Developing the protein quality control system in Streptomyces for selective translation of ultra-long full-length polyketide synthase mRNAs to enhance biosynthesis efficiency

Genes that encode type I polyketide synthases (PKSs), which synthesize an array of invaluable drugs, typically exceed 10 kilobases in length. Truncated messenger RNAs (mRNAs) have been demonstrated to constitute the majority of PKS mRNAs; However, a method for the selective translation of full-length PKS mRNAs has yet to be established. Here we developed a protein quality control system in <i>Streptomyces</i> to selectively translate ultra-long full-length PKS mRNAs for enhanced biosynthesis efficiency. The <i>Streptomyces</i> protein quality control (strProQC) system comprises the switch RNA, which encapsulates the start codon and the ribosome binding site within a secondary structure, and the trigger RNA, which is the complementary counterpart to the switch RNA. The trigger RNA is placed at the 3' end of the mRNA so that it can hybridize with the switch RNA to expose the translation-initiation region to ribosomes, enabling translation initiation. Truncated mRNAs do not contain the <i>cis</i>-trigger at their 3' ends, therefore, their translation cannot be initiated and full-length mRNAs are selectively translated. We initially identified strong terminators and switch-trigger pairs to establish the strProQC system. Then the switch sequences in the strProQC system were further optimized by exchanging ribosome binding sites to improve the ON state strength by 2.8 folds and the ON/OFF ratio by 31.6 folds. Finally, the refined strProQC system was utilized for the selective translation of the full-length mRNAs of the 7.8-kb spinosad PKS gene (<i>spnA</i>) and the 25.7-kb rapamycin PKS gene (<i>rapA</i>). This resulted in a 1.4-fold increase in spinosad yields and a 4.7-fold increase in rapamycin yields, respectively, when compared to the yields obtained from the nonselective translation of both full-length and truncated PKS mRNAs.