ARTP/NTG Compound Mutagenesis Improved the Spinosad Production and the Insecticidal Virulence of Saccharopolyspora Spinosa

Abstract

Spinosad is an efficient and broad-spectrum environmentally friendly biopesticide, but its low yield in wild-type Saccharopolyspora spinosa limits its further application. ARTP/NTG compound mutagenesis was used in this study to improve the spinosad titer of S. spinosa and obtain a high-yield mutant-NT24. Compared with the wild-type strain, the fermentation cycle of NT24 was shortened by 2 days and its maximum titer of spinosad reached 858.3 ± 27.7 mg/L, which is 5.12 times more than for the same-period titer of the wild-type strain. In addition, RT-qPCR, resequencing, and targeted metabolomics showed that the upregulation of the key differential genes accD6, fadD, sdhB, oadA, and gntZ caused increased metabolic flux in the tricarboxylic acid cycle and pentose phosphate pathway, suggesting that the accumulation of pyruvate and short-chain acyl-CoA was the primary cause of spinosad accumulation in NT24. This study demonstrates the effectiveness of ARTP mutagenesis in S. spinosa, and provides new insights for the mechanism of spinosad biosynthesis and metabolic engineering in S. spinosa.

Keywords: ARTP/NTG mutagenesis; Saccharopolyspora spinosa; biotechnology; spinosad.

Figure 1

ARTP/NTG compound mutagenesis in S. spinosa. (A) The lethality rates of S. spinosa under different durations of ARTP treatment. (B) Spinosad titer of high-yield mutants of S. spinosa from ARTP/NTG compound mutagenesis. (C) The lethality rates of S. spinosa under different concentrations of NTG for 1 h. (D) Subculture of NT24 and spinosad titer of each passage. Error bars show standard deviations. Univariate variance, *** p < 0.001.

Figure 2

Biological activity comparison of WT and NT24. (A) Toxic symptoms of H. armigera, S. exigua and S. litura after treated with the fermentation broth (0.5 μL/cm²) of WT and NT24 for 48 h. (B) The survival rates of H. armigera, S. exigua and S. litura after being treated with different fermentation broths of WT and NT24 for 48 h.

Figure 3

Effects of ARTP/NTG compound mutagenesis on strain growth and development. (A) Glucose consumption curve of WT and NT24. (B) Biomass accumulation curves of WT and NT24. (C) Mycelium comparison of WT and NT24 via SEM. The white arrows point to the differentiation nodes in WT. (D) Sporulation capacity comparison of WT and NT24. (E) Expression levels of genes related to sporulation and mycelium growth. Error bars are calculated from three independent determinations of mRNA abundance in each sample. Univariate variance, * p < 0.05, ** p < 0.005.

Figure 4

Transcription analysis of the spinosad biosynthetic gene cluster analyzed via RT-qPCR. (A) Main structure of the spn gene cluster and the transcription direction of each gene. (B) Transcript levels of representative spn genes. The error bars indicate the standard deviations of three biological replicates. Univariate variance, ^ns p > 0.05, ** p < 0.005, *** p < 0.001.

Figure 5

Effects of ARTP/NTG compound mutagenesis on the accumulation of spinosad and acyl-coA pool. (A) Spinosad accumulation of WT and NT24. (B) Acetyl-CoA accumulation of WT and NT24. (C) Malonyl-CoA accumulation of WT and NT24. (D) Methylmalonyl-CoA accumulation of WT and NT24. Univariate variance, ^ns p > 0.05, * p < 0.05, ** p < 0.005, *** p < 0.001.

Figure 6

Representative differential genes and KEGG enrichment pathways in NT24. (A) Bubble plot of the most significant pathways with KEGG enrichment of DEGs. (B) Transcription levels of differential genes in NT24. Univariate variance, * p < 0.05, ** p < 0.005, *** p < 0.001.

Figure 7

Effects of ARTP/NTG compound mutagenesis on primary and secondary metabolisms of NT24. The pyruvate and acyl-CoA pools are marked with a red box.