(132a) Rapid Characterization and Engineering of Natural Product Biosynthetic Pathways Via DNA Assembler

Microorganisms and plants have been evolved to produce a myriad array of complex molecules known as natural products or secondary metabolites of pharmaceutical and biological importance.  Sequenced genomes and metagenomes provide a tremendously rich source for discovery of new genes and pathways.  However, the discovery and sustainable production of valuable natural products are often hampered owing to our limited ability to manipulate the corresponding biosynthetic pathways.  Here we developed a synthetic biology strategy based on the DNA assembler method we developed previously for discovery, characterization, and engineering of natural product biosynthetic pathways.

By taking advantage of the highly efficient yeast in vivo homologous recombination mechanism, this strategy synthesizes an entire expression vector containing the target biosynthetic pathway and the genetic elements needed for DNA maintenance and replication in Saccharomyces cerevisiae, Escherichia coli, and a target heterologous expression host in a single-step manner.  Since all the fragments are prepared by PCR, various manipulations including site-directed mutagenesis, scar-less gene deletion and artificial gene cluster construction can be easily achieved.  Such a strategy with unprecedented flexibility and versatility has many applications in heterologous expression, pathway functional studies, and combinatorial biosynthesis.

As proof of concept, two important biosynthetic pathways from Streptomyces including the aureothin and spectinabilin biosynthetic pathways were selected for characterization and engineering.  Aureothin, isolated from Streptomyces thiolutes, is a nitro-aryl containing polyketide exhibiting antitumor, antifungal, and insecticidal activities.  Using DNA assembler, we reassembled the aureothin biosynthetic pathway in the heterologous host Streptomyces lividans, and mutagenized the active site residues of the dehydratase domains and the ketoreductase domains in AurA and AurB.  As a result, two aureothin derivatives were generated by the mutated pathways, with their corresponding polyketide chain modified.  Spectinablin, isolated from Streptomyces spectabilis and Streptomyces orinoci, is a nitro-phenyl substituted polyketide exhibiting antimalarial and antiviral activities.  Although both hosts were reported to produce spectinabilin, only the pathway from S. spectabilis can be functionally expressed in S. lividans.  Examining the two gene clusters revealed two major differences.  First of all, the cluster from S. spectabilis contains three extra genes, spnK, spnL and spnM, which are not found in S. orinoci.  Secondly, these two clusters undergo different regulations as the cluster from S. spectabilis contains SpnD as an activator and the cluster from S. orinoci contains NorD as a repressor.  Previous bioinformatics study showed that these three genes are only involved in up-regulating substrate concentration and product transport.  To investigate the mechanisms for the failure of heterologous spectinabilin production, spnK was inactivated and spnM and spnL were deleted from the cluster via DNA assembler.  As a result, all three mutants could still synthesize spectinabilin in S. lividans, indicating these three genes are not essential for spectinabilin biosynthesis, supporting our bioinformatics study.  Therefore, we hypothesized the silencing of the spectinabilin biosynthesis is due to the altered regulation in S. lividans.