A method for engineering actinomycetes for the production of purinemycin compounds

Abstract

This invention discloses a method for engineering actinomycetes for the production of puromycin compounds. The method includes: identifying the puromycin biosynthetic gene cluster (BGC) in the actinomycete genome; determining one or more engineering targets selected from modules of aminotransfer, methylation / post-modification, nucleotide treatment, dephosphorylation, acetylation / autoimmunity, transcriptional regulation, or product transport / autoimmunity based on the BGC and a metabolic network model connected to it; and performing genetic manipulation on the target to obtain engineered actinomycetes. This invention also provides specific target genes (such as arnB3, prmC_8, nudI, efpA_8, etc.) derived from the BGC of *Streptomyces HU182* and their combined modification schemes. The engineered strains constructed in this invention can effectively increase the fermentation yield of puromycin and its analogue *Puromycin E* by synergistically enhancing precursor supply, methylation modification, and product efflux, providing a systematic strain creation scheme for the green manufacturing of aminonucleoside antibiotics.

Description

Technical Field

[0001] This invention belongs to the field of actinomycete engineering modification technology, specifically, it relates to a method for engineering modification of actinomycetes for the production of purinemycin compounds. Background Technology

[0002] Puromycin is an important aminonucleoside antibiotic, its structure consisting of 3'-amino-3'-deoxyadenosine and an O-methyltyrosine residue linked by an amide bond. Puromycin acts as an analog of aminoacyl-tRNA, causing premature release of the peptide chain during protein translation, thereby effectively inhibiting protein synthesis. Based on this unique mechanism, puromycin is widely used in molecular biology and cell biology research as a selection marker (e.g., for encoding puromycin N-acetyltransferases). pac (Genes) or used to study the protein translation process.

[0003] Currently, the main methods for preparing puromycin include chemical synthesis and microbial fermentation. Chemical synthesis is complex, involving multiple protection and deprotection reactions, requiring large amounts of organic solvents, and suffers from high costs, low yields, and poor environmental compatibility, making it unsuitable for large-scale production. Microbial fermentation, due to its mild conditions, environmental friendliness, and controllable costs, has become a more ideal industrial production method. In nature, certain actinomycetes (such as...) Streptomyces alboniger It possesses the ability to biosynthesize puromycin, and its biosynthetic gene cluster (BGC) has been partially analyzed.

[0004] However, existing technologies for producing puromycin through fermentation of actinomycetes still face the following technical bottlenecks: 1. Low fermentation yield: The fermentation units of the known wild-type production strains or their preliminary mutant strains are generally low, which makes it difficult to meet the economic requirements of industrial production.

[0005] 2. Complex byproducts: The fermentation of puromycin is often accompanied by the production of various structural analogs, which increases the difficulty and cost of downstream separation and purification.

[0006] 3. Unclear genetic background and lack of systematic modification strategies: Most studies focus on the process optimization of single strains (such as culture medium, pH, and temperature), lacking a systematic understanding of the complete genome information of the production strains, the precise structure of puromycin BGC, regulatory mechanisms, and interactions with precursor metabolic networks. This results in strain modification work being highly empirical and random, lacking predictable and universally applicable rational design methods.

[0007] 4. Unclear Targets for Engineering Modification: Existing genetic engineering studies mainly focus on individual modifying enzyme genes within the BGC, without considering multiple bottleneck steps in the synthetic pathway (such as key precursor supply, cofactor balance, and product autotoxicity). In particular, the biosynthesis of puromycin not only depends on its BGC but also requires sufficient precursors (such as tyrosine, methionine / SAM, PRPP, etc.) and energy from primary metabolism. However, there are currently no reports combining BGC engineering with global metabolic network optimization.

[0008] 5. Product efflux and self-resistance mechanisms are not fully utilized: Feedback inhibition and intracellular toxicity caused by product accumulation are key factors limiting high production, but systematic engineering research on puromycin-specific efflux pumps (such as MFS family transporters) is still lacking.

[0009] In recent years, advancements in genome sequencing, bioinformatics (such as antiSMASH and KnownClusterBlast), and systems biology (such as genome-scale metabolic models, GEM) have offered new possibilities for overcoming the aforementioned challenges. By resolving the complete genome of target strains, locating their bounding genes (BGCs), and combining this with comparative genomics and orthogonal gene family analysis, it is hoped that potential engineering targets can be identified. Furthermore, constructing strain-specific GEMs and simulating the coupling relationship between precursor supply, energy metabolism, and product synthesis using methods such as flux balance analysis (FBA) holds promise for predicting modification strategies for target strains at the systemic level.

[0010] Therefore, there is an urgent need in this field to develop a systematic method for the engineering of actinomycetes based on puromycin BGC and its upstream metabolic network. This method should provide one or more clearly defined, cross-validated engineering targets and establish an operable, synergistic engineering scheme encompassing precursor supply, core synthesis modules, transcriptional regulation, and product efflux, thereby effectively increasing the fermentation yield of puromycin and its analogues, reducing byproducts, and providing a new solution for the green manufacturing of aminonucleoside antibiotics. Summary of the Invention

[0011] To address the problems existing in the prior art, this invention discloses a method for engineering actinomycetes for the production of puromycin compounds. The method includes: identifying puromycin biosynthetic gene clusters (BGCs) in the actinomycete genome; determining one or more engineering targets selected from modules such as aminotransfer, methylation / post-modification, nucleotide treatment, dephosphorylation, acetylation / autoimmunity, transcriptional regulation, or product transport / autoimmunity based on the BGCs and / or metabolic network models connected to them; and performing genetic manipulation on the targets to obtain engineered actinomycetes. This invention also provides specific target genes (such as arnB3, prmC_8, nudI, efpA8, etc.) derived from the BGCs of *Streptomyces HU182* (CGMCC 25619) and their combined engineering schemes. By integrating a multi-evidence strategy of genome mining, comparative genomics, orthogonal gene family analysis, and metabolic model throughput simulation, priority engineering targets can be screened. The engineered strain constructed in this invention can effectively increase the fermentation yield of puromycin and its analogue Puromycin E by synergistically enhancing precursor supply, methylation modification and product efflux, providing a systematic strain creation scheme for the green manufacturing of aminonucleoside antibiotics.

[0012] On one hand, the present invention provides a method for engineering the modification of actinomycetes for the production of purinemycin compounds, the method comprising the following steps: Step 1) Screening for engineered targets or combinations of engineered targets; the engineered targets include targets located in the following modules: amino transfer module; methylation / post-modification module; nucleotide treatment module; dephosphorylation module; acetylation / autoimmunity module; transcriptional regulation module; SAM methyl supply module, L-tyrosine module, PRPP nucleotide activation module, ATP energy turnover module, product transport / autoimmunity module; the combination of engineered targets includes at least two engineered targets.

[0013] Step 2) Perform genetic manipulation on the actinomycetes using the engineering target points or combinations of engineering target points to obtain the engineered actinomycetes.

[0014] This invention transforms the traditional, trial-and-error-based strain improvement process into an interpretable, predictable, and scalable rational design method based on genomic information and systems biology models. By first precisely locating the puromycin biosynthetic gene cluster (BGC) and then combining it with upstream metabolic network models (such as GEM), key bottleneck modules limiting yield (such as aminotransfer, methylation, precursor supply, or product efflux) can be identified from a global metabolic perspective and used for strain modification. This modular design allows for the combined regulation of multiple functional modules, avoiding metabolic imbalances or toxicity accumulation caused by single operations, thus making it more likely to achieve synergistic effects on products and significantly improve the construction efficiency and fermentation yield of engineered strains.

[0015] This invention, through complete genome, antiSMASH, KnownClusterBlast, comparative genome, orthogonal gene family, and KEGG precursor network analysis of actinomycetes, reveals that they possess a highly conserved puromycin BGC, and based on this, a systematic method for the engineering modification of actinomycetes can be proposed.

[0016] Complete genome sequencing and annotation of *HU182*, an actinomycete, yielded the following genomic characteristics: main chromosome approximately 7,989,773 bp; GC content approximately 71.16%; 6,970 predicted genes; 6,870 CDS; and 27 secondary metabolic BGCs identified by antiSMASH. Among these, a nucleoside BGC was identified in the ctg1:6,650,721-6,674,088 region, annotated as puromycin by antiSMASH, with a known cluster similarity of 100% (the nucleoside BGC in the ctg1:6,650,721-6,674,088 region of HU182 strain is 100% homologous to the reference gene cluster BGC0000878 in the MIBiG database, which has been verified to be responsible for puromycin biosynthesis). This BGC contains multiple modules including genes related to nucleotide processing, aminotransfer, methylation, dephosphorylation, acetylation, regulation, and transport.

[0017] Theoretically, an efficient strategy for engineering puromycin strains must simultaneously consider both the structural genes within the BGC and the precursor supply genes in the metabolic network. Therefore, this invention also analyzed the metabolic processes of actinomycetes, identifying the SAM methyl supply module, L-tyrosine module, PRPP nucleotide activation module, ATP energy turnover module, and product transport / self-resistance module as being related to puromycin synthesis. For example, L-tyrosine is a precursor to a key structural unit in the puromycin molecule and provides a cofactor for the methylation modification of puromycin; PRPP (phosphoribose-pyrophosphate) nucleotide activation provides the ribose and phosphate backbone for the nucleoside moiety of puromycin; ATP provides energy for the entire synthesis process; and the product transport / self-resistance module can enhance product efflux, alleviate product inhibition, and improve tolerance, etc.

[0018] This invention combines machine learning-assisted multi-gene knockout / weakening prediction, scoring, and experimental verification methods for prediction and analysis. It was found that the amino transfer module, methylation / post-modification module, nucleotide treatment module, dephosphorylation module, acetylation / self-resistance module, transcriptional regulation module, SAM methyl supply module, L-tyrosine module, PRPP nucleotide activation module, ATP energy turnover module, and product transport / self-resistance module in the actinomycete gene are all related to the levels of puromycin and Puromycin E produced by the actinomycete.

[0019] Machine Learning Scoring Verification: This invention employs a weakly supervised machine learning ensemble scorer (RandomForest / Gradient Boosting; training labels are constructed using real bioinformatics evidence when no available experimental label set is available) to rank candidate targets and combinations from 0 to 12. Input features include MIBiG BGC0000878 homology evidence, S. alboniger conservation, HU182-GEM virtual knockout / FSEOF-like flux evidence, OptKnock-like results, transport / regulation / precursor hierarchy, and construction risk. This score is a calculated prediction and experimental priority ranking, and does not represent experimentally verified yield increases.

[0020] Further, the target of the aminotransfer module includes the arnB_3 gene as shown in SEQ ID NO.4 or the alaA gene as shown in SEQ ID NO.1; the target of the methylation / post-modification module includes the prmC_8 gene as shown in SEQ ID NO.5 or the tcmN gene as shown in SEQ ID NO.8; the target of the nucleotide processing module includes the nudI gene as shown in SEQ ID NO.3; the target of the dephosphorylation module includes the hisN_2 gene as shown in SEQ ID NO.6; the target of the acetylation / autoimmunity module includes the mshD_10 gene as shown in SEQ ID NO.7; the target of the transcriptional regulation module includes the hdfR_6 gene as shown in SEQ ID NO.2; the target of the SAM methyl supply module includes the ctg_01102 gene as shown in SEQ ID NO.23; and the target of the product transport / autoimmunity module includes the efpA_8 gene as shown in SEQ ID NO.9.

[0021] This invention, based on complete genome annotation, antiSMASH analysis, KnownClusterBlast, comparative genomics, orthogonal gene family analysis, KEGG precursor network analysis, and wet experimental verification, precisely screened nine key targets (SEQ ID NO.1~SEQ ID NO.9) for the engineering modification of actinomycetes. These nine targets were obtained by screening for aminotransfer, methylation / post-modification, nucleotide processing, dephosphorylation, acetylation / anti-resistance, transcriptional regulation, and product transport / anti-resistance modules, respectively. Engineering actinomycetes targeting one or more of these nine targets in any combination can help increase the levels of puromycin and Puromycin E or reduce the levels of impurities.

[0022] It is understood that homologous sequences that have at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the nucleotide sequences of the nine key targets and have the same or similar functions can also achieve the effects of the present invention and are also within the scope of protection of the present invention.

[0023] Furthermore, the genetic manipulation includes any one or more of the following: enhancing target gene expression, downregulating target gene expression, or introducing mutations in the target gene; The enhanced target gene expression is achieved by using strong promoters, increasing gene copy number, or optimizing ribosome binding sites; The downregulation of target gene expression includes downregulating or knocking out the expression of the engineered target, preferably achieved through homologous recombination, CRISPR-Cas system or CRISPRi system.

[0024] Furthermore, the operation of enhancing target gene expression includes enhancing the expression of one or more genes selected from arnB_3, alaA, prmC_8, tcmN, nudI, ctg_01102, or efpA_8.

[0025] And / or the operation of downregulating target gene expression includes: knocking out or downregulating the expression of transcription regulatory factor genes hdfR_6 and / or mshD_10.

[0026] Furthermore, the engineering target combination includes the arnB_3, prmC_8, nudI, and efpA_8 genes; the genetic manipulation includes enhancing the expression of the arnB_3, prmC_8, nudI, and efpA_8 genes.

[0027] Studies have shown that enhancing the genes of these four target sites simultaneously based on actinomycete HU182 can achieve better engineering modification results.

[0028] Furthermore, the enhancement of the expression of the arnB_3, prmC_8, nudI, and efpA_8 genes is achieved by constructing a recombinant expression plasmid containing the gene coding sequence and an operational linker promoter, and then transferring the recombinant plasmid into an actinomycete host.

[0029] In some embodiments, the recombinant expression plasmid is a pSET152-derived integrative expression vector containing a four-gene CDS tandem expression insert, as shown in SEQ ID NO. 10. This expression insert is located at the multiple cloning site / expression cassette insertion site of the pSET152-derived vector and can be integrated into the HU182 genome via the φC31 attP / attB site. The four CDS in the expression insert are linked by a well-defined 10bp synthetic spacer sequence GGTACCGGCC. This spacer sequence is used to distinguish adjacent CDS and facilitates PCR / sequencing verification, and does not represent the pSET152 vector backbone sequence. Preferably, the expression plasmid contains a Streptomyces integrative vector backbone, a strong promoter, a ribosome binding site, the target gene coding sequence, a transcription terminator, and an antibiotic resistance selection marker.

[0030] Furthermore, the puromycin compounds include puromycin and its derivative Puromycin E; and / or the actinomycetes include Streptomyces strains, preferably Streptomyces sp. HU182, with accession number CGMCC25619.

[0031] The structural formulas of the puromycin and its analogue Puromycin E are shown in formula (1) and formula (2), respectively: Equation (1) Equation (2).

[0032] Our research team previously isolated a puromycin-producing actinomycete, HU182, from mountain soil in Fengming Town, Qishan County, Shaanxi Province. This strain can ferment to produce puromycin and its analogue Puromycin E (see CN116445317A). The puromycin-producing capacity is 3135 mg / L, and the puromycin E-producing capacity is 1021 mg / L. However, it also produces other byproducts along with the puromycin and Puromycin E production, making subsequent purification processes very cumbersome. Furthermore, the production levels of puromycin and Puromycin E need further improvement. Therefore, we aim to engineer this strain.

[0033] It is understood that the actinomycete modification method provided by the present invention is not only applicable to Streptomyces HU182 with accession number CGMCC25619, but can also be extended to other actinomycetes containing homologous puromycin BGC, and can be used to guide the improvement of strains with other puromycin structural analogs such as Puromycin E, and has good versatility.

[0034] On the other hand, the present invention provides an engineered actinomycete, which is a derivative strain obtained by engineering modification of actinomycetes according to the method described above; the actinomycete is a Streptomyces strain, preferably Streptomyces sp. HU182, with accession number CGMCC 25619.

[0035] In some embodiments, this invention is based on the modification of actinomycete HU182. The modified engineered actinomycete undergoes insertion, overexpression, promoter substitution, or CRISPRi at the target gene site; wherein hdfR_6 is preferably subjected to CRISPRi attenuation or promoter substitution and is not used as an unconditional knockout target. The target gene site is selected from one or more of arnB_3, prmC_8, nudI, hdfR_6, hisN_2, alaA, efpA_8, mshD_10, and tcmN.

[0036] Furthermore, the target gene loci include the arnB_3, prmC_8, nudI, and efpA_8 genes.

[0037] Furthermore, the engineered actinomycete has a corresponding edited sequence or verification fragment as shown in SEQ ID NO.11.

[0038] SEQ ID NO.11 is not used to represent the whole genome sequence of the engineered strain, but rather to characterize the four-gene expression cassette integration verification fragment.

[0039] Before modification, the four-gene tandem expression cassette was not present in the HU182 genome; after modification, the expression cassette was integrated into the HU182 genome via a pSET152-derived integration vector through the φC31 attP / attB site. SEQ ID NO.11 can be used as an inner-side validation fragment for designing PCR sequencing primers, and in conjunction with flanking primers at the vector or genome integration site, to confirm that the four-gene expression cassette has been integrated into the engineered actinomycete HU182.

[0040] In another aspect, the present invention provides a method for producing puromycin compounds, wherein the method uses engineered actinomycetes as described above to produce puromycin compounds through fermentation culture in a fermentation medium; the puromycin compounds include puromycin and its derivative Puromycin E.

[0041] Furthermore, the fermentation medium comprises: corn starch, glycerol, glucose, yeast powder, soybean powder, MgSO4·7H2O and CaCO3, with a pH of 7.2-7.6; and / or, the fermentation conditions are: inoculum size 3%-8%, temperature 27-29℃, rotation speed 180-250 r / min, and fermentation time 5-7 days.

[0042] In some embodiments, the fermentation medium comprises: 15-25 g / L corn starch, 40-60 g / L glycerol, 3-8 g / L glucose, 8-12 g / L yeast extract, 25-35 g / L soybean flour, 0.3-0.8 g / L MgSO4·7H2O, and 2-4 g / L CaCO3, with a pH of 7.2-7.6.

[0043] In another aspect, the present invention provides a method for screening biosynthetic engineering targets or combinations of engineering targets for puromycin compounds in actinomycetes, the method comprising the following steps: a) Provide the genomic information of the actinomycete to be modified, and identify functional gene clusters related to the biosynthesis of puromycin compounds based on the genomic information, and identify target genes that affect the biosynthesis of puromycin compounds from them; b) Construct a genome-scale metabolic network model of actinomycetes to identify key metabolic genes that affect the biosynthesis of puromycin compounds; c) Merge the target genes within the functional gene clusters identified in step a) with the key metabolic genes identified in step (c) to construct a candidate engineering target library. d) Based on the candidate engineering target library, select engineering targets or combinations of engineering targets to construct gene modification schemes; e) Using a computational model to score and rank the genetic modification schemes, wherein the scoring is based at least on predictions of product synthesis capacity, strain adaptability, and feasibility of genetic manipulation. f) Based on the sorting results, select engineering modification targets or combinations of engineering modification targets.

[0044] Furthermore, the genome-scale metabolic network model constructed in step b) is ensured to support the generation of spurious fluxes of ATP, cofactors, precursors, biomass, and / or target products through targeted gap filling and / or manual review.

[0045] Furthermore, in step b), pathway analysis is performed on a genome-scale metabolic network model to identify key metabolic genes that affect the biosynthesis of puromycin compounds. Furthermore, the flux analysis includes one or more of flux balance analysis, virtual knockout analysis, flux scan analysis, and / or growth coupling knockout analysis.

[0046] Further, the computational model in step e) is a machine learning model, which is selected from one or more of random forest, gradient boosting tree, XGBoost, LightGBM, support vector machine, neural network or Bayesian optimization model.

[0047] Further, the score in step e) is calculated using a comprehensive scoring function, which includes one or more of the following: product synthesis potential score, strain growth adaptability score, precursor supply score, product efflux capacity score, genetic manipulation feasibility score, and / or risk penalty item.

[0048] Furthermore, step e) also includes an active learning iteration step: feeding back the experimental verification results of the gene combination modification scheme to the machine learning model, updating the model parameters, and then re-scoring and sorting the remaining combinations.

[0049] The present invention has the following beneficial effects: 1. This invention is based on genes within the puromycin biosynthesis gene cluster and integrates multiple functional modules such as amino transfer, methylation / post-modification, nucleotide treatment, dephosphorylation, acetylation / autoresistance, transcriptional regulation, SAM methyl supply, L-tyrosine supply, PRPP nucleotide activation, ATP energy turnover and product transport / autoresistance for target screening, achieving full-level synergistic optimization from primary metabolism to secondary metabolism.

[0050] 2. Based on complete genome annotation, antiSMASH analysis, KnownClusterBlast, comparative genomics, orthogonal gene family analysis, and KEGG precursor network analysis, nine priority engineering targets were precisely screened from actinomycete HU182, providing directly operable molecular targets for increasing the production capacity of purines in actinomycetes or reducing byproducts.

[0051] 3. The preferred combination modification scheme provided by the present invention (such as simultaneously enhancing the expression of four target genes, arnB_3, prmC8, nudI and efpA_8) can synergistically enhance precursor transformation, methylation modification and product efflux, effectively relieve intracellular product inhibition, etc., significantly increase the fermentation yield of puromycin and Puromycin E compared with the starting strain, while reducing the accumulation of by-products and simplifying the subsequent separation and purification process.

[0052] 4. This invention innovatively combines the flux characteristics of metabolic models with machine learning scoring models (random forest, XGBoost, LightGBM, etc.) to comprehensively score single-gene and multi-gene combination modification schemes (considering product potential, growth adaptability, precursor supply, transport capacity, editing feasibility and risk penalty items), and achieves active learning iteration through experimental verification and result feedback, which significantly reduces the cost of trial and error and improves the success rate of engineering modification. Attached Figure Description

[0053] Figure 1 The complete genome assembly and basic characteristics of HU182 are shown in the figure below; the left figure shows the gene length overview; the middle figure shows the assembly continuity overview; and the right figure shows the GC content of the gene. Figure 2 The graph shows the collinearity of HU182 BGC and MIBiG BGC0000878 in KnownClusterBlast. This graph demonstrates a high degree of homology between the puromycin BGC in HU182 and known puromycin BGCs in terms of core gene arrangement and homology, providing evidence for subsequent engineering modifications based on this BGC.

[0054] Figure 3 The graph shows the significant hit scores of KnownClusterBlast, indicating that HU182 puromycin BGC has significant homology with known puromycin BGC.

[0055] Figure 4 Conservation of the HU182 puromycin BGC gene in orthogonal families in comparative genomes; Figure 5 Tissue structure diagram of the HU182 puromycin BGC gene; Figure 6 Data summary for building the HU182-GEM model based on KEGG / ModelSEED; Figure 7 Figure showing the results of targeted gap filling validation for the connectivity of ATP, SAM, L-tyrosine, biomass, and puromycin pseudoflux. Figure 8 A graph showing the sensitivity results of virtual knockout after targeted gap filling; Figure 9 A graph showing the throughput analysis results of overexpression candidate genes associated with enhanced puromycin synthesis; Figure 10 Evidence for the conservation and homology of key targets arnB_3, prmC_8, nudI, hdfR_6, hisN_2, alaA, and efpA_8. Detailed Implementation

[0056] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be noted that the embodiments described below are intended to facilitate the understanding of the present invention and do not limit it in any way.

[0057] Example 1: Screening of targets or combinations of targets for the engineering modification of actinomycetes This embodiment uses Streptomyces sp. HU182, with accession number CGMCC 25619, as the starting strain. Based on its complete genome information, puromycin biosynthesis gene cluster (BGC) analysis, comparative genomics, orthogonal gene family analysis, and throughput simulation of genome-scale metabolic network model (GEM), engineering targets and optimal combinations for improving the yield of puromycin and / or Puromycin E were screened.

[0058] Starting strain: Streptomyces sp. HU182, deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC 25619. This strain has been verified to produce puromycin and puromycin E through fermentation.

[0059] Genomic data: The complete genome profile of HU182 is as follows Figure 1 As shown. Based on the complete genome sequence of HU182, the following genomic characteristics were obtained: main chromosome approximately 7,989,773 bp; GC content approximately 71.16%; 6,970 predicted genes; 6,870 CDS; and 27 secondary metabolic BGCs identified by antiSMASH.

[0060] Internal target identification based on puromycin BGC 1. BGC Location and Annotation: The complete genome of HU182 was analyzed using antiSMASH software. A nucleoside antibiotic biosynthesis gene cluster was identified in the ctg1:6,650,721-6,674,088 region. AntiSMASH annotation results showed that this gene cluster is "puromycin" and has 100% similarity to the known puromycin BGC (MIBiG number BGC0000878).

[0061] 2. Collinearity and Conservation Validation: The KnownClusterBlast tool was used to compare the puromycin BGC of HU182 with BGC0000878 in the MIBiG database. The results showed that eight HU182 proteins and BGC0000878 proteins had BLAST hits, and several proteins achieved 100% identity and 100% coverage, demonstrating high collinearity between the two. Figure 2Furthermore, the first significant hit in HU182 region 19 was BGC0000878, with a cumulative BLAST score as high as 5634. Figure 3 ).

[0062] Comparative genomics and orthogonal gene family analysis were used to compare HU182 with known puromycin-producing strains such as *Streptomyces alboniger* DSM40043. The results showed that 21 of the 22 CDS in the HU182 puromycin BGC were orthogonally supported in *S. alboniger*, indicating that this BGC is highly conserved among puromycin-producing strains. Figure 4 ).

[0063] 3. Functional Module Division and Initial Target Screening: Based on the gene annotation information within the BGC, each gene is divided into different functional modules (see...). Figure 5 The study included modules for nucleotide processing, amino transfer, methylation / post-modification, dephosphorylation, acetylation / autoimmunity, transcriptional regulation, and transport. Representative genes from each module were initially screened as candidate targets. Based on BGC localization, KnownClusterBlast direct hits, S. alboniger orthogonal support, cross-strain conservation, functional modules, and annotation confidence, priority engineering targets were determined as shown in Table 1.

[0064] Table 1. Targets obtained from screening within the BGC The nucleotide sequences and corresponding protein sequences of the target genes obtained through screening are shown in Table 2.

[0065] Table 2. Nucleotide sequences and corresponding protein sequences of target genes obtained from screening within the BGC. II. Global Target Identification Based on Genome-Scale Metabolic Network Model (GEM) 1. Construction of HU182-GEM: Based on the KEGG functional annotation (EC number) of HU182, it was mapped to the public ModelSEED response database to construct a genome-scale metabolic model framework for HU182. Through targeted gap-filling, key responses related to ATP, SAM, L-tyrosine, biomass, and puromycin pseudoflux were preferentially added. After manual review, a high-quality model, HU182-GEM, suitable for designing modification schemes was finally obtained (see...). Figure 6The model includes 3,276 reactions, 3,334 metabolites, and 2,433 genes, with a gene-protein-response (GPR) association coverage of 98.84%.

[0066] 2. Flux Balance Analysis (FBA) and Target Connectivity Validation: FBA was performed on HU182-GEM under simulated HU182 fermentation medium conditions. Results showed that the model can support non-zero ATP, SAM, L-tyrosine, biomass, and puromycin pseudoflux. In the figure, the height of different modules represents the maximum achievable flux or relative flux capacity of each target metabolic module under given constraints; a higher column indicates a higher flux-carrying capacity of the module in the model, while a lower column suggests that the module may constitute a precursor supply bottleneck. These results demonstrate that HU182-GEM can be used for subsequent engineering target prediction and indicate that SAM supply, L-tyrosine supply, PRPP / nucleotide activation, and ATP turnover are priority optimization directions.

[0067] 3. Virtual Knockout Analysis (in silico): Each reaction in the model was virtually knocked out (with the upper and lower limits of reaction flux set to 0) to simulate its effect on the pseudo-flux of puromycin. Results ( Figure 8 The results showed that when key reactions in the shikimic acid-branched acid-L-tyrosine synthesis pathway (such as rxn01332, rxn02212, rxn01268, etc.) or key reactions in the SAM cycle (such as rxn00126, methionine adenosyltransferase) were knocked out, the puromycin pseudoflux was almost completely lost (relative flux approached 0). This indicates that the supply of L-tyrosine precursors and the supply of methyl donors in the SAM are absolutely dependent nodes in puromycin synthesis.

[0068] 4. Flux Scan Analysis (FSEOF-like): The pseudo-flux requirement for puromycin was progressively increased in the model, and the changing trends of flux in each metabolic reaction were observed. Reactions with a significant increase in flux with increasing target product requirement (high delta_flux value) corresponded to potential overexpression targets. Results ( Figure 9 The results showed that the fluxes of reactions such as adenosine kinase (rxn00097), methionine adenosine transferase (rxn00126), and PRPP synthase (rxn00770) increased significantly, indicating that ATP / AMP turnover, SAM synthesis, and PRPP (phosphoribosyl pyrophosphate) nucleotide activation are key nodes limiting the high production of puromycin.

[0069] The gene target information obtained by screening based on the genome-scale metabolic network model (GEM) is shown in Table 3.

[0070] Table 3. Targets obtained based on GEM screening 3. Constructing a candidate engineering modification target library and prioritizing it. 1. Merging target libraries: The targets identified from puromycin BGC in step one are merged with the key metabolic genes identified through GEM simulation in step two to construct a comprehensive candidate engineering target library that includes everything from local synthetic pathways to global metabolic networks.

[0071] 2. Multi-evidence weighted scoring: Each candidate target or module is assigned a priority score based on multiple pieces of evidence. The scoring model consists of BGC evidence, homology evidence, comparative genomic evidence, functional module evidence, GEM throughput evidence, and engineering feasibility evidence. Each piece of evidence is scored from 0 to 2: 0 points indicates no traceable evidence to support it; 1 point indicates indirect support, meaning that although the candidate gene is not directly located within the puromycin BGC, its annotation function, metabolic response, homology family, or GEM / FSEOF-like results are related to puromycin precursor supply, SAM supply, nucleotide metabolism, transport, or tolerance; 2 points indicate direct support, meaning that the candidate gene is located in or near the HU182 puromycin BGC, or has a direct homology with known puromycin biosynthesis, modification, or transport genes in MIBiG BGC0000878, or virtual knockout / FSEOF-like analysis shows that its corresponding response has a direct impact on puromycin pseudo-flux.

[0072] Table 4. Single Target Scoring Results Based on the results in the table, targets with a comprehensive score of 8 or higher are classified as high-priority engineering targets, targets with a comprehensive score of 6-7 are classified as medium-priority targets, and targets with a score below 6 are considered as alternatives or not to be validated for the time being. Therefore, arnB_3, prmC_8, nudI, hdfR_6, hisN_2, and ctg_01102 (a candidate related to methionine adenosyltransferase / SAM synthesis) are all priority targets for validation. Among them, prmC_8 is the most relevant key target for HU182 to produce puromycin compounds, followed by hdfR_6. Considering that the efflux of puromycin compounds is also very important in the synthesis and secretion process, efpA_8 is also considered a priority target for validation.

[0073] IV. Screening of Target Combinations When used for engineering modifications, multiple targets are usually selected for combined modification, and the modification effect is significantly better than that of a single target.

[0074] This embodiment designs a variety of preferred combination modification schemes as shown in Table 5, and selects the optimal synergistic modification combination based on the scoring analysis.

[0075] The scoring analysis method employs a combination of random forest, gradient boosting tree, XGBoost, LightGBM, or Bayesian optimization models as scorers. When the amount of experimental data is limited, a weakly supervised training set can be constructed using FBA / FVA virtual knockout results and prior literature data. After the first round of combined mutation validation is completed, real LC-MS / HPLC yield, cell dry weight, fermentation stability, and byproduct changes are fed back to the model to form an active learning iteration.

[0076] Multi-evidence weighted scoring: Each candidate target or module is assigned a priority score based on multiple evidence. This scoring requires the construction of a candidate target scoring model, which consists of BGC evidence, homology evidence, comparative genomic evidence, functional module evidence, GEM throughput evidence, and engineering feasibility evidence. Each piece of evidence is scored from 0 to 2: 0 points indicates no support, 1 point indicates indirect support, and 2 points indicates direct support. The total score is the sum of the scores of each item, and the weight can be increased according to the throughput analysis results. Specifically, it includes: (1) whether it is located in the puromycin BGC or its adjacent region; (2) whether it is directly homologous to MIBiG BGC0000878; (3) whether it has orthogonal family support in S. alboniger; (4) whether it is conserved in comparative genomics; (5) whether it belongs to a clearly defined biosynthetic, autoimmune, precursor supply, or transport module; and (6) whether it is critical in GEM virtual knockout or FSEOF-like analysis. The scoring results are used to determine priority engineering targets.

[0077] Table 5. Preferred Modification Scheme Based on the analysis results, compared with various different synergistic modification schemes, the multi-module synergistic modification combination of arnB_3+prmC_8+nudI+efpA_8 is particularly recommended. This combination achieves a high overall benefit in terms of core BGC reaction enhancement, nucleotide precursor processing, and product efflux, while avoiding the introduction of excessive regulatory perturbations and expression cassette burden. Although further adding targets such as hdfR_6 weakening, hisN_2, or ctg_01102 may improve some precursor or regulatory benefits, the complexity, growth burden, and genetic stability risk increase simultaneously. Therefore, it should be considered as a second priority or a stepwise validation combination.

[0078] To verify the effectiveness of the above screening strategy, subsequent embodiments will construct corresponding engineered strains targeting specific targets (such as arnB_3, prmC_8, nudI, efpA_8) and their combinations screened in this embodiment, and evaluate their actual impact on the yield of puromycin and Puromycin E through shake-flask fermentation, LC-MS / HPLC quantitative detection, and other methods.

[0079] Example 2: Validation of Machine Learning Scoring for Target Combinations This embodiment combines and modifies multiple targets obtained from screening in Example 1, representing candidate combinations as feature vectors X. These feature vectors include at least: whether the candidate gene is located in or near the puromycin BGC, changes in FBA flux of the corresponding reaction, biomass retention after knockout, changes in puromycin pseudo-flux, changes in SAM / L-tyrosine / PRPP / ATP precursor supply, transporter or resistance annotation, gene editing feasibility, historical fermentation yield, and LC-MS / HPLC peak area. The machine learning model outputs the yield increase probability P_yield and the combination priority score S_combo for the candidate combinations.

[0080] The combination priority score S_combo is normalized to a score of 0-100 and is calculated as follows: S_combo = 100 × [0.35 × P_yield + 0.20 × G_growth + 0.15 × M_precursor + 0.10 × E_export + 0.10 × F_feasibility + 0.10 × (1-R_risk)]. Where P_yield, G_growth, M_precursor, E_export, F_feasibility, and R_risk are all normalized to 0-1; the higher the R_risk, the lower the final score.

[0081] The yield increase probability P_yield is calculated using the output of a weakly supervised ensemble model and normalized by the Sigmoid function. The calculation method is: P_yield=1 / [1+exp(-(β0+β1F_flux+β2M_precursor+β3E_export+β4F_feasibility-β5R_risk))]; where β0-β5 are the model parameters obtained by fitting labels from weakly supervised training constructed from real bioinformatics evidence. P_yield is only used for priority ranking and is not equivalent to the measured yield increase rate.

[0082] G_growth is the growth retention score, calculated as follows: G_growth=min(1, biomass_retention / 0.8), where biomass_retention is the ratio of simulated biomass flux after engineering perturbation to simulated biomass flux in the wild type; when biomass_retention<0.5, regardless of the S_combo value, the combination is eliminated or significantly reduced in weight.

[0083] M_precursor is the supply score for SAM, L-tyrosine, PRPP, and ATP precursors. It is calculated as follows: M_precursor=(S_SAM+S_Tyr+S_PRPP+S_ATP) / 4. Each sub-item is assigned a value based on FSEOF-like flux change, virtual knockout sensitivity, and pathway annotation. 0 indicates no support, 0.5 indicates indirect support, and 1 indicates direct flux support.

[0084] E_export is the efflux / resistance score, calculated as follows: E_export=1 indicates that the combination contains efpA_8 or other transport / resistance modules supported by TCDB / KEGG annotations; E_export=0.5 indicates the presence of indirect transport or membrane protein annotations; E_export=0 indicates no evidence of efflux or resistance.

[0085] F_feasibility is the editing feasibility score, calculated as: F_feasibility = 1 - R_edit, where R_edit is determined by the number of genes, expression cassette length, whether essential metabolic nodes are involved, whether multi-site integration is required, and whether repetitive sequences are present. Single-gene or bigene expression enhancement is usually assigned a value of 0.8-1.0, while multi-gene combinations or perturbations of regulatory genes are assigned a value of 0.5-0.8.

[0086] R_risk is a risk score for byproduct accumulation, growth impairment, or genetic instability. It is calculated as follows: R_risk = (R_growth + R_byproduct + R_instability) / 3. R_growth is determined by the decrease in biomass_retention, R_byproduct is determined by the ratio of Puromycin E to puromycin and the risk of intermediate accumulation, and R_instability is determined by the number of expression cassettes, duplicate promoters / terminators, and vector stability.

[0087] The selection range for the combination priority score S_combo is shown in Table 6.

[0088] Table 6. Scoring range of the overall score S The modification schemes are shown in Table 7, and their scoring results are shown in Table 8, which are used to help determine the combination of priority engineering targets.

[0089] Table 7. Renovation Plan Table 8. Optimal Improvement Scheme and Scoring Results According to the combination priority scoring results in Table 8, the arnB_3+prmC_8+nudI+efpA_8 combination has the highest S_combo and is prioritized for Top-level experimental validation. While the hdfR_6 weakened / promoter replacement + arnB_3+prmC_8+nudI+hisN_2 +ctg_01102+efpA_8 combination covers more regulatory and precursor supply modules, its higher construction complexity, growth burden, and genetic stability risk result in a lower score than the four-gene core combination, making it a suitable second-priority or stepwise validation scheme. This ranking reflects a comprehensive balance of benefits, risks, and feasibility, rather than simply accumulating based on the number of targets.

[0090] Example 3: Actual effects of different modification schemes on the yield of puromycin and Puromycin E This embodiment uses a Streptomyces strain with accession number CGMCC 25619. Streptomyces Using sp.HU182 as the starting strain, multi-gene combination modified strains were constructed based on the priority engineering targets (arnB_3, prmC_8, nudI, hdfR_6, efpA_8, etc.) screened in Example 1. The actual effects of each modification scheme on the yield of puromycin and Puromycin E were verified by shake-flask fermentation and LC-MS / HPLC quantitative detection.

[0091] Starting strain and plasmid: Starting strain: Streptomyces sp. HU182 (CGMCC 25619).

[0092] Escherichia coli-Streptomyces shuttle integration expression vector pSET152 (containing attP Sites that can integrate into the Streptomyces genome attB (site).

[0093] The CRISPRi vector pCRISPomyces-2 (dCas9 expression cassette) is used for gene knockout or attenuation.

[0094] In this embodiment, the term "target CDS" refers to the HU182 source coding sequence listed in the sequence listing: alaA is SEQ ID NO:1, hdfR_6 is SEQ ID NO:2, nudI is SEQ ID NO:3, arnB_3 is SEQ ID NO:4, prmC_8 is SEQ ID NO:5, hisN_2 is SEQ ID NO:6, mshD_10 is SEQ ID NO:7, tcmN is SEQ ID NO:8, efpA_8 is SEQ ID NO:9, and ctg_01102 / metK is SEQ ID NO:23. All of the above sequences are listed in the "Sequence Listing" section of the specification and can be directly used for primer design, PCR amplification, artificial synthesis, or codon optimization synthesis.

[0095] For the key targets and multi-target combinations screened in Example 1, the integrative expression vector pSET152 was used to place the target gene in a strong promoter. ermE Under the control of p* (erythromycin promoter), specific genetic manipulation methods were designed to construct the following overexpression strains: (1) HU182-EO1: arnB_3 was overexpressed or its promoter was enhanced to strengthen the amino transfer module. Specifically, the CDS of arnB_3 (SEQ ID NO:4) was constructed as independent expression cassettes. Each expression cassette used the ermE*p promoter, Streptomyces RBS, corresponding CDS and fd terminator. The expression cassettes were tandemly linked in the direction of 5′-ermE*p-RBS-CDS-fd terminator-3′ and separated by a neutral spacer sequence of about 10 bp. The tandem insert fragment was inserted into the multiple cloning site of the pSET152-derived integrative vector through Gibson assembly, so that it could be integrated into the HU182 genome via the φC31 attP / attB site. The specific procedures were as follows: The target CDS (i.e., the nucleotide sequence shown in arnB_3 (SEQ ID NO:4)) listed in this segment was amplified using HU182 genomic DNA as a template, and a recombinant plasmid was constructed using Gibson assembly; the recombinant plasmid was transformed into the E. coli ET12567 / pUZ8002 donor strain and conjugated with HU182 spores; after conjugation, conjugates were screened using the vector-corresponding antibiotic and nalidixic acid; integration was confirmed by junction PCR, transexpression cassette PCR, and Sanger sequencing; target gene expression was detected by qRT-PCR; finally, using wild-type HU182 and empty vector control strains as controls, puromycin and puromycin E were detected by HPLC or LC-MS after batch fermentation.

[0096] (2) HU182-EO2: Overexpression or promoter enhancement of alaA to enhance the amino transfer module. Specifically, the CDS of alaA (SEQ ID NO:1) is constructed into independent expression cassettes. Each expression cassette uses the ermE*p promoter, Streptomyces RBS, corresponding CDS and fd terminator. The expression cassettes are tandemly linked in the 5′-ermE*p-RBS-CDS-fd terminator-3′ direction and separated by a neutral spacer sequence of about 10 bp. The tandem insert fragment is inserted into the multiple cloning site of the pSET152-derived integrative vector through Gibson assembly, so that it can be integrated into the HU182 genome via the φC31 attP / attB site. The specific procedures were as follows: The target CDS (i.e., the nucleotide sequence shown in alaA (SEQ ID NO:1)) listed in this segment was amplified using HU182 genomic DNA as a template, and a recombinant plasmid was constructed using Gibson assembly; the recombinant plasmid was transformed into the E. coli ET12567 / pUZ8002 donor strain and conjugated with HU182 spores; after conjugation, conjugates were screened using the vector-corresponding antibiotic and nalidixic acid; integration was confirmed by junction PCR, transexpression cassette PCR, and Sanger sequencing; target gene expression was detected by qRT-PCR; finally, using wild-type HU182 and empty vector control strains as controls, puromycin and puromycin E were detected by HPLC or LC-MS after batch fermentation.

[0097] (3) HU182-EO3: Overexpression or promoter enhancement of arnB_3 and alaA to enhance the amino transfer module. Specifically, the CDS of alaA (SEQ ID NO:1) and arnB_3 (SEQ ID NO:4) were constructed as independent expression cassettes. Each expression cassette used the ermE*p promoter, Streptomyces RBS, corresponding CDS and fd terminator. The expression cassettes were tandemly linked in the direction of 5′-ermE*p-RBS-CDS-fd terminator-3′ and separated by a neutral spacer sequence of about 10 bp. The tandem insert fragment was inserted into the multiple cloning site of the pSET152-derived integrative vector through Gibson assembly, so that it could be integrated into the HU182 genome via the φC31 attP / attB site. The specific procedure was as follows: using HU182 genomic DNA as a template, the target CDS (i.e., the nucleotide sequences shown in alaA (SEQ ID NO:1) and arnB_3 (SEQ ID NO:4)) listed in this segment were amplified, and a recombinant plasmid was constructed using Gibson assembly; the recombinant plasmid was transformed into E. coli ET12567 / pUZ8002 donor strain and conjugated with HU182 spores; after conjugation, conjugates were screened using the vector-corresponding antibiotic and nalidixic acid; integration was confirmed by junction PCR, transexpression cassette PCR, and Sanger sequencing; target gene expression was detected by qRT-PCR; finally, using wild-type HU182 and empty vector control strains as controls, puromycin and puromycin E were detected by HPLC or LC-MS after batch fermentation.

[0098] (4) HU182-EO4: prmC_8 was overexpressed and combined with the SAM supply enhancement strategy to enhance the methylation or post-modification module. The specific method is as follows: the CDS of prmC_8 (SEQ ID NO:5) were constructed into independent expression cassettes. Each expression cassette used the ermE*p promoter, streptomyces RBS, corresponding CDS and fd terminator. The expression cassettes were tandemly arranged in the 5′-ermE*p-RBS-CDS-fd terminator-3′ direction and separated by a neutral spacer sequence of about 10 bp. The tandem insert fragment was inserted into the multiple cloning site of the pSET152-derived integrative vector through Gibson assembly, so that it could be integrated into the HU182 genome via the φC31 attP / attB site. The specific procedures were as follows: The target CDS (i.e., the nucleotide sequence shown in prmC_8 (SEQ ID NO:5)) listed in this segment was amplified using HU182 genomic DNA as a template, and a recombinant plasmid was constructed using Gibson assembly; the recombinant plasmid was transformed into the E. coli ET12567 / pUZ8002 donor strain and conjugated with HU182 spores; after conjugation, conjugates were screened using the vector-corresponding antibiotic and nalidixic acid; integration was confirmed by junction PCR, transexpression cassette PCR, and Sanger sequencing; target gene expression was detected by qRT-PCR; finally, using wild-type HU182 and an empty vector control strain as controls, puromycin and puromycin E were detected by HPLC or LC-MS after batch fermentation.

[0099] (5) HU182-EO5: tcmN was overexpressed and combined with the SAM supply enhancement strategy to enhance the methylation or post-modification module. The specific method is as follows: the CDS of tcmN (SEQ ID NO:8) were constructed into independent expression cassettes. Each expression cassette used the ermE*p promoter, streptomyces RBS, corresponding CDS and fd terminator. The expression cassettes were tandemly arranged in the 5′-ermE*p-RBS-CDS-fd terminator-3′ direction and separated by a neutral spacer sequence of about 10 bp. The tandem insert fragment was inserted into the multiple cloning site of the pSET152-derived integrative vector through Gibson assembly, so that it could be integrated into the HU182 genome via the φC31 attP / attB site. The specific procedures were as follows: The target CDS (i.e., the nucleotide sequence shown in tcmN (SEQ ID NO:8)) listed in this segment was amplified using HU182 genomic DNA as a template, and a recombinant plasmid was constructed using Gibson assembly; the recombinant plasmid was transformed into the E. coli ET12567 / pUZ8002 donor strain and conjugated with HU182 spores; after conjugation, conjugates were screened using the vector-corresponding antibiotic and nalidixic acid; integration was confirmed by junction PCR, transexpression cassette PCR, and Sanger sequencing; target gene expression was detected by qRT-PCR; finally, using wild-type HU182 and an empty vector control strain as controls, puromycin and puromycin E were detected by HPLC or LC-MS after batch fermentation.

[0100] (6) HU182-EO6: prmC_8 and tcmN were overexpressed and combined with the SAM supply enhancement strategy to enhance the methylation or post-modification module. The specific method is as follows: the CDS of prmC_8 (SEQ ID NO:5) and tcmN (SEQ ID NO:8) were constructed as independent expression cassettes. Each expression cassette used the ermE*p promoter, streptomyces RBS, corresponding CDS and fd terminator. The expression cassettes were tandemly arranged in the 5′-ermE*p-RBS-CDS-fd terminator-3′ direction and separated by a neutral spacer sequence of about 10 bp. The tandem insert fragment was inserted into the multiple cloning site of the pSET152-derived integrative vector through Gibson assembly, so that it could be integrated into the HU182 genome via the φC31 attP / attB site. The specific procedures were as follows: Using HU182 genomic DNA as a template, the target CDS (i.e., the nucleotide sequences shown in prmC_8 (SEQ ID NO:5) and tcmN (SEQ ID NO:8)) listed in this segment were amplified, and a recombinant plasmid was constructed using Gibson assembly. The recombinant plasmid was then transformed into the E. coli ET12567 / pUZ8002 donor strain and conjugated with HU182 spores. After conjugation, conjugates were screened using the vector-corresponding antibiotic and nalidixic acid. Integration was confirmed by junction PCR, transexpression cassette PCR, and Sanger sequencing. Target gene expression was detected by qRT-PCR. Finally, using wild-type HU182 and an empty vector control strain as controls, puromycin and puromycin E were detected by HPLC or LC-MS after batch fermentation.

[0101] (7) HU182-EO7: The expression of nudI was optimized to enhance the nucleotide precursor treatment. The specific method was as follows: the CDS of nudI (SEQ ID NO:3) was constructed into independent expression cassettes. Each expression cassette used the ermE*p promoter, streptomyces RBS, corresponding CDS and fd terminator. The expression cassettes were tandemly arranged in the direction of 5′-ermE*p-RBS-CDS-fd terminator-3′ and separated by a neutral spacer sequence of about 10 bp. The tandem insert fragment was inserted into the multiple cloning site of the pSET152-derived integrative vector through Gibson assembly, so that it could be integrated into the HU182 genome via the φC31 attP / attB site. The specific procedure was as follows: using HU182 genomic DNA as a template, the target CDS (i.e., the nucleotide sequence shown in nudI (SEQ ID NO:3)) listed in this segment was amplified, and a recombinant plasmid was constructed using Gibson assembly; the recombinant plasmid was transformed into E. coli ET12567 / pUZ8002 donor strain and conjugated with HU182 spores; after conjugation, conjugates were screened using the vector-corresponding antibiotic and nalidixic acid; integration was confirmed by junction PCR, transexpression cassette PCR, and Sanger sequencing; target gene expression was detected by qRT-PCR; finally, using wild-type HU182 and empty vector control strains as controls, puromycin and puromycin E were detected by HPLC or LC-MS after batch fermentation.

[0102] (8) HU182-EO8: HisN_2 was overexpressed to regulate the dephosphorylation reaction. The specific method was as follows: HisN_2 (SEQ ID NO:6) CDS was constructed into independent expression cassettes. Each expression cassette used the ermE*p promoter, Streptomyces RBS, corresponding CDS and fd terminator. The expression cassettes were tandemly linked in the direction of 5′-ermE*p-RBS-CDS-fd terminator-3′ and separated by a neutral spacer sequence of about 10 bp. The tandem insert fragment was inserted into the multiple cloning site of the pSET152-derived integrative vector through Gibson assembly, so that it could be integrated into the HU182 genome via the φC31 attP / attB site. The specific procedure was as follows: using HU182 genomic DNA as a template, the target CDS (i.e., the nucleotide sequence shown in hisN_2 (SEQ ID NO:6)) listed in this segment was amplified, and a recombinant plasmid was constructed using Gibson assembly; the recombinant plasmid was transformed into E. coli ET12567 / pUZ8002 donor strain and conjugated with HU182 spores; after conjugation, conjugates were screened using the vector-corresponding antibiotic and nalidixic acid; integration was confirmed by junction PCR, transexpression cassette PCR, and Sanger sequencing; target gene expression was detected by qRT-PCR; finally, using wild-type HU182 and empty vector control strains as controls, puromycin and puromycin E were detected by HPLC or LC-MS after batch fermentation.

[0103] (9) HU182-EO9: Overexpression of ctg_01102 (metK, S-adenosylmethionine synthase) was performed to enhance the supply of SAM methyl donors. The specific method was as follows: the CDS of ctg_01102 (SEQ ID NO:23) was constructed into independent expression cassettes. Each expression cassette used the ermE*p promoter, Streptomyces RBS, corresponding CDS and fd terminator. The expression cassettes were tandemly arranged in the 5′-ermE*p-RBS-CDS-fd terminator-3′ direction and separated by a neutral spacer sequence of about 10 bp. The tandem insert fragment was inserted into the multiple cloning site of the pSET152-derived integrative vector through Gibson assembly, so that it could be integrated into the HU182 genome via the φC31attP / attB site. The specific procedures were as follows: The target CDS (i.e., the nucleotide sequence shown in ctg_01102 (SEQ ID NO:23)) listed in this segment was amplified using HU182 genomic DNA as a template, and a recombinant plasmid was constructed using Gibson assembly; the recombinant plasmid was transformed into E. coli ET12567 / pUZ8002 donor strain and conjugated with HU182 spores; after conjugation, conjugates were screened using the vector-corresponding antibiotic and nalidixic acid; integration was confirmed by junction PCR, transexpression cassette PCR, and Sanger sequencing; target gene expression was detected by qRT-PCR; finally, puromycin and puromycin E were detected by HPLC or LC-MS after batch fermentation, using wild-type HU182 and empty vector control strains as controls.

[0104] (10) HU182-EO10: hdfR_6 is weakened by CRISPRi or the promoter is replaced to regulate the expression of the entire puromycin BGC. The specific method is as follows: hdfR_6 (SEQ ID NO:2) is weakened by CRISPRi or the promoter is replaced, and unconditional strong knockout is not used; the CRISPRi target is selected from the unique 20 nt spacer of the 5′ coding region of hdfR_6, or the natural promoter is replaced with a weak promoter by approximately 800-1500 bp homologous arms upstream and downstream of hdfR_6. The specific procedure is as follows: A CRISPRi spacer was designed based on hdfR_6 (SEQ ID NO:2), or the hdfR_6 promoter was amplified using HU182 genomic DNA as a template to replace the required upstream and downstream homologous arms. Recombinant plasmids were constructed using Gibson assembly. The recombinant plasmid was then transformed into the E. coli ET12567 / pUZ8002 donor strain and conjugated with HU182 spores. After conjugation, conjugates were screened using the vector-corresponding antibiotic and nalidixic acid. Integration was confirmed by junction PCR, transexpression cassette PCR, and Sanger sequencing. Target gene expression was detected by qRT-PCR. Finally, using wild-type HU182 and an empty vector control strain as controls, puromycin and puromycin E were detected by HPLC or LC-MS after batch fermentation.

[0105] (11) HU182-EO11: efpA_8 was overexpressed to enhance puromycin efflux and strain tolerance. The specific method was as follows: the CDS of efpA_8 (SEQ ID NO:9) was constructed into independent expression cassettes. Each expression cassette used the ermE*p promoter, streptomycin RBS, corresponding CDS and fd terminator. The expression cassettes were tandemly arranged in the 5′-ermE*p-RBS-CDS-fdterminator-3′ direction and separated by a neutral spacer sequence of about 10 bp. The tandem insert fragment was inserted into the multiple cloning site of the pSET152-derived integrative vector through Gibson assembly, so that it could be integrated into the HU182 genome via the φC31 attP / attB site. The specific procedures were as follows: The target CDS (i.e., the nucleotide sequence shown in efpA_8 (SEQ ID NO:9)) listed in this segment was amplified using HU182 genomic DNA as a template, and a recombinant plasmid was constructed using Gibson assembly; the recombinant plasmid was transformed into the E. coli ET12567 / pUZ8002 donor strain and conjugated with HU182 spores; after conjugation, conjugates were screened using the vector-corresponding antibiotic and nalidixic acid; integration was confirmed by junction PCR, transexpression cassette PCR, and Sanger sequencing; target gene expression was detected by qRT-PCR; finally, using wild-type HU182 and empty vector control strains as controls, puromycin and puromycin E were detected by HPLC or LC-MS after batch fermentation.

[0106] (12) HU182-EO12: arnB_3, prmC_8 and nudI were overexpressed to enhance the expression of the three genes in the core synthesis module. The specific method was as follows: the CDS of arnB_3 (SEQ ID NO:4), prmC_8 (SEQ ID NO:5) and nudI (SEQ ID NO:3) were constructed as independent expression cassettes. Each expression cassette used the ermE*p promoter, streptomyces RBS, corresponding CDS and fd terminator. The expression cassettes were tandemly arranged in the direction of 5′-ermE*p-RBS-CDS-fd terminator-3′ and separated by a neutral spacer sequence of about 10 bp. The tandem insert fragment was inserted into the multiple cloning site of the pSET152-derived integrative vector through Gibson assembly, so that it could be integrated into the HU182 genome via the φC31 attP / attB site. The specific procedure was as follows: using HU182 genomic DNA as a template, the target CDS (i.e., the nucleotide sequences shown in arnB_3 (SEQ ID NO:4), prmC_8 (SEQ ID NO:5), and nudI (SEQ ID NO:3)) listed in this segment were amplified, and a recombinant plasmid was constructed using Gibson assembly; the recombinant plasmid was transformed into E. coli ET12567 / pUZ8002 donor bacteria and conjugated with HU182 spores; after conjugation, conjugates were screened using the vector-corresponding antibiotic and nalidixic acid; integration was confirmed by junction PCR, transexpression cassette PCR, and Sanger sequencing; target gene expression was detected by qRT-PCR; finally, using wild-type HU182 and empty vector control strains as controls, puromycin and puromycin E were detected by HPLC or LC-MS after batch fermentation.

[0107] (13) HU182-EO13: hdfR_6 was weakened by CRISPRi or the promoter was replaced, and efpA_8 was overexpressed to regulate the dephosphorylation reaction and simultaneously enhance the puromycin efflux capacity and strain tolerance. The specific method is as follows: the CDS of efpA_8 (SEQ ID NO:9) was constructed into independent expression cassettes. Each expression cassette used the ermE*p promoter, streptomyces RBS, corresponding CDS and fd terminator. The expression cassettes were tandemly arranged in the 5′-ermE*p-RBS-CDS-fd terminator-3′ direction and separated by a neutral spacer sequence of about 10 bp. The tandem insertion fragment was inserted into the multiple cloning site of the pSET152-derived integrative vector through Gibson assembly, so that it could be integrated into the HU182 genome via the φC31 attP / attB site. For hdfR_6 (SEQ ID NO:2), CRISPRi weakening or promoter replacement is used instead of unconditional strong knockout. The CRISPRi target is selected from the unique 20 nt spacer of the 5′ coding region of hdfR_6, or the natural promoter is replaced with a weak promoter by using approximately 800-1500 bp homologous arms upstream and downstream of hdfR_6. The specific procedures were as follows: The CDS (nucleotide sequence of efpA_8 (SEQ ID NO:9)) of the expression enhancement gene was amplified using HU182 genomic DNA as a template. A CRISPRi spacer was designed based on hdfR_6 (SEQ ID NO:2), or the hdfR_6 promoter was amplified to replace the required upstream and downstream homologous arms. A recombinant plasmid was constructed using Gibson assembly. The recombinant plasmid was transformed into the E. coli ET12567 / pUZ8002 donor strain and conjugated with HU182 spores. After conjugation, conjugates were screened using the vector-corresponding antibiotic and nalidixic acid. Integration was confirmed by junction PCR, transexpression cassette PCR, and Sanger sequencing. Target gene expression was detected by qRT-PCR. Finally, using wild-type HU182 and an empty vector control strain as controls, puromycin and puromycin E were detected by HPLC or LC-MS after batch fermentation.

[0108] (14) HU182-EO14: Overexpression of arnB_3, prmC_8, nudI and efpA_8 was carried out by the following method: the CDS of arnB_3 (SEQ ID NO:4), prmC_8 (SEQ ID NO:5), nudI (SEQ ID NO:3) and efpA_8 (SEQ ID NO:9) were constructed as independent expression cassettes. Each expression cassette used the ermE*p promoter, streptomycin RBS, corresponding CDS and fd terminator. The expression cassettes were tandemly arranged in the 5′-ermE*p-RBS-CDS-fd terminator-3′ direction and separated by a neutral spacer sequence of about 10 bp. The tandem insert fragment was inserted into the multiple cloning site of the pSET152-derived integrative vector through Gibson assembly, so that it could be integrated into the HU182 genome via the φC31 attP / attB site. The specific procedure was as follows: Using HU182 genomic DNA as a template, the target CDS (i.e., the nucleotide sequences shown in arnB_3 (SEQ ID NO:4), prmC_8 (SEQ ID NO:5), nudI (SEQ ID NO:3), and efpA_8 (SEQ ID NO:9)) were amplified, and a recombinant plasmid was constructed using Gibson assembly. The recombinant plasmid was then transformed into the E. coli ET12567 / pUZ8002 donor strain and conjugated with HU182 spores. After conjugation, conjugates were screened using the vector-corresponding antibiotic and nalidixic acid. Integration was confirmed by junction PCR, transexpression cassette PCR, and Sanger sequencing. Target gene expression was detected by qRT-PCR. Finally, using wild-type HU182 and an empty vector control strain as controls, puromycin and puromycin E were detected by HPLC or LC-MS after batch fermentation.

[0109] (15) HU182-EO15: Overexpression of arnB_3, prmC_8, nudI, hisN_2, and efpA_8, and CRISPRi weakening or promoter replacement of hdfR_6. The specific method is as follows: the CDS of arnB_3 (SEQ ID NO:4), prmC_8 (SEQ ID NO:5), nudI (SEQ ID NO:3), hisN_2 (SEQ ID NO:6), and efpA_8 (SEQ ID NO:9) are constructed as independent expression cassettes. Each expression cassette uses the ermE*p promoter, Streptomyces RBS, corresponding CDS, and fd terminator. The expression cassettes are tandemly arranged in the 5′-ermE*p-RBS-CDS-fd terminator-3′ direction and separated by a neutral spacer sequence of about 10 bp. The tandem insert fragment is inserted into the multiple cloning site of the pSET152-derived integrative vector through Gibson assembly, so that it can be inserted via φC31 The attP / attB sites were integrated into the HU182 genome. hdfR_6 (SEQ ID NO:2) was weakened using CRISPRi or promoter replacement, without unconditional strong knockout. CRISPRi targets were selected from the uniquely matching 20 nt spacer in the 5′ coding region of hdfR_6, or by replacing the natural promoter with a weak promoter using approximately 800-1500 bp homologous arms upstream and downstream of hdfR_6. The specific procedure is as follows: Using HU182 genomic DNA as a template, amplify the CDS (i.e., the nucleotide sequences shown in arnB_3 (SEQ ID NO:4), prmC_8 (SEQ ID NO:5), nudI (SEQ ID NO:3), hisN_2 (SEQ ID NO:6), and efpA_8 (SEQ ID NO:9)) of the expression enhancement gene. Based on hdfR_6 (SEQ ID NO:2), design a CRISPRi spacer or amplify the hdfR_6 promoter to replace the required upstream and downstream homologous arms. Construct a recombinant plasmid using Gibson assembly. Transform the recombinant plasmid into *E. coli* ET12567 / pUZ8002 donor bacteria and perform conjugation transfer with HU182 spores. After conjugation, select conjugates using the vector-specific antibiotic resistance and nalidixic acid. The conjugates are then subjected to junction. Integration was confirmed by PCR, transexpression cassette PCR, and Sanger sequencing; target gene expression was detected by qRT-PCR; and puromycin and puromycin E were detected by HPLC or LC-MS after fermentation in the same batch, using wild-type HU182 and empty vector control strains as controls.

[0110] (16) HU182-EO16: Overexpression of alaA, arnB_3, prmC_8, nudI, ctg_01102, and efpA_8 was performed, and hdfR_6 was weakened by CRISPRi or its promoter was replaced. Specifically, the CDS of alaA (SEQ ID NO:1), arnB_3 (SEQ ID NO:4), prmC_8 (SEQ ID NO:5), nudI (SEQ ID NO:3), efpA_8 (SEQ ID NO:9), and ctg_01102 (SEQ ID NO:23) were constructed as independent expression cassettes. Each expression cassette used the ermE*p promoter, Streptomyces RBS, corresponding CDS, and fd terminator. The expression cassettes were tandemly linked in the direction of 5′-ermE*p-RBS-CDS-fd terminator-3′ and connected at approximately 10 The tandem insert fragments are separated by neutral spacer sequences; the tandem insert fragments are inserted into the multiple cloning site of the pSET152-derived integrative vector via Gibson assembly, allowing them to integrate into the HU182 genome via the φC31 attP / attB site. hdfR_6 (SEQ ID NO:2) is weakened using CRISPRi or promoter replacement, without unconditional strong knockout; the CRISPRi target site is selected from the uniquely matching 20 nt spacer of the hdfR_6 5′ coding region, or the natural promoter is replaced with a weak promoter by replacing approximately 800-1500 bp homologous arms upstream and downstream of hdfR_6. The specific procedure is as follows: using HU182 genomic DNA as a template, amplify the CDS of the expression enhancement gene (i.e., the nucleotide sequences shown in alaA (SEQ ID NO:1), arnB_3 (SEQ ID NO:4), prmC_8 (SEQ ID NO:5), nudI (SEQ ID NO:3), efpA_8 (SEQ ID NO:9), ctg_01102 (SEQ ID NO:23)). Based on hdfR_6 (SEQ ID NO:2), design a CRISPRi spacer or amplify the hdfR_6 promoter to replace the required upstream and downstream homologous arms. Use Gibson assembly to construct the recombinant plasmid; then transform the recombinant plasmid into E. coli. The ET12567 / pUZ8002 donor strain was conjugated with HU182 spores for transfer. After conjugation, conjugates were screened using the vector-specific antibiotic and nalidixic acid. Integration was confirmed by junction PCR, transexpression cassette PCR, and Sanger sequencing. Target gene expression was detected by qRT-PCR. Finally, using wild-type HU182 and an empty vector control strain as controls, puromycin and puromycin E were detected by HPLC or LC-MS after batch fermentation.

[0111] The starting strain HU182 and 16 modified strains (HU182-EO1~HU182-EO16) were subjected to shake-flask fermentation. All engineered strains were compared with wild-type HU182 under the same batch culture conditions, and at least three biological replicates were performed.

[0112] The fermentation method is as follows: Slant culture: 4.0g yeast extract, 10.0g malt extract, 4.0g glucose, 0.005g CoCl2·6H2O, 18.0g agar, 1000ml distilled water, pH 7.0-7.2, sterilized at 121℃ for 20min, and incubated at 28℃ for 7 days after inoculation.

[0113] Seed culture medium (g / L): glucose 8, corn starch 10, glycerol 20, yeast powder 7.5, soybean flour 20, CaCO3 2, pH 7.2. Wash the Streptomyces spores from the slant agar with 10–15 ml of sterile water to prepare a spore suspension with a concentration of 1×10⁶–1×10⁹ spores / ml. Add 1 ml of spore suspension to each bottle, place on a shaker, and incubate at 200–250 rpm for 48 h at 28°C.

[0114] Fermentation medium (g / L): corn starch 20, glycerol 50, glucose 5, yeast powder 10, soybean flour 30, MgSO4·7H2O 0.5, CaCO3 3, pH 7.4, prepared with distilled water, sterilized at 121℃ for 20 min. Inoculate the seed culture at a rate of 5% into a 1L fermenter (250L capacity), and culture at 28℃ with a rotation speed of 150-250 r / min for 8 days. Purinemycin compounds are detected by HPLC. Detection method: Sample preparation: The fermentation broth was first centrifuged (8,000 rpm, 10 min), and the supernatant was filtered through a 0.22 μm filter membrane for the determination of extracellular puromycin compounds. The precipitated mycelium was extracted by ultrasonic disruption with an equal volume of methanol for 30 min, followed by centrifugation to collect the supernatant. The precipitate was then extracted once more with 5 mL of the same solvent, and the supernatants from both extractions were combined. The supernatant was concentrated to dryness under nitrogen at 40 °C, redissolved with 1 mL of the initial HPLC mobile phase, filtered through a 0.22 μm filter membrane, and the intracellular puromycin compounds were determined by HPLC / LC-MS.

[0115] Total puromycin content = extracellular puromycin content + intracellular puromycin content.

[0116] Total Puromycin E content = Extracellular Puromycin E content + Intracellular Puromycin E content.

[0117] Potency = Total puromycin content + Total puromycin E content.

[0118] The chromatographic column was a C18 column, 5 μm, 4.6 × 250 mm, with a column temperature of 25℃; mobile phase A: water containing 5 mM ammonium acetate; mobile phase B: 100% acetonitrile; gradient program: 0–30 min: 5%–100% (V / V) phase B; 30–33 min: 100% phase B; 33–34 min: 100%–5% (V / V) phase B; 34–40 min: 5% phase B; flow rate: 1 mL / min; detection wavelength: 254 nm; injection volume: 10–20 μL. LC-MS conditions: electrospray ionization (ESI), positive ion mode, scan range m / z 100–800.

[0119] Standards: Puromycin was purchased from Sigma (P8833), and Puromycin E was previously isolated, purified, and structurally identified in our laboratory (see CN116445317A for details).

[0120] The test results on day 8 are shown in Table 9.

[0121] Table 9. Comparison of Puromycin and Puromycin E yields of different engineered bacteria As shown in Table 9, overexpression of alaA, arnB_3, prmC_8, tcmN, nudI, and hisN_2, as well as attenuated expression of hdfR_6, can significantly increase the yield of puromycin compounds and reduce byproducts. arnB_3 The optimal effect was observed. Overexpression of efpA_8 increased the extracellular / intracellular product ratio from 1.8 to 3.2, confirming its effective enhancement of puromycin efflux capacity; however, efpA_8 alone had limited impact on overall yield. ctg_01102 (metK), as a target for SAM supply enhancement, should be moderately overexpressed using a gradient promoter; if overexpression leads to growth burden or a decrease in product ratio, the promoter strength should be reduced or it should be combined with the expression of the core BGC gene for balance.

[0122] All combined modified strains showed significantly higher yields than single-gene modified strains, confirming the advantages of the multi-module synergistic modification strategy. Among them, the HU182-EO14 four-gene synergistic combination showed the best modification effect: the puromycin yield reached 5132 mg / L, which is 1.64 times that of the wild type; the puromycin E yield reached 1411 mg / L, which is 1.38 times that of the wild type; and it effectively enhanced the efflux capacity of puromycin and reduced by-products.

[0123] To further verify the fermentation performance of strain HU182-EO14 (preferred combination), this example also compared its fermentation time progression with that of the wild type. It was found that regarding puromycin accumulation: wild-type HU182 reached a peak (3137 mg / L) on day 7, and then stabilized; HU182-EO14 showed rapid and continuous accumulation from days 4 to 8, reaching a peak (5132 mg / L) on day 8. The trend regarding puromycin E accumulation was similar to that of puromycin.

[0124] This embodiment also conducted tolerance tests on the HU182-EO14 strain (preferred combination) and the wild-type HU182: different concentrations of puromycin (0, 50, 100, 200 mg / L) were added exogenously to the fermentation medium. The growth inhibition rate (OD reduction) of the HU182-EO14 strain at 100 mg / L was 20%, significantly lower than the 45% of the wild-type, indicating that... efpA_8 Overexpression did indeed enhance the strain's tolerance to puromycin.

[0125] To verify the modification effect, total RNA was extracted from fermentation samples of strain C5 and wild-type on day 3, and the transcription levels of the target gene and other genes within the BGC were detected by RT-qPCR. The results showed that the transcription levels of arnB_3, prmC_8, nudI, and efpA_8 in strain C5 were 8.3-fold, 7.6-fold, 9.1-fold, and 12.4-fold higher than those in the wild-type, respectively, confirming successful overexpression. The transcription levels of other genes within the BGC that were not directly modified (such as tcmN and hisN_2) also showed a 1.5-2.5-fold increase, which is speculated to be related to changes in metabolic flux or feedback regulation.

[0126] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

[0127] sequence list SEQ ID NO:1 alaA nucleotide sequence SEQ ID NO:2 hdfR_6 nucleotide sequence GTGATTGTGGATCTGGACCTGGCGCTGGTGCGATCGTTCGCGATCACCGCGGAAGAGCTGCACTTCCGGCGCGCGGCCGAGCGGCTCTTCATCACGCAGCAGGCGCTGTCCCAGCGGATCCGGCGCCTCGAACTGCTGCTGGGCGTCTCGCTCTTCGTCCGCGGCAACCGCAGCGTCGCGCTCACCGAGGAGGGGCAGCGGTTCCTCGCGCCGGCCCGCCGGGTCATGGAGGCCGCCGACGCGGCCGTCCAGGCCGTCCAGCCCCGTGAGCGCCGCCCGCTGCGGGTCGACGTGGTCGACGGACGGCTCGCCCCGCTCCAACTGGTCCGCAAGCTCCTTGAGGAGGACCCCGAGCTGCCGCTGAACCTGAGCATGCGGCAGTCGCTCGCCGCCGCGCTGCCCGCGCTGGCCAGGGACGAGATCGACACGGCGTTCGGCCGGGTCCATGACGTGCAGGGCTGGTCGGCGGAGTTCTCGCACCGCCTCGTGCGCCTCGAACCGCTGCGAGCGCTGGTGCCGCGCACCCACCCGCTGGCCGGCCGCGACGAGGTGCGGATCGATGAGCTGCGGAGCTGCGGCATCTGGCAGCCGCGCAACGGAGTGGCGTCGGAGTGGGAGCACTATGTGCGCCGCCTGGCCGACTCCTTCGGTCTCTCGCTCGCCTTCTCGGGCGCGGCACTGAGCGATGAGCACTTCCTGGAACACCTGTGGAGCCACGGCGAGATGGCGTGCCTGGCCGGGGCGGACGTCTCCTACGCCTTCTCGCCGGACATCAGGAGCATCCCGCTCGTCGACCCCACGCCGGTCTACCCGTGGTCGATGGTGTGGCGTCGGCAGGCGGGCCATCCGCTCGTCGACCGGCTGGTCGGTCTGGCCAAGCGGTCGGCCGGAGACTGGCTGACCCACGTGCCCGGGGAGATCTGGCTGCCGGCGCCCGACCGGGCCCTGCTGCGCGGCGCGGGGGTCGACGTGGACGCCGTCCGGGGCTGA SEQ ID NO:3 nudI nucleotide sequence ATGGCTGACGGGACCGCGCCCAAGCTGGTGGTGGGTGCGCTCATCAGCGATCCGCTCGGCCGGATCTTCGTACAGCGCCGCTCGGCCGACCGGCGCCTGTTCCCCGCATGCTGGGACGTCGTGGGCGGCGCCGTGGAGGACGGCGAGACCCACCACGAGGCACTGGCCCGGGAGATCGCCGAGGAGACCGGCTGGCGGCTGCGCCGGGTGCTGGCCCGGGTGGTGCACGAGAGCTGGACGGCGGACGGCGTACGGCACCTCGAGTCGGACTACGTCGTCGAGGTGGACGGGGACCTGTCGTCACCCACGCTCGAACGGGACAAGCACCCGGAGTTCGCGTGGGTGGGGCCGCAGGACATCTCCCTGCTCGACGAGAACGTCCGGCGCGGCGCCGGCACCTTCATCAAGGACACGGTCGCCGCCGGACACGACTGGCTCGCCGGATCCACGCGCCGCTGA SEQ ID NO:4 arnB_3 nucleotide sequence SEQ ID NO:5 prmC_8 nucleotide sequence ATGACTGTCAAGGAATCCCCCGAGGCCGCCGCCAAGGTCCTCGAGCACGTCATGACCGGGGACGACCTGCCGGACGACTTCTCCCTGCTGGGCCGCGAGTGGACCCTGCACCGCGGCGTCTTCCCCGGCACCCTGACCGCCGCCACCGAGGTCATGGCCTCGATCGTGCCCTATCCCAAGGGCGGCTCCTTCCTGGAGGTCGGCTGCGGCACCGGCGTCATCTCCGTGACCGCGGCGCTCCACGGCAGCGACGTCACGGCGCTCGACATCAACGAGAAGGCGGTGGCGAACACCCTCGCCAATGCCGAGCGGCACGGCGTCGAGGACCGGGTGCGGGTGCTGCACAGCGACCTGTACGCGGCGCTCGAACCCACCGACCGGTTCGACGCGATCTTCTGGAACGTGCCCTGGACGTACGTGGAGGACGGCTTCCCGCTCTCCAGCGACCTGCACTCCGCGGTCTTCGACCCCGGCTACCAGGGCCAGTCCCGGTTCATCTCCGGGGCCCGCGACCACCTGGCCGACGGCGGCCGGCTGCTGATCGGCACGGCCGATCTGGGAGACCGCGAGCGGATCGACGCCTTCGCCGCCGAGGCCGGGATGACCGTCGAGATGCTGCGCCGCGTACGCCGTATCGAGGTCGTCCGGGTCATGGAGTACCACCTGCTCGAACTGCGCGCCAAGTGA SEQ ID NO:6 hisN_2 nucleotide sequence ATGCCACAGCAGGCCGACCTGGAACTGGCCATGCGACTCGCCGACACCGCCGACCGCATCACCACCCGCCGCTTCCAGGCGCGCGACCTGCGGATACGCGAGAAGCCCGACCGCACCCCCGTCACCGACGCGGACACCACCGTCGAGGCGGCCGTACGCGAGTCCGTGCGGGCCGCCCGGCCCGACGACGACTTCGCGGGAGAGGAGACCGGCGGCGAGGTGACCGCAGGACGCACCTGGATCGTCGACCCCATCGACGGCACCAAGAACTTCCTGCGCGGAGTGCCGGTCTGGGCCACGCTGATCGCGCTCCTGGAGGACGGCAGGCCCACCGTCGGCGTCGTCGCCGCGCCCGCCCTGCGCAGCCGCTGGTGGGCGGCGGCCGGGCACGGGGCCTGGCTGCGGCGCGGGTCCGCCGGCGCCGAACCGCTGCGGCTGCACGTCTCCGGTGTCGCCCGCCTCGAAAACGCCTATCTGTCCACCACCAACACCCGTACCTGGGACGCCTTCCACTCGCGCGCGGCGTATCTGCGACTCGCCGACGCCTGCTGGGAGGACCGCGCCTTCGGCGACTTCCTCCAGCACTGCATGGTCGCCGAGGGCACGGTCGACATCGCGGCCGAACCGGTCGTGAGCCCCTGGGACATCGCCGCCCTCCAGATCCTCGTGGAGGAGGCGGGCGGCGTCTGCACCGACCTGCTCGGCGGCTCGCCCCAGCGCGGGACGGGTGCCCTGTCCGCCAACCCGGAGCTGCACCGGCTCGCCGTGGAGGCCCTCGCCGCCCCCGCGGCCGCCACGACCGGTGCCGCCACCATCCCCTGA SEQ ID NO:7 mshD_10 nucleotide sequence ATGACCGAGTACAAGCCCACGGTGCGCCTCGCCACCCGCGACGACGTCCCCCGGGCCGTACGCACCCTCGCCGCCGCGTTCGCCGACTACCCCGCCACGCGCCACACCGTCGACCCGGACCGCCACATCGAGCGGGTCACCGAGCTGCAAGAACTCTTCCTCACGCGCGTCGGGCTCGACATCGGCAAGGTGTGGGTCGCGGACGACGGCGCCGCGGTGGCGGTCTGGACCACGCCGGAGAGCGTCGAAGCGGGGGCGGTGTTCGCCGAGATCGGCCCGCGCATGGCCGAGTTGAGCGGTTCCCGGCTGGCCGCGCAGCAACAGATGGAAGGCCTCCTGGCGCCGCACCGGCCCAAGGAGCCCGCGTGGTTCCTGGCCACCGTCGGCGTCTCGCCCGACCACCAGGGCAAGGGTCTGGGCAGCGCCGTCGTGCTCCCCGGAGTGGAGGCGGCCGAGCGCGCCGGGGTGCCCGCCTTCCTGGAGACCTCCGCGCCCCGCAACCTCCCCTTCTACGAGCGGCTCGGCTTCACCGTCACCGCCGACGTCGAGGTGCCCGAAGGACCGCGCACCTGGTGCATGACCCGCAAGCCCGGTGCCTGA SEQ ID NO:8 tcmN nucleotide sequence SEQ ID NO:9 efpA_8 nucleotide sequence SEQ ID NO:10 The core sequence of the recombinant expression insert is a four-gene CDS tandem (arnB_3-prmC_8-nudI-efpA_8; adjacent CDS are linked by a 10 bp synthetic spacer sequence GGTACCGGCC). SEQ ID NO:11 Four-gene expression cassette integration verification fragment ATGACCACCGCCGACAAGCTCGCCCTCCTCGGAGGCACCCCGGCCATCGACCGGCCGCTTCCCCACGAGATCTGGCCGCCCCGCGCGGACGAGGCCGAACTGGCCGCACTCACCGAGCAGCGCAACACCGACATCTCCATCAAGGGCAACAGCGGCCCCATCGGCCAACTGGAAGCCGAATTCCTCGCCTTCCTCGACCACGGCGTCCGCTACGCCGTCA SEQ ID NO:12 alaA protein sequence MEFRQSSKLSEVCYEIRGPVIEHANALEEAGHSVLRLNTGNPALFGFEAPEEIVQDMIRMLPQAHGYTDSRGVLSARRAVAQRYQALGLDEVSVDDVFLGNGVSELVTMAVQALLEDGDEILIPAPDFPLWTAVTTLSGGKAVHYVCDEAADWYPDLDDMASKITDRTKAVVIINPNNPTGAVYPKEILEGILDLARRHGLMVFADEIYDQILYDDAVHHSVAALAPDLVVLTFCGLSKTYRVAGFRSGWLVITGPRQHARDYLEGLTMLASMRLCPNAPAQFAIQAALGGRQSIRELTAPGGRLHEQRDRAWEKLNEIPGVSCVKPKGALYAFPRLDPKVHPIHDDEKFVLDLLLQEKIQVVQGTGFNWPRPDHFRILTLPHADDLDAAISRIGRFLSGYRQGR SEQ ID NO:13 hdfR_6 protein sequence MIVDLDLALVRSFAITAEELHFRRAAERLFITQQALSQRIRRLELLLGVSLFVRGNRSVALTEEGQRFLAPARRVMEAADAAVQAVQPRERRPLRVDVVDGRLAPLQLVRKLLEEDPELPLNLSMRQSLAAALPALARDEIDTAFGRVHDVQGWSAEFSHRLVRLEPLRALVPRTHPLAGRDEVRIDELRSCGIWQPRNGVASEWEHYVRRLADSFGLSLAFSGAALSDEHFLEHLWSHGEMACLAGADVSYAFSPDIRSIPLVDPTPVYPWSMVWRRQAGHPLVDRLVGLAKRSAGDWLTHVPGEIWLPAPDRALLRGAGVDVDAVRG SEQ ID NO:14 nudI protein sequence MADGTAPKLVVGALISDPLGRIFVQRRSADRRLFPACWDVVGGAVEDGETHHEALAREIAEETGWRLRRVLARVVHESWTADGVRHLESDYVVEVDGDLSSPTLERDKHPEFAWVGPQDISLLDENVRRGAGTFIKDTVAAGHDWLAGSTRR SEQ ID NO:15 arnB_3 protein sequence MTTADKLALLGGTPAIDRPLPHEIWPPRADEAELAALTEQRNTDISIKGNSGPIGQLEAEFLAFLDHGVRYAVTFNSGTSALLAAYFALGVREGVEVAGPALTYHAALSPVFALRGDVVLVDIDPVSRGLDPKALEAAITENTRVVTVVHQWGHPCDMDAILGVAERYGLRVLEDCSHAHGSRYKGKPVGTFGDAAVFSLQANKAVYAGEGGILVTDDALVQDRATLLGHYRDRSRDSVIDEELRSHWVTGFGLKLRMSPFNAIVARHSLAAFPARKEARHRCLRHFGELLREVDYLEPITVPDHVDMGAWYGYKPLYRPEALGGVPRSVLIEALRAEGMEVGAPSGPRLATLPLYARPENPLFPGTPKRASAPEPGSQAAHVEQHALSLPTFTDWPEDVALIEAYAEAFRKIGRHREALAAHAAETSR SEQ ID NO:16 prmC_8 protein sequence MTVKESPEAAAKVLEHVMTGDDLPDDFSLLGREWTLHRGVFPGTLTAATEVMASIVPYPKGGSFLEVGCGTGVISVTAALHGSDVTALDINEKAVANTLANAERHGVEDRVRVLHSDLYAALEPTDRFDAIFWNVPWTYVEDGFPLSSDLHSAVFDPGYQGQSRFISGARDHLADGGRLLIGTADLGDRERIDAFAAEAGMTVEMLRRVRRIEVVRVMEYHLLELRAK SEQ ID NO:17 hisN_2 protein sequence MPQQADLELAMRLADTADRITTRRFQARDLRIREKPDRTPVTDADTTVEAAVRESVRAARPDDDFAGEEETGGEVTAGRTWIVDPIDGTKNFLRGVPVWATLIALLEDGRPTVGVVAAPALRSRWWAAAGHGAWLRR GSAGAEPLRLHVSGVARLENAYLSTTNTRTWDAFHSRAAYLLRADACWEDRAFGDFLQHCMVAEGTVDIAAEPVVSPWDIAALQILVEEAGGVCTDLLGGSPQRGTGALSANPELHRLAVEALAAPAAATTGAATIP SEQ ID NO:18 mshD_10 protein sequence MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA SEQ ID NO:19 tcmN protein sequence MAPTEATRGGPADPAPAAHRGGHTEHADPAEHAAQFGAQERILTLVWGYISSEILDLATRLDLPDLMGTEERAAAELAASLDTDPVATLRLLRFAFAALGLAEETGAGRFRLTPAGHRLRTDVPDSLHAFVRQGMGVFRQAWSHFDHSIRTGEPAFDQVFGTDFFSYLSERPELSGTFTSSMREATRTMSTALAKEEEYDFSSYGTVVDIGGADGSLLAAVLSAHPGVEGVVFDSPEGARDAAATLAAGVGERGRVETGDFFTRVPGGGDLYVLKSILHDWSDARSADILRTVRAAMPAHARLLVVEVLLPDTVDSAHPLGYLSDLYMLVNMGGRERSERDLRSLLSDTGFRTTRVRTPPGLTPFSLIEAAPV SEQ ID NO:20 efpA_8 protein sequence MARKPDISAVPVESAACQGPDPRRWWGLVVILAAQLLVVLDGTVVNIALPSVQRDLGMSDTSRQWVITAYTLAFGGLLLLGGRVADAFGRRRIFAVGILGFGLASLLGGAAPDPGTLFLARALQGVFAAALAPAALALINTLFTEPGERGKAFGVYGAVSGGGAAVGLLAGGLLTEYLDWRWCLYVNAPVALLALLGCRLLPRDRRTGRAVRLDLPGTLLGCGGLVAIVYAFAEAESGWGDPLVVRLLVLGVLMLVAFALVERRVQDPLLPPGVVAHRVRGGSFLVVGLPQIGLFGLFLFLTYYLQGILDYSPVLTGVAFLPLGLGIAVGSSLIAARLLPRTRPRTLIVGALLAAAAGMALLTRLEPDTPQVYLTHLLPAQILIGLGIGCMMMPAMHTATARVAPHEAGAAAAVVNSAQQVGGALGVALLNTVSTGATAAYLADHGTSPAATVDGTVHGYTVAIAFAVGVLLLTAVLAWVLIDSRTEAADETGSASVTPARPR SEQ ID NO:21 ctg_05430 nucleotide sequence SEQ ID NO:22 ctg_06351 (aroF, a DAHP synthesis-related candidate) nucleotide sequence SEQ ID NO:23 ctg_01102 nucleotide sequence SEQ ID NO:24 ctg_01472 nucleotide sequence SEQ ID NO:25 ctg_04207 nucleotide sequence ATGCGAATCGTCCTCGTCGGGCCGCCCGGTGCGGGCAAGGGAACGCAGGCCGCGTTCCTCGCCAAGAACCTGTCGATCCCGCACATCTCCACGGGCGACCTCTTCCGTGCCAACATCAGCCAGGGCACGGAGCTGGGCAAGCGGGCGAAGGCGTTCATGGACGCCGGGAACCTCGTGCCCGACGAGGTGACGATCGGCATGGCGAAGGACCGCATGGCGCAGCCGGACGCCGAGCACGGTTTCCTGCTCGACGGGTTCCCGCGCAACGTGTCGCAGGCGGAGGCGCTGGACGTGGCGCTCAAGGCCGATGACGTGAAGCTCGACGCCGTCCTGGACCTGGAGGTCCCCGAGGACGAGGTCGTCAAGCGGATCGCCGGCCGCCGCATCTGCCGCAAGGACTCCAGCCACGTCTTCCACGTGACGTACTCGCAGCCGGAGCAGGAAGGTGTCTGCGACGTCTGCGGCGGCGAGCTGTACCAGCGCGAGGACGACAGCGAGGACACCGTCCGCAAGCGCCTTGAGGTCTACCACACGCAGACCGAGCCGATCATCGACTACTACCGGACCCAGGGTCTGGTCGTCACGATCTCGGCGCTCGGCAAGGTGGACGAGGTCACCGAGCGTGCGATGGACGCCCTGAAGGGCGACAAGTAG SEQ ID NO:26 ctg_02875 nucleotide sequence GTGACCGGGATCAAGACGACCGGCGAGAAGAAGTTGATGCTCTTCTCCGGCCGCGCCCACCCCGAGTTGGCCGAGGAGGTCGCACACAAGCTGGGTGTCGGCATCGTGCCGACGAAGGCCTTCGATTTCGCCAACGGCGAGATCTACGTCCGCTACCAGGAGTCGGCGCGTGGCGCCGACTGCTTCTTGATCCAGAGCCACACCGCTCCGATCAACAAGTGGATCATGGAGCAGCTCATCATGATCGACGCGCTGAAGCGCGCGTCGGCCCGCTCCATCACCGTCATCGTGCCGTTCTACGGTTACGCGCGGCAGGACAAGAAGCACAAGGGACGTGAACCGATCTCGGCGCGTCTGGTCGCCGACCTGATGAAGACCGCGGGTGCGGACCGCATCCTCACGGTCGACCTGCACACGGACCAGATCCAGGGCTTCTTCGACGGCCCGGTCGACCATCTCTTCGCGCTGCCGATCCTCGCGGACTACGTGGGCGCGAAGGTCGACCGCTCGAAGCTCACGGTGGTCTCCCCGGACGCCGGCCGCGTGCGCGTGGCCGACCGCTGGTGCGACCGTCTCGGCGCGCCGCTGGCGATCGTCCACAAGCGCCGCGACAAGGACGTCGCGAACCAGGTGACCGTGCACGAGGTCGTCGGTGAGGTCAAGGGCCGCGTCTGCGTCCTGGTCGACGACATGATCGACACGGGTGGCACGATCTGCGCCGCGGCCGACGCGCTGTTCGCGCACGGCGCGGAGGACGTCATCGTGACGGCGACGCACGGCGTGCTCTCCGGACCGGCGGCGGACCGGCTGAAGAACTCGAAGGTGAGCGAGTTCGTCTTCACCGACACGCTGCCCGACCCGGGCGCGCTCGAACTCGACAAGATCACGGTGCTCTCGATCGCGCCGACGATCGCGAACGCCGTGCGTGAGGTCTTCGAGGACGGCTCGGTGACGAGCCTCTTCGACGAGCAGTAG SEQ ID NO:27 ctg_05430 protein sequence MTTPLTLTSLAETDVTSDAEREGQWLRTLPAQQQPRWKDHRLAEQLSSELRDVPGLVAWDEVRQLRLLLAEVATGALQVLQAGDCAEDPADCVPKVLRRKAGMLDALAGAMQAQTGLSVVRVGRIAGQFAKPRSSDLERHGEIELPAFRGHLVNDPRPDPQLRQPDPARLLTGYRAAYTATEFLRRLGGAWGLPAGAPVWTSHEALVLDYELPLLRQDRQGLLLSSTHWPWIGDRTRQLDGAHVQMLARVHNPVACKVGPTMTPAELTQLCAVLDPARTPGRLTLISRMGAERIEEWLPPLMAAVREAGHPVIWLCDPMHGNTQRGPLGRKTRLLTSVTREVELFQEVAGAQNVVAGGLHLEATPDNVGECVADESRLDDLLDPVGYTTHCDPRLNIDQAVAVVSAWHR SEQ ID NO:28 ctg_06351 protein sequence MVVVMAPEATQKDVDAVVELVRTAGGDAFVSRGVTRTIVGLVGDVDAFAALNLSHLRGVLDVVRISVPYKLVSREHHVDRTAVTVAGVPIGPDTLTVIAGPCAVETPSQTLAAALMAKAAGASLLRGGAFKPRTSPYAYQGLGESGLRILADVKAETGLPLVTEVIDPAGVELVAPYADMLQIGTRNMHNFALLQEVGAAGMPVLLKRGFGATIEEWLMAAEYIAQRGNLDIVLCERGIRTFETATRNTLDISAVPVVQRLSHLPVIVDPSHSGGRRDLVLPLTRAALAAGADGVMIDVHPDPGTALCDGDQALTRPEIAEVADAVARLSPLMGRLPARPVAEVRAGRGE SEQ ID NO:29 ctg_01102 protein sequence MSRRLFTSESVTEGHPDKIADQISDTILDALLREDPTSRVAVETLITTGLVHVAGEVTTKAYAPIAQLVRDKILEIGYDSSKKGFDGASCGVSVSIGAQSPDIAQGVDTAYEKRVEGDEDELDKQGAGDQGLMFGYACDETPELMPLPIHLAHRLSRRLSEVRKNGTIPYLRPDGKTQVTIEYDGDKAVRLDTVVVSSQHASDIDLDSLLAPDIREFVVEAELKALLDDGIKLDTEGYRLLVNPTGRFEIGGPMGDAGLTGRKIIIDTYGGMARHGGGAFSGKDPSKVDRSAAYAMRWVAKNVVAAGLAARCEVQVAYAIGKAEPVGLFVETFGTATVDTDKIETAIGEVFDLRPAAIIRDLDLLRPIYAQTAAYGHFGRAIPDFTWERTDRVEALRKAAGL SEQ ID NO:30 ctg_01472 protein sequence MRTALVIGTGLIGTSAALALASRGVTVHLADHDPAQARTAAALGAGTDESPEGLVDLCVVAVPPAHVAATLADAMRRGTARAYLDVASVKGGPRRELEALGLDLTPYIGTHPMSGRERSGPLAATADLFEGRPWVLTPTRDTDTEVLNLALELVALCRAVPVVMDADAHDRAVALVSHMPHLVSSMVAARLENAEETAVRLCGQGIRDVTRIAASDPRMWIDILSANPGPVADLLAQVAGDLDETVTALRALQSADEQKRAEGAAGVESMLRRGNAGQTRVPGKHGTAPAAYEIVAVLIDDQPGQLARIFADADRAGVNVEDVRIEHATGQQAGLVQLMVEPRAASALSAALRERGWSIRQ SEQ ID NO:31 ctg_04207 protein sequence MRIVLVGPPGAGKGTQAAFLAKNLSIPHISTGDLFRANISQGTELGKRAKAFMDAGNLVPDEVTIGMAKDRMAQPDAEHGFLLDGFPRNVSQAELDVALKADDVKLDAVLDLEVPEDEVVKRIAGRRICRKDSSHVFHVTYSQPEQEGVCDVCGGELYQREDDSEDTVRKRLEVYHTQTEPIIDYYRTQGLVVTISALGKVDEVTERAMDALKGDK SEQ ID NO:32 ctg_02875 protein sequence MTGIKTTGEKKLMLFSGRAHPELAEEVAHKLGVGIVPTKAFDFANGEIYVRYQESARGADCFLIQSHTAPINKWIMEQLIMIDALKRASARSITVIVPPFYGYARQDKKHKGREPISARLVADLMKTAGADRILTVDLHTDQIQGFFDGPVDHLFALPILADYVGAKVDRSKLTVVSPDAGRVRVADRWCDRLGAPLAIVHKRRDKDVANQVTVHEVVGEVKGRVCVLVDDMIDTGGTICAAADALFAHGAEDVIVTATHGVLSGPAADRLKNSKVSEFVFTDTLPDPGALELDKITVLSIAPTIANAVREVFEDGSVTSLFDEQ

Claims

1. A method for engineering the modification of actinomycetes for the production of purinemycin compounds, characterized in that, Includes the following steps: Step 1) Screening for engineered targets or combinations of engineered targets; the engineered targets include targets located in one or more of the following modules: amino transfer module, methylation / post-modification module, nucleotide treatment module, dephosphorylation module, acetylation / autoimmunity module, transcriptional regulation module, SAM methyl supply module, L-tyrosine module, PRPP nucleotide activation module, ATP energy turnover module, and product transport / autoimmunity module; the combination of engineered targets includes at least two engineered targets. Step 2) Perform genetic manipulation on the actinomycetes using the engineering target points or combinations of engineering target points to obtain the engineered actinomycetes.

2. The method as described in claim 1, characterized in that, Step 1) The target of the amino transfer module includes the arnB_3 gene as shown in SEQ ID NO.4 or the alaA gene as shown in SEQ ID NO.1; the target of the methylation / post-modification module includes the prmC_8 gene as shown in SEQ ID NO.5 or the tcmN gene as shown in SEQ ID NO.8; the target of the nucleotide processing module includes the nudI gene as shown in SEQ ID NO.3; the target of the dephosphorylation module includes the hisN_2 gene as shown in SEQ ID NO.6; the target of the acetylation / autoimmunity module includes the mshD_10 gene as shown in SEQ ID NO.7; the target of the transcriptional regulation module includes the hdfR_6 gene as shown in SEQ ID NO.2; the target of the SAM methyl supply module includes the ctg_01102 gene as shown in SEQ ID NO.23; and the target of the product transport / autoimmunity module includes the efpA_8 gene as shown in SEQ ID NO.

9.

3. The method as described in claim 2, characterized in that, Step 2) The genetic manipulation includes any one or more of the following: enhancing target gene expression, downregulating target gene expression, or introducing mutations in the target gene; enhancing target gene expression includes using a strong promoter, increasing gene copy number, or optimizing ribosome binding sites; downregulating target gene expression includes downregulating or knocking out the expression of the engineered target, preferably through homologous recombination, CRISPR-Cas system, or CRISPRi system.

4. The method as described in claim 3, characterized in that, The operation of enhancing target gene expression includes: enhancing the expression of one or more genes among arnB_3, alaA, prmC_8, tcmN, nudI, ctg_01102 or efpA_8; and / or the operation of downregulating target gene expression includes: knocking out or downregulating the expression of transcription regulatory factor genes hdfR_6 and / or mshD_10.

5. The method as described in claim 4, characterized in that, The engineering target combination includes the arnB_3, prmC_8, nudI, and efpA_8 genes; the genetic manipulation includes enhancing the expression of the arnB_3, prmC_8, nudI, and efpA_8 genes.

6. The method as described in claim 1, characterized in that, The puromycin compounds include puromycin and its derivative Puromycin E; and / or the actinomycetes include Streptomyces strains, preferably Streptomyces sp. HU182, with accession number CGMCC 25619.

7. An engineered actinomycete, characterized in that, Based on actinomycetes, the derived strains are obtained by engineering modification according to the method described in any one of claims 1 to 6; The actinomycetes include Streptomyces strains, preferably Streptomyces sp. HU182, with accession number CGMCC25619.

8. A method for producing a purinemycin-like compound, characterized in that, Puromycin compounds are produced by fermentation of the engineered actinomycetes as described in claim 7 in a fermentation medium; the puromycin compounds include puromycin and its derivative Puromycin E.

9. The production method as described in claim 8, characterized in that, The fermentation medium comprises: corn starch, glycerol, glucose, yeast powder, soybean powder, MgSO4·7H2O and CaCO3, with a pH of 7.2-7.6; and / or, the fermentation conditions are: inoculum size 3%-8%, temperature 27-29℃, rotation speed 180-250 r / min, and fermentation time 5-7 days.

10. A method for screening biosynthetic engineering targets or combinations of engineering targets for puromycin compounds in actinomycetes, characterized in that, Includes the following steps: a) Provide the genomic information of the actinomycete to be modified, and identify functional gene clusters related to the biosynthesis of puromycin compounds based on the genomic information, and identify target genes that affect the biosynthesis of puromycin compounds from them; b) Construct a genome-scale metabolic network model of actinomycetes to identify key metabolic genes that affect the biosynthesis of puromycin compounds; c) Merge the target genes within the functional gene clusters identified in step a) with the key metabolic genes identified in step (c) to construct a candidate engineering target library. d) Based on the candidate engineering target library, select engineering targets or combinations of engineering targets to construct gene modification schemes; e) Using a computational model to score and rank the genetic modification schemes, wherein the scoring is based at least on predictions of product synthesis capacity, strain adaptability, and feasibility of genetic manipulation. f) Based on the sorting results, select engineering modification targets or combinations of engineering modification targets.

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