Anaerobic Xylose Fermentation by Recombinant Saccharomyces cerevisiae Carrying XYL1 , XYL2 , and XKS1 in Mineral Medium Chemostat Cultures

Abstract

For ethanol production from lignocellulose, the fermentation of xylose is an economic necessity. Saccharomyces cerevisiae has been metabolically engineered with a xylose-utilizing pathway. However, the high ethanol yield and productivity seen with glucose have not yet been achieved. To quantitatively analyze metabolic fluxes in recombinant S. cerevisiae during metabolism of xylose-glucose mixtures, we constructed a stable xylose-utilizing recombinant strain, TMB 3001. The XYL1 and XYL2 genes from Pichia stipitis, encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, and the endogenous XKS1 gene, encoding xylulokinase (XK), under control of the PGK1 promoter were integrated into the chromosomal HIS3 locus of S. cerevisiae CEN.PK 113-7A. The strain expressed XR, XDH, and XK activities of 0.4 to 0.5, 2.7 to 3.4, and 1.5 to 1.7 U/mg, respectively, and was stable for more than 40 generations in continuous fermentations. To obtain an economically feasible industrial process for ethanol production from lignocellulose, it is necessary to ferment all sugars present with high yields and productivities (53). The commonly used Saccharomyces cerevisiae has many advantages as an ethanol producer, such as fast sugar consumption, high ethanol yield from hexoses, and high resistance to inhibitory compounds that are present in the hydrolysates. However, a major drawback is that S. cerevisiae cannot utilize the pentose sugar xylose, only its isomer xylulose. In xylose-utilizing yeasts, the conversion from xylose to xylulose is a two-step process catalyzed by xylose reductase (XR) and xylitol dehydrogenase (XDH) (10), whereas bacteria perform the conversion in one step with xylose isomerase (XI) (23).Xylose fermentation by recombinant S. cerevisiae carrying heterologous XYL1 and XYL2 genes from Pichia stipitis, which encode XR and XDH, respectively, has resulted mainly in xylitol formation (24,44,48). Similarly, if xylA from Thermus thermophilus, which encodes XI, is introduced into S. cerevisiae, then only limited xylose fermentation is observed (47). Limited xylose fermentation by recombinant S. cerevisiae has been ascribed to poor xylose uptake (9, 24, 25), a cofactor imbalance generated by the discrepancy in cofactor usage by XR and XDH (8,24,49), limitations in the pentose phosphate pathway (12,24,38,48), and insufficient induction or activation of ethanologenic enzymes (5,17,20,29). When homologous XKS1, which encodes xylulokinase (XK), was overexpressed in a Saccharomyces sp. strain carrying XYL1 and XYL2, the ethanol yield and the xylose uptake rate increased under oxygenlimited conditions, but xylitol was still a major by-product (22).Although the shortcomings of xylose fermentation by recombinant S. cerevisiae have been investigated in several studies, data from anaerobic fermentations do not exist and quantitative data are sparse. Chemostat cultivations in which growth rate and concentrations of substrates and products are constant enable quantitative determinations of metabolic fluxes. Analysis o...

Enhanced biofuel production through coupled acetic acid and xylose consumption by engineered yeast

“…Engineered S. cerevisiae strains with the XR/XDH pathway grow extremely slowly when xylose serves as the sole carbon source under anaerobic conditions that are desirable for large-scale industrial fermentation 14,27,28 . We constructed an efficient xylose-fermenting S. cerevisiae strain SR8 by combining rational and combinatorial approaches 29 .…”

“…For the fermentation experiment to track acetate reduction to ethanol, 2 g l À 1 acetate-2-13 C (Sigma-Aldrich) was used instead of normal acetate. Ergosterol and Tween 80 were added to final concentrations of 0.01 and 0.42 g l À 1 , respectively 27 . The anaerobic condition was prepared as described above.…”

Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current status

“…The mechanism by which cofactor imbalances are prevented in these strains is unclear. A relevant factor in this respect may be the large variation in XR cofactor dependency observed in Candida tenuis (see Table 3): the Purdue strains express the xylose reductase gene from CBS 5773 (Chen and Ho 1993), whereas the TMB strains from Lund express that from CBS 6054 (Eliasson et al 2000;Walfridsson et al 1995). The cultivation method may also help to explain the low xylitol production found with the Purdue strains.…”

Characterization of the xylose-transporting properties of yeast hexose transporters and their influence on xylose utilization

“…Efforts to establish a xylose-utilizing pathway in S. cerevisiae by insertion of the genes encoding xylose reductase and xylitol dehydrogenase from Pichia stipitis or other organisms have resulted in only poor ethanol production from xylose (Ko$ tter & Ciriacy, 1993 ;Tantirungkij et al, 1993 ;Walfridsson et al, 1995). Various steps, including the uptake of xylose, have been suggested to limit the metabolism of xylose in metabolically engineered S. cerevisiae (Ko$ tter & Ciriacy, 1993 ;Eliasson et al, 2000). Uptake of xylose by S. cerevisiae has been proposed to be mediated more or less unspecifically by its hexose-transport system.…”