This paper describes the strategies used to develop recombinant Saccharomyces strains that can effectively ferment xylose and, in particular, can coferment glucose and xylose present in the same medium. Using Saccharomyces sp. strain 1400 as an example, we successfully demonstrated that by cloning XR, XD, and XK, each of which was fused to an efficient glycolytic promoter, a superior glucose-fermenting Saccharomyces strain can be transformed to a recombinant yeast strain that is able to effectively metabolize xylose aerobically for growth and anaerobically for the production of ethanol as the overwhelmingly major product. Furthermore, the resulting recombinant xylose-fermenting Saccharomyces strains can also effectively coferment glucose and xylose present in the same medium. This unique property should make the recombinant Saccharomyces strains particularly effective in large-scale production of ethanol from fermenting mixed sugars present in cellulosic biomass hydrolysates.
Although only the results obtained with strain 1400(pLNH32) are presented here, the transformants containing pLNH31, pLNH33, or pLNH34 do not differ significantly in their ability to coferment glucose and xylose or to utilize xylose for growth. The presence of either AR or A*R in the transformants also does not significantly affect their ability to ferment xylose or utilize it for growth.
Our recombinant Saccharomyces strains can effectively ferment xylose in the presence of glucose, as shown in Fig. . However, they do not depend on the presence of glucose to ferment xylose. They can effectively ferment xylose in the absence of glucose when they are cultured in xylose medium, as shown in Fig. .
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; (more ...)
As mentioned above, while our work was in progress, Kotter et al. (20
), Walfridsson et al. (25
), and Tantirungkij et al. (24
) described the development of their recombinant S. cerevisiae
strains, which was accomplished by cloning only the P. stipitis
XR gene and XDH gene. However the recombinant yeast strains of these groups ferment xylose extremely slowly and produce little ethanol. Furthermore, the major fermentation product produced by these recombinant yeast strains is not ethanol but xylitol. Most importantly, there are fundamental differences between our strategy and the strategies used by the other three groups to develop recombinant yeast strains. For example, as mentioned above, our recombinant Saccharomyces
strains not only contain the cloned XR gene and the XDH gene but also contain the cloned XK gene. As a result, our recombinant yeast strains can effectively ferment high concentrations of xylose almost completely to ethanol, and very little xylitol is produced as a by-product (Fig. A and ).
Our strategy to fuse the cloned XR, XD, and XK genes to highly effective glycolytic promoters (or expression signals) made our recombinant yeast strains better than the recombinant yeast strains developed by others, as well as naturally occurring microorganisms in fermenting substrates containing both glucose and xylose. The other groups did not include this strategy in their designs. As a result, the expression of the cloned XR, XD, and XK genes in our recombinant yeast strains does not require the presence of xylose for induction, and also the expression of the cloned genes is not repressed by glucose in the cultural medium. This allowed our recombinant Saccharomyces strains to effectively coferment glucose and xylose without a lag period, as shown in Fig. A. However, for the conversion of cellulosic biomass to ethanol, the fact that our yeast strains do not require xylose for induction and the fact that they are able to maintain expression of the xylose-metabolizing genes in the presence of glucose provide a much greater benefit than metabolizing xylose without a lag period. The naturally occurring xylose-fermenting yeasts, such as P. stipitis and C. shehatae, which ferment xylose as effectively as our recombinant xylose-fermenting Saccharomyces strains but do not have the properties mentioned above, are not able to ferment xylose at all when glucose is present in the medium, even though they effectively ferment xylose in the absence of glucose. Data comparing the ability of our recombinant yeast strains to ferment xylose and to coferment glucose and xylose with the ability of the naturally occurring xylose-fermenting yeasts P. stipitis and C. shehatae to ferment xylose and to coferment glucose and xylose will be published elsewhere.
The fact that we developed a set of broad-host-range pLNH plasmids by using the Kmr and Apr genes as the primary selection markers also contributed greatly to the development of effective recombinant xylose-fermenting Saccharomyces strains, such as 1400(pLNH32). The development of such plasmids was intended to provide a means by which wild-type yeast strains, particularly diploid or polyploid industrial strains (such as strain 1400) that may have superior properties desirable for ethanol fermentation, can easily be transformed by these vectors. Furthermore, since there are other factors besides the enzymes encoded by the cloned XYL genes that may seriously affect the effectiveness of yeast xylose fermentation, we do not expect that all Saccharomyces strains that receive the same cloned XYL genes will ferment xylose with the same efficiency. Thus, the pLNH plasmids can also be excellent tools for screening new Saccharomyces strains that may be superior for cofermenting glucose and xylose. Nevertheless, since we did not have to screen many yeast strains to obtain strain 1400, which is an effective host for the expression of the XYL genes, there may be other Saccharomyces strains that are as effective as or more effective than strain 1400 as hosts for expression of the XYL genes for efficient fermentation of xylose to ethanol.
It is desirable to design a broad-host-range selective mechanism that requires only inexpensive and nontoxic chemicals as the selective pressure to select and maintain Saccharomyces transformants, and our discovery that Saccharomyces strains require a carbon source to grow even in rich medium provided an answer. This discovery provided an ideal and effective mechanism that could be used to screen and maintain true transformants containing the cloned XYL genes. Furthermore, it also provided a rapid and effective method for differentiating xylose-fermenting Saccharomyces transformants from other xylose-metabolizing microorganisms, including the naturally occurring xylose-fermenting yeasts, such as P. stipitis and C. shehatae. This is because there are very few microorganisms that require the presence of a carbon source in rich medium to grow.
The fact that xylose can serve as a selective agent to maintain the pLNH plasmids even in rich medium, such as YEPX, coupled with the fact that the pLNH plasmids are stable, high-copy-number plasmids in Saccharomyces strains, allows the pLNH transformants of Saccharomyces strains to effectively ferment xylose to ethanol in the absence of selection for at least four or five generations, as shown in Fig. . This makes it possible for industry to use these recombinant yeast strains, such as 1400(pLNH32), to carry out large-scale fermentation of the glucose and xylose present in cellulosic biomass to ethanol in the absence of selection. To achieve this, the only requirements are that the process is a batch fermentation process and that the yeast cells are propagated in the early stages in medium containing xylose as the major or sole carbon source.
Several groups in the United States and Canada are using our xylose-fermenting recombinant strain 1400 yeasts to perform various studies, and they have all verified what we report here. One group that used 1400(pLNH32) to ferment corn fiber sugars to ethanol has published their results (22
Nevertheless, additional improved xylose-fermenting Saccharomyces strains can still be developed. For example, even though the Saccharomyces transformants carrying the pLNH plasmids are very stable and can be maintained by an ideal selection mechanism (in which xylose is used as the selection pressure), an ideal transformant should be completely stable and not require the use of selection pressure at any stage of growth or fermentation.
Recently, we have been very successful in developing stable derivatives of xylose-fermenting recombinant yeast strain 1400, designated 1400(LNH-ST) strains (or LNH-ST strains). The latter Saccharomyces
strains are completely stable and do not require any selection pressure to maintain their ability to ferment xylose to ethanol or their ability to utilize this sugar for growth. Furthermore, these stable xylose-fermenting recombinant yeast strains also ferment xylose to ethanol slightly more efficiently than 1400(pLNH32) or any other pLNH transformant of strain 1400. The stable strains were developed by integrating multiple copies of the XYL
genes into the yeast chromosome. Brief descriptions of the ability of these stable xylose-fermenting Saccharomyces
strains to ferment mixed sugars in the absence of any selection pressure have been presented previously (16
). The details concerning the development and characterization of such stable xylose-fermenting Saccharomyces
strains will be described elsewhere.