Heterologous Expression of Xylose Isomerase from Clostridium Phytofermentans in Saccharomyces Cerevisiae for Single Step Conversion of Xylose into Xylulose and Enhanced Ethanol Production

Abstract

Second generation bioethanol produced from lignocellulosic and hemicellulosic biomass presents more advantage as alternative to fossil fuels due to abundance of feed-stocks, its' renewable nature, less GHG emission and no concern of food vs. fuel unlike first generation bioethanol. Saccharomyces cerevisiae, an organism of choice for production of both first and second generation bioethanol is unable to use the pentose fraction (xylose) of lignocellulosic hydrolysates, the second most abundant sugar following glucose. The inability of S. cerevisiae to use pentose sugar on lignocellulosic hydrolysates may be due to lack of xylose specific transporter and enzyme system in yeast to drive xylose in central metabolic pathway. In this study, recombinant strain of S. cerevisiae MKY09 (MKY09B2) is generated by heterologous expression of codon optimized xylose isomerase (XI) gene from Clostridium phytofermentans in episomal plasmid construct pGPD2+XI, where XI gene is flanked by constitutive promoter GPD and CYC1 terminator (i.e. GPD-XI-CYC1). Transformation of yeast was confirmed by PCR, southern blotting and was also visualized by fluorescence microscopy using DAPI staining. Both control strain (MKY09D2) and XI-recombinant strain (MKY09B2) exhibited similar trend in glucose use and slightly different growth kinetics. The XI-recombinant strain used more amount of xylose accompanied by higher biomass and extended lag phase (and delayed stationary phase) in YNBX media. Strain MKY09B2 was found to yield slightly higher amount of ethanol than MKY09D2 irrespective of sugar supplemented in media. Strain MKY09B2 was also found to utilize higher amount of xylose may be through co-consumption with glucose when both sugars were provided in media followed by subsequent increase in ethanol yield.

Keywords: Bioethanol, Saccharomyces cerevisiae, Xylose, Xylose Isomerase.