Heterologous expression of a Clostridium minicellulosome in Saccharomyces cerevisiae

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261

This work describes novel genetic tools for use in Clostridium thermocellum that allow creation of unmarked mutations while using a replicating plasmid. The strategy employed counter-selections developed from the native C. thermocellum hpt gene and the Thermoanaerobacterium saccharolyticum tdk gene and was used to delete the genes for both lactate dehydrogenase (Ldh) and phosphotransacetylase (Pta). The ⌬ldh ⌬pta mutant was evolved for 2,000 h, resulting in a stable strain with 40:1 ethanol selectivity and a 4.2-fold increase in ethanol yield over the wild-type strain. Ethanol production from cellulose was investigated with an engineered coculture of organic acid-deficient engineered strains of both C. thermocellum and T. saccharolyticum. Fermentation of 92 g/liter Avicel by this coculture resulted in 38 g/liter ethanol, with acetic and lactic acids below detection limits, in 146 h. These results demonstrate that ethanol production by thermophilic, cellulolytic microbes is amenable to substantial improvement by metabolic engineering.

mentioning

confidence: 99%

High Ethanol Titers from Cellulose by Using Metabolically Engineered Thermophilic, Anaerobic Microbes

Argyros¹,

Tripathi²,

Barrett³

et al. 2011

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261

“…Toward the goal of creating a robust CBP microbe, two model microorganisms (Bacillus subtilis and Saccharomyces cerevisiae) have been engineered to display small artificial cellulosomes (i.e., minicellulosomes) (32)(33)(34)(35)(36)(37). In all of these systems, a miniscaffol-din containing one or more cohesin modules is covalently or noncovalently attached to the cell surface.…”

mentioning

confidence: 99%

Recombinant Bacillus subtilis That Grows on Untreated Plant Biomass

Anderson

Miller

Fierobe

et al. 2013

Self Cite

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.

“…Upon binding to three different types of cellulolytic enzymes on the surface-displayed scaffold, Tsai et al were able to show ethanol fermentation from these engineered yeasts using cellulosic substrates. Similarly, Lilly and collaborators engineered a chimeric scaffoldin protein Scaf3p from C. cellulolyticum that contains two different cohesin domains and targeted the Scaf3p to display on the S. cerevisiae cell surface (17). Wen and coworkers constructed yeast strains displaying a series of uni-, bi-, and trifunctional minicellulosomal scaffoldins composed of a cellulose binding domain and cohesins (27).…”

Section: Discussionmentioning

confidence: 99%

“…Several types of free designer cellulosomes (not bound to cell surfaces) have been proposed that mimic the natural cellulosomes (8,18). For display on the yeast surface, only relatively short and simple minicellulosomes that imitate truncated forms of the native Clostridium cellulosomes have been reported (17,(25)(26)(27).The native Clostridium scaffoldin contains multiple cohesin units joined covalently by disordered linker regions. Here, we report an alternative molecular design for synthetic scaffoldin.…”

mentioning

confidence: 99%

Self-Assembled Amyloid-Like Oligomeric-Cohesin Scaffoldin for Augmented Protein Display on the Saccharomyces cerevisiae Cell Surface

Han

Zhang

Wang

et al. 2012

In this study, a molecular self-assembly strategy to develop a novel protein scaffold for amplifying the extent and variety of proteins displayed on the surface of Saccharomyces cerevisiae is presented. The cellulosomal scaffolding protein cohesin and its upstream hydrophilic domain (HD) were genetically fused with the yeast Ure2p N-terminal fibrillogenic domain consisting of residues 1 to 80 (Ure2p 1-80 ). The resulting Ure2p 1-80 -HD-cohesin fusion protein was successfully expressed in Escherichia coli to produce self-assembled supramolecular nanofibrils that serve as a novel protein scaffold displaying multiple copies of functional cohesin domains. The amyloid-like property of the nanofibrils was confirmed via thioflavin T staining and atomic force microscopy. These cohesin nanofibrils attached themselves, via a green fluorescent protein (GFP)-dockerin fusion protein, to the cell surface of S. cerevisiae engineered to display a GFP-nanobody. The excess cohesin units on the nanofibrils provide ample sites for binding to dockerin fusion proteins, as exemplified using an mCherry-dockerin fusion protein as well as the Clostridium cellulolyticum CelA endoglucanase. More than a 24-fold increase in mCherry fluorescence and an 8-fold increase in CelA activity were noted when the cohesin nanofibril scaffold-mediated yeast display was used, compared to using yeast display with GFPcohesin that contains only a single copy of cohesin. Self-assembled supramolecular cohesin nanofibrils created by fusion with the yeast Ure2p fibrillogenic domain provide a versatile protein scaffold that expands the utility of yeast cell surface display. Yeast surface display provides a promising platform for protein engineering (9). The ability to anchor and display functional proteins on the yeast cell surface is also being exploited for developing novel whole-cell biocatalysts (11). Although whole-cell biocatalysis using yeast surface display is generally considered an established technology, challenges remain regarding the efficient display of protein complexes and the further increase in the amount of protein that can be displayed on the yeast surface without destabilizing the cell. To this end, unique properties of protein building blocks found in natural higher-order protein complexes may be exploited to create novel synthetic protein chimeras that self-assemble into nano-scaffolds on the yeast surface to display increased levels and varieties of proteins. One such protein building block is the cohesin (Coh) domain, which is found with multiple copies in cellulosomes.The cellulosome is an extracellular multienzyme complex found on the cell surface of anaerobic bacteria such as Clostridium cellulolyticum, facilitating highly efficient hydrolysis of amorphous and crystalline cellulose. It is composed of numerous cellulolytic enzymes and other functional units assembled, via highaffinity interactions between the cohesin and dockerin domains (22), on a protein scaffold (scaffoldin) that contains multiple copies of cohesin. Cellulases and r...