Expression Of Xylose Reductase Enzyme From Spathaspora passalidarum In Saccharomyces cerevisiae.

Abstract

Baker’s yeast (Saccharomyces cerevisiae) has been genetically engineered to
ferment the pentose sugar xylose present in lignocellulosic biomass. One of the
reactions controlling the rate of xylose utilization is catalyzed by xylose reductase
(XR).The current study describes xylose reductase from Spathasporapassalidarum
with NADH preference. According to JGI site the gene coding for this enzyme
contains 954 nucleotides and it consists of 317 amino acids. The restriction sites for
the enzymes SacII and NotI located on the 5P
´
Ptermini for both the forward and
reverse specific primers were designed using Lasergen 9.0 program. The genomic
DNA was isolated and purified from S .passalidarum. Polymerase chain reaction
(PCR) was used to amplify this gene. The amplified gene was cloned into pSN303
plasmid resulting of the pYIM1 plasmid and then transformed into Escherichia coli.
This plasmid was reisolated from E. coli, sequenced,and finally transformed into S.
cerevisiae. The yeast transformants carrying pYIM1 plasmid named YJTY1. The
specific activity of enzyme was 1.55 and 0.48 U/mg on NADH and NADPH
respectively for YJTY1. This enzyme has a natural preference for NADH which
makes it a good candidate for combination with NADP
+
Pdependent xylitol
dehydrogenase which may enable S. cerevisiae to utilize xylose under anaerobic
conditions and convert it to ethanol.