scholarly journals Rational engineering of industrial S. cerevisiae: towards xylitol production from sugarcane bagasse

2021 ◽  
Author(s):  
Fellipe da Silveira Bezerra de Mello ◽  
Carla Maneira ◽  
Frank Uriel Suarez Lizarazo ◽  
Sheila Nagamatsu ◽  
Beatriz de Oliveira Vargas ◽  
...  
Keyword(s):  
Sugarcane Bagasse ◽  
Xylose Reductase ◽  
Economic Value ◽  
Laboratory Strain ◽  
Building Blocks ◽  
Solid Particles ◽  
Xylitol Production ◽  
Inhibitor Tolerance ◽  
Xylose Consumption ◽  
Operational Conditions

BACKGROUND: Sugarcane hemicellulosic material is a compelling source of usually neglected xylose that could figure as feedstock to produce chemical building blocks of high economic value, such as xylitol. In this context, Saccharomyces cerevisiae strains typically used in the Brazilian bioethanol industry are a robust chassis for genetic engineering, given their robustness towards harsh operational conditions and outstanding fermentation performance. Nevertheless, there are no reports on the use of these strains for xylitol production using sugarcane hydrolysate. RESULTS: Potential single-guided RNA off-targets were analyzed in two preeminent industrial strains (PE-2 and SA-1), providing a database of 5'-NGG 20 nt sequences, and guidelines for the fast and cost-effective CRISPR-editing of such strains. After genomic integration of a NADPH-preferring xylose reductase (XR), FMYX (SA-1 hoΔ::xyl1) and CENPKX (CEN.PK-122 hoΔ::xyl1) were tested in varying cultivation conditions for xylitol productivity to infer the influence of the genetic background. Near-theoretical yields were achieved for all strains, however, the industrial consistently outperformed the laboratory strain. Batch fermentation of raw sugarcane bagasse hydrolysate with remaining solid particles represented a challenge for xylose metabolization and 3.65 ± 0.16 g/L xylitol titre was achieved by FMYX. Finally, quantification of NADPH - cofactor implied in XR activity - revealed that FMYX has 33% more available cofactors than CENPKX. CONCLUSIONS: Although widely used in several S. cerevisiae strains, this is the first report of CRISPR-Cas9 editing major yeast of the Brazilian bioethanol industry. Fermentative assays of xylose consumption revealed that NADPH availability is closely related to mutant strains' performance. We also pioneer the use of sugarcane bagasse as a substrate for xylitol production. Finally, we demonstrate how industrial background SA-1 is a compelling chassis for the second-generation industry, given its inhibitor tolerance and better redox environment that may favor the production of reduced sugars.

scholarly journals Assessing the Potential of Newly Isolated Pichia Fermentans for Xylitol Production Using Non-Detoxified Xylose Rich Pre-Hydrolysate Derived from Sugarcane Bagasse

2020 ◽  
Author(s):  
Ashish A Prabhu ◽  
Ekkarin Bosakornranut ◽  
Yassin Amraoui ◽  
Deepti Agarwal ◽  
Frederic Coulon ◽  
...  
Keyword(s):  
Sugarcane Bagasse ◽  
Type Strain ◽  
Shake Flask ◽  
Wild Type Strain ◽  
Xylose Reductase ◽  
Value Added ◽  
Xylitol Production ◽  
Wild Type ◽  
Conversion Yield ◽  
Cell Factories

Abstract Background: Integrated management of hemicellulosic fraction and its economical transformation to value-added products is the key driver towards sustainable second-generation biorefineries. In this aspect microbial cell factories are harnessed for sustainable production of biochemicals by valorising C5 and C6 sugars generated from agro-industrial waste. However, most of the strains can effectively consume C6 sugars but lacks pentose metabolism pathway. The effective utilization of both pentose and hexose sugars is key for economical biorefinery. Results: In the current study, the ability of a newly isolated xylose assimilating Pichia fermentans was explored for xylitol production. The wild type strain robustly grew on xylose and produced xylitol with >40% conversion yield. Mutagenesis with ethyl methanesulphonate (EMS) yielded seven mutants. The mutant obtained after 15 min exposure, exhibited best xylose bioconversion efficiency. This mutant under shake flask conditions produced maximum xylitol titre and yield of 34.0 g/L and 0.68 g/g, respectively. oweverHoHHHoHowever, under same conditions, the control wild type strain accumulated 27.0 g/L xylitol with a conversion yield of 0.45 g/g. Improved performance of the mutant was attributed to 34.6% activity enhancement in xylose reductase with simultaneous reduction of xylitol dehydrogenase activity by 22.9%. Later, the culture medium was optimized using statistical design and validated at shake flask and bioreactor level. Bioreactor studies affirmed the competence of mutant in xylitol accumulation. The xylitol titre and yield obtained with pure xylose were 98.9 g/L and 0.67 g/g, respectively while xylitol produced using non-detoxified xylose rich pre-hydrolysate from sugarcane bagasse was 79.0 g/L with an overall yield of 0.54 g/g. Conclusion: This study established the potential of P. fermentans in successfully valorising the hemicellulosic fraction for sustainable xylitol production.

scholarly journals Reduced Oxidative Pentose Phosphate Pathway Flux in Recombinant Xylose-Utilizing Saccharomyces cerevisiae Strains Improves the Ethanol Yield from Xylose

2002 ◽  
Vol 68 (4) ◽  
pp. 1604-1609 ◽  
Author(s):  
Marie Jeppsson ◽  
Björn Johansson ◽  
Bärbel Hahn-Hägerdal ◽  
Marie F. Gorwa-Grauslund
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Pentose Phosphate Pathway ◽  
Metabolic Flux ◽  
Ethanol Yield ◽  
Xylose Reductase ◽  
Pentose Phosphate ◽  
Xylitol Production ◽  
Xylose Consumption ◽  
Xylitol Yield ◽  
Pathway Flux

ABSTRACT In recombinant, xylose-fermenting Saccharomyces cerevisiae, about 30% of the consumed xylose is converted to xylitol. Xylitol production results from a cofactor imbalance, since xylose reductase uses both NADPH and NADH, while xylitol dehydrogenase uses only NAD+. In this study we increased the ethanol yield and decreased the xylitol yield by lowering the flux through the NADPH-producing pentose phosphate pathway. The pentose phosphate pathway was blocked either by disruption of the GND1 gene, one of the isogenes of 6-phosphogluconate dehydrogenase, or by disruption of the ZWF1 gene, which encodes glucose 6-phosphate dehydrogenase. Decreasing the phosphoglucose isomerase activity by 90% also lowered the pentose phosphate pathway flux. These modifications all resulted in lower xylitol yield and higher ethanol yield than in the control strains. TMB3255, carrying a disruption of ZWF1, gave the highest ethanol yield (0.41 g g−1) and the lowest xylitol yield (0.05 g g−1) reported for a xylose-fermenting recombinant S. cerevisiae strain, but also an 84% lower xylose consumption rate. The low xylose fermentation rate is probably due to limited NADPH-mediated xylose reduction. Metabolic flux modeling of TMB3255 confirmed that the NADPH-producing pentose phosphate pathway was blocked and that xylose reduction was mediated only by NADH, leading to a lower rate of xylose consumption. These results indicate that xylitol production is strongly connected to the flux through the oxidative part of the pentose phosphate pathway.

scholarly journals Identification of the major fermentation inhibitors of recombinant 2G yeasts in diverse lignocellulose hydrolysates

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Gert Vanmarcke ◽  
Mekonnen M. Demeke ◽  
Maria R. Foulquié-Moreno ◽  
Johan M. Thevelein
Keyword(s):  
Second Generation ◽  
Industrial Process ◽  
Superior Performance ◽  
Fermentation Inhibitors ◽  
Inhibitor Tolerance ◽  
Xylose Consumption ◽  
Process Economics ◽  
Partial Inhibition ◽  
Second Generation Bioethanol ◽  
Lignocellulose Hydrolysates

Abstract Background Presence of inhibitory chemicals in lignocellulose hydrolysates is a major hurdle for production of second-generation bioethanol. Especially cheaper pre-treatment methods that ensure an economical viable production process generate high levels of these inhibitory chemicals. The effect of several of these inhibitors has been extensively studied with non-xylose-fermenting laboratory strains, in synthetic media, and usually as single inhibitors, or with inhibitor concentrations much higher than those found in lignocellulose hydrolysates. However, the relevance of individual inhibitors in inhibitor-rich lignocellulose hydrolysates has remained unclear. Results The relative importance for inhibition of ethanol fermentation by two industrial second-generation yeast strains in five lignocellulose hydrolysates, from bagasse, corn cobs and spruce, has now been investigated by spiking higher concentrations of each compound in a concentration range relevant for industrial hydrolysates. The strongest inhibition was observed with industrially relevant concentrations of furfural causing partial inhibition of both D-glucose and D-xylose consumption. Addition of 3 or 6 g/L furfural strongly reduced the ethanol titer obtained with strain MD4 in all hydrolysates evaluated, in a range of 34 to 51% and of 77 to 86%, respectively. This was followed by 5-hydroxymethylfurfural, acetic acid and formic acid, for which in general, industrially relevant concentrations caused partial inhibition of D-xylose fermentation. On the other hand, spiking with levulinic acid, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid or vanillin caused little inhibition compared to unspiked hydrolysate. The further evolved MD4 strain generally showed superior performance compared to the previously developed strain GSE16-T18. Conclusion The results highlight the importance of individual inhibitor evaluation in a medium containing a genuine mix of inhibitors as well as the ethanol that is produced by the fermentation. They also highlight the potential of increasing yeast inhibitor tolerance for improving industrial process economics.

Use of Immobilized Candida Cells on Xylitol Production from Sugarcane Bagasse

2000 ◽  
Vol 55 (3-4) ◽  
pp. 213-217 ◽  
Author(s):  
Walter de Carvalho ◽  
Silvio Silvério da Silva ◽  
Michele Vitolo ◽  
Ismael Maciel de Mancilha
Keyword(s):  
Sugarcane Bagasse ◽  
Immobilized Cells ◽  
Alginate Beads ◽  
Candida Guilliermondii ◽  
Volumetric Productivity ◽  
Xylitol Production ◽  
Experimental Conditions ◽  
Work Volume ◽  
Yield Factor ◽  
Ca Alginate

Abstract In this study we used the yeast Candida guilliermondii FTI 20037 immobilized by entrapment in Ca-alginate beads (2 .5 -3 mm diameter) for xylitol production from concentrated sugarcane bagasse hemicellulosic hydrolysate in a repeated batch system. The fermentation runs were carried out in 125- and 250-ml Erlenmeyer flasks placed in an orbital shaker at 30 °C and 200 rpm during 72 h, keeping constant the proportion between work volume and flask total volume. According to the results, cell viability was substantially high (98%) in all fermentative cycles. The values of parameters xylitol yield and volumetric productivity increased significantly with the reutilization of the immobilized biocatalysts. The highest values of xylitol final concentration (11.05 g/1), yield factor (0.47 gig) and volumetric productivity (0.22 g/lh) were obtained in 250-ml Erlenmeyer flasks containing 80 ml of medium plus 20 mi of immobilized biocatalysts. The support used in this study (Ca-alginate) presented stability in the experimental conditions used. The results show that the use of immobilized cells is a promising approach for increasing the xylitol production rates.

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