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Applied and Environmental Microbiology, March 2008, p. 1748-1756, Vol. 74, No. 6
0099-2240/08/$08.00+0 doi:10.1128/AEM.00186-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Adaptive Evolution of a Lactose-Consuming Saccharomyces cerevisiae Recombinant
Pedro M. R. Guimarães,1
Jean François,2,3,4
Jean Luc Parrou,2,3,4
José A. Teixeira,1 and
Lucília Domingues1*
Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal,1 Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France,2 INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France,3 CNRS, UMR5504, F-31400 Toulouse, France4
Received 21 January 2008/ Accepted 22 January 2008
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 (β-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.
* Corresponding author. Mailing address: Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal. Phone: 351 253 604 400. Fax: 351 253 678 986. E-mail: luciliad{at}deb.uminho.pt
Published ahead of print on 28 January 2008.
Applied and Environmental Microbiology, March 2008, p. 1748-1756, Vol. 74, No. 6
0099-2240/08/$08.00+0 doi:10.1128/AEM.00186-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
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