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(672a) Computational and Experimental Exploration of Sequence-Structure-Function Relationships of a Medium-Chain Acyl-ACP Thioesterase

Jindra, M. - Presenter, The Ohio State University
Ghaffari, S., The Pennsylvania State University
Chowdhury, R., Harvard Medical School
Luke, T., The University of Wisconsin-Madison
Maranas, C. D., The Pennsylvania State University
Pfleger, B., University of Wisconsin-Madison
Enzymes which perform industrially relevant chemistry can provide selectivity otherwise difficult to achieve from chemical synthesis or agriculture. Engineering the acyl-ACP thioesterase in fatty acid biosynthesis has proven an effective approach to tailor the fatty acid distribution toward C8, C10, and C12 products. To date, engineering of acyl-ACP thioesterases with a preference for the corresponding medium-chain substrates has been successful with directed evolution and rational mutagenesis techniques. However, fundamental understanding of the factors driving thioesterase chain-length selectivity remains elusive. Here, we apply a computational method to tune substrate binding and explore the sequence-structure-function relationships of an acyl-ACP thioesterase from Umbellularia californica. We used the Iterative Protein Redesign and Optimziation (IPRO) algorithm to computationally design thioesterase variants with improved in silico binding to C8, C10, and C12 substrates. The pre-screened thioesterase variants were transformed in an Escherichia coli fatty acid production strain to test for changes in fatty acid chain-length distribution from the wild type thioesterase. In this talk, I will describe how we navigated through the design, build, and test cycle to enhance the learnings from each round of mutagenesis.