Functional Expression of Pichia Stipitis XR and XDH Genes into Starter Cultures of Saccharomyces cerevisiae Obtained from Degrading Cocoa (Theobroma cacao L.) Pod Husk Biomass

Abstract

Biobased energy and fuels are among the exercisable sustainable energy options mankind has in the not-so-distant future as issues pertaining to global warming and shortfall in fossil fuels loom dark over the planet. The environmental necessity to stop this development by switching to alternative strategies nowadays is generally undisputed. Biofuel made from biomass provide unique environmental economic strategic benefit and can be considered as safe and by and large, the cleanest liquid fuel alternative to fossil fuels. Biofuel produced from Agricultural waste biomass like cocoa (Theobroma cacao L) pod husk shows many potentials advantages in comparison with sugar or starch-based stocks since the latter materials are also food for human and animals. However, the complex nature of this biomass necessitates the use of genetic techniques to produce engineered organisms that are able to transform this polymer into the desired product. With Bioinformatics tools using NCBI BLAST programme, two genes XL1 and XL2 encoding pentose utilization were isolated from the genomic DNA of Pichia stipitis (CBS 6054) and two primers each were designed to span the full coding region of these genes with attached enzymes restriction sites using DNA strider 1.4f7 and Macplasmap programmes. PCR reactions were carried out on 120hg of the isolated genomic DNA for 30 cycles using the DNA Gotaq polymerase enzyme. The amplified PCR fragments were introduced into plasmid vectors pGAPZA and pVT100-U respectively and the constructs were then used to transform the selected ethanol-producing strain of S. cerevisiae (BY4743) isolated from degrading cocoa pod husk biomass meant to serve as starter for biofuel production from cocoa pod husk hydrolysate.