Recombinant Bacillus subtilis That Grows on Untreated Plant Biomass

Abstract

Lignocellulosic biomass is a promising feedstock to produce biofuels and other valuable biocommodities. A major obstacle to its commercialization is the high cost of degrading biomass into fermentable sugars, which is typically achieved using cellulolytic enzymes from Trichoderma reesei. Here, we explore the use of microbes to break down biomass. Bacillus subtilis was engineered to display a multicellulase-containing minicellulosome. The complex contains a miniscaffoldin protein that is covalently attached to the cell wall and three noncovalently associated cellulase enzymes derived from Clostridium cellulolyticum (Cel48F, Cel9E, and Cel5A). The minicellulosome spontaneously assembles, thus increasing the practicality of the cells. The recombinant bacteria are highly cellulolytic and grew in minimal medium containing industrially relevant forms of biomass as the primary nutrient source (corn stover, hatched straw, and switch grass). Notably, growth did not require dilute acid pretreatment of the biomass and the cells achieved densities approaching those of cells cultured with glucose. An analysis of the sugars released from acid-pretreated corn stover indicates that the cells have stable cellulolytic activity that enables them to break down 62.3% ؎ 2.6% of the biomass. When supplemented with beta-glucosidase, the cells liberated 21% and 33% of the total available glucose and xylose in the biomass, respectively. As the cells display only three types of enzymes, increasing the number of displayed enzymes should lead to even more potent cellulolytic microbes. This work has important implications for the efficient conversion of lignocellulose to value-added biocommodities.

Nanoscale Engineering of Designer Cellulosomes

“…The MiniCbpA scaffoldin from C.cellulovoran, was successfully expressed in B. subtilis, S. cerevisiae and Corynebacterium glutamicum. [234,239] Parallel to these activities in the production of minicellulsomes, Nanosized platforms, based on a variety of different types of materials, are currently being assessed as potential cellulosomal scaffolds. Kim and colleagues [240] used the high affinity streptavidin-biotin binding pair to form clusters of organic nanoparticles with expressed CBM modules and cellulosome enzymatic domains.…”

“…For example, cellulosomes from the mesophilic bacterium C. cellulovorans were able to completely degrade soft biomass (rice straw). [231] In addition, C. thermocellum cells were used to degrade the same substrate in an efficient manner [232] Others used cell-surface display of mini-cellulosomes on bacteria and yeasts to degrade cellulosic substrates [233,234] A synthetic cellulosome mimic for cellulose ethanol production was also constructed, [235] based on a similar previously published approach. [236] By combining the cohesin-dockerin interactions and advancements in protein re-engineering, new types of synthetic cellulosomes have been generated, an example of which is the thermostable group II chaperonin called a rosettazyme.…”

Engineering microbial surfaces to degrade lignocellulosic biomass

“…For simplicity, only strains capable of degrading insoluble forms of cellulose without the need for adding purified enzyme cocktails are considered. B. subtilis cells displaying a covalently attached self-assembling minicellulosome have the highest demonstrated activity 62 . They degrade the most complex forms of cellulose, both untreated and acid-treated lignocellulose.…”

“…Notably, these bacteria grow in minimal media containing industrially relevant forms of untreated lignocellulosic biomass as a primary nutrient source (corn stover, hatched straw, and switchgrass) to densities that are similar to those achieved by cells that are cultured in glucose. B. subtilis cells displaying an ex vivo assembled complex can degrade microcrystalline cellulose, but the need to add purified enzymes to construct the complex limits their ability to replicate using cellulose as a nutrient 62 , 65 . The most cellulolytic yeast strains reported to date display a complex that contains 6 enzymes (two copies of three types of enzymes) 48 .…”

Synthetic scaffold based on a cohesin–dockerin interaction for improved production of 2,3-butanediol in Saccharomyces cerevisiae

“…The spatial proximity of the tethered cellulases in cellulosome structure provides the efficiency of degrading cellulosic substrate (Fontes and Gilbert, 2010). The high affinity cohesin-dockerin interaction has been widely used not only to express synthetic mini-cellulosomes in heterologous bacteria (Anderson et al, 2013;Hyeon et al, 2011) and yeasts (Fan et al, 2012;Kim et al, 2013a;Tsai et al, 2010;Wen et al, 2010), but also for various applications such as protein purification (Craig et al, 2006;Demishtein et al, 2010), biosensor (Jeon et al, 2012), and * Corresponding author. Tel.…”