A TetR family transcriptional regulator, SP_2854 can affect the butenyl-spinosyn biosynthesis by regulating glucose metabolism in Saccharopolyspora pogona

Abstract

Background: Butenyl-spinosyn produced by Saccharopolyspora pogona exhibits strong insecticidal activity and a broad pesticidal spectrum. Currently, important functional genes involve in butenyl-spinosyn biosynthesis remain unknown, which leads to difficulty in efficiently understanding its regulatory mechanism, and improving its production by metabolic engineering.

Results: Here, we identified a TetR family transcriptional regulator, SP_2854, that can positively regulate butenyl-spinosyn biosynthesis and affect strain growth, glucose consumption, and mycelial morphology in S. pogona. Using targeted metabolomic analyses, we found that SP_2854 overexpression enhanced glucose metabolism, while SP_2854 deletion had the opposite effect. To decipher the overproduction mechanism in detail, comparative proteomic analysis was carried out in the SP-2854 overexpressing mutant and the original strain, and we found that SP_2854 overexpression promoted the expression of proteins involved in glucose metabolism.

Conclusion: Our findings suggest that SP_2854 can affect strain growth and development and butenyl-spinosyn biosynthesis in S. pogona by controlling glucose metabolism. The strategy reported here will be valuable in paving the way for genetic engineering of regulatory elements in actinomycetes to improve important natural products production.

Keywords: Butenyl-spinosyn; Metabolic engineering; Omics analysis; SP_2854; Saccharopolyspora pogona.

Fig. 1

Deletion and overexpression of SP_2854. A Transcription levels of SP_2854 at different time points via qRT–PCR analysis. B Neighbour-joining (NJ) distance tree constructed using the amino acid sequence SP_2854 from actinomycetes using MEGA6. C Schematic diagrams of the S. pogona-ΔSP_2854 mutant. D Schematic diagrams of the S. pogona::SP_2854 mutant. E PCR identification of the S. pogona-ΔSP_2854 mutant. Wild-type S. pogona genomic DNA was used as the control. Lane M, DL5000 DNA marker; Lanes 1 and 2, S. pogona-ΔSP_2854 mutant; Lane 3, control. The tested transformant showed a 2.3-kb PCR amplicon, whereas the control showed no band in that location. F PCR identification of the S. pogona::SP_2854 mutant. Wild-type S. pogona genomic DNA was used as the control. Lane M, DL2000 DNA marker; Lanes 1–4, S. pogona::SP_2854 mutant; Lane 5, control. The tested transformant showed a 1.3-kb PCR amplicon, whereas the control showed no band that location. G Sequencing of the S. pogona-ΔSP_2854 mutant PCR amplicon

Fig. 2

SP_2854 positively regulates butenyl-spinosyn production in S. pogona. A Comparison of butenyl-spinosyn production in wild-type S. pogona and the S. pogona-ΔSP_2854 and S. pogona::SP_2854 mutants via HPLC analysis. Eight-day fermentation liquid was pretreated for HPLC analysis. B Genetic organization of the bus cluster in S. pogona. C Effects of SP_2854 deletion and overexpression on the transcription level of the bus cluster. The cells of the different strains were inoculated into SFM and cultured at 30 °C for 4 days. Total RNA was then isolated and used for qRT–PCR assays. The control strain was wild-type S. pogona. 16S rRNA served as the normalization control

Fig. 3

Effects of SP_2854 deletion and overexpression on strain growth, glucose consumption and mycelial morphology. A Growth curve analysis of wild-type S. pogona and the S. pogona-ΔSP_2854 and S. pogona::SP_2854 mutants in SFM. B Comparison of glucose utilization in wild-type S. pogona and the S. pogona-ΔSP_2854 and S. pogona::SP_2854 mutants in SFM. C Effects of SP_2854 deletion on the transcription levels of PTS^Glc genes involved in glucose uptake. The cells of the different strains were inoculated into SFM and cultured at 30 °C for 4 days. Total RNA was then isolated and used for qRT–PCR assays. The control strain was wild-type S. pogona. 16S rRNA served as the normalization control. D Scanning electron micrographs of wild-type S. pogona and the S. pogona-ΔSP_2854 and S. pogona::SP_2854 mutants. Comparison of mycelial morphology between the mutants and wild-type S. pogona on the 2nd, 4th and 6th days

Fig. 4

Heatmap with hierarchical clustering of all samples. Different colours represent distinct relative abundances of metabolites

Fig. 5

Comparison of the abundance of intracellular metabolites in the S. pogona::SP_2854 mutant (purple) and wild-type S. pogona (pink) related to the glycolysis pathway and TCA cycle (mean values from four biological replicates)

Fig. 6

Comparison of the abundance of intracellular metabolites in the S. pogona-ΔSP_2854 mutant (green) and wild-type S. pogona (pink) related to the glycolysis pathway and TCA cycle (mean values from four biological replicates)

Fig. 7

Comparative proteomic analysis of the S. pogona::SP_2854 mutant and wild-type S. pogona. A Venn diagram of protein identification in three biological replicates. The number of proteins is shown in each area. B Statistics for differentially expressed proteins. C KEGG pathway analysis for differentially expressed proteins

Fig. 8

Effect of SP_2854 deletion on primary metabolism in S. pogona. This regulatory network is based on the analysis of differentially expressed proteins and metabolites associated with glucose transport, glycolysis, the pentose phosphate pathway and the TCA cycle. Reactions are reported according to KEGG metabolic pathway databases. A triangle represents a change in protein abundance, and an arrow represents a change in metabolite abundance

Fig. 9

Regulatory network schematic diagram of SP_2854. The schematic diagram showed that SP_2854 can regulate the glucose metabolism and the bus cluster expression to promote butenyl-spinosyn biosynthesis in S. pogona. Red arrow: the pathway was enhanced; Cyan arrow: the regulatory effect of SP_2854