Genetic Engineering of Xylose Fermentation in Yeasts

Thomas W. Jeffries, Brian P. Davis, Kristine Dahn, Jae-Yong Cho,
Nian-Qing Shi, Ping Lu and James Hendrick
USDA, Forest Service, Forest Products Laboratory, Madison, WI

Abstract

Genetic engineering of xylose fermentation has been carried out through the introduction of genes from Pichia stipitis into Saccharomyces cerevisiae. Unfortunately, this has not always had the desired effect of enabling efficient production of ethanol from xylose. In order to improve knowledge of xylose metabolism, we have developed a genetic system that enables overexpression or deletion of key enzymes in one of the best studied xylose-fermenting yeasts, Pichia stipitis. It uses P. stipitis URA3 and LEU2 as selectable markers, a native P. stipitis ARS (autonomous replication sequence) to maintain the DNA extrachromosomally, and recipient hosts with multiple lesions for transformation and mating. We have created both [[lambda]] and expression libraries of the P. stipitis genomic DNA, and can retrieve any P. stipitis gene that has similarity to corresponding genes from S. cerevisiae.

Introduction

Xylose is the second most abundant sugar found in hardwood and agricultural residues. Because of its prevalence, xylose utilization is essential in any process for the production of ethanol from these feedstocks. Xylose is relatively easily recovered by acid or enzymatic hydrolysis, but can be fermented to ethanol only by a few naturally-occurring or recombinant microbes. Despite study in many laboratories over the past 15 years, the xylose fermentation is not yet commercial.

Xylose metabolism in yeasts

Pichia stipitis takes up xylose by both high-affinity and low-affinity proton symport mechanisms[1][2]. In C. shehatae the low affinity transport employs facilitated diffusion which is a passive, low energy requiring mechanism[3] whereas in P. stipitis both the low and high affinity systems are based on proton symport which require the expenditure of metabolic energy. Glucose inhibits the transport of xylose by P. stipitis in a competitive manner, indicating that both sugars use the same transport system[4].

The enzymes for xylose metabolism in P. stipitis are inducible by xylose and repressible by glucose[5]. The first step in yeast xylose metabolism is carried out by xylose (aldose) reductase. The gene coding for this enzyme has been given the designation XYL1. We refer to the active enzyme protein as XYL1p or XOR. In most yeasts and fungi, this enzyme has a cofactor specificity for NADPH, but in Pichia stipitis, the enzyme shows 70% as much activity with NADH as with NADPH[6]. The gene for xylose reductase has been cloned from P. stipitis by at least three independent laboratories[7],8,9 and it has been cloned more recently from Kluyveromyces lactis[10] and Pachysolen tannophilus[11]. In both P. stipitis and P. tannophilus, XYL1 is induced by growth on xylose, but in K. lactis, it appears to be constitutive. The relative affinity of various xylose reductases for NADH and NADPH appears to vary widely, and it has recently been reported that the enzyme from Candida boidinii actually has higher activity with NADH than with NADPH[12].

Figure 1. Initial steps of xylose pathway in yeasts and filamentous fungi. XYL1 is xylose (aldose) reductase; XYL2 is xylitol dehydrogenase; XYL3 is xylulokinase.

The second step in xylose metabolism is coded for by xylitol dehydrogenase (XYL2). Unlike aldose reductase, this enzyme is always specific for NAD, but the cloned enzyme[13] has been modified to alter cofactor specificity[14]. Xylitol dehydrogenase has also been purified and characterized from Debaromyces hansenii[15] and Candida shehatae[16].

Genetic engineering of xylose metabolism in S. cerevisiae

Several laboratories have attempted to engineer a xylose-fermenting S. cerevisiae through the expression of XYL1 or both XYL1 and XYL2. Expression of XYL1 alone has not proven sufficient to enable S. cerevisiae to ferment or even to grow on xylose7,8, but in the presence of glucose, S. cerevisiae strains expressing XYL1 will produce xylitol from xylose at high yield9,[17]. Production of xylitol appears to be a consequence of redox imbalance in the cell, and is affected by glycerol production18-20[18].

Co-expression of both XYL1 and XYL2 has proven to be more successful. Kötter et al.13 first to reported construction of an S. cerevisiae strain expressing both XYL1 and XYL2 in 1990. Both XYL1 and XYL2 have coding regions composed of the preferred codons for highly-expressed S. cerevisiae genes, and both genes are expressed at a low level in S. cerevisiae using their native P. stipitis promoters. Expression of both XYL1 and XYL2 made it possible for S. cerevisiae to grow on xylose, but it formed only trace quantities (10 mM) ethanol. Kötter and Ciriacy studied the xylose fermentation in S. cerevisiae more extensively, and compared the fermentative activities to P. stipitis[21]. They found that in the absence of respiration, S. cerevisiae transformed with both XYL1 and XYL2 converts about half of the xylose present in the medium into xylitol and ethanol in roughly equimolar amounts. By comparison, P. stipitis produces only ethanol. They proposed, as had Hahn-Hägerdal et al. that in S. cerevisiae, ethanol production is limited by cofactor imbalance.

Tantirungkij et al.[22] took the approach one step further by subcloning P. stipitis XYL1 into S. cerevisiae under the control of the enolase promoter on a multicopy vector. This achieved two to three times the level of XYL1 expression as was observed in P. stipitis. XYL2 was also cloned and co-expressed in S. cerevisiae at about twice the level achieved in induced P. stipitis. Despite these higher levels of expression, only low levels of ethanol (on the order of 5 g/l) were observed under optimal conditions after 100 h. These researchers also selected mutants of S. cerevisiae carrying XYL1 and XYL2 that exhibited rapid growth on xylose medium[23]. The fastest growing strain showed a lower activity of XYL1p, but a higher ratio of XYL2p to XYL1p activity. Southern hybridization showed that the vector carrying the two genes had integrated into the genome resulting in increased stability of the cloned genes. the yield and production rate of ethanol increased 1.6 and 2.7 fold, respectively, but the maximum concentration of ethanol reported was only 7 g/l after 144 h.

Ho and Tsao have recently filed an international patent application on a recombinant Saccharomyces strain expressing XYL1, XYL2 and xylulokinase (XYL3)24. The parental yeast strain Saccharomyces 1400, is a fusion product of Saccharomyces diastaticus and Saccharomyces uvarum[25]. It exhibits high ethanol and temperature tolerance and a high fermentation rate. Cloning of the XYL3 gene from Pachysolen tannophilus was first reported in 1987[26], and cloning of Saccharomyces cerevisiae XYL3 by complementation of a xylulokinase deficient mutant of E. coli was reported in 1989[27]. Disruption of the S. cerevisiae XYL3 gene by LEU2 resulted in strains that were unable to grow on D-xylulose[28]. Overexpression of XYL3 in the Saccharomyces 1400 fusant along with XYL1 and XYL2 results in production of about 47 g/l of ethanol in 84% of theoretical yield from a 1:1 glucose/xylose mixture[29].

Fermentation of xylose by Pichia stipitis

Many different researchers have compared the various species and strains of xylose fermenting yeasts under different conditions, and several reviews of such comparisons have been published30-35. In one such study, after screening 56 yeast isolates, du Preez and Prior31 identified P. stipitis and Candida shehatae as the best species for xylose fermentation. Many other researchers have compared various strains of P. stipitis to C. shehatae and other xylose fermenting yeasts, and although the strains vary a bit from one to another, P. stipitis often comes out near the top. Slininger et al.[36]  found that P. stipitis NRRL Y-7124 could convert 150 g/l of xylose into 52 g/l of ethanol at a yield of 0.39 g/g. In our own laboratory, direct comparisons of xylose fermentations -- either with pure sugars in defined medium or with acid hydrolysates plus crude nutritional supplements such as corn steep liquor -- C. shehatae often performs better than P. stipitis. In one fed-batch fermentation, C. shehatae ATCC 22984 produced 56 g/l of ethanol from xylose within 48 h[37]. The yield with C. shehatae is a bit lower, however, because of xylitol production.

Fermentation of acid hydrolysates and sugar mixtures

In fermenting acid hydrolysates of wood[38],39 wheat straw[40] or sugar cane bagasse[41] yields are often lower because of the presence of toxic degradation products. The mixture of glucose and xylose present in hydrolysate also causes problems because glucose generally represses xylose fermentation[42]. This difficulty has been approached by using S. cerevisiae and P. stipitis to coferment glucose and xylose either with immobilized cells[43] or in successive reactors[44]. A flocculating strain of P. stipitis has also been used for this same purpose[45]

Optimization of fermentation by P. stipitis

Relatively early studies by du Preez et al.[46] showed that the optimum temperature for fermentation was around 30deg.C, and that the optimum pH was about 4 to 5.5. In subsequent studies in which optimization has aimed at maximum ethanol concentrations rather than formation rate, the temperature optimum has been found to be closer to 25deg.C[47]. The maximum ethanol productivity with both C. shehatae and P. stipitis is around 0.9 g l-1 h-1.

The principal factor that must be optimized for both P. stipitis and C. shehatae is aeration rate. This has been studied by numerous laboratories48-53. The optimum aeration rate varies with the cell density, but an oxygen uptake rate in the range of 3.75 to 5 mmol l-1 h-1 gives a maximum ethanol productivity of around 0.12 to 0.13 g g-1 h-1. The optimal aeration rate is about the same with glucose, but the productivity rate increases to 0.35 g g-1 h-1.

In a few laboratories, these studies have been taken a step further into the realm of physiology by examining the effects of aeration on the induction of enzyme levels. du Preez et al. found that under anoxic conditions, the levels of xylose reductase and xylitol dehydrogenase did not change, and their cofactor specificities did not shift, suggesting that only a single isozyme of each is present, and the activities are not affected by aeration. The enzyme activity of P. stipitis decreased significantly which possibly contributed to its weaker anoxic fermentation of xylose[54]. Skoog and Hahn-Hägerdal observed that as the aeration rate decreased, pyruvate decarboxylase activity of P. stipitis increased and malate dehydrogenase activity decreased[55]. This observation is consistent with the report of Alexander et al.[56] that as the specific aeration rate was decreased in continuous cultures of C. shehatae, the levels (and cofactor activities) of xylose reductase and xylitol dehydrogenase did not change significantly, but alcohol dehydrogenase activity was induced about 8- to 10-fold. This has led us to the hypothesis that in P. stipitis and C. shehatae fermentation is regulated by oxygen availability[57].

Genetic studies with P. stipitis and C. shehatae

For genetic studies P. stipitis is preferred because unlike C. shehatae, it will form spores, implying that under appropriate conditions, strains can be mated. Because of morphological similarities between P. stipitis and C. shehatae, the former was believed to be the teleomorph and the latter the anamorph of closely related species. Genome comparisons, however, have not borne this out[58]. The two yeasts belong to closely related, but distinct species[59]. This can be seen by both DNA homology and chromosomal electrophoretic banding patterns[60].

One of the more difficult questions concerning the genetics of P. stipitis is a rather basic one: Is it diploid or haploid? Unfortunately, calculation of cell death rates by UV or x-ray bombardment do not provide reliable answers[61]. Sporulation, mating and transformation results suggest, however, that P. stipitis is principally diploid in nature, but it may also exist in a haploid state.

There have been a number of studies of P. stipitis genetics that have focused on obtaining mutants of xylose or glucose metabolism. Hagedorn and Ciriacy[62] isolated several xyl mutants of P. stipitis and found that by immunological assays and revertant analysis, both mutant types could be attributed to structural genes XYL1 and XYL2. Revertant analysis of XYL1 also indicated that a second cryptic gene exists that codes for an NADPH-dependent aldose reductase. Laplace et al.[63] in an attempt to obtain strains that are carbon catabolite derepressed have selected P. stipitis that have a decreased ability to convert glucose to ethanol. Sreenath and Jeffries have selected for rapid growth of C. shehatae on L-xylose and xylitol in the presence of respiratory inhibitors and have obtained strains that exhibit higher specific fermentation rates[64].

Protoplast fusion between auxotrophic strains has been used by several researchers to create hybrids and polyploids of P. stipitis and other xylose fermenting yeasts. The earliest of these studies were done by Gupthar[65]. Polyethylene induced fusion led to strains with increased ploidy, but these were very unstable and segregated readily into lower DNA content. Protoplast fusion has been used to create polyploids between P. stipitis and C. shehatae[66],67 and between P. stipitis and S. cerevisiae[68], but these likewise were unstable and showed only marginal changes in ethanol production rates or tolerance[69]. Other fusions have been carried out between Pachysolen tannophilus and Saccharomyces cerevisiae with similar results[70],71.

Genetic transformation of xylose fermenting yeasts

Contemporary molecular genetics cannot be carried out without an efficient genetic transformation and expression system that enables the specific, introduction and modification of genomic DNA. One of the first attempts at obtaining a transformation system for P. stipitis used resistance to kanamycin (KmR)[72]. Unfortunately, this system only enabled a few transformants per microgram of DNA, and because the presence of kanamycin is required to maintain extrachromosomal plasmids, this would not be useful in large scale fermentations.

The first high-efficiency transformation system for P. stipitis employed the native P. stipitis gene for URA3[73] and ura3 auxotrophs selected by resistance to 5'-fluoro-orotic acid (5'-FOA)[74]. The P. stipitis URA3 gene was cloned by its homology to S. cerevisiae URA3 with which it is 69% identical in the coding region. The P. stipitis ARS2 contains features similar to the consensus ARS of S. cerevisiae and other ARS elements. Intact Escherichia coli/P. stipitis shuttle plasmids bearing the P. stipitis URA3 gene with various amounts of flanking sequences formed 600 to 8,600 transformants/ug DNA when introduced into P. stipitis ura3 auxotrophs by electroporation. Most transformants obtained with circular vectors bearing URA3 alone arise through gene conversion without integration. One vector yielded 1,300 to 12,500 transformants/ug DNA when it was linearized by restriction at various sites within the P. stipitis URA3 insert. Transformants arising from linearized vectors produced stable integrants that incorporated the entire plasmid DNA into the host genome and were site-specific for the genomic ura3 in 20% of the transformants examined. In contrast, plasmids bearing the S. cerevisiae URA3 gene led to less than 10 transformants/ug DNA, and we observed only gene conversion events. Plasmids bearing the P. stipitis URA3 gene and ARS2 element transformed P. stipitis ura3 auxotrophs at a frequency of more than 30,000 transformants/mg plasmid DNA. Exogenous plasmids were stable for at least 50 generations and were present at an average of 10 copies per haploid genome.

We have cloned and sequenced a number of other genes from P. stipitis. These include XYL1 and XYL2, genes for fermentation, ADH1, ADH2, PDC1 and PDC2, and some of the genes for respiration including CYC1 and PDH. Most of the fermentative and respirative P. stipitis genes have strong similarity to their S. cerevisiae homologues, but one, PsPDC1 includes a 27 amino acid insert into the structural protein that is not present in S. cerevisiae or any other known protein sequence. Disruption of the fermentative ADH results in overproduction of xylitol and diminished growth. Unlike S. cerevisiae where fermentative genes are induced and respirative genes are repressed by glucose. In P. stipitis, fermentative metabolism is induced in response to oxygen limitation.

The use of this transformation system to obtain improved xylose fermenting yeasts is still at an early stage. Preliminary experiments to determine the effects of overexpressing XYL1 in P. stipitis have resulted in higher levels of expression of this enzyme[75], and its expression from the multicopy pJM6 vector appears to increase production of XYL1p in the presence of glucose. This modification, however, did not significantly increased ethanol production in the P. stipitis TJ26 host strain. Expression of genes for several enzymes simultaneously is hindered by the use of a single selectable marker. For this reason, we have cloned the LEU2 gene and used the URA3 gene to create a null mutant. A site specific disruption was performed by using the disruption cassette to transform a ura3 mutant, and resultant strains were selected for the leu- URA+ phenotype. Several isolates were plated onto 5'-FOA and screened for the leu-.ura- phenotype. The double auxotrophs are stable and can been transformed by PsLEU2 or PsURA3 with a high frequency. As additional selectable markers, transformable hosts and genes become available, the genetic engineering of P. stipitis will proceed rapidly.

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