Flaviolin-Like Gene Cluster Deletion Optimized the Butenyl-Spinosyn Biosynthesis Route inSaccharopolyspora pogona

Abstract

Reduction and optimization of the microbial genome is an important strategy for constructing synthetic biological chassis cells and overcoming obstacles in natural product discovery and production. However, it is of great challenge to discover target genes that can be deleted and optimized due to the complicated genome of actinomycetes. Saccharopolyspora pogona can produce butenyl-spinosyn during aerobic fermentation, and its genome contains 32 different gene clusters. This suggests that there is a large amount of potential competitive metabolism in S. pogona, which affects the biosynthesis of butenyl-spinosyn. By analyzing the genome of S. pogona, six polyketide gene clusters were identified. From those, the complete deletion of clu13, a flaviolin-like gene cluster, generated a high butenyl-spinosyn-producing strain. Production of this strain was 4.06-fold higher than that of the wildtype strain. Transcriptome profiling revealed that butenyl-spinosyn biosynthesis was not primarily induced by the polyketide synthase RppA-like but was related to hypothetical protein Sp1764. However, the repression of sp1764 was not enough to explain the enormous enhancement of butenyl-spinosyn yields in S. pogona-Δclu13. After the comparative proteomic analysis of S. pogona-Δclu13 and S. pogona, two proteins, biotin carboxyl carrier protein (BccA) and response regulator (Reg), were investigated, whose overexpression led to great advantages of butenyl-spinosyn biosynthesis. In this way, we successfully discovered three key genes that obviously optimize the biosynthesis of butenyl-spinosyn. Gene cluster simplification performed in conjunction with multiomics analysis is of great practical significance for screening dominant chassis strains and optimizing secondary metabolism. This work provided an idea about screening key factors and efficient construction of production strains.

Mechanistic insight for improving butenyl-spinosyn production through combined ARTP/UV mutagenesis and ribosome engineering in Saccharopolyspora pogona

“…In recent decades, considerable efforts have been devoted through genetic manipulation to enhance the production of butenyl-spinosyn in S. pogona . Deleting the flaviolin-like gene cluster in S. pogona resulted in a 4.06-fold increase in the production of butenyl-spinosyn, reaching 107.3 mg/L in shake flask experiments ( He et al, 2021b ). Overexpressed polynucleotide phosphorylase gene using a strong ermE* promoter, which resulted in a 1.92-fold increase in butenyl-spinosyn yield ( Li et al, 2018 ).…”

Exploring a High-Efficiency Genetic Transformation System for Engineering Saccharopolyspora pogona ASAGF58 To Improve Butenyl-Spinosyn Production

“…Sa. pogona, which is an actinomycete that was initially isolated from the soil, is a Gram-positive bacterium and contains a genome with a high G + C content and many BGCs, most of which are usually silent or expressed at very low levels. For example, although butenyl-spinosyn is synthesized in Sa.…”