Plasmid Construction for Producing Ethanol Using the Phosphoketolase Pathway in Saccharomyces cerevisae

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The extreme climate change is correlated to the increased greenhouse gas emission. Finding new alternative energies such as biomass energy, solar energy, wind energy, and biothermal energy instead of using fossil fuel become an important issue these days. The ethanol production from glucose in Saccharomyces cerevisiae involves decarboxylation reaction of pyruvate in nature. This metabolic pathway produces two molecules of carbon dixoide per molecules of glucose, resulting in carbon loss. Theoretical yield of ethanol is about 0.51 g ethanol /g glucose. The carbon loss in producing bioethanol reduces its potential as compared with other regenerative energies. In this study, we amplified xfp gene from Bifidobacterium longum and pta and mhpf genes from Escherichia coli K12 by polymerase chain reaction (PCR) for future introducing a phosphoketolase pathway into Saccharomyces cerevisiae. The PCR-amplified genes were cloned into pTEF1/Zeo vector using the Gibson mixture reagent and were designated as pTEF1-xfp, pTEF1-pta, and pTEF1-mhpf, respectively. xfp, pta, and mhpf will be amplified to include a promoter and a terminator and insert into pTEF1/Zeo with 2μori. Once this metabolic pathway is introduced into Saccharomyces cerevisiae which can produce ethanol using a non-oxidative pathway, resulting in a higher ethanol yield. The additional need for NADH for producing ethanol could be supplemented using a portion of glycerol as substrate. Keywords: Saccharomyces cerevisiae, phosphoketolase