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

Setati, Mathabatha Evodia (2002-12)

Dissertation (PhD)--University of Stellenbosch, 2002.

Thesis

ENGLISH ABSTRACT: Mannan polysaccharides occur in the hemicellulose fraction of plant cell walls, either as structural polymers or as reserve carbohydrates. They are found predominantly in the seeds of leguminous plants in the form of galactomannan, and in softwoods as galactoglucomannan. Endo-I,4-I3-mannanases hydrolyze mannan polysaccharides to oligosaccharides of various lengths. These enzymes are secreted as single catalytic modules or as part of multi-modular proteins by fungi, bacteria, plants and animals. For example, the l3-mannanase of Aspergillus aculeatus, designated Aa-Man5A, is secreted as 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 individual hydrolytic enzymes in a host expression system devoid of related activities. Saccharomyces cerevisiae has a well-developed expression system and has frequently been used as a model organism for heterologous gene expression. A number of autoselection systems have been devised so that recombinant S. cerevisiae strains can be cultivated in any medium of choice without exerting selective pressure. An autoselection system based on defective chromosomal ura3 andfurl genes involved in the pyrimidine biosynthesis pathway of S. cerevisiae, and complementation of the ura3 gene with a multicopy plasmid-borne URA3 gene, were used in this study. The man1 of A. aculeatus gene encoding Aa-Man5A was cloned and expressed in autoselective 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 were comparable to the production levels of enzymes produced in Pichia pastoris, which presumably has a higher secretion capacity than S. cerevisiae. The recombinant yeast strain expressing man1 under the regulation of the PGK1p promoter displayed stunted biomass formation during the logarithmic phase, which was relieved when the native f3- mannanase secretion signal was replaced with the yeast MFuis secretion signal. The recombinant Aa-Man5A displayed biochemical properties similar to those of the native Aa-Man5A. The recombinant enzyme hydrolyzed unsubstituted mannan to predominantly mannose, mannobiose, and mannotriose. The expression of the man1 and man1 ácbd gene constructs of T reesei in S. cerevisiae fur 1::LEU2 strains under the regulation of the PGK1 PT promoter and terminator resulted in the production and secretion of Tr-Man5A and Tr-Man5A~CBD (lacking the cellulose binding module), respectively. However, the production levels of both proteins were approximately I5-fold lower than the production levels of Aa-Man5A. These levels did not improve after replacement of the native secretion signal with the MFuis secretion signal. Interestingly, reducing the cultivation temperature from 30°C to 20°C led to a five-fold increase in the secreted levels of Tr-Man5A, but a three-fold decrease in the production of Aa-Man5A. A preliminary investigation was performed to evaluate the possibility of using the recombinant Aa-Man5A in the processing of instant coffee. Arabica coffee extracts treated with Aa-Man5A displayed low viscosity in comparison to the untreated extract and showed better retention of volatile/aromatic compounds than the autoclaved extract. The results indicated that Aa-Man5A is capable of hydrolyzing coffee galactomannan and can be used for processing instant coffee.

AFRIKAANSE OPSOMMING: Mannaanpolisakkariede kom in die hemisellulose fraksie van plantselwande as strukturele polimere of reserwe koolstofbron voor. Mannaan word hoofsaaklik in die sade van peulplante, III die vorm van galaktomannaan, en III sagtehout as galaktoglucomannaan aangetref. Endo-I,4-j3-mannanase kan mannaanpolisakkariede na oligosakkariede van verskillende lengtes afbreek. Hierdie ensieme word deur fungi, bakterieë, plante en diere as enkele katalitiese modules of as deel van multi-modulêre proteïene uitgeskei. Die j3-mannanase (Aa-Man5A) van Aspergillus aculeatus is byvoorbeeld 'n enkele katalitiese module, maar die j3-mannanase (Tr-Man5A) van Trichoderma reesei bestaan uit 'n j3-mannanase katalitiese module gekoppel aan 'n sellulose-bindingsmodule deur middel van 'n Pro-Ser- Thr-ryke koppelstuk. Heteroloë geenuitdrukking in gIS bied die geleentheid om individuele hydrolitiese ensieme in 'n gasheer uitdrukkingsisteem sonder verwante aktiwiteite te produseer. Saccharomyces cerevisiae het 'n goed ontwikkelde uitdrukkingsisteem en word as model organisme vir heteroloë geenuitdrukking gebruik. 'n Aantal outoseleksiesisteme is ontwikkel, waardeur rekombinante S. cerevisiae-tese in enige medium sonder selektiewe druk gekweek kan word. 'n Outoseleksiesisteem, gebaseer op defektiewe chromosomale ura3 en furl gene wat vir ensieme in die pirimidien biosinteseweg kodeer, en komplementasie van die ura3-geen met die wilde-tipe URA3-geen wat op In multikopie plasmied teenwoordig is, is vir hierdie studie gebruik. Die manl-geen, wat vir die Aa-Man5A j3-mannanase van A. aculeatus kodeer, is gekloneer en in outoselektiewe S. cerevisiae onder die regulering van die alkoholdehidrogenase 2 (ADH2PT) en fosfogliseraatkinase 1 (POKl PT) promotor- en termineerderopeenvolgings uitgedruk. Uitdrukking van die manl-geen onder albei promotors het hoë produksievlakke van Aa-Man5A gelewer. Die produksievlakke was aansienlik hoër as die endo-I,4-j3-mannanase-vlakke wat deur heteroloë geenuitdrukking in Escherichia coli geproduseer was, en kon vergelyk word met die produksievlakke van ensieme in Pichia pastoris. P. pastoris is veronderstel om In hoër sekresiekapasiteit as S. cerevisiae te hê. Die rekombinante gisras wat die manl-geen onder beheer van die PGKl p promotor uitgedruk het, se biomassavorming was belemmer gedurende die laat logaritmiese fase. Die belemmering is opgehef nadat die natuurlike sekresiesein van 13-mannanasemet die MFais sekresiesein vervang is. Die rekombinante Aa-ManSA het soortgelyke biochemiese eienskappe as die natuurlike Aa-ManSA getoon. Die rekombinante ensiem het onvertakte mannaan tot hoofsaaklik mannose, mannobiose en mannotriose gehidroliseer. Die uitdrukking van die manl- en manl Licbd-geenkonstrukte van T. reesei in S. cerevisiae furl::LEU2-rasse onder regulering van die PGKlPT promotor en termineerder het tot die produksie en sekresie van onderskeidelik die Tr-ManSA en Tr-ManSAilCBD (sonder die sellulose-bindingsdomein) ensieme gelei. Die produksievlakke van beide proteïene was egter ongeveer IS-voudig laer as die vlakke van Aa-ManSA. Hierdie vlakke het egter nie verbeter nadat die natuurlike sekresiesein met die MFais sekresiesein vervang is nie. Interessant is die feit dat 'n afname in opkwekingstemperatuur vanaf 30°C tot 20°C tot 'n vyf-voudige toename m sekresievlakke van die Tr-ManSA gelei het, maar tot 'n drie-voudige afname in die produksie van Aa-ManSA. 'n Voorlopige ondersoek na die moontlike gebruik van rekombinante Aa-ManSA in kitskoffieprosessering is ondersoek.. Arabica koffie-ekstrak wat met Aa-ManSA behandel is, het 'n laer viskositeit in vergelyking met onbehandelde ekstrak getoon, asook beter behoud van vlugtige/aromatiese verbindings in vergelyking met geoutoklaveerde ekstrak. Hierdie resultate toon dat Aa-ManSA in staat is om koffee galaktomannaan te hidroliseer en dat dit vir die prosessering van kitskoffie gebruik kan word.

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