Studies Using a Genetically Engineered Saccharomyces Yeast

M. S. Krishnan1’2, Y. Xia1, N. W. Y. Ho1, and G. T. Tsao1-2

lLaboratory of Renewable Resources Engineering and 2School
of Chemical Engineering, Purdue University, West Lafayette, IN 47907

Fermentation studies of ethanol production from lignocellulosic sugars using the genetically engineered Saccharomyces yeast 1400 (pLNH33) and its parent Saccharomyces yeast strain 1400 are reported. While the parent strain 1400 is unable to ferment xylose, the recombinant yeast 1400 (pLNH33) ferments xylose and mixtures of glucose and xylose. High ethanol yields upto 84% were obtained by fermentation of glucose-xylose mixtures using the recombinant yeast. The kinetics of ethanol inhibition of yeast cell growth on glucose and xylose are presented. Results of ethanol production from com fiber and com cob by the simultaneous saccharification and fermentation (SSF) process are also reported.

Ethanol has received attention recently as an octane booster and a transportation fuel. The economics of fuel ethanol production are significantly influenced by the cost of the raw materials used in the production process. Lignocellulosic materials such as agricultural residues and municipal waste paper have been identified as potential feedstocks, in view of their ready availability and low cost (7). These lignocellulosic hydrolyzates that are produced either chemically or enzymatically contain both pentoses and hexoses. The pentoses are comprised of D-xylose and L-arabinose while the major hexose is D-glucose (2). While the glucose is readily fermented by using Saccharomyces yeasts, few microorganisms have the ability to ferment xylose. For the economics of the biomass to ethanol process, it is necessary to convert the xylose to ethanol as well. Pichia stipitis and Candida shehatae are the best wild type xylose fermenting yeasts that have been reported in the literature (3). Recent advances in molecular biology techniques have led to the development of genetically engineered microorganisms for xylose fermentation. These include recombinant bacterial strains of E. coli (4), Klebsiella oxytoca (5) and Zymomonas mobilis (6).

© 1997 American Chemical Society

Although these strains show good xylose fermentation performance, the low ethanol tolerance of these microorganisms is a limiting factor in the process. Saccharomyces yeasts have a relatively higher ethanol tolerance and hence attempts have been made to develop recombinant Saccharomyces yeasts that can ferment xylose (7,8). However, the ethanol yields and productivities are low. This has been attributed to the cofactor imbalance and an insufficient capacity for xylulose conversion through the pentose phosphate pathway.

A recombinant yeast denoted 1400 (pLNH33) has been developed by Nancy Ho and co-workers at the Laboratory of Renewable Resources Engineering, Purdue University (9,10). This strain was developed using the high ethanol tolerance Saccharomyces yeast 1400 (77) as the host and cloning the xylose reductase, xylitol dehydrogenase genes (both from Pichia stipitis) and xylulokinase gene (from S. cerevisiae) into yeast 1400. The recombinant yeast ferments glucose and xylose simultaneously to ethanol in high yields.

In this paper, we report the fermentation studies conducted on glucose, xylose and their mixtures using this recombinant yeast. Ethanol tolerance is a key factor influencing process economics, motivating us to investigate the kinetics of ethanol inhibition on these genetically engineered yeasts. Results of the simultaneous saccharification and fermentation (SSF) process using com fiber and com cob as model feedstocks are also presented.