Genetic Engineering of Yeast with Novel Properties Using Localized Hypermutation

Abstract

Bioethanol is a promising alternative to fossil fuels, but is currently too cost prohibitive. Bioethanol is produced from the fermentation of sugars derived from plant biomass by bacteria and yeasts such as S. cerevisiae. Improving the fermentation step by engineering more efficient microorganisms can help reduce costs. The gold standards for strain development in biofuel research are UV or chemical mutagenesis and laboratory evolution. These approaches are limited by their low mutation rates and nonspecificity, which make engineering of polygenic traits difficult. This study investigated using an inducible double-strand break and human APOBEC3A to generate localized hypermutation as a method for targeting genes of interest at high mutation rates in S. cerevisiae. I hypothesized that localized hypermutation can be a useful tool for strain development that complements traditional techniques. Expression of APOBEC3A in S. cerevisiae increased the single gene inactivation frequency in a reporter system by 2 orders of magnitude and the double gene inactivation frequency by 1 order of magnitude. S. cerevisiae expressing XYL1 and XYL2 from S. stipitis and subjected to localized hypermutation yielded mutants with 1.8-fold higher growth rate on xylose over adapted-only controls. Using localized hypermutation also facilitated the generation of glucose and galactose co-utilizing mutants. By producing mutants with improved growth on xylose and mixed-sugar media, these results demonstrate the applicability of localized hypermutation to not only the biofuel industry, but to biotechnology in general.