Heterologous production of family 5 fungal endo-1,4-B-mannanases in Saccharomyces cerevisiae

[摘要] ENGLISH ABSTRACT: Mannan polysaccharides occur in the hemicellulose fraction of plant cell walls, either asstructural polymers or as reserve carbohydrates. They are found predominantly in theseeds of leguminous plants in the form of galactomannan, and in softwoods asgalactoglucomannan. Endo-I,4-I3-mannanases hydrolyze mannan polysaccharides tooligosaccharides of various lengths. These enzymes are secreted as single catalyticmodules or as part of multi-modular proteins by fungi, bacteria, plants and animals. Forexample, the l3-mannanase of Aspergillus aculeatus, designated Aa-Man5A, is secretedas a single catalytic module, whereas that of Trichoderma reesei, designated Tr-Man5A,contains a l3-mannanase catalytic module linked to a cellulose-binding module by a Pro-Ser-Thr-rich linker.Heterologous gene expression in yeast provides the opportunity to produce individualhydrolytic enzymes in a host expression system devoid of related activities.Saccharomyces cerevisiae has a well-developed expression system and has frequentlybeen used as a model organism for heterologous gene expression. A number ofautoselection systems have been devised so that recombinant S. cerevisiae strains can becultivated in any medium of choice without exerting selective pressure. An autoselectionsystem based on defective chromosomal ura3 andfurl genes involved in the pyrimidinebiosynthesis pathway of S. cerevisiae, and complementation of the ura3 gene with amulticopy plasmid-borne URA3 gene, were used in this study.The man1 of A. aculeatus gene encoding Aa-Man5A was cloned and expressed inautoselective S. cerevisiae under the regulation of the alcohol dehydrogenase (ADH2PT)and the phosphoglycerate kinase (PGK1PT) promoter and terminator sequences.Expression of man1 under both promoters resulted in high production levels of Aa-Man5A. The production levels were significantly higher than the levels of endo-l,4-13-mannanases produced by heterologous expression in Escherichia coli, and werecomparable to the production levels of enzymes produced in Pichia pastoris, whichpresumably has a higher secretion capacity than S. cerevisiae. The recombinant yeaststrain expressing man1 under the regulation of the PGK1p promoter displayed stuntedbiomass formation during the logarithmic phase, which was relieved when the native f3-mannanase secretion signal was replaced with the yeast MFuis secretion signal. Therecombinant Aa-Man5A displayed biochemical properties similar to those of the nativeAa-Man5A. The recombinant enzyme hydrolyzed unsubstituted mannan topredominantly mannose, mannobiose, and mannotriose.The expression of the man1 and man1 ácbd gene constructs of T reesei in S. cerevisiaefur 1::LEU2 strains under the regulation of the PGK1 PT promoter and terminator resultedin the production and secretion of Tr-Man5A and Tr-Man5A~CBD (lacking the cellulosebinding module), respectively. However, the production levels of both proteins wereapproximately I5-fold lower than the production levels of Aa-Man5A. These levels didnot improve after replacement of the native secretion signal with the MFuis secretionsignal. Interestingly, reducing the cultivation temperature from 30°C to 20°C led to afive-fold increase in the secreted levels of Tr-Man5A, but a three-fold decrease in theproduction of Aa-Man5A.A preliminary investigation was performed to evaluate the possibility of using therecombinant Aa-Man5A in the processing of instant coffee. Arabica coffee extractstreated with Aa-Man5A displayed low viscosity in comparison to the untreated extractand showed better retention of volatile/aromatic compounds than the autoclaved extract.The results indicated that Aa-Man5A is capable of hydrolyzing coffee galactomannanand can be used for processing instant coffee.

[发布机构] Stellenbosch University