Regulation of biomass degrading enzymes in anaerobic gut fungi and their application in synthetic co- culture systems

John K. Henske, Kevin V. Solomon, Sean P. Gilmore, Jessica Sexton, Michael K. Theodorou, Michelle A.
Oâ??Malley
To support renewable technologies, it is necessary to develop more efficient methods to extract sugars from crude plant biomass (lignocellulose). Plants contain cellulose that depolymerizes into fermentable sugars for microbial biofuel production. However, in crude biomass, cellulose is trapped within lignin, hemicellulose and other biopolymers that complicate its hydrolysis. To address this issue, one can turn to nature, particularly to microbes that routinely degrade plant biomass. Many large herbivores, such as cows and horses, harbor a consortium of microbes in their digestive tracts that convert recalcitrant biomass into sugars. Within this consortium, anaerobic gut fungi are the primary colonizers of plant material, and represent a rich source of biomass degrading enzymes. We have isolated several novel strains of gut fungi from herbivores at the Santa Barbara Zoo to characterize their ability to release sugars from crude biomass. We have used transcriptomic analysis to identify specific enzymes required for the breakdown of plant material including cellullases (GH5, GH9, GH48), hemicellulases (GH10, GH11, GH43), and accessory enzymes (Polysaccharide deacetylases). Through examining the regulatory pattern of these enzymes during growth on a variety of carbon sources, those that are most important for degradation of crude plant material can be identified. We have also used transcriptomics to identify sensor proteins and transcription factors that may be responsible for the global regulation of these enzymes. Through examination of putative regulatory proteins and the environmental stimuli that trigger their response, we can develop methods to control gut fungal metabolism and the production of biomass degrading enzymes.
While tools to engineer gut fungi directly are severely underdeveloped, another way to incorporate them into industrial processes is to create co-culture systems. Natively, gut fungi maintain a syntrophic relationship with archaeal methanogens by which the fungi produce CO 2 and H2 that the methanogens convert into methane. This relationship results in enhanced biomass breakdown by the fungus. In a synthetic system gut fungi are used for their degrading power to release sugars from biomass (~5 g/L released from cellulosic substrates). This excess sugar is then used to fuel production of a value-added chemical in a model microbe, such as S. cerevisiae or E. coli. We have used the production of Flavin- based fluorescent proteins (FbFPs) to quantify growth in both systems and production of n-butanol in E. coli to assess the ability to produce a fuel molecule in this system. If methanogens are incorporated into this synthetic system, it is expected that the amount of sugar available for value-added chemical production will increase. By coupling the lignocellulose-degrading capabilities of the gut fungi with the production capacity of model microbes, many different products may be generated directly from biomass.