We demonstrate herein the ability of Kluyveromyces marxianus to be an efficient ethanol producer and host for expressing heterologous proteins as an alternative to Saccharomyces cerevisiae. Growth and ethanol production by strains of K. marxianus and S. cerevisiae were compared under the same conditions. K. marxianus DMKU3-1042 was found to be the most suitable strain for high-temperature growth and ethanol production at 45°C. This strain, but not S. cerevisiae, utilized cellobiose, xylose, xylitol, arabinose, glycerol, and lactose. To develop a K. marxianus DMKU3-1042 derivative strain suitable for genetic engineering, a uracil auxotroph was isolated and transformed with a linear DNA of the S. cerevisiae ScURA3 gene. Surprisingly, Ura ؉ transformants were easily obtained. By Southern blot hybridization, the linear ScURA3 DNA was found to have inserted randomly into the K. marxianus genome. Sequencing of one Lys ؊ transformant confirmed the disruption of the KmLYS1 gene by the ScURA3 insertion. A PCR-amplified linear DNA lacking K. marxianus sequences but containing an Aspergillus ␣-amylase gene under the control of the ScTDH3 promoter together with an ScURA3 marker was subsequently used to transform K. marxianus DMKU3-1042 in order to obtain transformants expressing Aspergillus ␣-amylase. Our results demonstrate that K. marxianus DMKU3-1042 can be an alternative cost-effective bioethanol producer and a host for transformation with linear DNA by use of S. cerevisiaebased molecular genetic tools.Ethanol production at high temperature has received much attention because fermentation processes conducted at elevated temperatures will significantly reduce cooling costs (14). Other advantages of elevated temperatures include more-efficient simultaneous saccharification and fermentation, a continuous shift from fermentation to distillation, reduced risk of contamination, and suitability for use in tropical countries (3,5,27). However, the temperatures suitable for conventional strains of Saccharomyces cerevisiae are relatively low (25 to 30°C). While screens for S. cerevisiae mutants able to produce ethanol efficiently at high temperature have been performed, only a modest increase in temperature has been obtained, 40°C maximum (35,40). Alternatively, attention has also focused on thermotolerant yeast species capable of producing ethanol at elevated temperatures. Isolates of Kluyveromyces marxianus appear to be particularly promising (3,5,20,27). This species has been reported to grow at 47°C (3), 49°C (20), and even 52°C (6) and to produce ethanol at temperatures above 40°C (14). Moreover, K. marxianus offers additional benefits (36) including a high growth rate (34) and the ability to utilize a wide variety of industrially relevant substrates such as sugar cane, corn silage juice, molasses, and whey powder (17,27,32,42,49). Because of these advantages, K. marxianus is currently being promoted as a viable alternative to S. cerevisiae as an ethanol producer. However, systematic comparison of the ethanol productivity of...

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The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non-homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR-amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour-intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ-mediated integrative transformation with PCR-amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus.

Overlap extension or fusion PCR is thought to be a simple and easy method to produce fusion DNA fragments without the need for restriction enzyme digestion and DNA ligation. However, this method has not been used frequently, probably as it is not always reliable. When natural sequences are used for overlap sequences, sometimes either no fusion DNA is produced or only faint DNA bands are detected owing to low annealing between the overlap sequences selected. Here, we introduce several artificial overlap sequences, most of which are GC-rich, that can be used for reliable fusion PCR. We describe how these overlap sequences can be used for fusion DNA construction, in-frame gene fusion, and cloning in yeast.

Expression of foreign enzymes in yeast is a traditional genetic engineering approach; however, useful secretory enzymes are not produced in every case. The hyperthermostable α-amylase encoded by the AmyL gene of Bacillus licheniformis was expressed in Saccharomyces cerevisiae; however, it was only weakly produced and was degraded by the proteasome. To determine the cause of low α-amylase production, AmyL was expressed in a panel of yeast mutants harboring knockouts in non-essential genes. Elevated AmyL production was observed in 44 mutants. The knockout genes were classified into six functional categories. Remarkably, all non-essential genes required for N-linked oligosaccharide synthesis and a gene encoding an oligosaccharyl transferase subunit were identified. Immunoblotting demonstrated that differently underglycosylated forms of AmyL were secreted from oligosaccharide synthesis-deficient mutants, while a fully glycosylated form was produced by wild-type yeast, suggesting that N-linked glycosylation of AmyL inhibited its secretion in yeast. Mutational analysis of six potential N-glycosylation sites in AmyL revealed that the N33Q and N309Q mutations remarkably affected AmyL production. To achieve higher AmyL production in yeast, all six N-glycosylation sites of AmyL were mutated. In wild-type yeast, production of the resulting non-glycosylated form of AmyL was threefold higher than that of the glycosylated form.