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 59