title: Adaptive evolution of a lactose-consuming Saccharomyces cerevisiae recombinant.
abstract: The construction of Saccharomyces cerevisiae strains that ferment lactose has biotechnological interest, particularly for cheese whey fermentation. A flocculent lactose-consuming S. cerevisiae recombinant expressing the LAC12 (lactose permease) and LAC4 (beta-galactosidase) genes of Kluyveromyces lactis was constructed previously but showed poor efficiency in lactose fermentation. This strain was therefore subjected to an evolutionary engineering process (serial transfer and dilution in lactose medium), which yielded an evolved recombinant strain that consumed lactose twofold faster, producing 30% more ethanol than the original recombinant. We identified two molecular events that targeted the LAC construct in the evolved strain: a 1,593-bp deletion in the intergenic region (promoter) between LAC4 and LAC12 and a decrease of the plasmid copy number by about 10-fold compared to that in the original recombinant. The results suggest that the intact promoter was unable to mediate the induction of the transcription of LAC4 and LAC12 by lactose in the original recombinant and that the deletion established the transcriptional induction of both genes in the evolved strain. We propose that the tuning of the expression of the heterologous LAC genes in the evolved recombinant was accomplished by the interplay between the decreased copy number of both genes and the different levels of transcriptional induction for LAC4 and LAC12 resulting from the changed promoter structure. Nevertheless, our results do not exclude other possible mutations that may have contributed to the improved lactose fermentation phenotype. This study illustrates the usefulness of simple evolutionary engineering approaches in strain improvement. The evolved strain efficiently fermented threefold-concentrated cheese whey, providing an attractive alternative for the fermentation of lactose-based media.
authors.affiliation: Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
authors.forename: Pedro M R
authors.initials: PM
authors.lastname: Guimarães
authors.forename: Jean
authors.initials: J
authors.lastname: François
authors.forename: Jean Luc
authors.initials: JL
authors.lastname: Parrou
authors.forename: José A
authors.initials: JA
authors.lastname: Teixeira
authors.forename: Lucília
authors.initials: L
authors.lastname: Domingues
mesh_terms: D019020:Directed Molecular Evolution; D005285:Fermentation; D005690:Galactose / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D007716:Kluyveromyces / Q000235:genetics / Q000378:metabolism; D007785:Lactose / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D026901:Membrane Transport Proteins; C021499:lactose permease; D001616:beta-Galactosidase; D005947:Glucose; D007785:Lactose; D005690:Galactose