Investigations on the hexose-phosphorylating enzymes in the pentose-fermenting yeast, Pachysolen tannophilus : a thesis presented in partial fulfilment of the requirements for the degree in Doctor of Philosophy in Microbiology at Massey University

Abstract

Mutants of Pachysolentannophilus, resistant to2- deoxyglucose, the toxic analogue of D-glucose, have been isolated and characterised. Their growth characteristics on hexose and pentosesugars, resistance to 2-deoxyglucose and cellular hexose-phosphorylating activities were investigated. Loss of hexose-ATP-kinase activity was found to correlate with loss of ability to grow on hexose sugars and increased resistance to 2-deoxyglucose. The growth of these mutants on D-xylose was not affected. A further series of fructose-negative and glucose-negative mutants were isolated by selecting for increased resistance To 2-deoxyglucose and by UV mutagenesis. Mutants, defective in each of the three hexose-phosphorylating enzymes found to be present in this yeast, were completely negative for growth on D-glucose,but could slowly convert this sugar to D-fructose. The conversion of D-glucose to D-fructose was hypothesised to be catalysed by the enzymes xylosereductase and xylitoldehydrogenase and experiments were conducted to investigate this possibility. Cell-free extracts from the wild type strain and several of The glucose-negative mutants were chromatographed on DEAE cellulose. The results of hexokinaseassays and anion exchange chromatography confirmed the existence of three hexose-phosphorylating enzymes in P.tannophilus. Two hexokinases which phosphorylated both D-glucose and Dfructose, exhibited F/Gratiosof1.3/1.0and3.0/1.0, while a glucokinase specific for D-glucose was also present.These enzymes were referred to ashexokinaseA and Bandglucokinase. Examination of the hexose-ATP-kinase profiles on DEAE- cellulose glucose, glucokinase of the wild type extract from cells grown on DO -xylose and glycerol indicated that the andhexokinaseB were constitutive, while hexokinaseA was inducible. Glucose repression ofxylosereductase and xylitol dehydrogenase was found to require an active hexokinaseA enzyme. This enzyme was purified from a glucokinase defective mutant by DEAE-cellulose chromatography, followed by affinity chromatography on CibacronBlueF3G-ASepharose (BlueSepharose) and examined further. The Km values for D-glucose and D-fructose were 0.36 and 2.28mM respectively. An estimated Vmaxfructose/Vmaxglucose was 1.5/1.0. When incubated with D-xylose in the presence of MgCl2 and ATP, the enzyme was inactivated. A strain of Pachysolentannophilus, defective in all three hexose-phosphorylating enzymes, was transformed with a plasmid carrying the cloned PII hexokinase gene from Saccharomycescerevisiae.The gene was expressed and the presence of the enzyme within the cells was demonstrated by DEAE-cellulose chromatography of a cell-free extract. As part of the overall plan to attempt genetic improvement in P.tannophilus, two superior ethanol producing mutants were hybridised and the segregants made available for fermentation trials at the Forest Research Institute. Hexose-negative mutants able to fermen D-xyloseinthe presence of D-glucose were selected for and subjected to fermentation trials. Several of these mutants produced promising concentrations and yields of ethanol from the fermentation of D-xylose, both as a sole carbon source and in a mixture of D-glucoseandD-xylose.