Biomedical Engineering Reference
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1400 (pLNH32) that could ferment high concentrations of xylose almost
completely to ethanol with little xylitol accumulated. In addition, the yeast could
co-ferment glucose and xylose without a significant lag period between the
fermentation of these two sugars [ 76 ].
The Purdue strain was developed by transforming an industrial strain, 1400,
with a high copy number 2l plasmid pLNH 32, which contains the cloned and
overexpressed XR, XD and xylulokinase (XK) genes [ 76 ]. The 2l plasmid is a
broad host plasmid, designed to be able to transform any S. cerevisiae, including
industrial wild-type strains. Such a plasmid can be used to screen better hosts for
cellulosic ethanol production. Furthermore, Ho's group developed a unique new
gene integration technique, facilitating effective integration of multiple genes into
the yeast chromosome in multiple copies [ 77 , 78 ], which is easy to perform and
guarantees that the genes cloned on the integration plasmid are transferred into the
host strains and integrated into their genome in as many copies as desired to
provide the highest activity of the cloned enzymes. This technique allows the
integration of the XR-XD-XK genes together as a cassette into the yeast chro-
mosome in sufficient copies for the resulting yeast to ferment xylose efficiently.
The best strain developed by Ho's group prior to 2007 is 424A (LNH-ST), which
was screened from 10 different strains of S. cerevisiae by first transforming each of
them with the 2l plasmid pLNH32, to make sure that these strains were able to
ferment xylose as well as co-ferment glucose/xylose effectively in the presence of the
plasmid, followed by integrating genes into the chromosomes of the selected yeast
strains to develop the ''stable yeast''. The co-fermentation of glucose/xylose by
424A(LNH-ST) is shown in Fig. 10 . This strain is currently available for industry to
produce cellulosic ethanol. 424A(LNH-ST) as well as other strains developed by the
integration technique have all been validated by ethanol producers to be able to
co-ferment glucose and xylose to ethanol and also able to ferment glucose and xylose
present in actual hydrolysates from different feedstocks [ 78 ]. 424A(LNH-ST) has
also been used by companies for the production of cellulosic ethanol from wheat
straw and other feedstocks in demonstration plants as early as 2004. Dr. Ho and her
coworkers have continued to improve the strain by making it co-ferment other sugars
like arabinose, together with glucose, xylose, mannose and galactose [ 79 ], and by
making it more resistant to ethanol and acetic acid inhibition [ 80 , 81 ]. A new and
improved derivative of 424A(LNH-ST) has been developed that can ferment all
sugars present in hydrolysates produced from any cellulosic biomass and produce
more than 10% ethanol without requiring special detoxification to remove inhibitors
in the hydrolysates [ 82 ]. It will be available for industrial production of cellulosic
ethanol in the near future.
4.3 Process Integration and Optimization
Various technologies for pretreatment, enzymatic hydrolysis and fermentation
strains have been developed in recent decades for bioethanol production from
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