UC Santa Barbara

The development of a renewable, bio-based economy requires efficient methods to extract fermentable sugars from complex plant material. Currently, bioprocessing from crude biomass requires multiple steps including pretreatment to separate lignin from sugar-rich cellulose and hemicellulose, enzymatic hydrolysis to release simple sugars, and microbial fermentation to produce value-added chemicals. Consolidated bioprocessing seeks to improve bioprocessing efficiency by reducing the number of steps required to get from plant biomass to chemical product. To address this challenge, we derived inspiration from natural microbial communities known for degrading biomass. Within the rumen microbiome of large herbivores, anaerobic gut fungi are the primary colonizers of plant material and present an untapped opportunity for consolidated bioprocessing. These unique microorganisms efficiently hydrolyze lignocellulosic biomass into simple sugars, but remain relatively uncharacterized in comparison to industrial production organisms. We implemented Next Generation Sequencing (NGS) technologies alongside biochemical studies to develop a deeper understanding of gut fungi, their metabolism, and the mechanisms by which they break down complex biomass to identify a path forward for their industrial application. We also developed simple, rapid methodologies for cryopreservation and DNA extraction that are critical for the development of industrial microbes.

Sequencing and functional annotation of transcriptomes and genomes of novel isolated species of gut fungi has elucidated their large repertoire of biomass degrading enzymes including cellulases, hemicellulases, and accessory enzymes. These enzymes allow them to efficiently degrade crude biomass, yielding similar growth rates on complex plant material and simple sugars. Remarkably, in isolated batch culture, the biomass degrading power of gut fungi is sufficient to generate surplus fermentable sugars for the growth of additional microorganisms. This ability has been exploited to develop a novel two-stage consolidated bioprocessing scheme that uses anaerobic gut fungi to consolidate the pretreatment and hydrolysis steps in traditional bioprocessing to hydrolyze sugars directly from crude biomass. These sugars can then be fed to the easily metabolically engineered model yeast, Saccharomyces cerevisiae, to support growth and bioproduction in a two-stage fermentation scheme.

Further, RNA sequencing studies have provided critical insight into the regulation of biomass degrading activity. Gene expression during growth on varying substrates and in response to a carbon catabolite repressor has revealed conditions required to optimize expression of biomass degrading enzymes. Unannotated sequences that co-regulate with predicted biomass degrading enzymes have also been identified as candidate genes that may host novel biomass degrading function. Together these results reveal important process considerations for the use of gut fungi in industrial bioprocessing to maximize the production of enzymes and the degradation of biomass. While challenges remain for the implementation of gut fungi in industrial bioprocessing, we have demonstrated their potential to consolidate pretreatment and hydrolysis either through engineered culturing schemes or development of improved enzyme cocktails for biomass hydrolysis.