Heterologous Gene Expression in Saccharomyces Cerevisiae for Higher Isobutanol Production

【Introduction】 The bio-production of isobutanol by Escherichia coli and Corynebacterium glutamicum has been attained by introducing the Ehrlich pathway (Atsumi et.al., 2008, Smith et.al., 2010). Saccharomyces cerevisiae is also one of the most promising hosts for isobutanol production since its stress tolerance against harsh conditions during fermentation is useful for a cost-effective biorefinery process.Although Ehrlich pathway was introduced into S. cerevisiae, the isobutanol production by the engineered strains were still insubstantial, indicating that further engineering was required considering intercellular cofactor balances and compartmentation of the metabolic pathways. In this study, metabolically engineered S. cerevisiaestrains for isobutanol production were constructed by the flux balance analysis (FBA)-based metabolic design and the expression of heterologous genes.

【Methods】 The FBA was performed using the backbone model of the carbon central metabolism of S. cerevisiae (iBKSce50) by MATLAB R2013a with glpk solver. The glucose and oxygen up take rates were set to 10 and 0.1 mmol gDW^-1h^-1, respectively, in all analyses. Engineered yeast strains were constructed by introducing the plasmid vectors harboring the multi-copy type 2μ ori sequence and target genes into the yeast laboratory strain YPH499 using lithium-acetate methods. The constructed strains were cultivated in 5 mL of synthetic dextrose medium under semi-anaerobic conditions. Isobutanol titers at 48 h after the start of cultivation was determined by using gas chromatography.

【Result】Introduction of bacterial pathways: Isobutanol is synthesized from the intermediate of valine biosynthesis, 2-ketoisovalate, through decarboxylation by 2-ketoacid decarboxylase and reduction by alcohol dehydrogenase. The isobutanol yield of S. cerevisiae strain over-expressing kivd gene from Lactococcus lactis, S. cerevisiae ADH6 and ILV2 were 0.78±0.17% mol/mol-glucose (YKP003 strain). The FBA-based metabolic simulation indicated that cofactor (NADPH) imbalance could be improved by additional introductions of phosphoenolpyruvate carboxylase (PPC). Based on the findings, YKP006 strain was constructed by introducing PPC coding gene from Synechocystissp. PCC 6803. The fermentation test indicated that isobutanol yield was 1.13±0.02% mol/mol-glucose and 1.4 times higher than that of control strain.

Pathway construction in cytosol: The precursor for isobutanol synthesis, 2-ketoisovalate, was produced from pyruvate via the valine biosynthesis pathway localized in mitochondria. For more improvement of isobutanol production in S. cerevisiae, a valine biosynthetic pathway has been constructed in the cytosol by expression of three S. cerevisiae genes, ILV2c, ILV5c, and ILV3c, lacking the mitochondria transit sequences (Brat et.al., 2012). For further activation of cytosolic pathway, genes for valine biosynthesis were cloned from various organisms and introduced into S. cerevisiae. Among the gene tested, overexpressions of ALS gene from Lactobacillus plantarum, KARI gene from Sulfolobus tokodaii (YKI024 strain: 1.10±0.04% mol/mol-glucose) and the DHAD gene from Zymomonas mobilis (YKI015 strain: 1.27±0.10% mol/mol-glucose) increased isobutanol yield two folds compared to the control strain (0.52±0.08% mol/mol-glucose), respectively.