Application in Biofuel

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The attempt to reduce environmental footprint has created the urge among scientists to produce biofuels. Biofuel is a renewable source of energy and is thus a promising alternative to non-renewable natural resources such as oils and gases. The renewable sources of biofuels include plants, animals, and their wastes and by-products. 

The conversion of biomass into combustible fuel in biofuel production involves chemical reaction and fermentation of biomass. The fermentation process involves the action of microbial organisms. CRISPR/Cas technique can be used to manipulate the genome of these microorganisms involved in fermentation to improve biofuel production, biofuel tolerance, thermotolerance, and inhibitor tolerance.

The type of biofuel produced largely depends on how the biomass has been processed. While fermentation of plant sugars generates ethanol, biodiesel is generated from the chemical reaction between grease and alcohol. The by-products and end products in biofuel production can often have a toxic effect on the metabolic pathways of the microorganisms involved in the biofuel production, thus reducing the efficiency and productivity of biofuels. 

CRISPR-Cas9-based site-directed mutagenesis is capable of increasing metabolic activities in the microbial organisms, thus enhancing biofuel production. Examples of such gene alterations include CRISPR/Cas-based manipulation of the cAMP receptor protein in Escherichia coli which results in an increase in the biofuel tolerance and oxygen stress tolerance. 

Similarly, activation of stress response genes or appropriate modification of heat shock and membrane proteins can help to reduce the antimicrobial toxicity of biofuels. For example, modification of a single amino acid in pyruvate kinase and NADH dehydrogenase increases thermotolerance in Zymomonas mobilis, a Gram-negative and facultative anaerobic bacterium. Likewise, the knock-out of the gene encoding Dfg5 glycosylphosphatidylinositol-anchored membrane protein can improve thermal stability in Saccharomyces cerevisiae. CRISPR/Cas technique has been found to be useful for introducing such genetic modifications.

Different microalgal species are capable of metabolising lipids to generate biofuels. CRISPR/Cas knock-out of multiple transcription factors in several phototropic microalgal species almost doubles the lipid production that leads to an increased amount of algae biofuel production.

The generation of biofuels usually involves the use of genetically modified yeast and bacteria. Yeasts find application in the fermentation of sugars to biofuels; however, they are susceptible to the toxicity of the chemicals present during the production of biofuels. CRISPR/Cas technology can be used to make genetic modifications in the yeasts that render them resistant to these chemicals. For example, CRISPR/Cas-based manipulation of MSN2, a transcription factor gene regulating stress in Saccharomyces cerevisiae can improve ethanol tolerance. Similarly, a single amino acid modification in ADH3, SKS1, GIS4, and ASG1 genes can improve acetic acid tolerance in S. cerevisiae.

The energy demand of the increasing global population continues to rise. Therefore, effective strategies are required to meet the increasing energy demand. Thus, in the future, research should be aimed at optimising different steps of biofuel production including pretreatment, hydrolysis, fermentation, and purification to improve the overall efficiency of biofuel production. Towards this end, the production of genetically altered microorganisms with high metabolic activity, tolerance towards different stresses, resistance to toxic chemicals involved in fermentation is of utmost importance for enhanced biofuel production.