Shen-Long Tsai

Functional Assembly of Minicellulosomes on the Saccharomyces cerevisiae Cell Surface for Cellulose Hydrolysis and Ethanol Production

ABSTRACT We demonstrated the functional display of a miniscaffoldin on the Saccharomyces cerevisiae cell surface consisting of three divergent cohesin domains from Clostridium thermocellum (t), Clostridium cellulolyticum (c), and Ruminococcus flavefaciens (f). Incubation with Escherichia coli lysates containing an endoglucanase (CelA) fused with a dockerin domain from C. thermocellum (At), an exoglucanase (CelE) from C. cellulolyticum fused with a dockerin domain from the same species (Ec), and an endoglucanase (CelG) from C. cellulolyticum fused with a dockerin domain from R. flavefaciens (Gf) resulted in the assembly of a functional minicellulosome on the yeast cell surface. The displayed minicellulosome retained the synergistic effect for cellulose hydrolysis. When a β-glucosidase (BglA) from C. thermocellum tagged with the dockerin from R. flavefaciens was used in place of Gf, cells displaying the new minicellulosome exhibited significantly enhanced glucose liberation and produced ethanol directly from phosphoric acid-swollen cellulose. The final ethanol concentration of 3.5 g/liter was 2.6-fold higher than that obtained by using the same amounts of added purified cellulases. The overall yield was 0.49 g of ethanol produced per g of carbohydrate consumed, which corresponds to 95% of the theoretical value. This result confirms that simultaneous and synergistic saccharification and fermentation of cellulose to ethanol can be efficiently accomplished with a yeast strain displaying a functional minicellulosome containing all three required cellulolytic enzymes.

Surface Display of a Functional Minicellulosome by Intracellular Complementation Using a Synthetic Yeast Consortium and Its Application to Cellulose Hydrolysis and Ethanol Production

ABSTRACT In this paper, we report the surface assembly of a functional minicellulosome by using a synthetic yeast consortium. The basic design of the consortium consisted of four different engineered yeast strains capable of either displaying a trifunctional scaffoldin, Scaf-ctf (SC), carrying three divergent cohesin domains from Clostridium thermocellum (t), Clostridium cellulolyticum (c), and Ruminococcus flavefaciens (f), or secreting one of the three corresponding dockerin-tagged cellulases (endoglucanase [AT], exoglucanase [EC/CB], or β-glucosidase [BF]). The secreted cellulases were docked onto the displayed Scaf-ctf in a highly organized manner based on the specific interaction of the three cohesin-dockerin pairs employed, resulting in the assembly of a functional minicellulosome on the yeast surface. By exploiting the modular nature of each population to provide a unique building block for the minicellulosome structure, the overall cellulosome assembly, cellulose hydrolysis, and ethanol production were easily fine-tuned by adjusting the ratio of different populations in the consortium. The optimized consortium consisted of a SC:AT:CB:BF ratio of 7:2:4:2 and produced almost twice the level of ethanol (1.87 g/liter) as a consortium with an equal ratio of the different populations. The final ethanol yield of 0.475 g of ethanol/g of cellulose consumed also corresponded to 93% of the theoretical value. This result confirms the use of a synthetic biology approach for the synergistic saccharification and fermentation of cellulose to ethanol by using a yeast consortium displaying a functional minicellulosome.

Microbial Biosensors: Engineered Microorganisms as the Sensing Machinery

Whole-cell biosensors are a good alternative to enzyme-based biosensors since they offer the benefits of low cost and improved stability. In recent years, live cells have been employed as biosensors for a wide range of targets. In this review, we will focus on the use of microorganisms that are genetically modified with the desirable outputs in order to improve the biosensor performance. Different methodologies based on genetic/protein engineering and synthetic biology to construct microorganisms with the required signal outputs, sensitivity, and selectivity will be discussed.

Functional display of complex cellulosomes on the yeast surface via adaptive assembly.

A new adaptive strategy was developed for the ex vivo assembly of a functional tetravalent designer cellulosome on the yeast cell surface. The design is based on the use of (1) a surface-bound anchoring scaffoldin composed of two divergent cohesin domains, (2) two dockerin-tagged adaptor scaffoldins to amplify the number of enzyme loading sites based on the specific dockerin-cohesin interaction with the anchoring scaffoldin, and (3) two dockerin-tagged enzymatic subunits (the endoglucanse Gt and the β-glucosidase Bglf) for cellulose hydrolysis. Cells displaying the tetravalent cellulosome on the surface exhibited a 4.2-fold enhancement in the hydrolysis of phosphoric acid swollen cellulose (PASC) compared with free enzymes. More importantly, cells displaying the tetravalent celluosome also exhibited an ~2-fold increase in ethanol production compared with cells displaying a divalent cellulosome using a similar enzyme loading. These results clearly indicate the more crucial role of enzyme proximity than just simply increasing the enzyme loading on the overall cellulosomal synergy. To the best of our knowledge, this is the first report that exploits the natural adaptive assembly strategy in creating artificial cellulosome structures. The unique feature of the anchoring and the adaptor scaffoldin strategy to amplify the number of enzymatic subunits can be easily extended to more complex cellulosomal structures to achieve an even higher level of enzyme synergy.

Biomolecular scaffolds for enhanced signaling and catalytic efficiency

Proteins inherently are not designed to be standalone entities. Whether it is a multi-step biochemical reaction or a signaling event that triggers several other cascading events, proteins are naturally designed to function cohesively. Several natural systems have been developed through evolution to co-localize the functional proteins of the same pathway in order to ensure efficient communication of signals or intermediates. This review focuses on some selected examples of where synthetic scaffolds inspired by nature have been used to enhance the overall biological pathway performance. Applications encompass both in vivo and in vitro systems that address two key biological events in cell signaling and biosynthesis will be discussed.

Synthetic scaffolds for pathway enhancement.

Controlling local concentrations of reactants, intermediates, and enzymes in synthetic pathways is critical for achieving satisfactory productivity of any desired products. An emerging approach to exert control over local concentrations is the use of synthetic biomolecular scaffolds to co-localize key molecules of synthetic pathways. These scaffolds bring the key molecules into close proximity by recruiting pathway enzymes via ligand binding and/or physically sequestrating enzymes and metabolites into isolated compartments. Novel scaffolds made of proteins, nucleic acids, and micro-compartments with increasingly complex architecture have recently been explored and applied to a variety of pathways, with varying degrees of success. Despite these strides, precise assembly of synthetic scaffolds remains a difficult task, particularly in vivo, where interactions both intended and unexpected can lead to unpredictable results. Additionally, because heterologous enzymes often have lowered activities in their new hosts, an ideal scaffold should provide a flexible platform that can adapt to kinetic imbalances in different contexts. In this review, we discuss some of the notable advances in the creation of these synthetic scaffolds and highlight the current challenges in their application.

Designed biomolecule–cellulose complexes for palladium recovery and detoxification

An efficient, selective and reusable biosorbent is important for precious metal recovery. This paper examines the recovery of palladium Pd(II) from wastewater on designed biomolecule–cellulose complexes. A genetically engineered fusion protein composed of palladium binding peptides (PdBP) and cellulose binding domains (CBD) was expressed in Escherichia coli and enabled the binding of cellulose for palladium recovery and detoxification. The results of this study indicated that the range of pH levels suitable for PdCBD–cellulose complexes is wide and that the effect of temperature on palladium recovery is insignificant. In addition, the PdCBD–cellulose complexes exhibited good reusability and a high selectivity for Pd(II) recovery. The maximum adsorption capacity of the PdCBD–cellulose was 175.44 mg g−1, indicating a high adsorption capacity for Pd(II). The Langmuir adsorption isotherm was applied to describe the processes for removing Pd(II). The kinetics of the Pd(II) removal were identified as following a pseudo-second order rate equation. Furthermore, the results of a Lemna minor growth inhibition test showed effective detoxification of the designed complexes. The results of this study revealed that the designed biomolecule–cellulose complexes can be used to develop a selective process for recovering and detoxifying precious metals.

Simultaneous silver recovery and bactericidal bionanocomposite formation via engineered biomolecules

Biomolecules natively possess specific interactions with metals and thus can be promising tools for precious metal recovery from solution phase. In this study, we genetically engineered a biomolecule consisting of a silver-binding peptide and a cellulose-binding domain for simultaneous silver recovery and bactericidal bionanocomposite formation. This is the first research demonstrating this rapid, green and eco-friendly concept for one-pot silver recovery and antimicrobial material synthesis.

Hemicellulose Degradation and Utilization by a Synthetic Saccharomyces cerevisiae Consortium

Since Saccharomyces cerevisiae does not inherently possess the capability to utilize pentose sugars released from hemicellulose degradation, the degradation and utilization of hemicellulose poses a conundrum to bioethanol production by consolidated bioprocessing (CBP) using S. cerevisiae. In this study, S. cerevisiae was exploited for its ability to degrade xylan, one of the major polysaccharide chains present in hemicellulose. Different hemicellulases from Trichoderma reesei, namely: endoxylanase (Xyn2), β-xylosidase (Bxl1), acetylxylan esterase (Axe1), α-D-glucuronidase (Glr1) and α-L-arabinofuranosidase (Abf1), were heterologously secreted by S. cerevisiae. A mixture experimental design was adapted to statistically describe the synergistic interactions between the hemicellulases and to determine the optimum formulations for the hydrolysis of xylan substrates. The hydrolytic activities of the hemicellulase mixtures were then improved by displaying the hemicellulases on the yeast surface to serve as whole-cell biocatalysts. The engineered yeast strains displaying hemicellulases were further engineered with xylose-utilization genes to enable abilities of utilizing xylose as a sole carbon source. The resulting consortia were then able to grow and produce ethanol from different xylan substrates.