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°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...

Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non‐homologous end joining‐mediated integrative transformation with genes from Saccharomyces cerevisiae

“…In contrast to S. cerevisiae and K. lactis, which are widely used for molecular biology and industrial applications (Mattiazzi et al, 2012;Schaffrath and Breunig, 2000;Schwimmer et al, 2006;Smith and Snyder, 2006;van Leeuwen et al, 2012), K. marxianus has received relatively little attention, although there are an increasing number of reports describing the potential application of K. marxianus for the production of ethanol, biomass, endogenous enzymes and aromatic compounds (Bansal et al, 2008;Fonseca et al, 2008;Gao and Daugulis, 2009;Limtong et al, 2007;Nonklang et al, 2008Nonklang et al, , 2009Rodrussamee et al, 2011;Wittmann et al, 2002) and for protein structural analysis (Watanabe et al, 2012;Yamaguchi et al, 2012aYamaguchi et al, , 2012b. Thus, in the present study, we generated and characterized 79 auxotrophic mutants and identified 35 complementing genes using a novel integrative transformation approach for the development of genetic tools in K. marxianus.…”

“…The strains were obtained from the Biological Resource Center, NITE, Japan (NBRC) and the National Center for Yeast Collection (NCYC), UK. K. marxianus RAK3605 (ura3-1) (Nonklang et al, 2008) was used as a parental strain to isolate double auxotrophic mutants. A flow-cytometric analysis indicated that the parental strain DMKU3 …”

“…To isolate auxotrophic mutants of K. marxianus, cells of parental strain RAK3605 (ura3-1 genotype) (Nonklang et al, 2008) were exposed to UV light and formed colonies were replica-plated on YPD and MMU plates. From approximately 15 000 colonies, the nutritional requirements of 79 colonies that failed to grow on MMU were first examined, based on growth on MM + 7aa medium.…”

“…The thermotolerant yeast Kluyveromyces marxianus is a non-conventional yeast species with the superior ability for high-temperature fermentation, a property that has been utilized for the development of cost-saving ethanol fermentation processes (Abdel-Banat et al, 2010a;Fonseca et al, 2008;Limtong et al, 2007;Nonklang et al, 2008). In addition, several K. marxianus proteins, including the autophagy-related proteins KmAtg7, KmAtg10 and KmHsv2, have been applied to crystal structural analysis because the formation of crystals of the orthologous S. cerevisiae proteins was difficult (Watanabe et al, 2012;Yamaguchi et al, 2012aYamaguchi et al, , 2012b.…”

“…Moreover, NHEJ-mediated integration was used for the complementation of a Kmura3 mutant with a ScURA3 gene PCR fragment without modification, such as promoter exchange (Nonklang et al, 2008), suggesting that other S. cerevisiae genes might be able to complement K. marxianus mutants without the need for plasmid construction or gene modification.…”