Improving polyketide biosynthesis by rescuing the translation of truncated mRNAs into functional polyketide synthase subunits

Abstract

Modular polyketide synthases (mPKSs) are multidomain enzymes in bacteria that synthesize a variety of pharmaceutically important compounds. mPKS genes are usually longer than 10 kb and organized in operons. To understand the transcriptional and translational characteristics of these large genes, here we split the 13-kb busA gene, encoding a 456-kDa three-module PKS for butenyl-spinosyn biosynthesis, into three smaller separately translated genes encoding one PKS module in an operon. Expression of the native and split busA genes in Streptomyces albus reveals that the majority ( >93%) of PKS mRNAs are truncated, resulting in a greater abundance of and a higher synthesis rate for the proteins encoded by genes closer to the operon promoter. Splitting the large busA gene rescues translation of truncated mRNAs into functional PKS subunits, and increases the biosynthetic efficiency of butenyl-spinosyn PKS by 13-fold. The truncated mRNA translation rescue strategy will facilitate engineering of multi-domain proteins to enhance their functions.

Fig. 1. Splitting BusA into smaller, separately translated PKS subunits improves biosynthetic efficiency.

a The Modular polyketide synthases (mPKSs) for butenyl-spinosyn and salinomycin. The modules in each mPKS protein are joined with linkers. The modules between mPKS proteins are organised by docking domains to form a megasynthase complex. The operons in the butenyl-spinosyn, and salinomycin PKS gene cluster are indicated with arrows. ^CDD_SlnA1, the C-terminal docking domain of the salinomycin PKS SlnA1. ^NDDSlnA2, the N-terminal docking domain of the salinomycin PKS SlnA2. ^CDD_SlnA7, the C-terminal docking domain of the salinomycin PKS SlnA7. ^NDD_SlnA8, the N-terminal docking domain of the salinomycin PKS SlnA8. TGA, stop codon. ATG, start codon. RBS, ribosomal binding site. b The three strategies used to split BusA. The linkers between the two modules were removed and replaced with exogenous docking domains from two adjacent salinomycin mPKSs. c Butenyl-spinosyn production by S. albus J1074 strains harbouring gene clusters containing wild-type busA (2.36 mg L⁻¹) or split busA indicated in (b). n = 3 independent fermentation samples. Data are presented as the mean ± S.D. The p-values of the two-tailed t test are indicated. Source data are provided as a Source Data file.

Fig. 2. Splitting AveA2 and EpoD into smaller, separately translated PKS subunits improves biosynthetic efficiency.

a The mPKSs for avermectin and the mPKS-NRPS hybrid for epothilone. The operon in the epothilone mPKS-NRPS gene cluster is indicated with an arrow. b The two strategies used to split AveA2. c Avermectin B1a production by S. coelicolor CH999 strains harbouring gene clusters containing wild-type aveA2 or split aveA2 indicated in (b). d The strategy used to split EpoD. ^CD_stiB, the C-terminal docking domain of the tigmatellin PKS StiB. ^NDD_stiC, the N-terminal docking domain of the stigmatellin PKS StiC. e Epothilone A production by S. albus J1074 strains harbouring gene clusters containing wild-type epoD or split epoD indicated in (d). n = 3 independent fermentation samples. Data are presented as the mean ± S.D. The p-values of the two-tailed t test are indicated. Source data are provided as a Source Data file.

Fig. 3. PKS mRNA truncation increases the abundance of transcripts and proteins encoded by genes closer to the operon promoter.

a The butenyl-spinosyn PKS gene cluster. The two operons are indicated with arrows. b Schematics of the wild-type and split busA genes fused with the gusA gene. Two loci on gusA indicated with asterisks are used for qRT‒PCR-based transcription analysis. c qRT‒PCR-based transcription analysis of gusA in J1074 strains harbouring butenyl-spinosyn gene clusters containing the wild-type or split busA genes from (b) using primers annealing to locus 1. d β-Glucuronidase activity assay of J1074 strains harbouring butenyl-spinosyn gene clusters containing the wild-type or split busA genes from (b). e Parallel reaction monitoring absolute quantification of BusA, BusA-1-1, and BusA-2-1 protein levels in Del14 strains expressing the busA, busA-1-1, or busA-2-1 genes. The positions of the unique peptide fragment, A₄₈₉ALVADDEPK₄₉₈, used for quantifying are indicated with #. n = 3 independent biological samples. Data are presented as the mean ± S.D. The p-values of the two-tailed t test are indicated. ns, not significant. Source data are provided as a Source Data file.

Fig. 4. Transcription analysis of the tmRNA–smpB ribosome-rescue system in S. albus J1074 harbouring the wild-type or split busA genes.

a qRT‒PCR transcription analysis of the ssrA gene (encoding the tmRNA in the tmRNA–smpB ribosome-rescue system) in J1074 strains harbouring butenyl-spinosyn gene clusters containing the wild-type or split busA genes indicated in Fig. 1b. b qRT‒PCR analysis of the smpB gene of the tmRNA–smpB ribosome rescue system in J1074 strains harbouring butenyl-spinosyn gene clusters containing the wild-type or split busA genes. n = 3 independent biological samples. Data are presented as the mean ± S.D. The p-values of the two-tailed t test are indicated. ns, not significant. Source data are provided as a Source Data file.

Fig. 5. mRNA truncation increases production rate of PKS proteins encoded by genes closer to the operon promoter.

a Schematics of the wild-type and split busA genes fused with the gusA gene under the control of the cumate-inducible promoter. b Real-time GusA (β-glucuronidase reporter) activity assay of J1074 strains containing the DNA constructs from (a). c Production rate of BusA-1-1-GusA and BusA-2-1-GusA proteins in J1074 strains after induction for 40–80 min. d Production rate of BusA-GusA, BusA-1-1-GusA, and BusA-2-1-GusA proteins in J1074 strains after induction for 80–160 min. e β-Glucuronidase activity assay of J1074 strains containing the DNA constructs from (a) after induction for 20 h. f Schematics of the wild-type and split busA genes fused with the gusA gene under the control of the cumate-inducible promoter. g Real-time GusA (the β-glucuronidase) activity of J1074 strains containing the DNA constructs from (f). h Production rate of BusA-1-2-GusA and BusA-2-2-GusA proteins in J1074 after induction for 20–30 min. i Production rate of BusA-GusA, BusA-1-2-GusA, and BusA-2-2-GusA proteins in J1074 strains after induction for 60–160 min. j β-Glucuronidase activity of J1074 strains expressing the DNA constructs from (a) after induction for 20 h. n = 3 independent biological samples. Data are presented as the mean ± S.D. The p-values of the two-tailed t test are indicated. ns, not significant. Source data are provided as a Source Data file.

Fig. 6. Transcriptional reinforcement of downstream PKS genes further increases butenyl-spinosyn production.

a Schematic of the kasOp* promoter insertion. b–d Butenyl-spinosyn production by S. albus J1074 strains harbouring gene clusters from (a). n = 3 independent fermentation samples. e qRT‒PCR analysis of the transcription of bus genes in J1074 strains harbouring the busA-3 butenyl-spinosyn gene cluster with the kasOp* promoter inserted upstream of busB, busC, busD, or busE. n = 3 independent biological samples. Data are presented as the mean ± S.D. The p-values of the two-tailed t test are indicated. Source data are provided as a Source Data file.

Fig. 7. The proposed model for transcription and translation of the native and split PKS genes.

a Transcription and translation of native PKS genes. b Transcription and translation of the split PKS genes.