BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS Construction of recombinant Kluyveromyces marxianus UFV-3 to express dengue virus type 1 nonstructural protein 1 (NS1) Caio Roberto Soares Bragança & Lívia Tavares Colombo & Alvaro Soares Roberti & Mariana Caroline Tocantins Alvim & Silvia Almeida Cardoso & Kledna Constancio Portes Reis & Sérgio Oliveira de Paula & Wendel Batista da Silveira & Flavia Maria Lopes Passos Received: 28 April 2014 /Revised: 15 July 2014 /Accepted: 16 July 2014 /Published online: 2 August 2014 # Springer-Verlag Berlin Heidelberg 2014 Abstract The yeast Kluyveromyces marxianus is a conve- nient host for industrial synthesis of biomolecules. However, despite its potential, there are few studies reporting the ex- pression of heterologous proteins using this yeast. Here, we report expression of a dengue virus protein in K. marxianus for the first time. The dengue virus type 1 nonstructural protein 1 (NS1) was integrated into the K. marxianus UFV-3 genome at the LAC4 locus using an adapted integrative vector designed for high-level expression of recombinant protein in Kluyveromyces lactis. The NS1 gene sequence was codon- optimized to increase the level of protein expression in yeast. The synthetic gene was cloned in frame with K. lactis α- mating factor signal peptide, and the recombinant plasmid obtained was used to transform K. marxianus UFV-3 by electroporation. The transformed cells, selected in yeast ex- tract peptone dextrose containing 200 μg mL -1 Geneticin, were mitotically stable. Analysis of recombinant strains by RT-PCR and protein detection using blot analysis confirmed both transcription and expression of extracellular NS1 poly- peptide. After induction with galactose, the NS1 protein was analyzed by sodium dodecyl sulfate-PAGE and immunogenic detection. Protein production was investigated under two conditions: with galactose and biotin pulses at 24-h intervals during 96 h of induction and without galactose and biotin supplementation. Protease activity was not detected in post- growth medium. Our results indicate that recombinant K. marxianus is a good host for the production of dengue virus NS1 protein, which has potential for diagnostic applications. Keywords Kluyveromyces marxianus . Heterologous expression . Dengue virus . NS1 protein Introduction The yeasts Kluyveromyces marxianus and Kluyveromyces lactis are phylogenetically related to the baker ’s yeast Saccharomyces cerevisiae, but they have unique advantages for biotechnological applications. Unlike S. cerevisiae, K. marxianus and K. lactis have the ability to assimilate lactose and use it as a sole carbon and energy source. Owing to this feature, these yeasts are common in dairy products such as milk, cheese, and yogurt (Lane and Morrissey 2010), giving Kluyveromyces the status of Generally Recognized as Safe (GRAS) and allowing its use in the pharmaceutical and food industries. Moreover, K. marxianus has been isolated from several other environments, which reflects its high met- abolic diversity. As a consequence, several biotechnological applications have been investigated for this yeast, for example the production of aromatic compounds and bioingredients, including cheese whey (Fonseca et al. 2008), ethanol (dos Santos et al. 2013; Silveira et al. 2005), and most recently as a host for heterologous protein synthesis (Rocha et al. 2010; Rocha et al. 2011). In addition, the potential of K. marxianus for industrial use is further supported by its thermotolerance, high growth rate, and broad substrate spectrum (Fonseca et al. C. R. S. Bragança : L. T. Colombo : A. S. Roberti : M. C. T. Alvim : W. B. da Silveira : F. M. L. Passos (*) Laboratório de Fisiologia de Micro-organismos, Departamento de Microbiologia, BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, Brazil e-mail: flpassos@ufv.br S. A. Cardoso : S. O. de Paula Laboratório de Imunovirologia Molecular, Departamento de Biologia Geral, Universidade Federal de Viçosa, Viçosa, MG, Brazil K. C. P. Reis Microbiologia Veterinária Especial - MICROVET, Viçosa, MG, Brazil Appl Microbiol Biotechnol (2015) 99:1191–1203 DOI 10.1007/s00253-014-5963-5