(127g) Applying the “DNA Assembler” Approach to Study Natural Product Biosynthetic Gene Clusters

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


The assembly of large recombinant DNA encoding a whole gene cluster represents a significant challenge in metabolic engineering and synthetic biology. We recently developed ?DNA assembler?, a highly efficient one-step method for rapid construction of a multi-gene biochemical pathway via in vivo homologous recombination in Saccharomyces cerevisiae. Here we further extended this synthetic biology approach to study natural product biosynthetic gene clusters.

In the new strategy, pathway fragments encoding the gene cluster and helper fragments carrying the genetic elements needed for DNA maintenance and replication in S. cerevisiae, E. coli, and the target heterologous expression host are amplified from the genome of the native producer and the corresponding vectors, respectively, and subsequently co-transformed to S. cerevisiae. Since PCR primers are designed to generate an overlap region between two adjacent fragments, these fragments will be assembled into a single DNA molecule, i.e. a heterologous expression plasmid, in S. cerevisiae. The isolated plasmids are transformed to E. coli for plasmid enrichment and verification, and the verified construct is directly transformed into the desired organism for heterologous expression of the target gene cluster. The desired organism could be a bacterium, a plant cell, or a mammalian cell, as long as the essential elements required to select successful transformants and maintain the exogenous DNA are present. In addition, other interesting genetic elements can be readily supplemented. For example, an integrase gene and an integration recognition site can be added if the target gene cluster must be integrated to the chromosome of the desired organism. All these features are assembled into one single plasmid in a single-step fashion.

To demonstrate the power of this approach, we studied a wide variety of gene clusters, including (i) the fosfomycin gene cluster from Streptomyces fradiae; (ii) the spectinabilin gene cluster from Streptomyces orinoci; (iii) multiple cryptic pathways from Streptomyces griseus. We demonstrated its unmatched flexibility and versatility by performing multiple tasks, including performing unmarked targeted gene disruption, locating functionally important residues, generating new, valuable metabolites through a combinatorial biology approach, and studying cryptic pathways for which the corresponding products have not been identified. Because the prerequisite for using such an approach is only the knowledge of the cluster sequence and it circumvents the limitations of the traditional approaches, our method represents a new powerful tool for discovery, characterization and engineering of natural product gene clusters.