Production of isobutanol by pyruvate decarboxylase-deficient Saccharomyces cerevisiae

Abstract

Global environmental problems and high oil prices are driving the development of technologies for synthesizing alternative liquid biofuels from renewable resources as transportation energy. Compared to ethanol traditionally used as a gasoline additive, branched-chain higher alcohols exhibit significant advantages such as higher energy density, lower hygroscopicity, lower vapor pressure and compatibility with existing transportation infrastructures. Isobutanol is regarded as a next generation transportation fuel of good quality and so microbial production of isobutanol from cellulosic biomass has been done extensively. In this study, Saccharomyces cerevisiae was metabolically engineered to produce isobutanol. This strain has been traditionally used for industrial production of ethanol because of high tolerance against alcohols and many genetic tools. Naturally S. cerevisiae produces a little isobutanol by the valine biosynthesis pathway and the Erlich pathway. To strengthen the isobutanol biosynthetic pathway, the modified endogenous ILV2 gene from S. cerevisiae without the mitochondria targeting sequence, the ilvC and ilvD genes from Escherichia coli and the kivD gene from Lactobacillus lactis were overexpressed in S. cerevisiae. ILV2 is coding the acetolactate synthase (ALS), ilvC and ilvD are ketoacid reductoisomerase (KARI) and dihydroxyacid dehydratase (DADH) and kivD is ketoacid decarboxylase (ADH). ALS, KARI, DADH and ADH are the enzymes necessary fo isobutanol biosynthesis. The constructed strain produced 120 mg/L isobutanol from glucose, along with production of ethanol as a major metabolite. To improve isobutanol production through eliminating ethanol production, a pyruvate decarboxylase (Pdc)-deficient mutant (SOS4) was used as a host for isobutanol production, which is a non-ethanol producing and pyruvate accumulating strain. Pyruvate is a key intermediate for isobutanol production. When the modified endogenous ILV2 gene, ilvC and ilvD genes from E. coli and kivD gene from L. lactis were overexpressed in the SOS4, the resulting strain was able to produce 283 mg/L isobutanol from glucose in 144 h. Acetohydroxyacid reductoisomerase and dihyroxyacid dehydratase encoded by ilvC and ilvD genes act in mitochondria of S. cerevisiae naturally. Also these enzymes are presumed to be expressed in mitochondria in the yeast because the ilvC and ilvD genes have the specific sequences for mitochondria targeting. So the modified ilvC and ilvD genes without the specific sequences were used and the resulting strain produced 326mg/L isobutanol from glucose in 144 h. Additionally, to increase an expression level of all four genes involved in the isobutanol biosynthetic pathway, an existing GPD promoter was replaced with the truncated HXT7 promoter known as a strong promoter. The resulting strain produced 446 mg/L isobutanol from glucose in 144 h, which was about 15-fold higher than the wild type strain. Isobutanol production from xylose that is abundant in lignocellulosic hydrolyzate would make the production of isobutanol more sustainable and economical. However S. cerevisiae cannot utilize xylose as a carbon source, the XYL1, XYL2 and XYL3 genes coding for xylose reductase (XR), xylitol dehydrogenase (XDH) and xylulokinase (XK) derivied from Schefferosomyces stipitis were introduced into the SOS4 for xylose fermentation. The resulting strain (SOS4X) accumulated pyruvate by utilizing xylose without ethanol production. By introducing the isobutanol biosynthetic system into the SOS4X, the resulting strain produced 121 mg/L isobutanol from xylose in 144h. These results suggest that S. cerevisiae might be a promising host for producing isobutanol from lignocellulosic biomass for industrial applications.