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Summary: Butenyl‐spinosyn, a promising biopesticide produced by Saccharopolyspora pogona, exhibits stronger insecticidal activity and a broader pesticidal spectrum. However, its titre in the wild‐type S. pogona strain is too low to meet the industrial production requirements. Deletion of non‐target natural product biosynthetic gene clusters resident in the genome of S. pogona could reduce the consumption of synthetic precursors, thereby promoting the biosynthesis of butenyl‐spinosyn. However, it has always been a challenge for scientists to genetically engineer S. pogona. In this study, the Latour gene knockout system (linear DNA fragment recombineering system) was established in S. pogona. Using the Latour system, a hybrid NRPS‐T1PKS cluster (˜20 kb) which was responsible for phthoxazolin biosynthesis was efficiently deleted in S. pogona. The resultant mutant S. pogona‐Δura4‐Δc14 exhibited an extended logarithmic phase, increased biomass and a lower glucose consumption rate. Importantly, the production of butenyl‐spinosyn in S. pogona‐Δura4‐Δc14 was increased by 4.72‐fold compared with that in the wild‐type strain. qRT‐PCR analysis revealed that phthoxazolin biosynthetic gene cluster deletion could promote the expression of the butenyl‐spinosyn biosynthetic gene cluster. Furthermore, a TetR family transcriptional regulatory gene that could regulate the butenyl‐spinosyn biosynthesis has been identified from the phthoxazolin biosynthetic gene cluster. Because dozens of natural product biosynthetic gene clusters exist in the genome of S. pogona, the strategy reported here will be used to further promote the production of butenyl‐spinosyn by deleting other secondary metabolite synthetic gene clusters. [ABSTRACT FROM AUTHOR]

Spinosyns, the secondary metabolites produced by S accharopolyspora spinosa, are the active ingredients in a family of insect control agents. Most of the S . spinosa genes involved in spinosyn biosynthesis are found in a contiguous c. 74-kb cluster. To increase the spinosyn production through overexpression of their biosynthetic genes, part of its gene cluster ( c. 18 kb) participating in the conversion of the cyclized polyketide to spinosyn was obtained by direct cloning via Red/ ET recombination rather than by constructing and screening the genomic library. The resultant plasmid pUCAmT-spn was introduced into S . spinosa CCTCC M206084 from E scherichia coli S17-1 by conjugal transfer. The subsequent single-crossover homologous recombination caused a duplication of the partial gene cluster. Integration of this plasmid enhanced production of spinosyns with a total of 388 (± 25.0) mg L−1 for spinosyns A and D in the exconjugant S . spinosa trans1 compared with 100 (± 7.7) mg L−1 in the parental strain. Quantitative real time polymerase chain reaction analysis of three selected genes ( spnH, spnI, and spnK) confirmed the positive effect of the overexpression of these genes on the spinosyn production. This study provides a simple avenue for enhancing spinosyn production. The strategies could also be used to improve the yield of other secondary metabolites. [ABSTRACT FROM AUTHOR]

Background: Butenyl-spinosyn, produced by Saccharopolyspora pogona, is a promising biopesticide due to excellent insecticidal activity and broad pesticidal spectrum. Bacterioferritin (Bfr, encoded by bfr) regulates the storage and utilization of iron, which is essential for the growth and metabolism of microorganisms. However, the effect of Bfr on the growth and butenyl-spinosyn biosynthesis in S. pogona has not been explored. Results: Here, we found that the storage of intracellular iron influenced butenyl-spinosyn biosynthesis and the stress resistance of S. pogona, which was regulated by Bfr. The overexpression of bfr increased the production of butenyl-spinosyn by 3.14-fold and enhanced the tolerance of S. pogona to iron toxicity and oxidative damage, while the knockout of bfr had the opposite effects. Based on the quantitative proteomics analysis and experimental verification, the inner mechanism of these phenomena was explored. Overexpression of bfr enhanced the iron storage capacity of the strain, which activated polyketide synthase genes and enhanced the supply of acyl-CoA precursors to improve butenyl-spinosyn biosynthesis. In addition, it induced the oxidative stress response to improve the stress resistance of S. pogona. Conclusion: Our work reveals the role of Bfr in increasing the yield of butenyl-spinosyn and enhancing the stress resistance of S. pogona, and provides insights into its enhancement on secondary metabolism, which provides a reference for optimizing the production of secondary metabolites in actinomycetes. [ABSTRACT FROM AUTHOR]

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-ΔacuC 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. [ABSTRACT FROM AUTHOR]

Butenyl-spinosyn produced by Saccharopolyspora pogona exhibits strong insecticidal activity and broad pesticidal spectrum. However, its synthetic level was low in the wild-type strain. At present, important functional genes involved in butenyl-spinosyn biosynthesis remain unknown, which leads to difficulty in efficiently editing its genome to improve the butenyl-spinosyn yield. To accelerate the genetic modification of S. pogona, we conducted comparative proteomics analysis to screen differentially expressed proteins related to butenyl-spinosyn biosynthesis. A TetR family regulatory protein was selected from the 289 differentially expressed proteins, and its encoding gene (SP_1288) was successfully deleted by CRISPR/Cas9 system. We further deleted a 32-kb polyketide synthase gene cluster (cluster 28) to reduce the competition for precursors. Phenotypic analysis revealed that the deletion of the SP_1288 and cluster 28 resulted in a 3.10-fold increase and a 35.4% decrease in the butenyl-spinosyn levels compared with the wild-type strain, respectively. The deletion of cluster 28 affected the cell growth, glucose consumption, mycelium morphology, and sporulation by controlling the expression of ptsH, ptsI, amfC, and other genes related to sporulation, whereas SP_1288 did not. These findings confirmed not only that the CRISPR/Cas9 system can be applied to the S. pogona genome editing but also that SP_1288 and cluster 28 are closely related to the butenyl-spinosyn biosynthesis and growth development of S. pogona. The strategy reported here will be useful to reveal the regulatory mechanism of butenyl-spinosyn and improve antibiotic production in other actinomycetes. Key points: • SP_1288 deletion can significantly promote the butenyl-spinosyn biosynthesis. • Cluster 28 deletion showed pleiotropic effects on S. pogona. • SP_1288 and cluster 28 were deleted by CRISPR/Cas9 system in S. pogona. [ABSTRACT FROM AUTHOR]

Lipopeptides (such as iturin, fengycin, and surfactin) from Bacillus possess antibacterial, antifungal, and antiviral activities and have important application in agriculture and pharmaceuticals. Although unremitting efforts have been devoted to improve lipopeptide production by designing gene regulatory circuits or optimizing fermentation process, little attention has been paid to utilizing multi-omics for systematically mining core genes and proteins during the bacterial growth cycle. Here, lipopeptide bacillomycin Lb from new Bacillus amyloliquefaciens X030 was isolated and first found to have anticancer activity in various cancer cells (such as SMMC-7721 and MDA-MB-231). A comprehensive genomic and growth proteomic analysis of X030 revealed bacillomycin Lb biosynthetic gene cluster, key enzymes and potential regulatory proteins (PerR, PhoP, CcpA, and CsfB), and novel links between primary metabolism and bacillomycin Lb production in X030. The antitumor activity of the fermentation supernatant supplemented with amino acids (such as glutamic acid) and sucrose was significantly increased, verifying the role of key metabolic switches in the metabolic regulatory network. Quantitative real-time PCR analysis confirmed that 7 differential expressed genes exhibited a positive correlation between changes at transcriptional and translational levels. The study not only will stimulate the deeper and wider antitumor study of lipopeptides but also provide a comprehensive database, which promotes an in-depth analysis of pathways and networks for complex events in lipopeptide biosynthesis and regulation and gives great help in improving the yield of bacillomycin Lb (media optimization, genetic modification, or pathway engineering). [ABSTRACT FROM AUTHOR]

Polynucleotide phosphorylase is a highly conserved protein found in bacteria and fungi that can regulate the transcription of related enzymes involved in amino acid metabolism, organic acid metabolism, and cell biosynthesis. We studied the effect of polynucleotide phosphorylase on Saccharopolyspora pogona (S. pogona) growth and the synthesis of secondary metabolites. First, we generated the overexpression vector pOJ260-PermE-pnp via overlap extension PCR. The vector pOJ260-PermE-pnp was then introduced into S. pogona by conjugal transfer, thereby generating the recombination strain S. pogona-Pnp. Results showed that engineering strains possessed higher biomass than those of the wild-type strains. Moreover, the ability of these strains to produce spores on solid medium was stronger than that of the wild-type strains. HPLC results revealed that the butenyl-spinosyn yield in S. pogona-Pnp increased by 1.92-fold compared with that of S. pogona alone. These findings revealed that overexpression of polynucleotide phosphorylase effectively promoted butenyl-spinosyn biosynthesis in S. pogona. This result may be extended to other Streptomyces for strain improvement. [ABSTRACT FROM AUTHOR]

Spinosyn and its analogs, produced by Saccharopolyspora spinosa, are the active ingredients in a family of insect control agents. They are macrolides with a 21-carbon, 12-membered tetracyclic lactones that are attached to two deoxysugars, tri- O-methylrhamnose and forosamine. Labeling studies, analysis of the biosynthetically blocked mutants, and the genetic identification of the spinosyn gene cluster have provided detailed information concerning the mechanism of spinosyn biosynthesis and have enabled combinatorial biosynthesis of a large group of new spinosyns. The following developments have recently impacted the field of spinosyn biology: (1) A second-generation spinosyn called spinetoram (XDE-175) was launched in late 2007; it is a semisynthesized spinosyn derivative produced through the modification of 3′- O-methyl group of rhamnose and the double bond between C5 and C6 of spinosyn J and L. This molecule was shown to have improved insecticidal activity, enhanced duration of control, and an expanded pest spectrum. (2) A new class of spinosyns, the butenyl-spinosyns, was discovered from Saccharopolyspora pogona. The butenyl-spinosyns are similar to spinosyns, but differ in the length of the side chain at C-21. In addition to structural similarities with the spinosyns, the butenyl-spinosyns exhibit a high level of similarity in insecticidal activity to spinetoram. (3) Spinosyn analogs, 21-cyclobutyl-spinosyn A and 21-cyclobutyl-spinosyn D were generated by metabolic engineering of the spinosyn biosynthetic gene cluster. They showed better insecticidal activities against cotton aphid and tobacco budworm than that of spinosyn A and D. Future progress toward the development of more potent spinosad analogs, as well as enhancements in production yields will likely result from these recent advances in the genetics and biochemistry of spinosyns. [ABSTRACT FROM AUTHOR]

• The sensor kinase LytS-L influence the virulence against Helicoverpa armigera. • The expression of LytS-L influence growth and butenyl-spinosyn biosynthesis. • The LytS-L regulates the energy, carbon metabolism, and secondary metabolism. The LytTR family two-component system widely exists in bacterial cells and plays an important role in metabolic regulation. The lytS-L gene that encodes for a LytTR family sensor kinase was knocked out to study its influence on the growth, phenotype, and the biosynthesis of the insecticidal polyketide butenyl-spinosyn in Saccharopolyspora pogona NRRL 30141 (S. pogona). High performance liquid chromatography (HPLC) results showed that the butenyl-spinosyn yield of the lytS-L knockout mutant decreased by 58.9% compared with that of the parental strain. This is manifested by a weak toxicity of the mutant against the insect Helicoverpa assulta (H. armigera). Comparative proteomic analysis revealed the expression characteristics of the proteins in S. pogona and S. pogona- Δ lytS-L : a total of 14 proteins involved in energy metabolism were down-regulated, 9 proteins related to carbon metabolism such as glycolysis, and tricarboxylic acid cycle (TCA) were up-regulated, while 13 proteins involved in the biosynthesis of butenyl-spinosyn were down-regulated (fold change >1.2 or< 0.83). The qRT-PCR (Quantitative Real-time PCR) analysis illustrated that the changes in the expression levels of transcription and translation of the identified genes were consistent. This study explores the function of the two-component system of the LytTR family in S. pogona and shows that the lytS-L gene has an important influence on regulating primary metabolism and butenyl-spinosyn biosynthesis of S. pogona. [ABSTRACT FROM AUTHOR]