Enhanced production of isobutanol in engineered Saccharomyces cerevisiae by blocking competitive pathways

Abstract

As the global warming and climate change caused by petroleum-based fuels are intensifying, the research has actively conducted to develop bio-based fuels. Among the various candidates for ideal biofuels, isobutanol has valuable properties as a fuel such as high energy density, high octane number, low hygroscopicity and low vapor pressure. Also, isobutanol is an important platform chemical used as precursors of various chemicals. In this study, Saccharomyces cerevisiae, which has various advantages for industrial production, was used as a host strain to produce isobutanol. Isobutanol is synthesized via the L-valine biosynthesis pathway and Ehrlich pathway in S. cerevisiae. In previous studies, engineered S. cerevisiae, which expresses the enzymes involved in isobutanol biosynthesis in the cytosol, was constructed. But an isobutanol yield of the engineered S. cerevisiae was still low. Also, there are several competitive pathways for isobutanol production in S. cerevisiae

biosynthetic pathways for L-valine, L-isoleucine, L-leucine, isobutyrate and ethanol biosynthesis. In this study, the competitive pathways were eliminated by the CRISPR-Cas9 gene editing method and by overexpressing acetolactate synthase (ALS) from Bacillus subtilis, ketolacid reductoisomerase (KARI) from Escherichia coli, dihydroxyacid dehydratase (DHAD) and ketoacid decarboxylase (KDC) from Lacococcus lactis, an isobutanol yield based on glucose was improved a 8.6-fold higher than the corresponding value of the control strain. In batch cultivation in a bioreactor with gas trapping, this strain produced 662 mg/L isobutanol with 6.7 mg/g of yield. This study indicates that competitive pathways are one of the causes for the low production of isobutanol in S. cerevisiae. By totally blocking the ethanol synthesis and screening another KDC with higher enzyme activity, it would be possible to improve the production of isobutanol in S. cerevisiae.