(12c) Development of a Synthetic Biology Approach to Demystify the Target Cryptic Pathway for Novel Natural Product Discovery

The increasing availability of whole genome sequences has revised our view of the metabolic capabilities of microorganisms. Analyses of more than 500 microbial genome sequences currently in the publicly-accessible databases have revealed numerous examples of gene clusters encoding enzymes similar to those known to be involved in the biosynthesis of many important natural products. We recently developed a highly efficient one-step method, so-called “DNA assembler”, for rapid construction of a multi-gene biochemical pathway via in vivo homologous recombination in Saccharomyces cerevisiae. Here we combined this synthetic biology approach with metabolic engineering to study cryptic biosynthetic pathways. As proof of concept, one pathway from Streptomyces griseus, SGR810-815, containing a PKS/NRPS hybrid gene and several genes encoding potential tailing enzymes, was chosen as a model system. To decipher this target gene cluster, a library of promoters with different strengths was discovered and characterized and the promoter-gene cassettes were assembled by using DNA assembler. Verified constructs were selected and transformed into Streptomyces lividans for heterologous expression of the target gene cluster. qRT-PCR was used to track the transcription of each gene. It was found that the original gene cluster showed poor expression of every gene and the native promoter(s) were inactive. In contrast, our refactored gene cluster showed enhanced levels of gene expression for each gene and the levels were comparable to the native producers’. LC-MS analysis of the cell extracts from the constructs indicated that a peak with molecular weight of 510 may be the potential product of the gene cluster. The compound was purified and the structure determination by NMR is in progress. Meanwhile, gene deletion was performed to elucidate the biosynthetic mechanism of the target pathway and the minimal set of genes involved in the target pathway.