scholarly journals Engineering Pichia pastoris with surface-display minicellulosomes for carboxymethyl cellulose hydrolysis and ethanol production

2020 ◽  
Author(s):  
Ce Dong ◽  
Jie Qiao ◽  
Xinping Wang ◽  
Wenli Sun ◽  
Lixia Chen ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Ethanol Production ◽  
Carboxymethyl Cellulose ◽  
Cellulosic Ethanol ◽  
Consolidated Bioprocessing ◽  
Surface Display ◽  
Cell Factory ◽  
Carbohydrate Binding ◽  
Surface Assembly

Abstract Backgrounds: Engineering yeast as a consolidated bioprocessing (CBP) microorganism by surface assembly of cellulosomes has been aggressively utilized for cellulosic ethanol production. However, most of the previous studies focused on Saccharomyces cerevisiae, achieving efficient conversion of phosphoric acid-swollen cellulose (PASC) or microcrystalline cellulose (Avicel) but not carboxymethyl cellulose (CMC) to ethanol, with an average titer below 2 g/L. Results: Harnessing an ultra-high-affinity IM7/CL7 protein pair, here we describe a method to engineer Pichia pastoris with minicellulosomes by in vitro assembly of three recombinant cellulases including an endoglucanase (EG), an exoglucanase (CBH) and a β-glucosidase (BGL), as well as a carbohydrate binding module (CBM) on the cell surface. For the first time, the engineered yeasts enable efficient and direct conversion of CMC to bioethanol, observing an impressive ethanol titer of 5.1 g/L. Conclusions: The research promotes the application of P. pastoris as a CBP cell factory in cellulosic ethanol production and provides a promising platform for screening the cellulases from different species to construct surface-assembly celluosome.

scholarly journals Engineering Pichia pastoris with surface-display minicellulosome for carboxymethyl cellulose hydrolysis and ethanol production

2020 ◽  
Author(s):  
Ce Dong ◽  
Jie Qiao ◽  
Xinping Wang ◽  
Wenli Sun ◽  
Lixia Chen ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Cell Surface ◽  
Ethanol Production ◽  
Carboxymethyl Cellulose ◽  
Consolidated Bioprocessing ◽  
Surface Display ◽  
Cellulose Hydrolysis ◽  
Cell Factory ◽  
Yeast Cell Surface

Abstract Backgrounds: Engineering yeast with cell surface immobilized cellulosome is a promising strategy for consolidated bioprocessing (CBP) to produce bioethanol from the conversion of cellulose. However, previous studies mostly focused on utilization of Saccharomyces cerevisiae , which was able to directly convert phosphoric acid-swollen cellulose (PASC) or microcrystalline cellulose (Avicel) but not carboxymethyl cellulose (CMC) to ethanol, with an average titer below 2 g/L. Results: Harnessing an ultra-high-affinity IM7/CL7 protein pair, here we describe a method to engineer Pichia pastoris with minicellulosome through in vitro assembly of various recombinant cellulases on the cell surface. For the first time, the yeast can efficiently convert CMC to bioethanol, achieving an impressive ethanol titer of 5.1 g/L. Further, the engineered yeasts were lyophilized to powders that can be utilized as compound cellulases. Conclusions: This research promotes the application of P. pastoris as CBP cell factory in cellulosic ethanol production and provides a promising platform for screening optimal cellulase species and ratios to construct celluosome on the yeast cell surface.

A novel bifunctional aldehyde/alcohol dehydrogenase mediating ethanol formation from acetyl-CoA in hyperthermophiles

2020 ◽  
Author(s):  
Qiang Wang ◽  
Chong Sha ◽  
Hongcheng Wang ◽  
Kesen Ma ◽  
Juergen Wiegel ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Alcohol Dehydrogenase ◽  
Ethanol Production ◽  
Ethanol Fermentation ◽  
Cellulosic Ethanol ◽  
Decarboxylase Activity ◽  
Aldh Activity ◽  
Acetyl Coa ◽  
Ethanol Formation

Abstract Background: Hyperthermophilic fermentation at temperatures above 80 °C allows in situ product removal to mitigate the ethanol toxicity, and reduces microbial contamination without autoclaving/cooling of feedstock. Many species of Thermotoga grow at temperatures up to 90 °C, and have enzymes to degrade and utilize lignocelluloses, which provide advantages for achieving consolidated processes of cellulosic ethanol production. However, no CoA-dependent aldehyde dehydrogenase (CoA-Aldh) from any hyperthermophiles has been documented in literature so far. The pyruvate ferredoxin oxidoreductases from hyperthermophiles have pyruvate decarboxylase activity, which convert about 2% and 98% of pyruvate to acetaldehyde and acetyl-CoA (ac-CoA), respectively. Acetyl-CoA can be converted to acetic acid, if there is no CoA-Aldh to convert ac-CoA to acetaldehyde and further to ethanol. Therefore, the current study aimed to identify and characterize a CoA-Aldh activity that mediates ethanol fermentation in hyperthermophiles.Results: In Thermotoga neapolitana (Tne), a hyperthermophilic iron-acetaldehyde/alcohol dehydrogenase (Fe-AAdh) was, for the first time, revealed to catalyze the ac-CoA reduction to form ethanol via an acetaldehyde intermediate, while the annotated aldh gene in Tne genome only encodes a CoA-independent Aldh that oxidizes aldehyde to acetic acid. Three other Tne alcohol dehydrogenases (Adh) exhibited specific physiological roles in ethanol formation and consumption: Fe-Adh2 mainly catalyzed the reduction of acetaldehyde to produce ethanol, and Fe-Adh1 showed significant activities only under extreme conditions, while Zn-Adh showed special activity in ethanol oxidation. In the in vitro formation of ethanol from ac-CoA, a strong synergy was observed between Fe-Adh1 and Fe-AAdh. The Fe-AAdh gene is highly conserved in Thermotoga spp. and in Pyrococus sp., which is probably responsible for ethanol metabolism in hyperthermophiles.Conclusions: Hyperthermophilic Thermotoga spp. are excellent candidates for biosynthesis of cellulosic ethanol fermentation strains. The finding of a novel hyperthermophilic CoA-Aldh activity of Tne Fe-AAdh revealed the existence of a hyperthermophilic fermentation pathway from ac-CoA to ethanol, which offers a basic frame for in vitro synthesis of a highly active AAdh for effective ethanol fermentation pathway in hyperthermophiles, which is a key element for the approach to the consolidated processes of cellulosic ethanol production.

Using a microorganism consortium for consolidated bioprocessing cellulosic ethanol production

Biofuels ◽  
2011 ◽  
Vol 2 (5) ◽  
pp. 569-575 ◽  
Author(s):  
Ran Du ◽  
Shizhong Li ◽  
Xiaoqing Zhang ◽  
Chenyu Fan ◽  
Li Wang
Keyword(s):  
Ethanol Production ◽  
Cellulosic Ethanol ◽  
Consolidated Bioprocessing

Enhanced cellulosic ethanol production via consolidated bioprocessing by Clostridium thermocellum ATCC 31924☆

2018 ◽  
Vol 250 ◽  
pp. 860-867 ◽  
Author(s):  
Nisha Singh ◽  
Anshu S. Mathur ◽  
Ravi P. Gupta ◽  
Colin J. Barrow ◽  
Deepak Tuli ◽  
...  
Keyword(s):  
Ethanol Production ◽  
Cellulosic Ethanol ◽  
Clostridium Thermocellum ◽  
Consolidated Bioprocessing

scholarly journals Consolidated bioprocessing of corn cob-derived hemicellulose: engineered industrial Saccharomyces cerevisiae as efficient whole cell biocatalysts

2020 ◽  
Author(s):  
Joana T. Cunha ◽  
Aloia Romaní ◽  
Kentaro Inokuma ◽  
Björn Johansson ◽  
Tomohisa Hasunuma ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ethanol Production ◽  
Large Scale ◽  
Consolidated Bioprocessing ◽  
Surface Display ◽  
Total Biomass ◽  
Economic Costs ◽  
Corn Cob ◽  
Direct Production ◽  
Whole Cell

AbstractConsolidated bioprocessing, which combines saccharolytic and fermentative abilities in a single microorganism, is receiving increased attention to decrease environmental and economic costs in lignocellulosic biorefineries. Nevertheless, the economic viability of lignocellulosic ethanol is also dependent of an efficient utilization of the hemicellulosic fraction, which is mainly composed of xylose and may comprise up to 40 % of the total biomass. This major bottleneck is mainly due to the necessity of chemical/enzymatic treatments to hydrolyze hemicellulose into fermentable sugars and to the fact that xylose is not readily consumed by Saccharomyces cerevisiae – the most used organism for large-scale ethanol production. In this work, industrial S. cerevisiae strains, presenting robust traits such as thermotolerance and improved resistance to inhibitors, were evaluated as hosts for the cell-surface display of hemicellulolytic enzymes and optimized xylose assimilation, aiming at the development of whole-cell biocatalysts for consolidated bioprocessing of corn cob-derived hemicellulose. These modifications allowed the direct production of ethanol from non-detoxified hemicellulosic liquor obtained by hydrothermal pretreatment of corn cob, reaching an ethanol titer of 11.1 g/L corresponding to a yield of 0.328 gram per gram of potential xylose and glucose, without the need for external hydrolytic catalysts. Also, consolidated bioprocessing of pretreated corn cob was found to be more efficient for hemicellulosic ethanol production than simultaneous saccharification and fermentation with addition of commercial hemicellulases. These results show the potential of industrial S. cerevisiae strains for the design of whole-cell biocatalysts and paves the way for the development of more efficient consolidated bioprocesses for lignocellulosic biomass valorization, further decreasing environmental and economic costs.

scholarly journals Cellulosic ethanol production via consolidated bioprocessing at 75 °C by engineered Caldicellulosiruptor bescii

2015 ◽  
Vol 8 (1) ◽  
Author(s):  
Daehwan Chung ◽  
Minseok Cha ◽  
Elise N. Snyder ◽  
James G. Elkins ◽  
Adam M. Guss ◽  
...  
Keyword(s):  
Ethanol Production ◽  
Cellulosic Ethanol ◽  
Consolidated Bioprocessing ◽  
Caldicellulosiruptor Bescii

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

2010 ◽  
Vol 76 (22) ◽  
pp. 7514-7520 ◽  
Author(s):  
Shen-Long Tsai ◽  
Garima Goyal ◽  
Wilfred Chen
Keyword(s):  
Ethanol Production ◽  
Ethanol Yield ◽  
Surface Display ◽  
Specific Interaction ◽  
Cellulose Hydrolysis ◽  
Surface Assembly ◽  
Engineered Yeast ◽  
Yeast Surface ◽  
Different Populations ◽  
Synthetic Biology Approach

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.

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