[CCoE Notice] Dissertation Defense: Engineering Recombinant Escherichia coli for Improved Triacetic Acid Lactone (TAL) Production by High-throughput Screening Using Endogenous TAL Sensor

[Grayson, Audrey A]

PhD DEFENSE STUDENT: Ye Li
DATE: April 4, 2016
TIME: 8:30 AM
PLACE: Chemical Engineering Conference Room
DISSERTATION CHAIR: Dr. Patrick Cirino
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TITLE:
Engineering Recombinant Escherichia coli for Improved Triacetic Acid Lactone (TAL) Production by High-throughput Screening Using Endogenous TAL Sensor
Triacetic acid lactone (TAL) is a signature byproduct of many polyketide synthases, resulting from offloading of the corresponding unreduced triketide. TAL is a precursor to a wide variety of commodity and higher-value chemicals, and TAL biosynthesis therefore represents renewable production of a platform chemical. In a previous study by our group, an endogenous TAL sensor-reporter system was developed by engineering the Escherichia coli regulatory protein AraC to activate gene expression in response to TAL. The sensor-reporter system was applied in a high-throughput screen to evolve Gerbera hybrida 2-pyrone synthase (2-PS) for improved TAL production when expressed in E. coli. In this thesis, high-TAL-titer E.coli expressing the 2-PS S1 variant was used as the parent strain to evolve for further improvement of TAL production. In detail, culturing conditions were firstly optimized and some osmolytes were found to be helpful for improve TAL production. Next, performance of the endogenous sensor-reporter system was improved by tuning reporter expression level for improved ON/OFF ratio. This system was then used to screen: a) a host strain genomic library with random transposon insertions, which helped for identification of three non-intuitive gene targets that led to TAL titer improved up to 30%; b) genomic overexpression libraries, which helped for identification of a pathway enzyme and two membrane proteins that led to TAL titer improved up to 50% when overexpressed. In contrast, efforts of rational modifications of several pathway genes to improve availability of precursors for 2-PS enzyme based on the stoichiometric metabolic model failed to improve TAL production from the parent strain.
In general, this work shows the optimization of high-throughput screening by fine tuning of the reporter expression level and highlights the applicability of this system to screen and isolate novel gene targets for improved biosynthesis of important platform chemicals.
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