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
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
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.
Content removed due to copyright restrictions: pp. 175-176.
Clark, T. T., Wedlock, N. N., & James, A. P. (1986). Strain improvement of the xylose-fermenting yeast Pachysolen tannophilus by hybridisation of two mutant strains. Biotechnology Letters, 8(11), 801-806.