2:00pm - 3:00pm
The Miller Room 328 Havemeyer
New York, NY 10027
Metabolic engineering of microbes promises to be a cost-effective and green way to produce compounds of pharmaceutical, agricultural and industrial importance. An efficient approach to improve compound production or to create novel analogs is to build and test combinatorial libraries of the biosynthetic pathway. However, it remains technically challenging to assemble long (10s – 100s kb) and complex multi-gene pathways, and to engineer them to create pathway libraries. This is especially so in host cells with a limited toolkit for genetic engineering. One strategy is to build the pathway/library in a genetically tractable host such as the yeast Saccharomyces cerevisiae, followed by shuttling of the constructs into a heterologous production host for testing. In this talk, we present the de novo assembly of a ~ 100 kb Type I modular polyketide synthase (mPKS) pathway in the yeast chromosome using Reiterative Recombination. CRISPR/Cas9 was used to engineer the pathway to create promoter libraries in an attempt to increase polyketide production. We then developed FLIP for the efficient recovery of the pathway/library from the yeast chromosome onto shuttle plasmids. Our next step is to transfer these into Streptomyces lividans for production and testing. Finally, we established a high-throughput fluorescence polarization (FP) assay, which we plan to use to screen the promoter library to identify strains producing higher titres of the polyketide. The FP assay is high-throughput, quantitative, sensitive, cheap and easy to perform. We envision that it can be readily applied to overcome the testing bottleneck in metabolic engineering for a wide variety of target molecules.
Wednesday, June 22, 2016 at 2:00pm
Miller Room 328 Havemeyer
Department of Chemistry, Columbia University, Havemeyer Hall, 3000 Broadway, New York, NY 10027, USA | 212-854-2202 | http://chem.columbia.edu/