High level expression of Glomerella cingulata cutinase in dense cultures of Pichia pastoris grown under fed-batch conditions
Introduction
Cutinases (EC 3.1.1.74) are the smallest members of the α/β hydrolase family (Longhi and Cambillau, 1999). Cutins, polymers of hydroxyl- and epoxy-fatty acids, form the cuticle of higher plants and are the natural substrates of cutinases (Purdy and Kolattukudy, 1975). Structural analyses of cutinase crystals show that a serine residue forms the active site. In contrast to lipases, this serine is not buried under an amphipathic loop (Longhi and Cambillau, 1999). Since cutinases are able to hydrolyse a variety of short- and long-chain esters, are highly active and stable even in the presence of oxidising agents, detergents and proteases, they are widely used in agriculture, detergents, foods, pharmaceuticals and so on. They are even used as surfactants in detergents (Kolattukudy and Poulose, 1988). Cutinases also modify and degrade biodegradable and synthetic plastics. The enzymatic modification of synthetic and natural polymers allows highly specific and environmentally friendly alternatives to harsh chemicals (Baker and Montclare, 2010). Recently, cutinases have been used in the degradation of polyethylene terephthalate (PET), a high-molecular-weight synthetic polyester widely used in textile fibre and plastic industries (Donelli et al., 2010). To enhance the properties of Fusarium solani pisi cutinase, the enzyme was modified to increase its activity towards PET by creating more space in the active site (Araújo et al., 2007). Cutinases also possess properties that make them superior to lipases in enzyme-catalysed reactions including the ability to hydrolyse both water-soluble esters as well as emulsified, insoluble triacylglycerols (Lauwereys et al., 1991). In these senses cutinases, seem to be intermediate between esterases and lipases (Martinez et al., 1992).
Most extracellular cutinases have been isolated from fungi, particularly, fungal phytopathogens, although bacterial cutinolytic and cutinase-like enzymes are also known (Chen et al., 2013). The cutinase of the phytopathogen F. solani pisi is the most studied cutinase thus far (Carvalho et al., 1999, Chen et al., 2013, Dutta et al., 2009, Egmond and de Vlieg, 2000, Lauwereys et al., 1991). The F. solani pisi cutinase has been cloned and expressed in several hosts including Aspergillus awamori (van Gemeren et al., 1996), Escherichia coli (Griswold et al., 2003), Fusarium venenatum (Sorensen et al., 2007), Pichia pastoris (Kwon et al., 2009) and Saccharomyces cerevisiae (Calado et al., 2002).
Meeting industrial requirements for large-scale production of catalytically efficient cutinases is very challenging. Scaling-up the production of cutinases in heterologous hosts is by far the best approach as expression levels in their original sources are usually very low (Chen et al., 2013). As a system for expressing foreign proteins, the methylotrophic yeast P. pastoris has many advantages (Cereghino and Cregg, 2000). Alcohol oxidase (EC 1.1.3.13—AOX1) catalyses the first step of methanol catabolism and is strongly induced by methanol. Thus genes of interest cloned under the control of the AOX1 promoter are highly expressed when methanol is the sole carbon source. Heterologous proteins are also properly post-translationally modified and secreted in large quantities by P. pastoris. Indeed, Kwon et al. (2009) claimed expression levels of 340 mg L−1 F. solani pisi cutinase in shaken-flasks without rigorously optimising the conditions.
The Glomerella cingulata anamorph Colletotrichum gloeosporioides is a phytophatogenic fungus that causes anthracnose in a wide variety of plants including tropical and subtropical fruits. The cutinase gene of G. cingulata was identified from a genomic library, its corresponding cDNA isolated from papaya cutin-induced cells, the enzyme encoded by the cDNA expressed in E. coli and its three dimensional structure and function characterised (Abu Bakar et al., 2001, Nyon et al., 2008, Nyon et al., 2009). Expression levels of G. cingulata cutinase in E. coli (at 9 mg L−1) were low however but the enzyme preferred the medium chain triglyceride substrate tricaprylin (C8) rather than the shorter length triglycerides of the F. solani cutinase (Nyon et al., unpublished).
Here we optimised the pH, temperature and time-course of production of the G. cingulata cutinase in P. pastoris by carefully controlling the time course, pH and temperature in both shaken-flasks and an automated 5 L bioreactor. Properties of the enzyme were also investigated and we were able to confirm that it prefers medium to long chain p-nitrophenyl esters and is able to depolymerise the high molecular weight polyester, PET.
Section snippets
Microorganisms, plasmids and culture media
Plasmid pFD4 carrying the G. cingulate cutA cDNA was used as the source of template DNA for PCR (Abu Bakar et al., 2001). E. coli DH5α was used for plasmid amplification and other DNA manipulations. P. pastoris X-33 (Invitrogen/Life Technologies, Carlsbad, CA, USA) was used as a host for cutinase gene expression. pGEMT® Easy vector (Promega, Madison, WI, USA) was used for cloning PCR products and pPICZαC (Invitrogen/Life Technologies) was used as the expression plasmid in P. pastoris. All media
Vector construction
The recombinant pPICZαC-cutA plasmid shown in Fig. 1 contains the inducible promoter AOX1, the S. cerevisiae α-factor secretion signal and a transcription termination signal. Transformation using linear pPICZαC-cutA favoured insertion into the yeast genome via homologous recombination as described in the EasySelect™ Pichia Expression System Manual (Invitrogen/Life Technologies). Selection for recombinants on YPD-zeocin plates gave 59 transformants, 25 of which may harbour multi-copy
Discussion
Since it became commercially available, P. pastoris has been widely used to express bacterial and fungal enzymes. Advantages of the P. pastoris expression system include: easy selection for secreted proteins, availability of strong promoters (e.g. AOX1), multiple copy-number expression vectors and post-translational modification activity. Thus, this system is particularly suitable for the expression of proteins that form inclusion bodies in E. coli, and/or whose production levels are low in
Conclusions
The P. pastoris expression system used here permitted easy selection of secreted proteins, was driven by the strong AOX1 promoter and yielded large amounts of recombinant G. cingulata cutinase in a eukaryote. A fed-batch bioreactor increased cutinase production and activity seven-fold and 188-fold, respectively, over that attained in shaken-flasks. The G. cingulata cutinase has a broad substrate-specificity, preferring medium to long carbon chain ρ-nitrophenyl esters and in this sense contrasts
Acknowledgements
This research was funded by the Ministry of Science, Technology and Innovation (MOSTI), Malaysia, under a research grant UKM-MGI-NBD-0012-2007 and the National Science Fellowship (NSF), Malaysia.
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2021, Journal of Biological ChemistryCitation Excerpt :The activity maximum (“100%” activity, Fig. 2) was reached on shorter chain substrates: pNP-C2 (PbCut), pNP-C4 (CcCut, ScSub, ScCut, McCut, FsCut, and CspCut), or pNP-C8 (AnCut, TtCut). While enzymes with activity maxima at pNP-C2 to C6 seem to be common (14–21), the preference of AnCut and TtCut for pNP-C8 appears to be less common as only a few cutinases have been described to prefer these longer chain fatty acid esters (22). With the longest chain length substrates, pNP-C12 and pNP-C16, all nine cutinases had lower activity than on the short substrates (Fig. 2), an effect also observed in other studies (15–22).