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.

scholarly journals Expression and Display of Glycoengineered Antibodies and Antibody Fragments with an Engineered Yeast Strain

Antibodies ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 38
Author(s):  
Anjali Shenoy ◽  
Srisaimaneesh Yalamanchili ◽  
Alexander R. Davis ◽  
Adam W. Barb
Keyword(s):  
Surface Display ◽  
Yeast Surface Display ◽  
Surface Receptors ◽  
Mammalian Expression ◽  
Er Retention ◽  
Sds Page ◽  
Engineered Yeast ◽  
Yeast Surface ◽  
Protein Disulfide ◽  
Retention Signal

Interactions with cell surface receptors enhance the therapeutic properties of many important antibodies, including the low-affinity Fc γ Receptors (FcγRs). These interactions require proper processing of the immunoglobulin G Fc N-glycan, and eliminating the N-glycan abolishes binding, restricting antibody production to mammalian expression platforms. Yeasts, for example, generate extensively mannosylated N-glycans that are unsuitable for therapeutics. However, Fc with a specifically truncated N-glycan still engages receptors with considerable affinity. Here we describe the creation and applications of a novel Saccharomyces cerevisiae strain that specifically modifies the IgG1 Fc domain with an N-glycan consisting of a single N-acetylglucosamine residue. This strain displayed glycoengineered Fc on its surface for screening yeast surface display libraries and also served as an alternative platform to produce glycoengineered Rituximab. An IgG-specific endoglycosidase (EndoS2) truncates the IgG1 Fc N-glycan. EndoS2 was targeted to the yeast ER using the signal peptide from the yeast protein disulfide isomerase (PDI) and a yeast ER retention signal (HDEL). Furthermore, >99% of the yeast expressed Rituximab displayed the truncated glycoform as determined by SDS-PAGE and ESI-MS analyses. Lastly, the yeast expressed Rituximab engaged the FcγRIIIa with the expected affinity (KD = 2.0 ± 0.5 μM) and bound CD20 on Raji B cells.

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.

scholarly journals Development and Characterization of Two Types of Surface Displayed Levansucrases for Levan Biosynthesis

Catalysts ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 757
Author(s):  
Huiyi Shang ◽  
Danni Yang ◽  
Dairong Qiao ◽  
Hui Xu ◽  
Yi Cao
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
Large Scale ◽  
Surface Display ◽  
Yeast Surface Display ◽  
Fusion Partner ◽  
Whole Cell ◽  
Food Industries ◽  
Yeast Surface ◽  
The Stability

Levan has wide applications in chemical, cosmetic, pharmaceutical and food industries. The free levansucrase is usually used in the biosynthesis of levan, but the poor reusability and low stability of free levansucrase have limited its large-scale use. To address this problem, the surface-displayed levansucrase in Saccharomyces cerevisiae were generated and evaluated in this study. The levansucrase from Zymomonas mobilis was displayed on the cell surface of Saccharomyces cerevisiae EBY100 using a various yeast surface display platform. The N-terminal fusion partner is based on a-agglutinin, and the C-terminal one is Flo1p. The yield of levan produced by these two whole-cell biocatalysts reaches 26 g/L and 34 g/L in 24 h, respectively. Meanwhile, the stability of the surface-displayed levansucrases is significantly enhanced. After six reuses, these two biocatalysts retained over 50% and 60% of their initial activities, respectively. Furthermore, the molecular weight and polydispersity test of the products suggested that the whole-cell biocatalyst of levansucrase displayed by Flo1p has more potentials in the production of levan with low molecular weight which is critical in certain applications. In conclusion, our method not only enable the possibility to reuse the enzyme, but also improves the stability of the enzyme.

scholarly journals Enzymatic Process for Cystoseira barbata Valorization: Ethanol Production and Additional By-Products

Processes ◽  
2021 ◽  
Vol 9 (5) ◽  
pp. 741
Author(s):  
Doinita-Roxana Cioroiu Tirpan ◽  
Ancaelena Eliza Sterpu ◽  
Claudia Irina Koncsag ◽  
Alina Georgiana Ciufu ◽  
Tănase Dobre
Keyword(s):  
Ethanol Production ◽  
Alcoholic Fermentation ◽  
Ethanol Yield ◽  
Product Yield ◽  
Liquid Ratio ◽  
Experimental Conditions ◽  
Factors Affecting ◽  
Cystoseira Barbata ◽  
Main Factors ◽  
Process Factors

The aim of this study is to evaluate the potential of dried Cystoseira barbata alga for ethanol production through alcoholic fermentation. The influence of the main factors affecting the fermentation are studied in the frame of a 23 factorial experimental plan. The main factors influencing the process are the fermentation temperature (t from 25 °C to 35 °C), the solid to liquid ratio (S/L from 0.040 g/g to 0.080 g/g), and the cellulase ratio (R from 8 U/g d.m to 16 U/g d.m.). The maximum volatile compounds yield of 0.2808 g/g d.m and ethanol yield of 0.0158 g/g d.m were favored by the following experimental conditions: process temperature of 35 °C, solid to liquid ratio of 0.0415, and enzyme ratio of 16 U/g d.m. A statistical model was used to correlate the product yield with the process factors. Additionally, 19 interesting bioactive compounds were found in the enzymatic hydrolysis and alcoholic fermentation broths which seem likely to maintain natural defence mechanisms against diseases and physical disorders.

scholarly journals Hemicellulosic Bioethanol Production from Fast-Growing Paulownia Biomass

Processes ◽  
2021 ◽  
Vol 9 (1) ◽  
pp. 173
Author(s):  
Elena Domínguez ◽  
Pablo G. del Río ◽  
Aloia Romaní ◽  
Gil Garrote ◽  
Lucília Domingues
Keyword(s):  
Ethanol Production ◽  
Ethanol Concentration ◽  
Ethanol Yield ◽  
Xylose Reductase ◽  
Energy Crop ◽  
Scheffersomyces Stipitis ◽  
Fractionation Process ◽  
Renewable Source ◽  
Fast Growing ◽  
Engineered Strain

In order to exploit a fast-growing Paulownia hardwood as an energy crop, a xylose-enriched hydrolysate was obtained in this work to increase the ethanol concentration using the hemicellulosic fraction, besides the already widely studied cellulosic fraction. For that, Paulownia elongata x fortunei was submitted to autohydrolysis treatment (210 °C or S0 of 4.08) for the xylan solubilization, mainly as xylooligosaccharides. Afterwards, sequential stages of acid hydrolysis, concentration, and detoxification were evaluated to obtain fermentable sugars. Thus, detoxified and non-detoxified hydrolysates (diluted or not) were fermented for ethanol production using a natural xylose-consuming yeast, Scheffersomyces stipitis CECT 1922, and an industrial Saccharomyces cerevisiae MEC1133 strain, metabolic engineered strain with the xylose reductase/xylitol dehydrogenase pathway. Results from fermentation assays showed that the engineered S. cerevisiae strain produced up to 14.2 g/L of ethanol (corresponding to 0.33 g/g of ethanol yield) using the non-detoxified hydrolysate. Nevertheless, the yeast S. stipitis reached similar values of ethanol, but only in the detoxified hydrolysate. Hence, the fermentation data prove the suitability and robustness of the engineered strain to ferment non-detoxified liquor, and the appropriateness of detoxification of liquor for the use of less robust yeast. In addition, the success of hemicellulose-to-ethanol production obtained in this work shows the Paulownia biomass as a suitable renewable source for ethanol production following a suitable fractionation process within a biorefinery approach.

scholarly journals Effects of Ultrasound on Fermentation of Glucose to Ethanol by Saccharomyces cerevisiae

Fermentation ◽  
2019 ◽  
Vol 5 (1) ◽  
pp. 16 ◽  
Author(s):  
Luis Huezo ◽  
Ajay Shah ◽  
Frederick Michel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mass Transfer ◽  
Glucose Uptake ◽  
Ethanol Production ◽  
Ethanol Yield ◽  
Biofuel Production ◽  
Inhibitory Effects ◽  
Negative Effects ◽  
Intraparticle Mass Transfer ◽  
Improve Mass Transfer

Previous studies have shown that pretreatment of corn slurries using ultrasound improves starch release and ethanol yield during biofuel production. However, studies on its effects on the mass transfer of substrates and products during fermentation have shown that it can have both beneficial and inhibitory effects. In this study, the effects of ultrasound on mass transfer limitations during fermentation were examined. Calculation of the external and intraparticle observable moduli under a range of conditions indicate that no external or intraparticle mass transfer limitations should exist for the mass transfer of glucose, ethanol, or carbon dioxide. Fermentations of glucose to ethanol using Saccharomyces cerevisiae were conducted at different ultrasound intensities to examine its effects on glucose uptake, ethanol production, and yeast population and viability. Four treatments were compared: direct ultrasound at intensities of 23 and 32 W/L, indirect ultrasound (1.4 W/L), and no-ultrasound. Direct and indirect ultrasound had negative effects on yeast performance and viability, and reduced the rates of glucose uptake and ethanol production. These results indicate that ultrasound during fermentation, at the levels applied, is inhibitory and not expected to improve mass transfer limitations.

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