How to Find New Enzymes for Making Cellulosic Ethanol

by Emil Morhardt

Nonedible agricultural waste plant material is the most abundant ready source of biomass for making ethanol. But this “cellulosic” ethanol is expensive because breaking down the lignocellulose in plant waste so it can be fermented to ethanol requires either large amounts of energy, or specialized enzymes that are costly to manufacture. Furthermore, the enzymes discovered so far are not as resistant as they might be to the high temperature and high solids (low water) environments that work best for industrial processing.

One way to discover new candidate enzymes is to look for them where they are being produced naturally in an abundant source of agricultural waste; in this case, composting rice straw greenwaste. Genetic engineering technology makes it efficient to look not for the enzymes (proteins) directly, but for the messenger RNA that codes for them—the mRNA that is being actively transcribed from the various microbial genomes present. To distinguish the appropriate genes, a comparison can be made between the RNA products from room temperature (mesophilic) and heated (thermophilic) cultures degrading rice straw. The actual technique is to collect the mRNA, use it to create its complimentary DNA, then sequence the cDNA, looking for genes that code for protein families likely to be involved in degrading lignocellulose.

Simmons et al. (2014), a team from the University of California at Davis, Lawerence Berkley Laboratory, and Lawerence Livermore Laboratory, processed millions of RNA transcriptions and discovered that almost all of the enzymes that were overexpressed in the thermophilic microbial communities—compared to those in the mesophilic communities—are from the single Actinobacteria genus, Micromonospora, also thought to contribute to the lignocellulolytic activity found in termite guts. The next step is to produce enzymes from these cellulase and polysaccharide monooxygenase genes, and test them to see if they are better than the current round oflignocellulolytic enzymes.

Simmons, C.W., Reddy, A.P., D’haeseleer, P., Khudyakov, J., Billis, K., Pati, A., Simmons, B.A., Singer, S.W., Thelen, M.P., VanderGheynst, J.S., 2014. Metatranscriptomic analysis of lignocellulolytic microbial communities involved in high-solids decomposition of rice straw. Biotechnology for Biofuels 7, 495.

http://www.biotechnologyforbiofuels.com/content/pdf/s13068-014-0180-0.pdf

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