Kluyveromyces marxianus.
Artigo publicado na revista World Journal of
A simple and rapid method for lithium acetate-mediated transformation of Kluyveromyces marxianus cells
Daiane Felberg Antunes, ClaÂudio GalvaÄo de Souza Junior and Marcos Antonio de Morais Junior* Setor de Biologia Molecular/LIKA and Departamento de GeneÂtica, Universidade Federal de Pernambuco, Recife, PE, Brasil
*Author for correspondence: Setor de Biologia Molecular/LIKA, Universidade Federal de Pernambuco. Av. Moraes Rego, s/n, Cidade UniversitaÂria, 50670-901, Recife, PE, Brasil
Fax: +55 81 271 8485, E-mail: morais@lika.ufpe.br, URL: www.lika.ufpe.br/setores/biomol.html
Received 30 May 2000; accepted 19 September 2000
Keywords: Dithiothreitol, lithium acetate, Kluyveromyces marxianus, yeast transformation Summary
Ecient plasmid transformation of Kluyveromyces marxianus cells of 1.9 ´ 103transformant lg)1DNA with an episomal plasmid was achieved by the use of a simple lithium acetate method with the addition of 10 mM DTT and an increased heat shock temperature of 47 °C. This method is shown to be also ecient for replicative plasmids. Therefore, we suggest its use as a routine method to transform K. marxianus cells.
Introduction
Kluyveromyces marxianus is a budding yeast with in- creasing applications in the production of food biomass, several hydrolytic enzymes and useful products, specially used in the food and pharmaceutical industries (reviewed by BeleÂm & Lee 1998). Some papers have reported the use of K. marxianus as an ecient host for gene heterologous expression and protein production (Berg- kamp et al. 1993). However, only a few reports can be found in the literature on the construction of ecient cloning vectors and speci®c transformation methodol- ogy. The episomal vectors based on the K. drosophilarum 2-lm-like endogenous plasmid pKD1, like the pE1 plasmid (Bergkamp et al. 1993), and replicative vectors based on the K. lactis replication origins have been used (Iborra 1993). Recently, we reported that the Schizosac- charomyces pombe plasmid pDblet was eciently intro- duced and stable in K. marxianus cells (Souza Jr & Morais Jr 2000). Moreover, the lithium acetate (LiAc) procedure described for S. cerevisiae or the laborious and expensive electroporation method have been used for K. marxianus cell transformation (Meilhoc et al. 1990; Bergkamp et al. 1993; Iborra 1993). In this context, we describe a rapid and simple LiAc-mediated transforma- tion procedure to produce K. marxianus transformants. Results and Discussion
We have used the procedure described by Soni et al. (1993) for S. cerevisiae to transform stationary
K. marxianus cells with the episomal plasmid pE1. After approximately 16 h of incubation, the cultures reached around 2 ´ 108cell ml)1 and the transformation eciency was 7 ´ 102 transformant lg)1 pE1 plasmid using the basic procedure. This last value was the same as described by Souza Jr & Morais Jr (2000) for exponentially growing K. marxianus cells. Therefore, we tested the eect of dithiothreitol (DTT) or dimethyl sulphoxide (DMSO) added to Polyethyleneglycol (PEG)/LiAc solution and ethanol (EtOH) added during the heat shock on the transformation eciency of yeast cells.
The incubation of the cells in PEG/LiAc solution in the presence of DMSO decreased cell transformation for values below the basic procedure, and at a concentration of 10% (v/v) practically no transformants could be observed (Figure 1). The use of DMSO was based on the ®nding that this compound can stimulate lithium acetate-mediated whole-cell transformation in S. cerevi- siae, although it decreased cell viability (Hill et al. 1991; Soni et al. 1993). In this context, it could be suggested that if DMSO is eective in increasing cell wall porosity it may be very toxic to K. marxianus cells. On the other hand, the presence of 10 mM DTT during lithium acetate incubation period promoted a 2.5-fold increase in the transformation eciency over the basic protocol to 1.9 ´ 103 transformant lg)1 DNA, while some inhibitory eect was observed at a higher DTT concen- tration of 15 mM (Figure 1). It has been reported that DTT increases yeast competence and transformation (Meilhoc et al. 1990; Chen et al. 1992; Iborra 1993) and that an inhibitory eect was also observed for
K. marxianus cells using electroporation procedure by both higher DTT concentration or prolongated period of incubation (Iborra 1993).
Post-incubation with ethanol during heat-shock treat- ment also increased the transformation eciency in the range of 2-fold over the basic procedure and practically no dierence was found whether 5 or 10% (v/v) ethanol was used (Figure 1). Ethanol at a concentration of 10% (v/v) was also found to increase cell transformation in S. cerevisiae (Lauermann 1991; Soni et al. 1993), probably by increasing yeast cell membrane ¯uidity (Jones 1987). Combined addition of pre-incubation agents and ethanol had a synergistic eect on cell toxicity that resulted in a decrease of the transformation eciency (Figure 1). The same eect was also observed in S. cerevisiae cells by the combination of DMSO and ethanol (Soni et al. 1993).
The results presented here suggest for use of 10 mM DTT during the incubation in LiAc solution as the best condition for ecient transformation of K. marxianus in the stationary phase of growth. Additionally, prepara- tion of LiAc/PEG solution in Tris-buered solution and the increase of heat shock temperature from 42 to 47 °C promoted a 2-fold increase in the eciency of transfor- mation (data not shown). We used another plasmid, the replicative vector pDBlet and its recombinant forms pDBKan and pDBaAM, to transform K. marxianus cells with our improved protocol. The transformation eciency was around 1.3 ´ 103transformant lg)1DNA for the three plasmids, which was 10 times lower than that described for pDBlet (Souza Jr & Morais Jr 2000) but double that obtained for the replicative plasmid pKL2 (Iborra 1993), both using LiAc-mediated trans- formation of exponential K. marxianus cells.
The optimized procedure described here is as follows: cells from 1.5 ml overnight culture were washed with sterile water and resuspended by vortexing in 20 ll (200 lg) of carrier DNA, transforming DNA at mini- mum volume, 0.4 ml of PEG solution (40% PEG 4000, 0.1 M LiAc, 10 mM Tris-HCl pH 7.5, 1 mM EDTA) and DTT to 10 mM at ®nal concentration. The mixture was incubated for 15 min at room temperature and submitted to heat shock at 47 °C for 15 min. The cells were centrifuged and resuspended in 1 ml sterile water before plating 200 ll on selective plates.
Acknowledgements
This work was supported by grants from the Conselho Nacional de Desenvolvimento Cienti®co e Tecnologico (CNPq) and Fundacao de Amparo a Ciencia e Tecno- logia do Estado de Pernambuco (Facepe). The authors thank Dr W.M. Ledingham, St Andrews University, UK, for his critical review of the manuscript.
References
BeleÂm, M.A. & Lee, B.H. 1998 Production of bioingredients from Kluyveromyces marxianus grown on whey: an alternative. Critical Reviews in Food Science and Nutrition 38, 565±598.
Bergkamp, R.J.M., Bootsman, T.C., Tschka, H.Y., Mooren, A.T.A., Kox, L., Verbakel, J.M.A., Geerse, R.H. & Planta, R.J. 1993 Expression of an a-galactosidase gene under control of the homologous inulinase promoter in Kluyveromyces marxianus. Applied Microbiology and Biotechnology 40, 309±317.
Chen, D.C., Yang, B.C. & Kuo, T.T. 1992 One-step transformation of yeast in stationary phase. Current Genetics 21, 83±84.
Hill, J., Donald, K.A., Griths, D.E., Donald, G. & Donald, K.A. 1991 DMSO-enhanced whole cell yeast transformation. Nucleic Acids Research 19, 5791.
Iborra, F. 1993 High eciency transformation of Kluyveromy- ces marxianus by a replicative plasmid. Current Genetics 24, 181± 183.
Jones, R.P. 1987 Factors in¯uencing deactivation of yeast cells exposed to ethanol. Journal of Applied Bacteriology 63, 153±164. Lauermann, V. 1991 Ethanol improves the transformation eciency of
intact yeast cells. Current Genetics 20, 1±3.
Meilhoc, E., Masson, J.M. & TeissieÂ, J. 1990 High eciency transformation of intact yeast cells by electric ®eld pulses. Biotechnology 8, 223±227.
Soni, R., Carmichael, J.P. & Murray, J.A.H. 1993 Parameters aecting lithium acetate-mediated transformation of Saccharomyces cerevi- siae and development of a rapid and simpli®ed procedure. Current Genetics 24, 455±459.
Souza Jr, C.G. & Morais Jr, M.A. 2000 The use of the replicating pDblet plasmid as a cloning vector with enhanced stability in Kluyveromyces marxianus. Biotechnology Letters 22, 43±45. Figure 1. Transformation eciency of the K. marxianus KMS2 strain
by the plasmid pE1 using modi®ed LiAc-mediated procedure with the permeabilization agents at indicated concentrations. Details of the procedure are described in the text and the values represent the mean of triplicates.
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