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Comparative transcriptomic analysis of two Saccharopolyspora spinosa strains reveals the relationships between primary metabolism and spinosad production.

ABSTRACT: 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.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC8292330 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundSaccharopolyspora spinosa is an important producer of antibiotic spinosad with clarified biosynthesis pathway but its complex regulation networks associated with primary metabolism and secondary metabolites production almost have never been concerned or studied before. The proteomic analysis of a novel Saccharopolyspora spinosa CCTCC M206084 was performed and aimed to provide a global profile of regulatory proteins.ResultsTwo-dimensional-liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 1090, 1166, 701, and 509 proteins from four phases respectively, i.e., the logarithmic growth phase (T1), early stationary phase (T2), late stationary phase (T3), and decline phase (T4). Among the identified proteins, 1579 were unique to the S. spinosa proteome, including almost all the enzymes for spinosad biosynthesis. Trends in protein expression over the various time phases were deduced from using the modified protein abundance index (PAI), revealed the importance of stress pathway proteins and other global regulatory network proteins during spinosad biosynthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis followed by one-dimensional LC-MS/MS identification revealed similar trend of protein expression from four phases with the results of semi-quantification by PAI. qRT-PCR analysis revealed that 6 different expressed genes showed a positive correlation between changes at translational and transcriptional expression level. Expression of three proteins that likely promote spinosad biosynthesis, namely, 5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase (MHSM), glutamine synthetase (GS) and cyclic nucleotide-binding domain-containing protein (CNDP) was validated by western blot, which confirmed the results of proteomic analysis.ConclusionsThis study is the first systematic analysis of the S. spinosa proteome during fermentation and its valuable proteomic data of regulatory proteins may be used to enhance the production yield of spinosad in future studies.

Project description:BackgroundSaccharopolyspora spinosa produces the environment-friendly biopesticide spinosad, a mixture of two polyketide-derived macrolide active ingredients called spinosyns A and D. Therefore considerable interest is in the improvement of spinosad production because of its low yield in wild-type S. spinosa. Recently, a spinosad-hyperproducing PR2 strain with stable heredity was obtained from protoplast regeneration of the wild-type S. spinosa SP06081 strain. A comparative proteomic analysis was performed on the two strains during the first rapid growth phase (RG1) in seed medium (SM) by using label-free quantitative proteomics to investigate the underlying mechanism leading to the enhancement of spinosad yield.ResultsIn total, 224 proteins from the SP06081 strain and 204 proteins from the PR2 strain were unambiguously identified by liquid chromatography-tandem mass spectrometry analysis, sharing 140 proteins. A total of 12 proteins directly related to spinosad biosynthesis were identified from the two strains in RG1. Comparative analysis of the shared proteins revealed that approximately 31% of them changed their abundance significantly and fell in all of the functional groups, such as tricarboxylic acid cycles, glycolysis, biosynthetic processes, catabolic processes, transcription, translation, oxidation and reduction. Several key enzymes involved in the synthesis of primary metabolic intermediates used as precursors for spinosad production, energy supply, polyketide chain assembly, deoxysugar methylation, and antioxidative stress were differentially expressed in the same pattern of facilitating spinosad production by the PR2 strain. Real-time reverse transcriptase polymerase chain reaction analysis revealed that four of five selected genes showed a positive correlation between changes at the translational and transcriptional expression level, which further confirmed the proteomic analysis.ConclusionsThe present study is the first comprehensive and comparative proteome analysis of S. spinosa strains. Our results highlight the differentially expressed proteins between the two S. spinosa strains and provide some clues to understand the molecular and metabolic mechanisms that could lead to the increased spinosad production yield.

Project description:Butenyl-spinosyn, a secondary metabolite produced by Saccharopolyspora pogona, exhibits strong insecticidal activity than spinosyn. However, the low synthesis capacity and unknown metabolic characteristics of butenyl-spinosyn in wild-type S. pogona limit its broad application and metabolic engineering. Here, we showed that S. pogona exhibited increased glucose consumption ability and growth rate compared with S. spinosa, but the production of butenyl-spinosyn was much lower than that of spinosyn. To further elucidate the metabolic mechanism of these different phenotypes, we performed a comparative proteomic and metabolomic study on S. pogona and S. spinosa to identify the change in the abundance levels of proteins and metabolites. We found that the abundance of most proteins and metabolites associated with glucose transport, fatty acid metabolism, tricarboxylic acid cycle, amino acid metabolism, energy metabolism, purine and pyrimidine metabolism, and target product biosynthesis in S. pogona was higher than that in S. spinosa. However, the overall abundance of proteins involved in butenyl-spinosyn biosynthesis was much lower than that of the high-abundance protein chaperonin GroEL, such as the enzymes related to rhamnose synthesis. We speculated that these protein and metabolite abundance changes may be directly responsible for the above phenotypic changes in S. pogona and S. spinosa, especially affecting butenyl-spinosyn biosynthesis. Further studies revealed that the over-expression of the rhamnose synthetic genes and methionine adenosyltransferase gene could effectively improve the production of butenyl-spinosyn by 2.69- and 3.03-fold, respectively, confirming the reliability of this conjecture. This work presents the first comparative proteomics and metabolomics study of S. pogona and S. spinosa, providing new insights into the novel links of phenotypic change and metabolic difference between two strains. The result will be valuable in designing strategies to promote the biosynthesis of butenyl-spinosyn by metabolic engineering.

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Comparative transcriptomic analysis of two Saccharopolyspora spinosa strains reveals the relationships between primary metabolism and spinosad production.

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