The PurR family transcriptional regulator promotes butenyl-spinosyn production in Saccharopolyspora pogona

Abstract

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 by ⁶⁰Co 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.

Keywords: Saccharopolyspora pogona; Butenyl-spinosyn; PurR family transcriptional regulator; Transcriptomic.

Fig. 1

Fermentation profiles of ASAGF58 and high-yield strain H2. a The process of mutant strain screening and validation. b Lethality rate and mutation rate of S. pogona. c Comparison of butenyl-spinosyn production in ASAGF58 and H2. d Comparison of glucose consumption between ASAGF58 and H2. e The biomass of ASAGF58 and H2

Fig. 2

Transcription analysis of ASAGF58 and H2. a Comparative transcriptome analysis of the DEGs between ASAGF58 and H2. b-d Bubble plot of the significant pathways with KEGG enrichment of DEGs at 72, 120, and 192 h, respectively

Fig. 3

The differences in pivotal metabolic pathways between H2 and ASAGF58 in the ribosome, oxidative phosphorylation, and purine metabolism pathways. The heatmap colors represent gene expression levels (Log2FC) for each sample, as indicated in the legend on the right. PRPP: phosphoribosyl pyrophosphate, PRA: 5-phosphoribosylamine, GAR: 5’-phosphoribosylglycinamide, FGAR: 5’-phosphoribosyl-N-formylglycinamide, FGAM: 2-(formamido)-N1-(5’-phosphoribosyl) acetamidine, AIR: aminoimidazole ribotide, CAIR: 1-(5-phospho-D-ribosyl)−5-amino-4-imidazolecarboxylate, SAICAR: 1-(5’-phosphoribosyl)−5-amino-4-(N-succinocarboxamide)-imidazole, AICAR: 1-(5’-phosphoribosyl)−5-amino-4-imidazolecarboxamide, FAICAR: 1-(5’-phosphoribosyl)−5-formamido-4-imidazolecarboxamide, IMP: inosine monophosphate, XMP: xanthosine monophosphate, GMP: guanosine monophosphate, GDP: guanosine diphosphate, GTP: gnosine 5’-triphosphate, AMP: adenosine monophosphate, ADP: adenosine 5’-diphosphate, ATP: adenosine 5’-triphosphate, purF: gutamine phosphoribosyl pyrophosphate amidotransferase, purD: phosphoribosylglycinamide synthetase, purN: phosphoribosylglycinamide formyltransferase, purS/L/Q: phosphoribosylformylglycinamidine synthase, purM: phosphoribosylaminoimidazole synthetase, purC: phosphoribosylaminoimidazole-succinocarboxamide synthetase, purB: adenylosuccinate lyase, purH: phosphoribosylaminoimidazolecarboxamide formyltransferase, guaB: IMP dehydrogenase, guaA: guanylate synthetase, gmk: guanylate kinase, adk: adenylate kinase, pyk: pyruvate kinase, hpt: hypoxanthine phosphoribosyltransferase, apt: adenine phosphoribosyltransferase, rpsA/B/C/D/E/F/G/H/I/J/K/L/M/N/O/P/Q/R2/S/T: 30S ribosomal protein, atpA/B/C/D/E/F/G/H: ATP synthase, sdhA/B/C/D: succinate dehydrogenase, nuoA/B/C/D/E/F/G/H/I/J/K/L/M/N: NADH dehydrogenase, cydA/B: cytochrome d oxidase subunit, cyoE: heme o synthase, qcrB/C: cytochrome c reductase, coxA/B/C: cytochrome c oxidase

Fig. 4

The function validation of 20 transcriptional regulators. a Flowchart of the functional validation of transcriptional regulators. b The production of butenyl-spinosyn in transcriptional regulator overexpression strain

Fig. 5

The effect of TF00350 on the growth and metabolism of S. pogona. a Comparison of butenyl-spinosyn production between ASAGF58 and RS00350. b Gene expression levels of the butenyl-spinosyn biosynthetic gene. busA/B/C/D/E: polyketide synthase, busJ: dehydrogenase, busM/L: cyclase, busF: [4 + 2]-carbocyclases, busG: rhamnosyltransferase, busH/I/K: O-methyl-transferase, busN: 3-ketoreductase, busO: 2,3-dehydratase, busP: forosamyltransferase, busQ: 3,4-dehydratase, busR: transaminase, busS: dimethyltransferase, epi: 3′5’-epimerase, gdh: NDP-glucose-dehydratase, gtt: NDP-glucose synthase, kre: 4’-ketoreductase. c Mycelial morphology comparison between ASAGF58 and RS00350 at 72 and 120 h. d Growth curve analysis and (e) Glucose consumption of ASAGF58 and RS00350

Fig. 6

Transcription analysis between ASAGF58 and RS00350. a Schematic illustration of gene expression in precursor synthesis, protein name colored as red corresponds to upregulated genes. BCAT: branched-chain amino acid aminotransferase, BCKDHA: 2-oxoisovalerate dehydrogenase E1 component, BCKDHB: 2-oxoisovalerate dehydrogenase E2 component, ACAD: acyl-CoA dehydrogenase, OXCT1: 3-oxoacid CoA-transferase subunit, ACAA: acetyl-CoA acyltransferase, HIBADH: 3-hydroxyisobutyrate dehydrogenase, GPI: glucose-6-phosphate isomerase, PFK: ATP-dependent phosphofructokinase, ALDO: 1,6-diphosphofructose aldolase, G: glucose-1P, G6P: glucose 6-phosphate, F6P: fructose 6-phosphate, FBP: fructose 1,6-bisphosphate, G3P: glyceraldehyde 3-phosphate, 4-MO: 4-methyl-2-oxopentanoate, 3-ME: S-(3-methylbutanoyl)-dihydrolipoamide-E, 3-MA: 3-methyl-1-hydroxybutyl-ThPP, 3-MeA: 3-methylcrotonyl-CoA, 3-MgA: 3-methylglutaconyl-CoA, 3-M-2-OP: 3-methyl-2-oxopentanoic acid, 2-ME: S-(2-methylbutanoyl)-dihydrolipoamide-E, 2-MBC: 2-methylbutanoyl-CoA, T-2-MA: 2-methylbut-2-enoyl-CoA, 3-H-2-MA: 3-hydroxy-2-methylbutyryl-CoA, 2-MAA: 2-methylacetoacetyl-CoA, 3-MO: 3-methyl-2-oxobutanoic acid, 3-HA: 3-hydroxyisobutyryl-CoA, MS: methylmalonate semialdehyde. b The concentration of propionyl-CoA, malonyl-CoA, and methylmalonyl-CoA in ASAGF58 and RS00350 at 72 and 120 h. c–d Upregulated and downregulated genes were detected in RS00350 and ASAGF58. e GO functional enrichment analysis of downregulated genes

Fig. 7

The regulatory mechanism of PurR on S. pogona. The concentration of (a) c-di-GMP and (b) c-di-AMP in H2 and ASAGF58. c Schematic illustration of gene expression related to purine metabolic pathway and strain growth. whiA/B: sporulation regulatory protein, whiG: RNA polymerase sigma factor, ftsK/W: cell division protein, ssgA: spore division protein, bldC: developmental transcriptional regulator