Prevotella ruminicola xylose isomerase and co-expression with xylulokinase in yeast for xylose fermentation
Inventors
Hector, Ronald E. • Dien, Bruce S. • Cotta, Michael A.
Assignees
US Department of Agriculture USDA
Publication Number
US-8748152-B1
Publication Date
2014-06-10
Expiration Date
2032-09-14
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Abstract
A xylose isomerase (XI) enzyme which exhibits increased activity and affinity for xylose is produced by strain TC2-24 of the rumen bacterium, Prevotella ruminicola. The gene encoding this enzyme may be used to produce improved recombinant yeast capable of utilizing xylose. The recombinant yeast are preferably transformed with heterologous polynucleotide sequences coding both the P. ruminicola XI, and the xylulokinase (XKS) of a Prevotella species. Yeast transformed with the polynucleotide sequences coding both of these XI and XKS exhibit significantly increased xylose utilization and cell growth on a culture medium containing xylose as the sole carbon source, in comparison to yeast transformed with XKS and XI from other sources.
Core Innovation
The invention provides a novel xylose isomerase (XI) enzyme produced by strain TC2-24 of the rumen bacterium Prevotella ruminicola, which exhibits increased catalytic activity and affinity for xylose. The gene encoding this enzyme has been isolated, and recombinant yeast strains transformed with this gene are capable of efficiently utilizing xylose. Preferably, these recombinant yeasts are also transformed with heterologous polynucleotide sequences encoding a xylulokinase (XKS) enzyme from a Prevotella species. Yeasts expressing both the P. ruminicola XI and Prevotella XKS demonstrate significantly improved xylose utilization and cell growth on xylose as the sole carbon source compared to yeasts transformed with XI and XKS from other sources.
The background identifies a critical problem: while Saccharomyces cerevisiae yeasts ferment hexose sugars like glucose, they lack the natural ability to ferment pentose sugars such as xylose, which are abundant in lignocellulosic biomass. Existing native xylose-fermenting yeasts have technical limitations including poor anaerobic growth on xylose, low tolerance to toxic inhibitors, and low productivity compared to glucose fermentation. Prior engineering strategies introducing xylose reductase and xylitol dehydrogenase had cofactor imbalances leading to xylitol production, limiting ethanol yields. Attempts to express heterologous xylose isomerase genes in Saccharomyces also suffered from low enzyme activity. Therefore, improved enzymes and yeast strains that efficiently ferment xylose remain a technical need.
This invention solves these problems by providing a xylose isomerase isolated from P. ruminicola strain TC2-24 which displays unexpectedly high activity and affinity for xylose, as measured by Michaelis-Menten kinetics. Recombinant expression of the codon-optimized gene in Saccharomyces cerevisiae results in enhanced enzyme kinetics and improved fermentation performance. In addition, co-expression of the P. ruminicola XI with an XKS enzyme from a Prevotella species, preferably from the same genus, further enhances xylose utilization and yeast growth compared to XI and XKS from other organisms. This dual gene transformation represents an improved strategy for engineering yeast capable of efficient xylose fermentation from biomass-derived sugars.
Claims Coverage
The patent contains two independent claims covering nucleic acid constructs and yeast cells transformed with sequences encoding the novel xylose isomerase and optionally the xylulokinase.
Nucleic acid construct encoding the P. ruminicola xylose isomerase
A nucleic acid construct comprising an isolated nucleic acid molecule encoding a xylose isomerase that has the amino acid sequence of SEQ ID NO: 1, operably linked to expression control sequences. The sequence may be selected from SEQ ID NOs: 2 or 3.
Yeast transformed with nucleic acid encoding the P. ruminicola xylose isomerase and optionally xylulokinase
A yeast cell transformed with the nucleic acid construct encoding the P. ruminicola xylose isomerase, optionally further transformed with a nucleic acid construct encoding a xylulokinase, where the xylulokinase has the amino acid sequence of SEQ ID NO: 4, and the encoding sequence is selected from SEQ ID NOs: 5 or 6. The yeast species may be Saccharomyces, preferably Saccharomyces cerevisiae.
The independent claims cover nucleic acid constructs encoding the isolated P. ruminicola xylose isomerase and optionally a xylulokinase, as well as yeast cells transformed with these constructs, conferring enhanced xylose metabolism and growth.
Stated Advantages
The novel xylose isomerase exhibits increased catalytic activity and affinity for xylose compared to prior enzymes.
Yeasts co-transformed with P. ruminicola xylose isomerase and Prevotella xylulokinase show significantly increased xylose utilization and cell growth on xylose as the sole carbon source.
The engineered yeasts exhibit improved performance for xylose fermentation, including higher ethanol yields.
Documented Applications
Recombinant yeast capable of fermenting xylose to ethanol using xylose as the sole carbon source.
Use of the yeasts to ferment sugars derived from lignocellulosic biomass or agricultural wastes to produce ethanol.
Potential further transformation of yeasts with enzymes for hydrolyzing complex lignocellulosic substrates to release fermentable sugars.
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