Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Combinatorial Library Design for Improving Isobutanol Production in Saccharomyces cerevisiae

Authors:

Joshua Dietrich1,2* (jdietrich3@wisc.edu), Francesca Gambacorta1,2, Justin Baerwald1, Stephanie Brown1, Yun Su1, Jeremy Cortez3,4, Brian Pfleger1,2, Jose Avalos3,4, Timothy J. Donohue2

Institutions:

1University of Wisconsin–Madison; 2Great Lakes Bioenergy Research Center, University of Wisconsin–Madison; 3Princeton University; 4Center for Advanced Bioenergy and Bioproducts Innovation, Princeton University

Goals

Improvement of isobutanol production in the yeast Saccharomyces cerevisiae by screening a combinatorial library of isobutanol pathway genes.

Abstract

The branched-chain alcohol isobutanol absorbs less water and has a higher energy density than ethanol, making it a promising next-generation biofuel. Isobutanol can also be catalytically upgraded to produce sustainable aviation fuel. The yeast Saccharomyces cerevisiae is well-suited for bioproduction of isobutanol given its tolerance to stressors, fast growth rate, and extensive genetic toolkit. However, the native, robust ethanol fermentation of S. cerevisiae hinders isobutanol production. To increase isobutanol production in S. cerevisiae, researchers created a combinatorial library from a diverse set of isobutanol pathway genes. For each of the five enzymatic reactions in the isobutanol pathway, the library varied both enzyme variant and promoter strength, for a combined library size of ~1 billion unique members. Though its large size prevented comprehensive screening, researchers identified several sets of genes enabling high isobutanol production. These genes were then inserted into a strain of S. cerevisiae that cannot produce ethanol. The resulting strains produced isobutanol at yields close to the highest reported in academic literature, without producing ethanol. The inability to completely screen this library led researchers to design and assemble a smaller library of just the first three genes in the isobutanol pathway. This new library will be screened completely by using a combination of growth-coupling and a S. cerevisiae strain containing a fluorescent biosensor for isopentanol, which shares a key intermediate compound with the isobutanol pathway. Next-generation sequencing of the enriched library will reveal further insights into which gene combinations from the library are best for isobutanol production.

Funding Information

This material is based upon work supported by the Great Lakes Bioenergy Research Center, U.S. DOE, Office of Science, BER Program under Award Number DE-SC0018409.