Journal of Biotechnology 189 (2014) 136–142 Contents lists available at ScienceDirect Journal of Biotechnology j ourna l ho me pa ge: www.elsevier.com/locate/jbiotec Upstream regulatory regions controlling the expression of the Candida utilis maltase gene Hana Boˇ nková, Michaela Osadská, Ján Krahulec ∗ , Veronika Liˇ sková, Stanislav Stuchlík, Ján Tur ˇ na Comenius University in Bratislava, Faculty of Natural Sciences, Department of Molecular Biology, Mlynská dolina, 842 15 Bratislava 4, Slovak Republic a r t i c l e i n f o Article history: Received 26 May 2014 Received in revised form 5 September 2014 Accepted 8 September 2014 Available online 16 September 2014 Keywords: Candida utilis Maltase promoter Cre-loxP system -Galactosidase qPCR a b s t r a c t Candida utilis represents a promising expression host, generating relatively high levels of recombinant proteins. The current study presents preliminary characterization of the upstream regulatory regions controlling the carbon source-dependent expression of the C. utilis maltase gene. Cellobiose and soluble starch were recognised as inducers of maltase promoter, besides maltose. We successfully applied the Cre-loxP system to acquire a null mutant strain with disrupted maltase gene and promoter in polyploid yeast C. utilis. Furthermore, the strength and minimal functional region of the promoter was evaluated by measuring -galactosidase activity. Our results suggest, the qPCR was shown itself a very smart method for relatively easy characterization of the transformants and correlation of the expression levels with gene dosage. © 2014 Elsevier B.V. All rights reserved. 1. Introduction For more than six decades, Candida utilis has represented an industrially important yeast, being classified as GRAS (Generally Recognized as Safe) by regulatory authorities. Owing to its high protein content and its safety, it is considered to be a fodder yeast and a potential microbial source of various endogenous products (Boze et al., 1992). Cultivation cost is very acceptable, comparable to that of Escherichia coli and C. utilis can adapt to a number of carbon and nitrogen sources. Moreover, secretome of C. utilis is without any proteases (Buerth et al., 2011). Recently, the whole genome sequencing and phylogenetic analysis of C. utilis was published (Buerth et al., 2011; Tomita et al., 2012). The development of genetic tools, including the Cre-loxP recombination system, has allowed the efficient production of recombinant proteins in C. utilis, such as monellin (Kondo et al., 1997), -amylase (Miura et al., 1999), carotenoids (Miura et al., 1998), biotin (Hong et al., 2006), xylanase (Wei et al., 2010) and l-lactate (Ikushima et al., 2009a). Further- more, the dried yeast powder of recombinant protein-producing strain C. utilis may have the potential to be used directly as food ∗ Corresponding author. Tel.: +421 2 602 96 509. E-mail addresses: halaszovah@fns.uniba.sk (H. Boˇ nková), osadska@fns.uniba.sk (M. Osadská), krahulec@nic.fns.uniba.sk, krahulec@fns.uniba.sk (J. Krahulec), liskova.veronikaa@gmail.com (V. Liˇ sková), stuchlik@fns.uniba.sk (S. Stuchlík), turna@fns.uniba.sk (J. Tur ˇ na). additive, cosmetic additive, or animal feed without further expen- sive processes to isolate and purify recombinant product from the fermentation broth. However, extensive use of C. utilis for the expression of het- erologous proteins is hampered, especially due to its polyploidy (Ikushima et al., 2009b). The current paper presents a study of the promoter region upstream of the C. utilis maltase gene in order to compare its regulation. The main aims of this work were con- struction of C. utilis strain with functionless maltase gene and promoter, identification of minimum-length promoter region of maltase gene and screening for multiple copy integrants using qPCR based method. 2. Material and methods 2.1. Strains and growth conditions Candida utilis CCY 39-38-18 strain was obtained from the Slovak collection of yeasts and yeast-like microorganisms (Insti- tute of Chemistry, SAS Bratislava, Slovakia). For yeast cultivation was used minimal medium (1.34% yeast nitrogen base, 4 × 10 -5 % biotin, and 2% carbon source) or YPD medium supplemented with 200 mg dm -3 of G418 (Serva, Heidelberg, Germany) or 30 mg dm -3 of zeocin (Invitrogen, Carlsbad, CA), unless otherwise specified. Escherichia coli strain DH5 (Invitrogen, Carlsbad, CA) served for plasmids maintenance and construction. LB medium supple- mented with 100 mg dm -3 of ampicillin, 10 mg dm -3 of zeocin or http://dx.doi.org/10.1016/j.jbiotec.2014.09.006 0168-1656/© 2014 Elsevier B.V. All rights reserved.