Enhancement of isobutanol production in engineered Saccharomyces cerevisiae by optimizing cytosolic valine biosynthesis

Abstract

Global environmental problems due to fossil fuel and its depletion have promoted the development of microorganisms for synthesizing alternative liquid biofuels. Compared to ethanol, a traditional biofuel, biobutanol has significant advantages such as higher energy density, lower hygroscopicity and compatibility with existing transportation infrastructures. Especially, isobutanol has a higher octane number, a standard measure of the performance of gasoline. Also, it has been applied in various industrial fields and used as an important platform chemical. In this study, Saccharomyces cerevisiae which has been traditionally used for industrial ethanol production was engineered to produce isobutanol. Naturally S. cerevisiae produces isobutanol at low concentrations by the valine biosynthesis pathway and the Ehrlich pathway. However, there are some problems because of different location of the valine biosynthesis pathway and the Ehrlich pathway. Therefore, this thesis was focused on optimizing the cytosolic valine biosynthesis pathway for improving isobutanol production. First, investigations of various genes from bacteria involved in the valine biosynthesis pathway were conducted. The alsS gene from Bacillus subtilis which has high specificity to pyruvate was selected for expression of acetolactate synthase (ALS). The genes coding for ketolacid reductoisomerase (KARI) and dihydroxyacid dehydratase (DHAD) from Escherichia coli and Corynebacterium glutamicum were cloned and introduced to the D452-2 strain with keto acid decarboxylase (KDC) of Lactococcus lactis. All of genes which have the mitochondria targeting sequences were modified. The yeast strain containing the endogenous genes of ALS, KARI and DHAD was used as the control strain. Flask fermentations with 40 g/L glucose was carried out under micro-aerobic conditions. As a result, the isobutanol titer and yield of the strain expressing alsS from B. subtilis, ilvC and ilvD from C. glutamicum and kivD from L. lactis were the highest among engineered strains used in this study. Second, to improve the activity of DHAD, engineering of the Fe-S cluster was conducted. The Grx3 protein controls iron sensing. Cfd1 is an important factor of the cytosolic Fe-S cluster assembly. The CRISPR/Cas9 system was used to replace the GRX3 gene in the chromosome of S. cerevisiae with the CFD1 gene to construct the D_FeS strain. The D_FeS-SK_CCMDC strain was constructed by introducing alsS from B. subtilis, ilvC and ilvD from C. glutamicum and kivD from L. lactis. Compared to the D-SK_CCMDC, the strain D_FeS-SK_CCMDC produced isobutanol titer more than 60%. Finally, the D_FeS-SK_CCMDC was cultivated in a reactor with gas trapping and the fermentation condition was controlled by altering agitation speed and aeration. The final concentration of isobutanol of the D_FeS-SK_CCMDC was 246 mg/L with isobutanol yield (6.15 mg isobutanol/g glucose), corresponding to a 25% increase in titer and a 32% increase in yield than those obtained in flask fermentation. These results suggested that modulation of the cytosolic valine biosynthetic pathway in combination with optimization of a fermentation process can be a successful strategy for producing isobutanol in S. cerevisiae.