A bacterial pioneer produces cellulase complexes that persist through community succession.

Cultivation of microbial consortia provides low-complexity communities that can serve as tractable models to understand community dynamics. Time-resolved metagenomics demonstrated that an aerobic cellulolytic consortium cultivated from compost exhibited community dynamics consistent with the definition of an endogenous heterotrophic succession. The genome of the proposed pioneer population, 'Candidatus Reconcilibacillus cellulovorans', possessed a gene cluster containing multidomain glycoside hydrolases (GHs). Purification of the soluble cellulase activity from a 300litre cultivation of this consortium revealed that ~70% of the activity arose from the 'Ca. Reconcilibacillus cellulovorans' multidomain GHs assembled into cellulase complexes through glycosylation. These remarkably stable complexes have supramolecular structures for enzymatic cellulose hydrolysis that are distinct from cellulosomes. The persistence of these complexes during cultivation indicates that they may be active through multiple cultivations of this consortium and act as public goods that sustain the community. The provision of extracellular GHs as public goods may influence microbial community dynamics in native biomass-deconstructing communities relevant to agriculture, human health and biotechnology.

Predictive modeling to de-risk bio-based manufacturing by adapting to variability in lignocellulosic biomass supply.

Commercial-scale bio-refineries are designed to process 2000tons/day of single lignocellulosic biomass. Several geographical areas in the United States generate diverse feedstocks that, when combined, can be substantial for bio-based manufacturing. Blending multiple feedstocks is a strategy being investigated to expand bio-based manufacturing outside Corn Belt. In this study, we developed a model to predict continuous envelopes of biomass blends that are optimal for a given pretreatment condition to achieve a predetermined sugar yield or vice versa. For example, our model predicted more than 60% glucose yield can be achieved by treating an equal part blend of energy cane, corn stover, and switchgrass with alkali pretreatment at 120°C for 14.8h. By using ionic liquid to pretreat an equal part blend of the biomass feedstocks at 160°C for 2.2h, we achieved 87.6% glucose yield. Such a predictive model can potentially overcome dependence on a single feedstock.

A homogeneous FRET assay system for multiubiquitin chain assembly and disassembly.

Ubiquitin (Ub, 76aa) is a small highly conserved protein present universally in eukaryotic cells. Covalent attachment of (Ub)(n) to target proteins is a well-known posttranslational modification that has been implicated in a wide array of cellular processes including cell biogenesis. Ubiquitin polymerization by the Ub activation-conjugation-ligation cascade and the reverse disassembly process catalyzed by Ub isopeptidases largely regulate substrate protein targeting to the 26S proteasome. Ub chains of four or more subunits attached by K48 isopeptide linkages have been shown to be necessary for the 26S proteasome association and subsequent degradation of protein molecules. To better understand this protein degradation event, it is important to develop Ub polymerization and depolymerization assays that monitor every reaction step involved in Ub attachment to, or detachment from, substrate protein molecules. In this chapter, we describe homogeneous, easy-to-use, nonradioactive, complementary continuous fluorescence assays capable of monitoring the kinetics of Ub chain formation by E3 Ub ligases, and their hydrolysis by isopeptidases, which rely on mixing a 1:1 population of fluorophore-labeled Ub molecules containing a FRET pair. The proximity of fluorescein (donor) and tetramethylrhodamine (acceptor) in Ub polymers results in fluorescein quenching on ligase-induced Ub chain assembly. Conversely, a dramatic enhancement of fluorescein emission was observed on Ub chain disassembly because of isopeptidase activity. These assays thus provide a valuable tool for monitoring Ub ligase and isopeptidase activities using authentic Ub monomers and polymers as substrates. Screening of a large number of small molecule compound libraries in a high-throughput fashion is achievable, warranting further optimization of these assays.