High-temperature ethanol fermentation and transformation with linear DNA in the thermotolerant yeast Kluyveromyces marxianus DMKU3-1042

Abstract

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 degrees 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 alpha-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 alpha-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. cerevisiae-based molecular genetic tools.

FIG. 1.

Growth properties of K. marxianus and S. cerevisiae strains. (A) Strains of K. marxianus and S. cerevisiae were streaked on YPD plates and incubated at the indicated temperatures for 2 days. (B) Strains were streaked as described for panel A and grown at 28°C on MM supplemented with 2% of the indicated sugars. (C) K. marxianus DMKU3-1042 (closed symbols) and S. cerevisiae NCYC3233 (open symbols) were grown in liquid MM supplemented with 2% lactose (circles), cellobiose (squares), or glycerol (triangles) for the graph on the left and with xylitol (circles), xylose (squares), or arabinose (triangles) for the graph on the right. OD, optical density.

FIG. 2.

Ethanol fermentation. (A) Ethanol production (top) and glucose consumption (bottom) by S. cerevisiae NCYC3233 (closed circles) and K. marxianus strains DMKU3-1042 (closed triangles), NCYC587 (closed squares), NCYC1429 (open triangles), and NCYC2791 (open circles) in YP medium supplemented with 5% glucose at 30°C (left) and 45°C (right). (B) Growth of K. marxianus DMKU3-1042 (Km) and S. cerevisiae NCYC3233 (Sc) on YPD and YP supplemented with 2% xylose (YP+xylose) under aerobic or anaerobic conditions (3 or 5 days, respectively).

FIG. 3.

Transformation of a K. marxianus ura3 mutant with a linear DNA of S. cerevisiae URA3 (ScURA3). (A) The K. marxianus ura3 mutant was not transformed with the intact ScURA3 plasmid (pRS316) but with SmaI-digested pRS316 (4.8 kb) and the ScURA3 PCR fragment (1.7 kb). (B) Southern blot hybridization of chromosomal DNA of S. cerevisiae BY4704 (lane 1), K. marxianus wild-type (WT) DMKU3-1042 (lane 2), ura3 mutant RAK3605 (lane 3), and nine ScURA3 transformants from strain RAK3605 (lanes 4 to 12). Chromosomal DNA was digested with BamHI, run on a 0.8% agarose gel, transferred to a nylon membrane, and hybridized with a digoxigenin-labeled ScURA3 probe. The bands indicated by an arrowhead are likely the authentic K. marxianus URA3 that cross-hybridizes with ScURA3. M, DNA size marker.

FIG. 4.

Screening for auxotrophic mutants following transformation with ScURA3. (A) Sequence flanking the ScURA3 insertion was obtained by TAIL-PCR (KmLYS1) and was found to be highly homologous to that of K. lactis (KlLYS1). An arrow indicates the insertion site. (B) PCR amplification using genomic DNA from DMKU3-1042 (wild type [WT]) and the Lys⁻ mutant RAK3627 (LYS⁻) as templates confirmed the disruption of KmLYS1 by ScURA3. ScURA3 was inserted in an inverse direction relative to KmLYS1. The absence of a wild-type band in the lys1 disruptant suggests a haploid chromosomal constitution.

FIG. 5.

Construction of an α-amylase expression strain in K. marxianus by transformation with a linear DNA. An ScTDH3 promoter-driven TAA (Aspergillus oryzae α-amylase gene) was constructed at the URA3 locus of S. cerevisiae and the PCR-amplified construct was then used without additional manipulation to transform a K. marxianus ura3 mutant. The appearance of clear zones around colonies indicates that the α-amylase gene was expressed under the control of the S. cerevisiae TDH3 promoter in K. marxianus.