Growth and fermentation of D-xylose by Saccharomyces cerevisiae expressing a novel D-xylose isomerase originating from the bacterium Prevotella ruminicola TC2-24

Abstract

BackgroundSaccharomyces cerevisiae strains expressing D-xylose isomerase (XI) produce some of the highest reported ethanol yields from D-xylose. Unfortunately, most bacterial XIs that have been expressed in S. cerevisiae are either not functional, require additional strain modification, or have low affinity for D-xylose. This study analyzed several XIs from rumen and intestinal microorganisms to identify enzymes with improved properties for engineering S. cerevisiae for D-xylose fermentation.ResultsFour XIs originating from rumen and intestinal bacteria were isolated and expressed in a S. cerevisiae CEN.PK2-1C parental strain primed for D-xylose metabolism by over expression of its native D-xylulokinase. Three of the XIs were functional in S. cerevisiae, based on the strain’s ability to grow in D-xylose medium. The most promising strain, expressing the XI mined from Prevotella ruminicola TC2-24, was further adapted for aerobic and fermentative growth by serial transfers of D-xylose cultures under aerobic, and followed by microaerobic conditions. The evolved strain had a specific growth rate of 0.23 h-1 on D-xylose medium, which is comparable to the best reported results for analogous S. cerevisiae strains including those expressing the Piromyces sp. E2 XI. When used to ferment D-xylose, the adapted strain produced 13.6 g/L ethanol in 91 h with a metabolic yield of 83% of theoretical. From analysis of the P. ruminicola XI, it was determined the enzyme possessed a Vmax of 0.81 μmole/min/mg protein and a Km of 34 mM.ConclusionThis study identifies a new xylose isomerase from the rumen bacterium Prevotella ruminicola TC2-24 that has one of the highest affinities and specific activities compared to other bacterial and fungal D-xylose isomerases expressed in yeast. When expressed in S. cerevisiae and used to ferment D-xylose, very high ethanol yield was obtained. This new XI should be a promising resource for constructing other D-xylose fermenting strains, including industrial yeast genetic backgrounds.

Novel two‐stage fermentation process for bioethanol production using Saccharomyces pastorianus

“…Although the immobilized xylose isomerase with urea system had a very high ethanol to sugar yield for the fermentation, it would also have significant operating costs for the immobilization process (Figure C) . The highest ethanol yield for many recent recombinant system publications was 0.32 g/g, where the most recent works have reported up to 0.42 g/g . Earlier reports for recombinant S. cerevisiae systems that overexpressed xylose isomerase, xylulokinase, ribose 5‐phosphate isomerase, ribulose 5‐phosphate epimerase, transketolase, and transaldolase, and contained a deleted aldose reductase gene, reported ethanol yields as high as 0.40 to 0.43 g/g; and ethanol productivities of 1.21 g/L h .…”

Unraveling the genetic basis of xylose consumption in engineered Saccharomyces cerevisiae strains

“…Xylose isomerase activity in LVY34.4 cell extracts was determined as described elsewhere65 adapted for microplate assay, using 100 mM Tris-HCl (pH 7.5) buffer, 0.001 mM FeSO 4 .7H2O, 0.15 mM NADH, 2 U sorbitol dehydrogenase (Sigma-Aldrich), and the cell lysate. The reaction started with the addition of 500 mM xylose.…”

A Bibliometric Study of Scientific Publications regarding Hemicellulose Valorization during the 2000–2016 Period: Identification of Alternatives and Hot Topics

“…The most practical approach proposes the introduction of the pentose-utilizing capability into efficient ethanol producers such as S. cerevisiae and Z. mobilis [135,188,189]. The introduction of xylose isomerases that function well in other xylose-fermenting yeasts is the preferred solution to overcome the limitations caused by xylose to these host organisms [190,191].…”