(132d) Uncovering Cryptic Biosynthetic Pathways Using Synthetic Biology

Authors: 
Luo, Y. - Presenter, University of Illinois at Urbana-Champaign
Shao, Z. - Presenter, University of Illinois at Urbana-Champaign


Computational analysis of sequenced genomes and metagenomes has revealed numerous gene clusters that are potentially involved in the biosynthesis of natural products.  However, many of these gene clusters produce unknown products.  Hence they are called cryptic biosynthetic pathways. 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 extended this synthetic biology approach to study cryptic biosynthetic pathways.  As proof of concept, one pathway from Streptomyces griseus, SGR810-815, containing a PKS/NRPS hybrid gene, was chosen as a model system.  To decipher this target gene cluster, seven overlapping DNA fragments covering the entire gene cluster and three helper DNA fragments carrying the genetic elements needed for DNA maintenance and replication in S. cerevisiae, E. coli, and the target heterologous expression host- Streptomyces lividans were PCR-amplified from the genomic DNA of the native producer and the corresponding vectors, respectively.  All of the resulting DNA products were subsequently co-transformed into S. cerevisiae for assembly into a single DNA molecule.  The isolated plasmids were transformed into E. coli for plasmid enrichment and verification.  Three verified constructs were selected and transformed into Streptomyces lividans for heterologous expression of the target gene cluster.  At the same time, two modified constructs, one with an ErmE promoter added in front of the whole gene cluster, the other with a different promoter added in front of each of the six pathway genes, were assembled.  First, qPCR was performed to track the transcription of each gene.  The results indicated that the original gene cluster showed poor expression of every gene and the native promoter was in-active while the ErmE promoter construct showed enhanced level of gene expression and was compatible to the native producer.  LC-MS analysis of the cell extracts from all the constructs indicated that a peak with molecular weight of 510 may be the final product of the gene cluster.  Further production and structural characterization is in progress.
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