African Journal of Biotechnology Vol. 9(15), pp. 2173-2184, 12 April, 2010 Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2010 Academic Journals Review Carbon source feeding strategies for recombinant protein expression in Pichia pastoris and Pichia methanolica Raúl A. Poutou-Piñales1, 4*, Henry A. Córdoba-Ruiz2, Luis A. Barrera-Avellaneda3 and Julio M. Delgado-Boada4 1Laboratorio de Biotecnología Aplicada, Grupo de Biotecnología Ambiental e Industrial (GBAI), Departamento de Microbiología. Facultad de Ciencias, Pontificia Universidad Javeriana (PUJ). Bogota, D.C., Colombia. 2Departamento de Química. Facultad de Ciencias, Pontificia Universidad Javeriana (PUJ). Bogota, D.C., Colombia. 3Instituto de Errores Innatos del Metabolismo (IEIM), Pontificia Universidad Javeriana (PUJ). Bogota D.C., Colombia. 4Biotechnova Ltda. Bogota, D.C. Colombia. Accepted 28 December, 2009 Pichia pastoris and Pichia methanolica have been used as expression systems for the production of recombinant protein. The main problems of the production are the slow hierarchic consumption of ethanol and acetate which cause toxicity problems due to methanol accumulation when this surpasses 0.5 gl-1. In some cases, the laboratory scale cultures does not show the methanol accumulation problems because the cells are usually washed before changing the depleted carbon source culture media, by a fresh one, supplemented with methanol; a strategy that is clearly inapplicable in the industrial productions. Other authors use to feed the methanol before the depletion of glycerol or D- glucose, but this practice does not guaranty the ethanol and acetate fast consumption; leading to methanol accumulation and toxicity problems. It was concluded that pre-induction stage strategy should be studied in detail and that it is very important to start the induction stage with a concentration of biomass as great as possible. On the other side, it is essential that there should be a monitoring of ethanol and acetate until reaching non-toxic methanol stable-concentration and these conditions should be maintained till the end of the process. Key words: Carbon source, protein expression, Pichia pastoris, Pichia methanolica. INTRODUCTION Yeasts are beneficial microorganisms which are used to such as single cell protein (SCP), recombinant vaccines produce food by employing them in fermentation process, or recombinant proteins, enzymes, growth factors, ethanol like in the production of goods such as wine, beer, bread, and many other biochemical compounds (Cinza et al., among other leavened products. Recently, these micro 1991; Nigam, 2001; Vassileva et al., 2001; Sevo et al., organism have been used for production of compounds 2002; Teramoto et al., 2005; Cordoba-Ruiz et al., 2009). These microorganisms are one of the host cells most frequently used for heterologous proteins expression. Initially the utilization of yeasts for this purpose was *Corresponding author. E-mail: [email protected]. Tel/ focused on Saccharomyces cerevisiae, although other Fax: (57-1) 320 83 20 ext 4070. species like Pichia sp., Kluyveromyces sp. or Hansenula sp. have been considered up to date (Gellisen and Abbreviations: SCP, Single cell protein; Mut pathway, Hollenberg, 1997; Kang et al., 2001; Wang et al., 2007). methanol utilization pathway; AO, alcohol oxidase; MO, Different yeast genera, except S. cerevisiae and methanol oxidase, PTS, peroxisomal targeting signals; DHAS, dihydroxyacetone synthase, CAT, catalase; MOD2 (AUG2), Schizosaccharomyces pombe, were classified by Wolf, alcohol utilizing genes; AOX, alcohol oxidase genes; MutS, slow as non-conventional yeasts (Wolf, 1996). methanol growth phenotype; Mut+, wild type; Mut-, negative Methylotrophic yeasts, like Pichia pastoris, Pichia methanol utilization. angusta (Hansenula polymorpha), Pichia methanolica 2174 Afr. J. Biotechnol. (Pichia pinus) or Candida boidini among other methylo- studied up to date, the most common, the PTS1 [(S/A/V/ trophic yeasts, were included in the non-conventional G)(K/R/H)(L/I/M/V/Y/F)] is located at the C-terminus group according to their capacity to use methanol as sole region of many peroxisomal proteins in animals, plants carbon and energy source, which is given by the presence and yeasts (van der Klei et al., 2006). Other peroxisomal of genes coding enzymes related with the methanol proteins (named as PST2) is a nonapeptide containing a utilization pathway (Mut pathway), (Poutou et al., 2005b; signal at the N-terminus [(R/K) (L/V/I) (X5) (H/Q) (L/A)], Hartner and Glieder, 2006). which is commonly found in mammalians and yeasts (the Several studies have reported that methanol meta- most frequent amino acid are shown in bold for both bolism in methylotrophic yeasts requires the expression PTS1 and 2), (Poutou et al., 2005b; van der Klei et al., of alcohol oxidase (AO) (E.C.1.1.3.13) or methanol oxidase 2006). Several peroxisomal proteins, called peroxins, which (MO) from P. pastoris, P. methanolica or P. angusta participate in the process of importing proteins, have (Couderc and Baratti, 1980; Nakagawa et al., 1996). The been associated with the cytosolic face of the peroxisome AO and MO promoters and transcription termination membrane or cytosol containing PTS1 or PTS2 receptors sequences has been used to develop different protein (also know as putative receptors or shuttle-receptors). expression systems (Kang et al., 2001; Guo et al., 2006; These receptors are responsible of selective recognition Landazuri et al., 2009) to regulate the heterologous of proteins PTS and to allow importation (Marzioch et al., protein expression with the influence of the carbon 1994; Rachubinski and Subramani, 1995; Zhang and source feeding strategies (Chauhan and Khanna, 1999; Lazarow, 1996; Johnson et al., 1999; Koller et al., 1999; Cordoba et al., 2003). Snyder et al., 1999; Ozimek et al., 2003; van der Klei et This review summarizes the carbon source addition al., 2006). strategies for the pre-induction stage when P. pastoris In methylotrophic yeasts, the peroxisomes biogenesis and P. methanolica were used for the expression of is induced abundantly, when the cells grow in methanol, recombinant proteins. There is the necessity of deeper pectin or oleic acid as sole carbon and energy source studies of the pre-induction stage’s metabolites as (Nakagawa et al., 2005; Poutou et al., 2005b); being ethanol and acetate. clear that the importation of AO, CAT and the oxidase into the organelle has occurred, but when the methylo- trophic yeasts cells grows in D-glucose or ethanol the METHYLOTROPHIC YEASTS peroxisome´s active degradation is promoted through autophagy process (mediated by proteolysis), (Holzer, Essential steps of the Mut pathway, occurs in the per- 1976; Veenhuis et al., 1978; Yuan et al., 1997; Guan et oxisomes. Consequently yeasts experiments a process al., 2001; Stromhaug et al., 2001). called peroxisome biogenesis, when methanol is present in the culture media, increasing the peroxisomes numbers for detoxifying the cell from H2O2 generated from the Methanol metabolism in methylotrophic yeast methanol metabolism (Subramani, 1993; Wiemer et al., 1996; Johnson et al., 1999; Snyder et al., 1999). Methylotrophy in eukaryotic cells refers to the ability of an organism to use C1-compounds like methanol as energy sources, having the capacity to derive all energy and cell Peroxisome and methanol metabolism carbon from reduced molecules that have no C - C bond. The general mode to assimilated methanol is to convert Peroxisomes are typical organelles present in almost all three C1 molecules into a C3 compound via a cyclic path- eukaryotic cells, characterized because they contain way (Poutou et al., 2005b; van der Klei et al., 2006).The oxidative enzymes such as catalase (CAT) (E.C.1.11.1.7) methanol metabolism inside peroxisomes starts with the and oxidase, which is the enzyme responsible of gene- oxidation of methanol into CO2 by AO, resulting in the rating hydrogen peroxide (H2O2). All the peroxisomal formation of formaldehyde and H202 and the decomposition enzymes are codified in the nuclear DNA and imported of hydrogen peroxide into H2O and O2 by the action of into the organelle after the synthesis of proteins. The per- CAT. The peroxisomal dihydroxyacetone synthase (DHAS) oxisomal targeting signals (PTS) are marks for importation (E.C.2.2.1.3) catalyses the formaldehyde metabolism into the peroxisome and it have to be present in each one forming two C3 compounds (dihydroxyacetone (DHA) and of the immature proteins that should be imported (de glyceraldehyde-3-phosphate (GAP)) which are derived Hoop and Ab, 1992; Glover et al., 1994; Rachubinski and from the condensation reaction between formaldehyde Subramani, 1995; Johnson et al., 1999). and xylulose-5-P (Xu-5p), (Figure 1-I). It is important to mention that other enzymes required for methanol utili- zation are cytosolic (dissimilation pathway) in yeast, Targeting of peroxisomal proteins which includes the enzymes for the oxidation of formald- ehyde to CO2 (that is, formaldehyde dehydrogenase Two peroxisomal targeting signals (PTS) have been (E.C.1.2.1.1), S-formylglutathione hydrolase (E.C.3.1.2.12) Poutou-Piñales et al. 2175 Glycerol Glucose Methanol ATP Pi 3NADP+ H O PEROXISOME GSH CYTOSOL 38 11 2 ATP CH30H 3NADPH ADP 2 O2 3(H2O) ADP 13 I 19 1 15 16 3(6FGL) (3) G6P H2O2 HCHO HCHO GS-CH2OH HCOOH CO2 + 2 3(NADP ) ATP 14 FAD+ 20 G3P F6P NAD+ NAD+ 3(6FG) Pi ½O2 + H20 39 ADP 3 12 21 H2O 17 NADH NADH2 CO 2 2 1,6FBP FADH2 3(Ru5P) 22 (1) NADPH2 4 23