Xinying Li’s research while affiliated with Beijing Institute of Technology and other places

Butenyl-spinosyn discovered from Saccharopolyspora pogona, is a broad-spectrum bioinsecticide. In order to further improve the production, the fermentation medium of a high-production strain Sa. pogona ASAGF30A11 obtained by mutagenesis, was optimized by adding different species and concentrations vegetable oil. In our study, the effect of peanut oil on the growth and production was proved by monitoring the growth curves, key gene transcription level and content of acyl-CoA. After adding 10 g/L of peanut oil, the additional carbon sources redirected the carbon flux toward strain growth, inhibiting the synthesis of butenyl-spinosyn, while increasing biomass by approximately 1.5-fold. However, when adding 1 g/L of peanut oil, it functions as a surfactant, greatly promoting the synthesis of butenyl-spinosyn, resulting in a 1.52-fold increase in production. The research provides a promising strategy to improve butenyl-spinosyn production.

Butenyl-spinosyn, derived from Saccharopolyspora pogona, is a broad-spectrum and effective bioinsecticide. However, the regulatory mechanism affecting butenyl-spinosyn synthesis has not been fully elucidated, which hindered the improvement of production. Here, a high-production strain S. pogona H2 was generated by Cobalt-60 γ-ray mutagenesis, which showed a 2.7-fold increase in production compared to the wild-type strain S. pogona ASAGF58. A comparative transcriptomic analysis between S. pogona ASAGF58 and H2 was performed to elucidate the high-production mechanism that more precursors and energy were used to synthesize of butenyl-spinosyn. Fortunately, a PurR family transcriptional regulator TF00350 was discovered. TF00350 overexpression strain RS00350 induced morphological differentiation and butenyl-spinosyn production, ultimately leading to a 5.5-fold increase in butenyl-spinosyn production (141.5 ± 1.03 mg/L). Through transcriptomics analysis, most genes related to purine metabolism pathway were downregulated, and the butenyl-spinosyn biosynthesis gene was upregulated by increasing the concentration of c-di-GMP and decreasing the concentration of c-di-AMP. These results provide valuable insights for further mining key regulators and improving butenyl-spinosyn production. Key points • A high production strain of S. pogona H2 was obtained by⁶⁰Co γ-ray mutagenesis. • Positive regulator TF00350 identified by transcriptomics, increasing butenyl-spinosyn production by 5.5-fold. • TF00350 regulated of butenyl-spinosyn production by second messengers.

As a plant‐derived pentacyclic triterpenoid, β‐amyrin has been heterogeneously synthesized in Saccharomyces cerevisiae. However, β‐amyrin is intracellularly produced in a lower gram scale using recombinant S. cerevisiae, which limits the industrial applications. Although many strategies have been proven to be effective to improve the production of β‐amyrin, the intracellularly accumulation is still a challenge in reaching higher titer and simplifying the extraction process. To solve this problem, the amphiphilic β‐cyclodextrin (β‐CD) has been previously employed to aid the efflux of β‐amyrin out of the cells. Nevertheless, the supplemented β‐CD in the medium is not consistent with β‐amyrin synthesis and has the disadvantage of rather high cost. Therefore, an aided‐efflux system based on in situ synthesis of β‐CD was developed in this study to enhance the biosynthesis of β‐amyrin and its efflux. The in situ synthesis of β‐CD was started from starch by the surface displayed cyclodextrin glycosyltransferase (CGTase) on yeast cells. As a result, the synthesized β‐CD could capture 16% of the intracellular β‐amyrin and improve the total production by 77%. Furthermore, more strategies including inducing system remodeling, precursor supply enhancement, two‐phase fermentation and lipid synthesis regulation were employed. Finally, the production of β‐amyrin was increased to 73 mg/L in shake flask, 31 folds higher than the original strain, containing 31 mg/L of extracellular β‐amyrin. Overall, this work provides novel strategies for the aided‐efflux of natural products with high hydrophobicity in engineered S. cerevisiae.