Expression and characterization of an intracellular cellobiose phosphorylase in Saccharomyces cerevisiae

Sadie, Christa J. (Christiena Johanna) (Stellenbosch : Stellenbosch University, 2007-03)

Thesis (MSc)--University of Stellenbosch, 2007.

Thesis

ENGLISH ABSTRACT: Cellulose, a glucose polymer, is considered the most abundant fermentable polymer on earth. Agricultural waste is rich in cellulose and exploiting these renewable sources as a substrate for ethanol production can assist in producing enough bioethanol as a cost-effective replacement for currently used decreasing fossil fuels. Saccharomyces cerevisiae is an excellent fermentative organism of hexoses; however the inability of the yeast to utilize cellulose as a carbon source is a major obstruction to overcome for its use in the production of bio-ethanol. Cellobiose, the major-end product of cellulose hydrolysis, is hydrolyzed by -glucosidase or cellobiose phosphorylase, the latter having a possible metabolic advantage over -glucosidase. Recently, it has been showed that S. cerevisiae is able to transport cellobiose. The construction of a cellulolytic yeast that can transport cellobiose has the advantage that end-product inhibition of the extracellular cellulases by glucose and cellobiose is relieved. Furthermore, the extracellular glucose concentration remains low and the possibility of contamination is decreased. In this study the cellobiose phosphorylase gene, cepA, of Clostridium stercorarium was cloned and expressed under transcriptional control of the constitutive PGK1 promoter and terminator of S. cerevisiae on a multicopy episomal plasmid. The enzyme was expressed intracellulary and thus required the transport of cellobiose into the cell. The fur1 gene was disrupted for growth of the recombinant strain on complex media without the loss of the plasmid. The recombinant strain, S. cerevisiae[yCEPA], was able to sustain aerobic growth on cellobiose as sole carbon source at 30°C with Vmax = 0.07 h-1 and yielded 0.05 g biomass per gram cellobiose consumed. The recombinant enzyme had activity optima of 60°C and pH 6-7. Using Michaelis-Menten kinetics, the Km values for the colorimetric substrate p-nitrophenyl-b-D-glucopyranoside (pNPG) and cellobiose was estimated to be 1.69 and 92.85 mM respectively. Enzyme activity assays revealed that the recombinant protein was localized in the membrane fraction and no activity was present in the intracellular fraction. Due to an unfavourable codon bias in S. cerevisiae, CepA activity was very low. Permeabilized S. cerevisiae[yCEPA] cells had much higher CepA activity than whole cells indicating that the transport of cellobiose was inadequate even after one year of selection. Low activity and insufficient cellobiose transport led to an inadequate glucose supply for the yeast resulting in low biomass formation. Cellobiose utilization increased when combined with other sugars (glucose, galactose, raffinose, maltose), as compared to using cellobiose alone. This is possibly due to more ATP being available for the cell for cellobiose transport. However, no cellobiose was utilized when grown with fructose indicating catabolite repression by this sugar. To our knowledge this is the first report of a heterologously expressed cellobiose phosphorylase in yeast that conferred growth on cellobiose. Furthermore, this report also reaffirms previous data that cellobiose can be utilized intracellularly in S. cerevisiae.

AFRIKAANSE OPSOMMING: Sellulose, ‘n homopolimeer van glukose eenhede, word beskou as die volopste suiker polimeer op aarde. Landbou afval produkte het ‘n hoë sellulose inhoud en benutting van diè substraat vir bio-etanol produksie kan dien as ‘n koste-effektiewe aanvulling en/of vervanging van dalende fossielbrandstof wat tans gebruik word. Die gis, Saccharomyces cerevisiae, is ‘n uitmuntende organisme vir die fermentasie van heksose suikers, maar die onvermoë van die gis om sellulose as koolstofbron te benut is ‘n groot struikelblok in sy gebruik vir die produksie van bio-etanol. Sellobiose, die hoof eindproduk van ensiematiese hidrolise van sellulose, word afgebreek deur -glukosidase of sellobiose fosforilase. Laasgenoemde het ‘n moontlike metaboliese voordeel bo die gebruik van -glukosidase vir sellobiose hidrolise. Daar was onlangs gevind dat S. cerevisiae in staat is om sellobiose op te neem. Die konstruksie van ‘n sellulolitiese gis wat sellobiose intrasellulêr kan benut, het die voordeel dat eindproduk inhibisie van die ekstrasellulêre sellulases deur sellobiose en glukose verlig word. Verder, wanneer die omsetting van glukose vanaf sellobiose intrasellulêr plaasvind, word die ekstrasellulêre glukose konsentrasie laag gehou en die moontlikheid van kontaminasie beperk. In hierdie studie was die sellobiose fosforilase geen, cepA, van Clostridium stercorarium gekloneer en uitgedruk onder transkripsionele beheer van die konstitutiewe PGK1 promoter en termineerder van S. cerevisiae op ‘n multikopie episomale plasmied. Die ensiem is as ‘n intrasellulêre proteïen uitgedruk en het dus die opneem van die sellobiose molekuul benodig. Die disrupsie van die fur1 geen het toegelaat dat die rekombinante ras op komplekse media kon groei sonder die verlies van die plasmied. Die rekombinante ras, S. cerevisiae[yCEPA], het aërobiese groei by 30°C op sellobiose as enigste koolstofbron onderhou met mmax = 0.07 h-1 en ‘n opbrengs van 0.05 gram selle droë gewig per gram sellobiose. Die rekombinante ensiem het optima van 60°C en pH 6-7 gehad. Die K m waardes vir die kolorimetriese substraat pNPG en sellobiose was 1.69 en 92.85 mM onderskeidelik. Ondersoek van die ensiem aktiwiteit het getoon dat die rekombinante proteïen gelokaliseer was in die membraan fraksie en geen aktiwiteit was teenwoordig in die intrasellulêre fraksie nie. CepA aktiwiteit was laag as gevolg van ‘n lae kodon voorkeur in S. cerevisiae. Verder het geperforeerde S. cerevisiae[yCEPA] selle aansienlik beter CepA aktiwiteit getoon as intakte selle. Hierdie aanduiding van onvoldoende transport van sellobiose na binne in die sel tesame met die lae aktiwiteit van die CepA ensiem het gelei tot onvoldoende glukose voorraad vir die sel en min biomassa vorming. Sellobiose verbruik het toegeneem wanneer dit tesame met ander suikers (glukose, galaktose, raffinose, maltose) gemeng was, heelwaarskynlik deur die vorming van ekstra ATP’s vir die sel wat ‘n toename in sellobiose transport teweeg gebring het. Fruktose het egter kataboliet onderdrukking veroorsaak en sellobiose was nie benut nie. Sover ons kennis strek, is hierdie die eerste verslag van ‘n heteroloë sellobiose fosforilase wat in S. cerevisiae uitgedruk is en groei op sellobiose toegelaat het. Verder, bewys die studie weereens dat S. cerevisiae wel sellobiose kan opneem.

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