Researchers have made significant strides in enhancing the efficiency of polyketide biosynthesis through innovative genetic engineering approaches. By splitting the large busA gene, which encodes a polyketide synthase responsible for producing the insecticidal compound butenyl-spinosyn, scientists have successfully increased the biosynthetic output by 13-fold.
Modular polyketide synthases (mPKSs) are complex, multi-domain enzymes found in bacteria, and they play a pivotal role in the synthesis of various key pharmaceuticals, including antibiotics and anti-cancer drugs. One of the challenges faced with these enzymes stems from their size—often exceeding 10 kb—which can lead to the production of truncated messenger RNAs (mRNAs) and non-functional proteins, thereby reducing biosynthetic efficiency.
To address this issue, researchers focused on the busA gene, which is integral to the butenyl-spinosyn synthesis pathway. They discovered through experimentation with the bacterial host Streptomyces albus, the majority of mPKS mRNAs produced are truncated, leading to reduced translation rates for the enzymes necessary for effective polyketide synthesis.
To mitigate these effects, the researchers split the busA gene to create smaller, separately translated units, maintaining the overall genetic framework necessary for functionality. This strategic alteration not only rescued the translation of previously truncated mRNAs but also amplified the production of functional PKS subunits.
Notably, the study found, "Splitting the large busA gene rescues translation of truncated mRNAs... increases the biosynthetic efficiency of butenyl-spinosyn PKS by 13-fold." This aligns with their observations of increased expression and enzyme activity associated with the genes closer to the operon promoter.
The researchers employed advanced techniques to measure the transcription levels of the busA genes before and after splitting the gene. Their findings revealed how truncated mRNAs predominantly made up the mRNA population, affirming the need for fully intact open reading frames for reliable protein translation.
“The presence of truncated mRNAs results in greater abundance and production rate of the proteins encoded by genes closer to the operon promoter,” they noted. This discovery has important ramifications for improving the productivity of bacterial systems used for drug synthesis.
Further validating their approach, the researchers indicated this gene-splitting method enhances not only the efficiency of butenyl-spinosyn production but also opens pathways for similar strategies to optimize the biosynthesis of other significant compounds.
The research presents groundbreaking potential for future engineering of multi-domain proteins within various biosynthetic pathways, showcasing the versatility of synthetic biology applications. By addressing the long-standing challenge of truncated mRNAs, this work will likely lead to more effective and efficient production methods for pharmaceuticals derived from polyketides.
Integrative approaches such as these, combining genetic alteration with detailed mechanistic insight, provide promising avenues for advancements not only within the field of polyketide biosynthesis but also within broader biological engineering disciplines.