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The growing demand for sustainable agricultural pest control solutions has increased the use of microbial bioinput. This study characterized an industrial bioinput produced by Saccharopolyspora spinosa , focusing on identifying metabolites and quantifying spinosyns A and D by HPLC-MS. The concentrations of these compounds were 29.4 ± 3.6 mg L −1 and 13.3 ± 1.5 mg L −1 , respectively, for a total of 42.7 mg L −1 . The limit of quantification (LOQ) and limit of detection (LOD) values obtained were 13.7 ng L −1 and 4.16 ng L −1 for spinosyn A and 7.63 ng L −1 and 2.31 ng L −1 for spinosyn D, respectively. The bioinput contained essential amino acids (leucine, phenylalanine, and tryptophan), plant cell death inducers (sphinganine), diketopiperazines with insecticidal potential (Cyclo(Met-Val)), larvicides ( N -stearoyl tryptophan), antimicrobials (Penicitrinol D), and phospholipids associated with cell defense and stress responses (phosphatidylethanolamine, phosphatidylserine, ceramides, and mycolactones). In vitro trials demonstrated mortalities of 82.5% for Spodoptera frugiperda , 100.0% for Dalbulus maidis , and 64.1% for Ceratitis capitata . These results are statistically equivalent to those obtained using commercial products, which cost up to five times more. This bioinput’s chemical diversity suggests multifunctional action, probable synergism, and a lower risk of resistance, which reinforces its potential in biological agricultural management. Key points •  Identification of a diverse set of bioactive compounds, including spinosyns A and D. •  Bioinput achieves statistical efficacy equal to commercial products in in vitro tests. •  A sustainable, cost-effective alternative for large-scale production and application.

Spinosad is an efficient and broad-spectrum environmentally friendly biopesticide, but its low yield in wild-type Saccharopolyspora spinosa limits its further application. ARTP/NTG compound mutagenesis was used in this study to improve the spinosad titer of S. spinosa and obtain a high-yield mutant—NT24. Compared with the wild-type strain, the fermentation cycle of NT24 was shortened by 2 days and its maximum titer of spinosad reached 858.3 ± 27.7 mg/L, which is 5.12 times more than for the same-period titer of the wild-type strain. In addition, RT-qPCR, resequencing, and targeted metabolomics showed that the upregulation of the key differential genes accD6, fadD, sdhB, oadA, and gntZ caused increased metabolic flux in the tricarboxylic acid cycle and pentose phosphate pathway, suggesting that the accumulation of pyruvate and short-chain acyl-CoA was the primary cause of spinosad accumulation in NT24. This study demonstrates the effectiveness of ARTP mutagenesis in S. spinosa, and provides new insights for the mechanism of spinosad biosynthesis and metabolic engineering in S. spinosa.

Background Spinosad, a secondary metabolite produced by Saccharopolyspora spinosa , is a polyketide macrolide insecticide with low toxicity and environmental friendliness. Owing to the high level of DNA methylation and unclear regulatory mechanisms, gene engineering to increase spinosad production is challenging. Limited improvements in yield have been observed with heterologous expression or partial overexpression of the 74-kb spinosyn gene cluster ( spn ), and research on the overexpression of the complete spinosyn gene cluster is lacking. Results The plasmid pCM265-spn was constructed using CRISPR/Cas9-mediated Transformation-Associated Recombination cloning to enable the overexpression of the complete spn gene cluster in Sa. spinosa . The engineered strain Sa. spinosa-spn achieved a 124% increase in spinosad yield (693 mg/L) compared to the wild type (309 mg/L). The overexpression of the spn gene cluster also delayed spore formation and reduced hyphal compartmentalization by influencing the transcription of related genes ( bldD , ssgA , whiA , whiB , and fstZ ). Transcriptional analysis revealed significant upregulation of genes in the spn gene cluster, thereby enhancing secondary metabolism. Additionally, optimization of the fermentation medium through response surface methodology further increased spinosad production to 920 mg/L. Conclusions This study is the first to successfully overexpress the complete spn gene cluster in Sa. spinosa , significantly enhancing spinosad production. These findings have significance for further optimization of spinosad biosynthesis.

In this study, a new isolated strain YJY-12 with high yield of spinosad was isolated and its fermentation conditions for spinosad production was optimized. It was preliminary identified by morphological and molecular biology analysis to be S. spinosa YJY-12, and it demonstrated stable high-yield trait, with a variation of less than 5% in fermentation levels across five generations. Subsequently, its fermentation condition for producing spinosad was optimized in shake flask experiments. The process involved single-factor tests, Plackett–Burman (PB) experiments, steepest ascent experiments, and central composite design (CCD) methodology. After optimization, the spinosad fermentation level by strain YJY-12 reached up to 4.38 g/L. The fermentation process was further scaled up to a 30-L fermentor by fed-batch fermentation, achieving a spinosad fermentation level of 6.22 ± 0.12 g/L after 16-day. Finally, the efficacy of spinosad in controlling Eggplant Thrips was investigated and the technical spinosad produced by strain YJY-12 exhibited superior efficacy of thrips control. Overall, this study provided a methodological foundation for enhancing spinosad fermentation efficiency and offers guidance for future spinosad fermentation optimization research.

Pirin family proteins perform a variety of biological functions and widely exist in all living organisms. A few studies have shown that Pirin family proteins may be involved in the biosynthesis of antibiotics in actinomycetes. However, the function of Pirin-like proteins in S. spinosa is still unclear. In this study, the inactivation of the sspirin gene led to serious growth defects and the accumulation of H2O2. Surprisingly, the overexpression and knockout of sspirin slightly accelerated the consumption and utilization of glucose, weakened the TCA cycle, delayed sporulation, and enhanced sporulation in the later stage. In addition, the overexpression of sspirin can enhance the β-oxidation pathway and increase the yield of spinosad by 0.88 times, while the inactivation of sspirin hardly produced spinosad. After adding MnCl2, the spinosad yield of the sspirin overexpression strain was further increased to 2.5 times that of the wild-type strain. This study preliminarily revealed the effects of Pirin-like proteins on the growth development and metabolism of S. spinosa and further expanded knowledge of Pirin-like proteins in actinomycetes.Key points• Overexpression of the sspirin gene possibly triggers carbon catabolite repression (CCR)• Overexpression of the sspirin gene can promote the synthesis of spinosad• Knockout of the sspirin gene leads to serious growth and spinosad production defects

Saccharopolyspora spinosa is a well-known actinomycete for producing the secondary metabolites, spinosad, which is a potent insecticides possessing both efficiency and safety. In the previous researches, great efforts, including physical mutagenesis, fermentation optimization, genetic manipulation and other methods, have been employed to increase the yield of spinosad to hundreds of folds from the low-yield strain. However, the metabolic network in S. spinosa still remained un-revealed. In this study, two S. spinosa strains with different spinosad production capability were fermented and sampled at three fermentation periods. Then the total RNA of these samples was isolated and sequenced to construct the transcriptome libraries. Through transcriptomic analysis, large numbers of differentially expressed genes were identified and classified according to their different functions. According to the results, spnI and spnP were suggested as the bottleneck during spinosad biosynthesis. Primary metabolic pathways such as carbon metabolic pathways exhibited close relationship with spinosad formation, as pyruvate and phosphoenolpyruvic acid were suggested to accumulate in spinosad high-yield strain during fermentation. The addition of soybean oil in the fermentation medium activated the lipid metabolism pathway, enhancing spinosad production. Glutamic acid and aspartic acid were suggested to be the most important amino acids and might participate in spinosad biosynthesis.

Background Acetoin utilization protein (acuC) is a type I histone deacetylase which is highly conserved in bacteria. The acuC gene is related to the acetylation/deacetylation posttranslational modification (PTM) system in S. spinosa. Spinosyns, the secondary metabolites produced by Saccharopolyspora spinosa , are the active ingredients in a family of insect control agents. However, the specific functions and influences of acuC protein in S. spinosa are yet to be characterized. Results The knockout strain and overexpression strain were constructed separately with the shuttle vector pOJ260. The production of spinosyns A and D from S. spinosa-acuC were 105.02 mg/L and 20.63 mg/L, which were 1.82-fold and 1.63-fold higher than those of the wild-type strain (57.76 mg/L and 12.64 mg/L), respectively. The production of spinosyns A and D from S. spinosa- Δ acuC were 32.78 mg/L and 10.89 mg/L, respectively. The qRT-PCR results of three selected genes ( bldD , ssgA and whiA ) confirmed that the overexpression of acuC affected the capacities of mycelial differentiation and sporulation. Comparative proteomics analysis was performed on these strains to investigate the underlying mechanism leading to the enhancement of spinosad yield. Conclusions This study first systematically analysed the effects of overexpression acuC on the growth of S. spinosa and the production of spinosad. The results identify the differentially expressed proteins and provide evidences to understand the acetylation metabolic mechanisms which can lead to the increase of secondary metabolites.

This review highlights the importance of natural product research and industrial microbiology for product development in the agricultural industry, based on examples from Dow AgroSciences. It provides an overview of the discovery and development of spinetoram, a semisynthetic insecticide derived by a combination of a genetic block in a specific O-methylation of the rhamnose moiety of spinosad coupled with neural network-based QSAR and synthetic chemistry. It also emphasizes the key role that new technologies and multidisciplinary approaches play in the development of current spinetoram production strains.

Dissolved oxygen (DO) is an important influencing factor in the process of aerobic microbial fermentation. Spinosad is an aerobic microbial-derived secondary metabolite. In our study, spinosad was used as an example to establish a DO strategy by multi-scale analysis, which included a reactor, cell and gene scales. We changed DO conditions that are related to the characteristics of cell metabolism (glucose consumption rate, biomass accumulation and spinosad production). Consequently, cell growth was promoted by maintaining DO at 40% in the first 24h and subsequently increasing DO to 50% in 24h to 96h. In an in-depth analysis of the key enzyme genes (gtt, spnA, spnK and spnO), expression of spinosad and specific Adenosine Triphosphate (ATP), the spinosad yield was increased by regulating DO to 30% within 96h to 192h and then changing it to 25% in 192h to 240h. Under the four-phase DO strategy, spinosad yield increased by 652.1%, 326.1%, 546.8%, and 781.4% compared with the yield obtained under constant DO control at 50%, 40%, 30%, and 20% respectively. The proposed method provides a novel way to develop a precise DO strategy for fermentation. We provide a model of four-stage dissolved oxygen strategy which based on multi-scale analysis for improving spinosad yield by Saccharopolyspora spinosa ATCC49460,The proposed method provides a novel way to develop a precise DO strategy for fermentation.

Spinosad (a mixture of spinosyns A and D) is a macrocyclic lactone green bioinsecticide produced by Saccharopolyspora spinosa. It is known for its high efficiency, low toxicity, and broad‐spectrum activity. Although numerous strategies have been employed to enhance spinosad production, intricate regulation of secondary metabolism and inefficient genetic manipulation impede systematic and comprehensive metabolic engineering in this spinosad‐producing strain. In this study, a genome‐scale metabolic model (GEM) for Sa. spinosa NHF132 is developed to dissect the intricate secondary metabolic pathways of spinosad biosynthesis, analyzing interactions among precursors, key enzymes, and competing or bypass pathways. Guided by the model, the impact of rhamnose precursor overexpression, gene cluster amplification, short‐chain acyl‐CoA enhancement, and chassis optimization on spinosad production is systematically evaluated. By integrating these metabolic engineering strategies, engineered strain NHF132‐BAC‐SP43‐NCM achieved a spinosad titer of 1816.8 mg L−1, a 553.3% increase over the starting strain, with substantial improvements in yield and product proportion. The model‐driven framework for metabolic engineering of complex secondary metabolites in actinomycetes substantially increased spinosad production and offered valuable insights for other complex natural products. A genome‐scale model guided the systematic metabolic engineering of Saccharopolyspora spinosa, enabling a 5.5‐fold boost in the green bioinsecticide spinosad to 1.8 g L−1 and establishing an actinomycete framework for complex natural products overproduction.