stnYp pMTI_6538- stnYp pMTI- stnYp Using pMTI-skeleton-fw as a template stnYp -rev obtain pMTI- via PCR stnYp Backbone; then PCR (using primer pairs) stnYp -1737-fw / 1737-rev and stnYp Gene fragments with optimized codons (-6538-fw / 6538-rev) were obtained, and finally constructed through in vitro recombination. sp44 The construction of promoter-derived plasmids is similar.

[0039] 1.3 Conjugation and transfer test: efficiency of pMTI series plasmids

[0040] Recombinant plasmids were introduced into the model Streptomyces leucis via conjugation transfer. Streptomyces albusJ1074, using the classic PhiC31 integration system as a positive control, systematically evaluated the integration efficiency, promoter activity, and host compatibility of various MTI systems. Finally, based on integration efficiency and target tolerance, the optimal system was selected for MNGE method development. The specific conjugation transfer procedure for Streptomyces is as follows: First, a single clone of E. coli containing the target plasmid was cultured overnight in LB liquid medium containing the corresponding antibiotic (ampromycin 50 mg / L), and then transferred to fresh LB medium for culture until OD. 600 The concentration reached 0.4-0.6. Simultaneously, a suspension of Streptomyces recipient spores was prepared. The donor and recipient bacteria were mixed at a 1:1 ratio and spread on the surface of MS solid medium containing 10 mM MgCl2. The medium was co-cultured at 30 °C for 16-18 hours to promote conjugation transfer. Subsequently, 1 mL of sterile aqueous solution containing nalidixic acid (to inhibit donor bacterial growth) and the corresponding plasmid-resistant antibiotic (ampromycin) was placed on the plate. The medium was then cultured at 30 °C for 5-7 days. The conjugates were verified by colony PCR (using ID-MTI-fw / ID-MTI-rev primers to identify the MTI gene). Finally, an engineered strain with the target gene integrated was obtained.

[0041] 2. Results

[0042] Experimental results showed that, among the four MTI systems, MTI_1737, MTI_2871, and MTI_6538 all successfully integrated exogenous plasmids into *Streptomyces whiteum* J1074. ermEp Integration efficiency driven by the promoter is significantly better than stnYp Promoter. This demonstrates that the three MTI systems described above can mediate exogenous DNA integration into the bacterial genome. Among them, MTI_2871 exhibits the most relaxed integration sequence specificity and the best integration efficiency, and was identified as the core tool enzyme for the MNGE method and named MTI1. The MNGE method constructed based on this method employs a "single MTI-multiple natural" approach. attB The innovative "site" strategy eliminates the need for pre-installation of artificial... attB The site is theoretically applicable to a variety of bacteria other than actinomycetes, successfully overcoming the limitations of existing MSGE (which requires multiple rounds of pre-installation). attB Overcoming the technical bottlenecks of methods such as αMSGE (limited to actinomycetes) provides a more universal technical tool for microbial genome engineering (Li et al., 2017; Li et al., 2019). The relevant sequences are as follows: the amino acid sequence of MTI1 (MTI_2871), the nucleotide sequence obtained after codon optimization, and the native sequence of the phage to which it belongs. attPThe nucleotide sequences at the sites are shown in SEQ ID NO: 1-3. MTI_1737 corresponds to SEQ ID NO: 4-6. MTI_6538 corresponds to NO: 7-9. MTI_Cp36 corresponds to SEQ ID NO: 10-12.

[0043] Example 2: Application of MNGE technology in Streptomyces for multicopy, random integration, and metabolic engineering

[0044] 1. Materials and Methods

[0045] The strains and plasmids used in Example 2 are shown in Table 3-4.

[0046] Table 3. Strains used in Example 2

[0047] Table 4. Plasmids used in Example 2

[0048] 1.1 Construction of the Indigo Reporter System and Whole Genome Sequencing

[0049] Firstly, through bioinformatics analysis, in Clostridium innocuum VE303-07 and Amedibacterium intestinale Evidence was found in the genomes of strains such as JCM30884 that the MTI system naturally mediates the integration of multiple copies of the same mobile genetic element. To construct a reporter system, [further details were provided]. XbaI Enzyme digestion of pMTI and pMTI- stnYp Obtain a linearized vector skeleton using Tn315- idgS As a template (primer pair XbaI- idgS -fw / idgS PCR amplification (-ter-rev) idgS-sfp The reporter gene cassette (4.8 kb, provided by Professor Lu Yinhua's research group at Shanghai Normal University) (Li et al., 2015) was used to obtain pMTI- through in vitro recombination. idgS Series of report plasmids; introduction of recombinant plasmids into Streptomyces hosts via conjugation transfer, using site-specific PhiC31 integration plasmid pLC01- idgS and random transposon plasmid Tn315- idgS Used as a control. Conjugates were screened on R5A solid medium containing apramycin, and samples were collected after 2 days of culture. S. albus J1074 / pMTI- idgSBacterial cells (0.1-0.2 g) were collected by centrifugation, and genomic DNA was extracted. Next-generation sequencing was performed using the DNBSEQ-T7 platform. Raw data underwent quality control using Trimmomatic v0.39 (removing bases with a quality value <20 and retaining sequences ≥75 bp). De novo assembly was performed using SPAdes v3.15 software based on the isolate model to obtain the final whole genome sequence for identification. idgS- sfp The chromosomal integration site and copy number.

[0050] 1.2 Indigo fermentation and quantification

[0051] The above-mentioned strains were inoculated into 5 mL TSB seed culture medium (13 mL shaker tubes) and cultured at 30℃ for 24-30 hours. 2 mL of the seed culture was then transferred to 50 mL R5A fermentation medium (components g / L: sucrose 100, glucose 10, yeast extract 5, MgCl2·6H2O 10.12, K2SO4 0.25, casein amino acids 0.1, MOPS 21, NaOH 2, trace element solution 2 mL, pH 6.85; trace element solution components: CaCl2 5.88 mg / L, ZnCl2 80 μg / L, FeCl3·6H2O 400 μg / L, MnCl2 20 μg / L, CuCl2 20 μg / L, Na2B4O7·10H2O 20 μg / L, (NH4)6Mo7O 24 In a 250 mL shake flask, incubate 20 μg / L of 4H₂O at 30 °C and 200 rpm for 1-5 days. Dilute 200 μL of the fermentation broth with 1.8 mL of methanol, centrifuge at 12000 rpm for 5 minutes, and measure the OD600 of the supernatant for quantification. The yield of indigo is then compared with... idgS-sfp Association analysis was performed on the chromosome integration sites and copy numbers, and yield changes were analyzed after supplementing the engineered bacteria with 5 mM L-glutamine precursor.

[0052] 1.3 Non-model industrial Streptomyces introduction test

[0053] Three MTI1 system plasmids (pMTI, pMTI-) driven by different promoters were used. sp44 pMTI- stnYp Three industrial Streptomyces strains were introduced following the above-described Streptomyces conjugation transfer steps: Streptomyces venabilis (Streptomyces venabilis) Streptomyces

[0054] venezuelae ATCC10712, chloramphenicol-producing bacteria), Streptomyces boswellia ( Streptomyces peucetius ATCC27952, epirubicin-producing strain) and avermectin ( Streptomyces avermitilisNRRL8165, (Avermectin-producing bacteria).

[0055] 2. Results

[0056] MTI1 and MTI_1737 can mediate the integration of multiple copies of the same mobile genetic elements in their original chromosome (e.g. Clostridium innocuum The presence of two identical mobile genetic elements demonstrates that this type of system naturally possesses the ability to integrate multiple targets. Figure 2 b). The MTI1 system was successfully implemented in all three model Streptomyces strains. idgS-sfp Effective integration, with integration sites randomly distributed, up to three copies ( Figure 2 c and e). Whole-genome sequencing revealed that MTI1 has a more relaxed role in Streptomyces. attB Sequence specificity (requiring only a conserved TT dinucleotide core), and the significant difference between its 5' and 3' end sequences and those of the original host due to the high GC characteristics of the host, mechanistically explain its broad applicability. Furthermore, the indigo yield of multi-copy integrated strains was significantly increased, with the double-copy engineered strain J1074 / pMTI- idgS -2-29 After supplementation with 5 mM L-glutamine, the yield reached 457.9 mg / L, compared to single-copy bacteria.

[0057] An increase of 15.7% ( Figure 3 d). Furthermore, the MTI1 system is effective against various non-model industrial Streptomyces (such as...). S. venezuelae ATCC10712 S. peucetius ATCC27952, S. avermitilis It was also successfully integrated into NRRL8165, proving its broad applicability. Figure 4 ).

[0058] Example 3: Heterogeneous production of fungicide UK-2 using the MNGE method and breaking the production record.

[0059] 1. Materials and Methods

[0060] The strains and plasmids used in Example 3 are shown in Tables 5-6.

[0061] Table 5. Strains used in Example 3

[0062] Table 6. Plasmids used in Example 3

[0063] 1.1 Whole-genome resequencing and UK-2 synthetic gene cluster capture

[0064] First, the whole genome of *Streptomyces huili* GDMCC 4.215 was resequencing using the third-generation PacBio RSII sequencing platform and the second-generation DNBSEQ-T7 platform. The assembled genome was analyzed for biosynthetic gene clusters using antiSMASH 7.1.0 software to determine the complete coding sequence of UK-2 BGC. A complete 41 kb UK-2 BGC was captured from *Streptomyces huili* GDMCC 4.215 using CRISPR / Cas9-assisted TAR cloning technology (Lee et al., 2015) and cloned into the pCAP01 vector containing the PhiC31 system to construct the plasmid. Specifically, the upstream and downstream homologous arms of the UK-2 BGC were amplified using primer pairs UK-HA-up-fw / rev and UK-HA-down-fw / rev, and then recombinated in vitro with the KpnI / SpeI linearized pCAP01 vector to construct pCAP-UK-HA. sgRNA targeting the UK-2 BGC boundary was prepared using primer pairs UK-up-sRNA-fw / UK-sgRNA-rev and UK-down-sRNA-fw / UK-sgRNA-rev. Genomic DNA digested with sgRNA / Cas9 was co-transformed into yeast VL6-48 (Leeet al., 2015) with PmeI-linearized pCAP-UK-HA and screened using SD-Trp (synthetic tryptophan-deficient agar) plates. Positive clones were verified using primer pairs ID-pCAP-UK-1-fw / rev, ID-pCAP-UK-2-fw / rev, and ID-pCAP-UK-3-fw / rev. Finally, the pCAP-UK plasmid was amplified and verified by enzyme digestion using E. coli EPI300.

[0065] 1.2 Construction of Heterologous Expression and Detection System

[0066] Subsequently, it was introduced into four Streptomyces model hosts, Streptomyces albopictus, via conjugation transfer. Streptomyces albus J1074, Streptomyces cerevisiae ( Streptomyces lividans SBT5, Streptomyces cerevisiae ( Streptomyces lividans RedStrep 1.7, Marine Streptomyces ( Streptomyces atratusZH16 was cultured and screened using five different fermentation media. Specifically, their spore suspensions were inoculated into 50 mL TSB seed medium (250 mL shake flask) and cultured at 30°C and 200 rpm for 24 hours. Take 2 mL of seed culture and transfer it to 50 mL of fermentation medium (including R5A, MS medium (soybean meal 20 g / L, mannitol 20 g / L), A3M medium (glucose 5 g / L, glycerol 20 g / L, starch 20 g / L, cottonseed meal 15 g / L, yeast extract 3 g / L, pH 7.0-7.2), GYM medium (yeast extract 4 g / L, malt extract 10 g / L, glucose 4 g / L, peptone 1 g / L, NaCl 2 g / L, pH 7.2-7.4), and ISP2 medium (yeast extract 4 g / L, malt extract 10 g / L, glucose 4 g / L)) in 250 mL shake flasks and incubate at 30℃ and 200 rpm for 2-6 days. Extract 750 μL of fermentation broth with 750 μL of ethyl acetate / petroleum ether (1:1 v / v) and centrifuge at 4000 rpm for 5 minutes. 500 μL of the supernatant was concentrated under vacuum and reconstituted with 100 μL of DMSO for HPLC analysis (Agilent 1260 series). An XBridge C18 column (4.6 × 100 mm, 5 μm) was used with a water-acetonitrile gradient elution (0 min 50% acetonitrile, 2 min 50% acetonitrile, 23 min 95% acetonitrile, 27 min 15% acetonitrile, 29 min 50% acetonitrile, 30 min 50% acetonitrile), a flow rate of 1.0 mL / min, a detection wavelength of 231 nm, and a column temperature of 30 °C. UK-2A, UK-2B, and UK-2CD were preliminarily identified by LC-MS.

[0067] 1.3 Scale-up fermentation and purification of UK-2 compounds

[0068] After confirming the optimal combination, *Streptomyces albopictus* J1074 / pCAP-UK-C1 spores were inoculated into TSB medium and cultured at 30°C and 200 rpm. The culture was then scaled up to MS fermentation medium. 4.5 L of fermentation broth was collected, extracted with ethyl acetate / petroleum ether (1:1, v / v), and concentrated to obtain a crude extract. The target component was enriched by Sephadex LH-20 column chromatography (methanol elution), followed by preparative HPLC (XBridge BEH Prep C18 column, water-acetonitrile gradient elution) to obtain UK-2A (21.8 mg).t R = 9.1 min), UK-2B (2.9 mg, t R = 10.4 min) and UK-2CD (10.5 mg, t R = 11.1 min). The purity of the product was verified by HPLC, the structure was confirmed by LC-MS, and quantitative analysis was performed based on the standard curve.

[0069] 1.4 Construction of pCAP-UK-BT1 and pCAP-UK-MTI1 plasmids

[0070] Using iCASRED (Zheng et al., 2025) gene editing technology, the PhiC31 system in pCAP-UK was replaced with either the PhiBT1 (Gregory, 2003) or MTI1 system, constructing pCAP-UK-BT1 and pCAP-UK-MTI1 plasmids, respectively. First, linear edited fragments containing different integration systems (PhiBT1 or MTI1) and the apramycin resistance gene were amplified by PCR. acc(3)IV -BT1 (primer pair) acc(3)IV -BT1-fw / acc(3)IV -BT1-rev) or acc(3)IV -MTI1 (primer pair) acc(3)IV -MTI1-fw / acc(3)IVThe pCAP-UK plasmid pCB003-C31 was constructed by in vitro recombination. The plasmid pCB003-C31 contained an sgRNA transcriptome targeting the PhiC31 gene. The vector backbone was amplified using the primer pair pCB003-skeleton-fw / pCB003-skeleton-rev, and the DNA template targeting the sgRNA was amplified using the primer pair PhiC31-sgRNA-fw / sgRNA-rev, followed by in vitro recombination as described in Example 1. Subsequently, the original pCAP-UK plasmid was electroporated into *E. coli* BL23 containing the λ-Red recombination system, and the recombination system was induced to express with arabinose to prepare competent cells. Finally, the edited plasmid pCB003-C31 and the corresponding linear edited fragment were co-electroporated into the competent cells. Through homologous recombination mediated by the λ-Red system, the original PhiC31 system in pCAP-UK was precisely replaced with the PhiBT1 or MTI1 system. Positive clones were screened on double antibiotic plates containing apramycin and spectinomycin, and then verified by colony PCR and sequencing. Finally, the resistant and integrated system-edited plasmids pCAP-UK-BT1 and pCAP-UK-MTI1 were successfully obtained.

[0071] 1.5 Comparative Analysis of UK-2 Yield Optimization Based on Traditional Genome Engineering Methods and MNGE Method

[0072] Utilizing the real and pseudo-PhiC31 systems present in the J1074 genome attB At the first round of conjugation transfer, single-copy integrated strain J1074 / pCAP-UK-C1 and double-copy integrated strain J1074 / pCAP-UK-C2 were screened using kanamycin. Utilizing alternating use of resistance and integration sites, double- or triple-copy strains with further increased copy numbers were screened using apramycin at the second round of conjugation transfer. Specifically, using the single-copy integrated strain J1074 / pCAP-UK-C1 as the starting strain, the double-copy strains J1074 / pCAP-UK-C1-B1 and J1074 / pCAP01-UK-C1-MTI1 can be obtained by introducing pCAP-UK-BT1 and pCAP-UK-MTI1; similarly, using the double-copy integrated strain J1074 / pCAP-UK-C2 as the starting strain, the triple-copy strains J1074 / pCAP-UK-C2-B1 and J1074 / pCAP01-UK-C2-MTI1 can be obtained. Fermentation and quantitative analysis were performed according to the above method.

[0073] 2. Results

[0074] This invention identified *Streptomyces albopictus* J1074 as the optimal source expression host among four screened *Streptomyces* hosts (Figure 5); based on the presence of one true and one pseudo-PhiC31- in the J1074 genome... attB Two types of conjugates were obtained: the UK-2 yield of the single-copy engineered strain J1074 / pCAP-UK-C1 was 262.1 mg / L, and the yield of the double-copy engineered strain J1074 / pCAP-UK-C2 reached 449.2 mg / L, which was 7 times that of the original strain. This indicates that J1074 is the optimal host and that increasing the copy number can effectively optimize UK-2 biosynthesis (Figures 6c and 6d).

[0075] Subsequently, the non-specific MTI1 integration system and the site-specific PhiBT1 system (as positive controls) were used to further increase the copy number of UK-2 BGC. After introducing pCAP-UK-MTI1 into J1074 / pCAP-UK-C1, 12 out of 50 conjugates showed higher yields than J1074 / pCAP-UK-C1-B1, with one strain achieving a yield of 502.7 mg / L, superior to the 438.5 mg / L of the double-copy strain. This demonstrates that the MNGE method can achieve non-specific genome integration of 41-kb UK-2 BGC and improve fermentation levels based on chromosomal position effects (Figure 7).

[0076] This experiment screened 100 conjugates and obtained 9 strains with yields exceeding those of the parents. Among them, 7 strains showed a stable increase in yield after repeated fermentation. The optimal strain, J1074 / pCAP01-UK-C2-MTI1-7, achieved a UK-2 yield of 590.3 mg / L, which was 31.4% higher than that of the two-copy parent J1074 / pCAP-UK-C2. This indicates that the non-specific integration of the MTI1 system not only promotes UK-2 biosynthesis through chromosomal position effects but also overcomes the metabolic burden caused by the introduction of multiple copies of BGC (Figure 8).

[0077] Example 4: Extending MNGE technology to difficult-to-operate Gram-positive non-pattern bacteria (Saccharomyces cerevisiae)

[0078] 1. Materials and Methods

[0079] The strains and plasmids used in Example 4 are shown in Tables 7-8.

[0080] Table 7. Strains used in Example 4

[0081] Table 8. Plasmids used in Example 4

[0082] 1.1 Analysis of the integration efficiency of the MTI1 system in *Polyspora sacchariformis* 301

[0083] Empty plasmids (pMTI, pMTI-) for testing the MTI1 system sp44 pMTI- stnYp The basic information about *Polyspora sacchariformis* 301 was introduced. The plasmid was transferred from the donor bacterium *Escherichia coli* S17-1 into the mycelium of the recipient bacterium *Polyspora sacchariformis* 301 via conjugation transfer. Similar to the conjugation transfer procedure for *Streptomyces*, the bacteria were co-cultured on 20 mM MgCl2-containing 2 CMC agar medium. Screening was performed on plates containing nalidixic acid and apramycin. Due to the slow growth of *Polyspora sacchariformis*, culturing for 12-14 days was required to obtain conjugates, which were then identified by colony PCR.

[0084] 1.2 pCL01-spi-MTI1-Δ spnK plasmid construction

[0085] Using the iCASRED seamless gene editing method (Zheng et al., 2025), plasmid pCL01-spi-int32-Δ spnK The Int32 system in the original (containing the Int32 integration system) was replaced with the MTI1 system, thereby constructing the BGC expression plasmid pCL01-spi-MTI1-Δ for random integration. spnK Specifically, using pMTI1 as a template, a linear MTI1 fragment was obtained by high-fidelity enzyme PCR amplification using primer pairs spi-MTI1-fw / spi-MTI1-rev; using pCB003 as a template, a DNA template targeting sgRNA was amplified by PCR using primer pairs spi-sgRNA-fw / sgRNA-rev; and using pCL01-spi-int32-Δ spnK Using a template, PCR amplification was performed using spi-up-fw / spi-up-rev and spi-down-fw / spi-down-rev to obtain the upstream and downstream homologous arms of the edited region. These arms were then ligated using overlapping PCR, and finally assembled with the pCB003 linear backbone to obtain the editing plasmid pCB003-spi-MTI1. This plasmid was then electroporated into a matrix containing pCL01-spi-int32-Δ spnKThe expression of the λ-Red recombinant system has been induced in *E. coli* BL23 competent cells. The MTI1 expression cassette is designed with terminals designed to interact with pCL01-spi-int32-Δ. spnK The arm sequences homologous to the Int32 system region on the plasmid are replaced with the MTI1 system through homologous recombination. The corresponding conjugates are then obtained using the method described above.

[0086] 1.3 Sponsauce J / L Fermentation Detection

[0087] The fermentation method of Polysporum oryzae and the detection method of spinosad J / L fermentation are as follows: 1 cm 2 Agar cultures were inoculated into 30 mL of BYT seed culture medium (components g / L: beef extract 1, yeast extract 5, tryptone 5, glucose 5, MgSO4 2, pH 7.4) and cultured in 250 mL shake flasks at 28℃ and 220 rpm for 3-4 days. 3 mL of the seed culture was transferred to 30 mL of fermentation medium (components g / L: yeast extract 6, corn steep liquor powder 10, oilseed meal 15, cottonseed meal 10, glucose 6, FeSO4 0.05, soybean oil 6, CaCO3 5, pH 7.4) and fermented under the same conditions for 10 days. 200 μL of the fermentation broth was mixed with 800 μL of ethanol, sonicated at room temperature for 30 minutes, and centrifuged at 12000 rpm for 10 minutes. The supernatant was analyzed by HPLC (Agilent 1260 series) using a ZORBAX Eclipse XDB-C18 column (4.6 × 10⁻⁶). The mobile phase was methanol / acetonitrile / 2% ammonium acetate (volume ratio 45:45:10), with a flow rate of 1.0 mL / min and a detection wavelength of 246 nm.

[0088] At nm, column temperature 30℃, the retention times of spinosad J and L were 6.9 min and 7.8 min, respectively.

[0089] 2. Results

[0090] All tested empty plasmids containing the MTI system (pMTI, pMTI-) sp44 pMTI- stnYp All of them can be successful

[0091] Introducing *Saccharopolysporum erythrosporum* 301, which contains a promoter derived from the same genus *Saccharopolysporum erythrosporum*. ermEp The pMTI plasmid driven by the highest conjugation efficiency showed significantly higher conjugation efficiency, indicating its superior compatibility in this host (Figure 9d). The constructed BGC expression plasmid pCL01-spi-MTI1-Δ was imported into the pMTI plasmid. spnK Subsequently, fermentation analysis of 30 conjugates revealed that 7 strains achieved spinosad J / L yields exceeding those of the previously optimal engineered strain using the site-specific Int32 system. Particularly noteworthy was the engineered strain 301 / pCL01-spi-MTI1-Δ. spnK The yield of No. 19 reached 925 mg / L, compared to 301 / pCL01-spi-int32-Δ spnK Compared with the basic strain 301 / pSI01, the improvement was significantly increased by 94.7% and 149.3%, respectively (Figures 9e and f).

[0092] Example 5: Extending the MNGE method to Gram-negative bacteria and solving the expression toxicity problem.

[0093] 1. Materials and Methods

[0094] The strains and plasmids used in Example 5 are shown in Table 9-10.

[0095] Table 9. Strains used in Example 5

[0096] Table 10. Plasmids used in Example 5

[0097] 1.1 Construction of pHZ-FK228 based on library screening

[0098] Using bacterial artificial chromosome library construction technology (Luo et al., 2003 and Xu et al., 2016), the FK228 synthetic gene cluster was cloned from the genome of Chromobacterium Beijing: genomic DNA was prepared into agarose plugs, and then... BamHI After partial enzyme digestion, the DNA fragments were separated by pulsed-field gel electrophoresis and high molecular weight DNA fragments were recovered using a Bio-Rad 422 electroelution instrument. The linearized and dephosphorylated pHZ vector was ligated with the enzyme-digested fragments, and after desalting and concentration, it was electroporated into E. coli DH10B. By screening 768 BAC clones and verifying them using primer pairs ID-depA-fw / rev and ID-depGH-fw / rev, the pHZ-FK228 plasmid containing the complete FK228 BGC was finally obtained.

[0099] 1.2 Construction and Toxicity Assessment of Weakly Inducible pMTI

[0100] In vector construction, multiple promoters were first used to drive the expression of MTI1 integrase, including strong constitutive promoters. ermEp , sp44 and stnYp and weakly inductive promoters tipAp (Thiosericin-induced) and Rhap (Rhamnose induction). Following a similar plasmid construction method as described in Example 1, using pKCCas9dO as a template, primer pair MTI- tipAp -fw / tipAp -MTI-rev obtained tipAp Promoter. Using pBBR1-Rha-Km-Redγ-BAS as a template, primer pair MTI- Rhap -fw / Rhap -MTI-rev obtained Rhap Promoter. Subsequently, the pMTI backbone was recombined in vitro with different promoter fragments to generate plasmid pMTI- tipAp and pMTI- Rhap Chromobacterium was obtained through conjugation transfer. Chromobacterium sp. Beijing and Burkholderia ( Burkholderia gladioli The ATCC10248 conjugate was used to assess the relationship between its expression level and cytotoxicity based on its growth. Specifically, the constructed plasmid was first electroporated into the donor strain *Escherichia coli* of a diaminopimelic acid auxotrophic strain. ​ In WM3064, the grown donor and recipient bacterial cultures were then mixed, and an appropriate amount of the mixed culture was spread onto LB solid medium containing 0.5 mM diaminopimelic acid and co-cultured at 37 °C for approximately 6 hours. Afterward, it was spread onto LB (Cytobacter Beijing) or CYMG (Burkholderia ATCC10248) plates containing apramycin (50 mg / L) and incubated at 30 °C to select conjugates for apramycin resistance. Finally, randomly selected resistant clones were further validated by colony PCR.

[0101] 1.3 pHZ-FK228-MTI1- ​ Build

[0102] Using the iCASRED gene editing technology described above (Zheng et al., 2025), the entire PhiC31 system in the original plasmid pHZ-FK228 (which was cloned from Chromobacterium Beijing using a BAC library to obtain FK228 BGC and has PhiC31 system integration) was replaced with a system composed of... ​ The promoter-controlled MTI1 integration system was used to construct the engineered plasmid pHZ-FK228-MTI1-. ​ The specific steps are as follows: The helper plasmid pCB006, containing the λ-Red recombination system, is transferred into a plasmid already carrying pHZ-FK228. ​ In the DH10B strain, after inducing λ-Red systemic expression with arabinose (10 mM), it contained... ​ -MTI1 expression cassette linear editing donor fragment (via pMTI- ​ Using primer MTI1- as a template ​ (High-fidelity PCR was performed using the -fw / rev fragment) and electrotransformed into competent cells. The linear fragment was designed with arms at both ends homologous to the PhiC31 system region on pHZ-FK228, precisely replacing the original PhiC31 element through homologous recombination. Finally, screening was performed on triple-antibody plates containing apramycin (50 mg / L), kanamycin (50 mg / L), and spectinomycin (50 mg / L), and colony PCR and sequencing were performed using the identification primer pair (ID-FK228-MTI1-fw / rev) to verify the correct edited plasmid pHZ-FK228-MTI1- was successfully obtained. ​ .

[0103] 1.4 Fermentation and Quantitative Analysis of FK228

[0104] After obtaining the corresponding contractitons via the above-described conjugation transfer, fermentation was performed. The fermentation products were adsorbed onto HP20 resin and extracted with methanol. Qualitative and quantitative analyses were then performed using high-performance liquid chromatography (HPLC) and high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS). An Agilent 1260 series HPLC system equipped with a Waters XBridge C18 column (4.6 × 100 mm, 5 μm) was used. Gradient elution was performed using water-acetonitrile (ACN) as the mobile phase (0–1 min: 5% ACN; 15 min: 50% ACN; 20 min: 95% ACN; hold for 5 min before returning to initial equilibrium). The column temperature was 30 °C, the flow rate was 1.0 mL / min, and the detection wavelength was 210 nm. By comparison with standards, the retention time of FK228 was approximately 20.6 min, and quantification was performed based on peak area.

[0105] 2. Results

[0106] use ​ , ​ and ​ When MTI1 was expressed using a strong promoter, the conjugates of Chromobacterium and Burkholderia were severely inhibited or unable to grow in both solid and liquid media, indicating that overexpression of MTI1 has significant toxicity to the host bacteria (Figures 10 and 11); while using a different promoter... ​ and ​ After the promoter was introduced, the toxicity issue was effectively resolved, and the conjugates grew well (Figures 11 and 12). In terms of yield, pHZ-FK228-MTI1- was successfully introduced. ​ Among the engineered bacteria with plasmids, 7 strains (14% of the total screened strains) showed an increased FK228 yield, with the best strain No. 25 reaching a yield of 7.8 mg / L, approximately 2.8 times that of the original strain (2.8 mg / L). This indicates that random integration using the MTI1 system can utilize chromosomal position effects to screen for high-yielding strains. Furthermore, control experiments revealed that engineered bacteria containing pHZ-FK228 (retaining the PhiC31 system) did not produce FK228 at all. Further analysis suggests that this is most likely due to random homologous recombination between the FK228 BGC carried on the plasmid and the endogenous BGC in the genome, leading to the destruction or loss of the gene cluster (Figure 12). This result highlights the unique advantages and reliability of the MNGE method of this invention in integrating large heterologous gene clusters.

[0107] Industrial prospects of this invention: 1. Technical Advantages: The MNGE method of this invention is based on a multi-target integrase (MTI) system, which has significant advantages such as broad host universality (covering both Gram-positive and Gram-negative bacteria), ease of operation (no need for pre-installation of specific attachment sites), and support for random integration of multiple copies and large DNA fragments. Through promoter engineering optimization, inducible weak promoters are used in non-natural hosts such as Chromobacterium and Burkholderia. ​ , ​ Precise regulation of MTI1 expression levels successfully resolved its expression toxicity issues in non-natural hosts; a strong homology promoter was used in *Saccharomyces cerevisiae*. ​ This technology drives efficient MTI1 expression, ensuring the integration efficiency of large gene clusters. Simultaneously, it can systematically screen rare clones with ultra-high expression performance due to the "chromosomal position effect" (favorable environment such as transcriptional activity and regulatory elements around the integration site) from random integration events, effectively buffering or offsetting the metabolic stress caused by multiple copies. This fundamentally solves the industry pain points of low efficiency, limited tools, and host-restricted limitations of traditional methods.

[0108] 2. Performance Indicators: The MNGE method of this invention has been proven to achieve stable integration of at least 3 copies with high integration efficiency, significantly shortening the development cycle for obtaining high-performance engineered bacteria. The MNGE method has achieved breakthroughs in the production of several important drugs: in heterologous hosts, the yield of the fungicide UK-2A was increased to 590 mg / L, the highest level reported to date; in naturally occurring production bacteria, by introducing ultra-large-scale biosynthetic gene clusters such as the 79 kb spinosad J / L BGC and the 86 kb FK228 BGC, the fermentation yields of the insecticide spinosad J / L and the antitumor drug FK228 were significantly increased. The spinosad J / L yield was 94.7% higher than the optimal strain in the traditional Int32 system and 149.3% higher than the original strain. Furthermore, this technology avoids the problem of natural gene cluster destruction caused by random homologous recombination due to the lack of corresponding integration sites during integration mediated by the traditional PhiC31 system, demonstrating its advantages in Gram-negative bacteria.

[0109] 3. Production Implementation: The MNGE method of this invention is highly compatible with existing industrial microbial breeding platforms and requires no special equipment. This technology lays a solid foundation for large-scale fermentation production by constructing genetically stable, antibiotic-free, and metabolically low-burden engineered strains, effectively reducing production costs and resulting in significant economic benefits.

[0110] In summary, the MNGE genome engineering technology platform provided by this invention has strong versatility and high efficiency, providing a powerful and universal tool for constructing microbial cell factories and efficiently producing high-value compounds through metabolic engineering. It has broad prospects for industrial application in the manufacturing fields of pharmaceuticals, pesticides, energy, and chemicals.

[0111] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A method for bacterial multi-target non-specific genome engineering, characterized in that, Includes the following steps: Step 1: The MTI integrase encoding gene, optimized with codons from the target bacteria, and the native MTI-related phage... attP The site, adaptor promoter, and target functional gene or biosynthetic gene cluster are cloned into the plasmid backbone through in vivo or in vitro recombination to form a recombinant integrative plasmid; the MTI integrase encoding gene is selected from at least one of the MTI_1737 gene, MTI_2871 gene, or MTI_6538 gene; the nucleotide sequences of the MTI_1737 gene, MTI_2871 gene, and MTI_6538 gene are shown in SEQ ID NO:4, SEQ ID NO:1, and SEQ ID NO:7, respectively; Among them, the integrase encoded by the MTI_1737 gene can recognize the GG conserved dinucleotide core sequence of the host genome; the integrase encoded by the MTI_2871 gene can recognize the TT conserved dinucleotide core sequence of the host genome; and the integrase encoded by the MTI_6538 gene can recognize the AA conserved dinucleotide core sequence of the host genome. The adapted promoter is ermEp , stnYp , sp44 , tipAp or Rhap ; The plasmid backbone is derived from pLC01, pCAP01, pCL01 or pHZ series plasmids; The codon optimization is based on the codon usage frequency table of the target bacteria; Step 2: The recombinant integrative plasmid is introduced into the target bacteria via conjugation transfer. Positive conjugates are cultured and screened on a selection medium containing apramycin to obtain engineered bacteria with multiple copies of the target functional gene or biosynthetic gene cluster randomly integrated into the host genome.

2. The bacterial multi-target non-specific genome engineering method as described in claim 1, characterized in that, In step two, the target bacteria include at least one of UK-2 actinomycetes and FK228 chromobacteria; wherein the UK-2 actinomycetes is Streptomyces huili ( Streptomyces huiliensis (The accession number is GDMCC 4.215).

3. The bacterial multi-target non-specific genome engineering method as described in claim 1, characterized in that, In step two, the engineered bacteria are *Streptomyces albopictus* J1074 / pCAP-UK-C2-MTI1-7 or *Polyspora spicata* 301 / pCL01-spi-MTI1-Δ spnK No.

19.

4. The bacterial multi-target non-specific genome engineering method as described in claim 1, characterized in that, In step one, when the target bacteria are Gram-positive bacteria, the appropriate method is to select... ermEp , stnYp or sp44 Strongly constitutive promoters; When the target bacteria are Gram-negative bacteria, select tipAp or Rhap Inducible promoters; For Gram-negative bacteria, the strong constitutive promoter can be selected in the initial testing phase.

5. The bacterial multi-target non-specific genome engineering method as described in claim 1, characterized in that, In step one, if the plasmid backbone originally carries the PhiC31 or Int32 integration system, the integration system is first precisely removed using CRISPR / Cas9 gene editing technology before being used for in vivo recombination.

6. The bacterial multi-target non-specific genome engineering method as described in claim 1, characterized in that, In step two, the conditions for conjugation transfer are: when the target bacterium is a Gram-positive bacterium, the donor bacterium is Escherichia coli S17-1 or... E. coli ET12567 / pUZ8002, the recipient bacteria are spore suspensions or mycelia, co-cultured in a solution containing 10 mM MgCl2. MS or 2CMC agar medium, co-cultured at 30°C for 18 hours, and covered with a selection plate containing 50 μg / mL apramycin and 25 μg / mL nalidixic acid to screen for conjugates; When the target bacteria are Gram-negative bacteria, the donor bacteria are Escherichia coli WM3064, which are co-cultured on LB solid medium containing 0.5 mM diaminopimelic acid at 37°C for 6 hours, and then transferred to LB medium containing 100 μg / mL apramycin.

7. The bacterial multi-target non-specific genome engineering method as described in claim 1, characterized in that, The target functional gene or biosynthetic gene cluster is idgS-sfp Reporter genes, UK-2 biosynthetic gene cluster, spinosad J / L biosynthetic gene cluster, or FK228 biosynthetic gene cluster.

8. The application of the bacterial multi-target non-specific genome engineering method according to claim 1 in bacterial genome engineering, characterized in that, Used to achieve non-specific multi-target integration of exogenous functional genes or biosynthetic gene clusters.

9. A recombinant integrative plasmid, characterized in that, Include: 1) Plasmid backbone, derived from pLC01, pCAP01, pCL01 or pHZ series plasmids, containing the origin of replication of Streptomyces / Gram-negative bacteria and a amprolium resistance selection marker; if the plasmid backbone originally carried the PhiC31 or Int32 integration system, the integration system has been removed by gene editing technology; 2) Functional expression units, consisting of adaptor promoters, MTI integrase-encoded genes, and natural... attP The site and target functional gene / biosynthetic gene cluster are sequentially tandemly arranged; wherein: the MTI integrase encoding gene is selected from one of the MTI_1737 gene, MTI_2871 gene, or MTI_6538 gene; the integrase encoded by the MTI_1737 gene can recognize the GG conserved dinucleotide core sequence of the host genome, and its corresponding natural attP The site is intrinsic to the phage to which MTI_1737 belongs. attP The two sites form a specific recognition pair; the integrase encoded by the MTI_2871 gene can recognize the TT conserved dinucleotide core sequence of the host genome, whose corresponding natural... attP The site is intrinsic to the phage to which MTI_2871 belongs. attP The two sites form a specific recognition pair; the integrase encoded by the MTI_6538 gene can recognize the AA conserved dinucleotide core sequence of the host genome, whose corresponding natural... attP The site is intrinsic to the phage to which MTI_6538 belongs. attP The two sites form a specific recognition pair; The adapted promoter is ermEp , stnYp , sp44 , tipAp or Rhap .

10. The recombinant integrative plasmid as described in claim 9, characterized in that, Default value pMTI_2871 and pMTI- sp44 、pMTI- stnYp 、pMTI- tipAp 、pMTI- Rhap 、pMTI- idgS 、pMTI- stnYp - idgS 、 、 pCAP-UK-MTI1, pCL01-spi-MTI1-Δ spnK , pHZ-FK228-MTI1- tipAp , pMTI_1737, pMTI_1737- stnYp , pMTI_6538 or pMTI_6538- stnYp .

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