Abstract
Objectives: Isobutanol is regarded as a next-generation biofuel for its higher octane number and higher energy density than ethanol. However, during isobutanol biosynthesis, ethanol and glycerol are major unwanted byproducts. In order to improve isobutanol production in Saccharomyces cerevisiae, we used molecular biology and genetic recombination technologies to eliminate ethanol and glycerol titers. Methods: In this study, GPD2 and PDC6 were deleted to increase isobutanol production in microaerobic fermentation of Saccharomyces cerevisiae. Engineered strain HZAL–13 (PGK1p–BAT2 gpd2Δ::RYUR) was constructed by overexpressing of BAT2 (which encodes a branched-chain amino-acid aminotransferase) and deleting GPD2 (which encodes glycerol-3-phosphate dehydrogenase). Engineered strain HZAL–14 (PGK1p–BAT2 pdc6Δ::R gpd2Δ::RYUR) was obtained by further deleting PDC6 (which encodes pyruvate decarboxylase) in HZAL– 13 pILV2. Then we tested the fermentation performances of engineered strains and control strain. During microaerobic fermentation, cultures were performed at 30°C in the unbaffled shake flasks kept at constant stirring speed of 100 rev/min with 100 ml medium for 48 hours. Results: The maximum isobutanol titers of control strain, HZAL–13 pILV2 and HZAL–14 pILV2 were 29.8 mg/l, 162.3 mg/l and 309.3 mg/l, respectively. These results demonstrate that decreasing glycerol formation and ethanol biosynthesis in combination through deletion of PDC6 and GPD2 could increase dramatically the isobutanol titer in S. cerevisiae. Conclusion: Overexpression of related genes in isobutanol biosynthesis pathway and deletion of key genes that encode glycerol and ethanol biosynthesis is a promising strategy to increase isobutanol titer in Saccharomyces cerevisiae.
Highlights
Environmental friendly biofuels gain great interests due to climate change and the need for renewable transportation fuels in recent years [1]
YEplac181), HZAL–13 pILV2 and HZAL–14 pILV2 were studied under microaerobic condition
The growth rate of the two-engineered strains HZAL–13 pILV2 and HZAL–14 pILV2 were both slower than that of control strain. These results were further more supported by the fact that the consumption rates of glucose in HZAL–13 pILV2 and HZAL–14 pILV2 lagged a little compared to control strain (Figure 2b)
Summary
Environmental friendly biofuels gain great interests due to climate change and the need for renewable transportation fuels in recent years [1]. Ethanol has been received more attention as the most widely used biofuel [2]. Compared to ethanol, higher alcohols have several advantages as next-generation transport fuels. They are compatible with current infrastructure [3]. Isobutanol exhibits superior physicochemical properties such as higher energy density and lower hygroscopicity than ethanol [4,5]. It has higher octane number than the isomer n-butanol [6,7]
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