Ingenieur Vol. 75 ingenieur July 2018-FA | Page 61

Figure 2: Strategy of inverse metabolic engineering
(SSF) and consolidated bioprocessing (CBP)
simplifies the conventional process by involving
less equipment, which reduces the cost of
investment. As shown in Figure 1, a conventional
process requires two separate operations to
cater for both saccharification and fermentation.
SSF simplifies the process by combining
saccharification and fermentation into a single
unit operation.
However, the major limitations of SSF
are the different operating temperatures
between the enzymes (45-50°C) and the
fermenting microorganism (30°C). Thus, the
use of thermotolerant yeast strains such as
Kluyveromyces marxianus is necessary to solve
the incompatibility problem between the optimal
fermentation temperatures in the SSF. In addition
to high temperature tolerance, a micro-organism in
SSF must also tolerate ethanol stress, and extreme
pH and inhibitors present after lignocellulosic
biomass pre-treatment. In order to construct a
multi-stress tolerant micro-organism, there is a
need to understand the different stresses that
the micro-organism needs to withstand during
SSF, right down to the gene level. These stress
tolerant phenotypes can be accomplished through
mutagenesis, adaptation or genetic engineering.
For the purpose of elucidating the underlying
cause of the obtained phenotype, the study of
gene expression is necessary as demonstrated via
an inverse metabolic engineering (IME) strategy
(Figure 2). The analysis points to genes, whose
changes in expression influence the phenotype (cell
behaviour) positively or negatively. This then leads
to identification of one or a combination of target
genes that may cause the desired phenotype. The
identified genes and factors can then be introduced
into another strain or manipulated (deleted or
overexpressed) to achieve a similar phenotype.
New CBP initiatives include creating enzyme
production in the same tank, instead of
supplementing an external enzyme. A microbe can
be genetically modified to be able to self-produce
cellulase, hydrolyse the lignocellulosic material
and produce ethanol. It can be designed to uptake
a broad range of sugars and tolerate fermentation
stresses. Currently research is advancing towards
the microbes for CBP to make them more robust
for industrial application.
Genetically Modified Microbes for
Ethanol Production
There are a number of wild type bacteria and
fungi suitable as ethanol producers. The most
well known bacteria is Zymomonas mobilis. Both
Z. mobilis and S. cerevisiae can produce ethanol
from glucose, but not from xylose. On the other
hand, Candida shehatae, Scheffersomyces stipitis
and Pachysolen tannophilus are recognised for
being able to convert xylose to ethanol. If genetic
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