Deciphering the Metabolic Pathway Difference Between Saccharopolyspora pogona and Saccharopolyspora spinosa by Comparative Proteomics and Metabonomics

Abstract

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 3.03fold, 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.

Bacterioferritin: a key iron storage modulator that affects strain growth and butenyl-spinosyn biosynthesis in Saccharopolyspora pogona

“…The side chain groups of butenyl-spinosyn are easily modified to generate many derivatives, and more than 30 derivatives have been isolated and identified so far [ 4 ]. However, the low yield of wild-type butenyl-spinosyn under natural conditions hinders its industrial production and application [ 8 , 9 ]. Initially, culture media optimization, physical or chemical mutagenesis, and gene rearrangement have been used to explore the potential productivity of secondary metabolites from Streptomyces [ 10 , 11 ].…”

“…Preliminary studies have been conducted on the metabolic pathways and regulatory mechanisms of butenyl-spinosyn biosynthesis [ 14 ]. Genome sequencing comparison has revealed that the PKS gene in the butenyl-spinosyn biosynthetic gene cluster is very similar to spinosyn (91%–94%), and there are many similarities in their biosynthetic and metabolic pathways [ 8 ].The main difference is that of the 5 functional domains encoded by busA in the butenyl-spinosyn gene cluster, a butenyl group replaces the ethyl group at position C21 [ 15 , 16 ]. Short-chain acyl-CoAs, such as acetyl-CoA, malonyl-CoA, methylmalonyl-CoA and propanoyl-CoA, are important precursors for the synthesis of many polyketide secondary metabolites, including butenyl-spinosyn [ 15 , 17 ].…”

“…Previous studies have shown that the tetR family of transcriptional regulators play an important regulatory role in the growth and biosynthesis of butenyl-spinosyn in S. pogona [ 18 , 19 ]. Analysis of metabolic pathways, targeted overexpression of key modules in the biosynthetic process or deletion of competitive PKS gene clusters can significantly increase the production of butenyl-spinosyn [ 8 ]. Song, et al used RedEx technology to replace different modules based on the gene cluster of spinosyn and performed heterologous expression in Streptomyces albicans , successfully synthesizing butenyl-spinosyn [ 20 ].…”

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

“…Additionally, though lots of studies such as proteomics and metabolomics have been done on spinosyns biosynthesis process, the linkage between spinosyns formation and primary metabolism network still remained un-revealed 32 – 36 . In this study, we try to find out the different metabolism processes in wild-type strain S. spinosa ATCC_49460 and spinosad high-yield strain S. spinosa S3-3.…”

rpoB and efp are stable candidate reference genes for quantitative real-time PCR analysis in Saccharopolyspora spinosa

“…To identify the key steps in spinosad biosynthesis of S. spinosa, the most commonly used reference gene was DNA polymerase sigma factor subunit A (sigA) [15][16][17]. Also, 16S rRNA was selected as the reference gene for revealing the metabolism difference between Saccharopolyspora pogona and S. spinosa, as well as seeking the link between primary metabolism and spinosad biosynthesis [18,19]. And ribosome protein L13 (rbL13) was also selected to determine the transcript levels of rex, ctyA and ctyb, which were related to the redox control in S. spinosa [20].…”