Genetically engineered Saccharomyces yeast capable of effective cofermentation of glucose and xylose

Abstract

Xylose is one of the major fermentable sugars present in cellulosic biomass, second only to glucose. However, Saccharomyces spp., the best sugar-fermenting microorganisms, are not able to metabolize xylose. We developed recombinant plasmids that can transform Saccharomyces spp. into xylose-fermenting yeasts. These plasmids, designated pLNH31, -32, -33, and -34, are 2 microns-based high-copy-number yeast-E. coli shuttle plasmids. In addition to the geneticin resistance and ampicillin resistance genes that serve as dominant selectable markers, these plasmids also contain three xylose-metabolizing genes, a xylose reductase gene, a xylitol dehydrogenase gene (both from Pichia stipitis), and a xylulokinase gene (from Saccharomyces cerevisiae). These xylose-metabolizing genes were also fused to signals controlling gene expression from S. cerevisiae glycolytic genes. Transformation of Saccharomyces sp. strain 1400 with each of these plasmids resulted in the conversion of strain 1400 from a non-xylose-metabolizing yeast to a xylose-metabolizing yeast that can effectively ferment xylose to ethanol and also effectively utilizes xylose for aerobic growth. Furthermore, the resulting recombinant yeasts also have additional extraordinary properties. For example, the synthesis of the xylose-metabolizing enzymes directed by the cloned genes in these recombinant yeasts does not require the presence of xylose for induction, nor is the synthesis repressed by the presence of glucose in the medium. These properties make the recombinant yeasts able to efficiently ferment xylose to ethanol and also able to efficiently coferment glucose and xylose present in the same medium to ethanol simultaneously.

FIG. 1

Construction of high-copy-number yeast-E. coli shuttle plasmids pLNH31, pLNH32, pLNH33, and pLNH34. containing the XYL three-gene cassette KK-AR (or A*R)-KD. The XhoI DNA fragment containing KK-AR (or A*R)-KD was inserted into pUCKm10 at its SalI site.

FIG. 2

Construction of E. coli plasmids containing the XYL gene cassette KK-AR (or A*R)-KD. KK, S. cerevisiae XK gene fused to the promoter of the S. cerevisiae pyruvate kinase gene; AR (or A*R), P. stipitis XR gene fused to the promoter of the S. cerevisiae alcohol dehydrogenase gene; KD, P. stipitis XDH gene fused to the promoter of the S. cerevisiae pyruvate kinase gene. The double dagger (‡) indicates that the XhoI site was regenerated after ligation, and the asterisks (∗) indicate an intact ADC1 promoter.

FIG. 3

Growth of S. cerevisiae in rich medium with or without a carbon source (sugar). (A) S. cerevisiae AH22 cultured in YEP or YEPD. (B) S. cerevisiae AH22 cultured in YEP or YEPXylu (1% yeast extract, 2% peptone, 2% xylulose). Symbols: ○, YEP; •, YEPD; ▴, YEPXylu.

FIG. 4

Comparison of the abilities of recombinant Saccharomyces sp. strain 1400(pLNH32) and parent strain 1400 to coferment glucose and xylose. (A) Strain 1400(pLNH32). (B) Strain 1400. Utilization of glucose and xylose and production of ethanol, xylitol, and glycerol were determined. Symbols: ▪, glucose; •, xylose; ▴, ethanol; □, xylitol; ▵, glycerol.

FIG. 5

Fermentation of xylose by recombinant Saccharomyces sp. strain 1400(pLNH32) cultured in the xylose-containing medium YEPX. Utilization of xylose and production of ethanol, xylitol, and glycerol were determined. Symbols: •, xylose; ▴, ethanol; □, xylitol; ▵, glycerol.