The Missing Link in the Fungal L-Arabinose Catabolic Pathway, Identification of the L-Xylulose Reductase Gene †,‡ Peter Richard,* Mikko Putkonen, Ritva Va ¨a ¨na ¨nen, John Londesborough, and Merja Penttila ¨ VTT Biotechnology, P.O. Box 1500, FIN-02044 VTT, Finland ReceiVed January 9, 2002; ReVised Manuscript ReceiVed February 28, 2002 ABSTRACT: The fungal L-arabinose pathway consists of five enzymes, aldose reductase, L-arabinitol 4-dehydrogenase, L-xylulose reductase, xylitol dehydrogenase, and xylulokinase. All the genes encoding the enzymes of this pathway are known except for that of L-xylulose reductase (EC 1.1.1.10). We identified a gene encoding this enzyme from the filamentous fungus Trichoderma reesei (Hypocrea jecorina). The gene was named lxr1. It was overexpressed in the yeast Saccharomyces cereVisiae, and the enzyme activity was confirmed in a yeast cell extract. Overexpression of all enzymes of the L-arabinose pathway in S. cereVisiae led to growth of S. cereVisiae on L-arabinose; i.e., we could show that the pathway is active in a heterologous host. The lxr1 gene encoded a protein with 266 amino acids and a calculated molecular mass of 28 428 Da. The LXRI protein is an NADPH-specific reductase. It has activity with L-xylulose, D-xylulose, D-fructose, and L-sorbose. The highest affinity is toward L-xylulose (K m ) 16 mM). In the reverse direction, we found activity with xylitol, D-arabinitol, D-mannitol, and D-sorbitol. It requires a bivalent cation for activity. It belongs to the protein family of short chain dehydrogenases. The enzyme is catalytically similar and homologous in sequence to a D-mannitol:NADP 2-dehydrogenase (EC 1.1.1.138). L-Arabinose is a major constituent of plant material (1). An L-arabinose catabolic pathway is therefore a relevant pathway for microorganisms which live on decaying plant material and also for biotechnology when cheap raw materi- als are used. An example is to make fuel ethanol from agricultural waste residues such as corn fiber, which is particularly rich in L-arabinose. Approximately a third of the corn fiber xylan is L-arabinose (2). In this context, it is interesting to have an L-arabinose catabolic pathway in an ethanol- and inhibitor-tolerant organism like Saccharomyces cereVisiae. Two distinct bacterial and fungal pathways are known for the catabolism of L-arabinose. The bacterial pathway consists of L-arabinose isomerase (EC 5.3.1.4), ribulokinase (EC 2.7.1.16), and L-ribulose phosphate 4-epimerase (EC 5.1.3.4). The pathway and the genes of this pathway are well- established (3). About the fungal pathway only little is known. The fungal pathway was first described by Chiang and Knight (4). It consists of aldose reductase (EC 1.1.1.21), L-arabinitol 4-dehydrogenase (EC 1.1.1.12), L-xylulose re- ductase (EC 1.1.1.10), D-xylulose reductase (EC 1.1.1.9), and xylulokinase (EC 2.7.1.17) which convert L-arabinose to L-arabinitol, L-xylulose, xylitol, D-xylulose, and D-xylulose 5-phosphate, respectively. The path from L-arabinose to D-xylulose is shown in Figure 1. The path consists of two reducing and two oxidizing steps with the reducing steps being NADPH-linked and the oxidizing steps being NAD-linked. Fungal aldose reductases which have activity with L-arabinose are known from Pichia stipitis (5) and S. cereVisiae (6), and their corresponding genes are known (7, 8). The gene for an L-arabinitol 4-dehydrogenase is known from Trichoderma reesei (9). Genes for D-xylulose reductases are known from P. stipitis (10), S. cereVisiae (8), and T. reesei (11). A D-xylulokinase gene is known for S. cereVisiae (12). A fungal gene for L-xylulose reductase is not known. In this paper, we describe the identification of a gene from the filamentous fungus T. reesei (Hypocrea jecorina) encod- ing an L-xylulose reductase and the functional expression of this gene in S. cereVisiae. We also expressed the gene together with all the other genes of the L-arabinose pathway, which led to a functional pathway, i.e., growth of S. cereVisiae on L-arabinose. EXPERIMENTAL PROCEDURES Yeast Strain for Screening. Although S. cereVisiae has the genes for aldose reductase, D-xylulose reductase, and xylu- lokinase in the genome, it cannot grow on xylose, because these genes are not adequately overexpressed. The yeast strain used for screening was therefore constructed from S. cereVisiae strain CEN.PK2 (VW1b) as follows. First, the XYL1 and XYL2 genes from P. stipitis encoding aldose reductase and D-xylulose reductase, respectively, and the XKS1 gene from S. cereVisiae encoding xylulokinase were added. The XYL1 and XYL2 genes under constitutive promot- ers (13) and the XKS1 gene under a consititutive promoter (14) were integrated into the chromosomes by targeted integration. The resulting strain (H2217) is auxotrophic for leucine and uracil. The L-arabinitol 4-dehydrogenase (lad1) was cloned as described previously (9). The EcoRI, BamHI † This work was supported by the Sustainable Use of Natural Resources (SUNARE) program of the Academy of Finland and the research program VTT Industrial Biotechnology (Academy of Finland; Finnish Centre of Excellence program, 2000-2005, Project 64330). ‡ The nucleotide sequence reported in this paper has been submitted to GenBank (accession number AF375616). * To whom correspondence should be addressed: VTT Biotechnol- ogy, Tietotie 2, Espoo, P.O. Box 1500, FIN-02044 VTT, Finland. Telephone: 358-9-456-7190. Fax: 358-9-455-2103. E-mail: Peter.Richard@vtt.fi. 6432 Biochemistry 2002, 41, 6432-6437 10.1021/bi025529i CCC: $22.00 © 2002 American Chemical Society Published on Web 04/25/2002