title: The metabolic burden of cellulase expression by recombinant Saccharomyces cerevisiae Y294 in aerobic batch culture.
abstract: Two recombinant strains of Saccharomyces cerevisiae Y294 producing cellulase using different expression strategies were compared to a reference strain in aerobic culture to evaluate the potential metabolic burden that cellulase expression imposed on the yeast metabolism. In a chemically defined mineral medium with glucose as carbon source, S. cerevisiae strain Y294[CEL5] with plasmid-borne cellulase genes produced endoglucanase and β-glucosidase activities of 0.038 and 0.30 U mg dry cell weight(-1), respectively. Chromosomal expression of these two cellulases in strain Y294[Y118p] resulted in no detectable activity, although low levels of episomally co-expressed cellobiohydrolase (CBH) activity were detected. Whereas the biomass concentration of strain Y294[CEL5] was slightly greater than that of a reference strain, CBH expression by Y294[Y118p] resulted in a 1.4-fold lower maximum specific growth rate than that of the reference. Supplementation of the growth medium with amino acids significantly improved culture growth and enzyme production, but only partially mitigated the physiological effects and metabolic burden of cellulase expression. Glycerol production was decreased significantly, up to threefold, in amino acid-supplemented cultures, apparently due to redox balancing. Disproportionately higher levels of glycerol production by Y294[CEL5] indicated a potential correlation between the redox balance of anabolism and the physiological stress of cellulase production. With the reliance on cellulase expression in yeast for the development of consolidated bioprocesses for bioethanol production, this work demonstrates the need for development of yeasts that are physiologically robust in response to burdens imposed by heterologous enzyme production.
authors.affiliation: Department of Process Engineering, Stellenbosch University, Private Bag X1, Stellenbosch, 7602, South Africa.
authors.forename: Eugéne
authors.initials: E
authors.lastname: van Rensburg
authors.forename: Riaan
authors.initials: R
authors.lastname: den Haan
authors.forename: Justin
authors.initials: J
authors.lastname: Smith
authors.forename: Willem H
authors.initials: WH
authors.lastname: van Zyl
authors.forename: Johann F
authors.initials: JF
authors.lastname: Görgens
mesh_terms: D000332:Aerobiosis; D000596:Amino Acids / Q000378:metabolism; D061249:Batch Cell Culture Techniques; D018533:Biomass; D002244:Carbon / Q000378:metabolism; D002480:Cellulase / Q000096:biosynthesis*; D015825:Chromosomes, Fungal; D003470:Culture Media / Q000737:chemistry; D004734:Energy Metabolism; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism* / Q000502:physiology
chemical_list: D000596:Amino Acids; D003470:Culture Media; D002244:Carbon; D002480:Cellulase; D005947:Glucose; D005990:Glycerol