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Transcriptomic Studies of an Amorphadiene Producing E. Coli Strain in an Acka/Pta/Poxb Deletion Background

Robert H. Dahl II, Yisheng Kang, and Jay D. Keasling. Chemical Engineering, University of California, Berkeley, 201 Gilman Hall, Berkeley, CA 94720

The engineered production of the precursors to the anti-malarial drug, artemisinin, via the exogenous mevalonate pathway in Escherichia coli branches from the central carbon metabolic pathway at acetyl Co-A. In an attempt to increase production by directing carbon flow through the pathway, knockouts of the pathways converting acetyl Co-A to acetate, ackA/pta/poxB, were created in a DH1 background. Acetate is also a major fermentation product under conditions of excess glucose, which leads to lower pH and slower growth. Thus, an acetate pathway knockout might be a suitable host strain for large-scale production. This system presents a unique opportunity to study the transcriptional burden of simultaneously expressing a full exogenous pathway to produce a secondary metabolite. The coordinated overexpression of an entire pathway presents a large burden on the cell's physiology, both in depleting nutrients and introducing foreign intermediates which could have toxic side-effects. Transcriptomic tools, including microarrays and quantitative PCR, were used to study the transcriptional responses of both the endogenous and exogenous genes as a result of inducing the pathway in both the wild type and the acetate knockout DH1 strain. We present the time course and inter-strain gene expression comparisons along with the concentrations of the pathway endpoint and intermediate, acetate, and growth rate.