Bioethanol from Lignocellulosic Biomass - Biotechnology in China: Biofuels and Bioenergy - page 44

species. For example, ethanol tolerance of S. cerevisiae is the highest, and more
than 20% ethanol can be tolerated by the species [ 73 ], which not only saves energy
consumption for ethanol distillation, but also for the treatment of distillage due to
the significant reduction in distillage discharged from the distillation system [ 48 ].
Moreover, S. cerevisiae prefers an acidic environment with a pH value below 4.5,
which can effectively prevent ethanol fermentation from microbial contamination,
since fermentors used by the industry for ethanol fermentation are too large to be
sterilized by vapor. In addition, although the natural Saccharomyces yeast is
unable to ferment xylose, there are other yeast species such as Pichia stipitis able
to ferment xylose.
Since the 1980s, substantial research efforts have been focused on the devel-
opment of genetically engineered Saccharomyces yeast to effectively ferment
xylose, the most abundant pentose in the hydrolysate of lignocellulosic biomass.
This was due in part to the failed attempts to discover new yeast species or strains
that could effectively co-ferment glucose and xylose to ethanol. Fortunately, the
remarkable advances in recombinant DNA techniques have provided the necessary
tools to genetically modify the yeast and made it able to co-ferment both glucose
and xylose to ethanol as described below.
Early studies had shown that S. cerevisiae can ferment xylulose to ethanol,
albeit not efficiently. Therefore, theoretically the yeast is only missing the
enzyme(s) to convert xylose to xylulose in order to be able to ferment xylose.
It was known that bacteria could convert xylose to xylulose with a single enzyme
that does not require co-factors. In contrast, the xylose-to-xylulose system from
xylose-fermenting yeasts such as P. stipitis required two enzymes, as illustrated in
Fig. 9 , which not only were very difficult to clone at that time, but also not an ideal
system as stated above.
Initially, there were nearly ten laboratories worldwide attempting to clone a
bacterial xylose isomerase gene into the yeast. Ho and co-workers at Purdue
University were the first group to accomplish the cloning of the xylose isomerase
gene from E. coli into the yeast (unpublished). However, the protein molecules
synthesized in S. cerevisiae by the cloned gene had no xylose isomerase activity.
Subsequently, other isomerase genes from different bacteria were cloned and
similar negative results were obtained. Failing to produce an active xylose
isomerase in S. cerevisiae by cloning the xylose isomerase genes, there was only
one potential approach remaining to make the yeast ferment xylose into ethanol:
cloning the xylose reductase (XR) and xylitol dehydrogenase (XD) genes from
P. stipitis. However, scientists predicted that any recombinant yeast containing
these cloned genes encoding the imperfect enzyme system would not be able to
sustain the fermentation of xylose to ethanol, and the result would only be the
production of xylitol!
In the early 1990s, three groups reported the successful cloning of the XR and
XD genes into S. cerevisiae to make the yeast ferment xylose [ 74 , 75 ]. However,
the recombinant yeast fermented xylose extremely slowly and produced little
ethanol and the main product was xylitol as predicted. In 1993, Ho's group
reported the successful development of the recombinant Saccharomyces yeast