| 1. |
- Adamo, Giusy M, et al.
(författare)
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Amplification of the CUP1 gene is associated with evolution of copper tolerance in Saccharomyces cerevisiae.
- 2012
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Ingår i: Microbiology (Reading, England). - : Microbiology Society. - 1465-2080 .- 1350-0872. ; 158:Pt 9, s. 2325-35
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Tidskriftsartikel (refereegranskat)abstract
- In living organisms, copper (Cu) contributes to essential functions but at high concentrations it may elicit toxic effects. Cu-tolerant yeast strains are of relevance for both biotechnological applications and studying physiological and molecular mechanisms involved in stress resistance. One way to obtain tolerant strains is to exploit experimental methods that rely on the principles of natural evolution (evolutionary engineering) and allow for the development of complex phenotypic traits. However, in most cases, the molecular and physiological basis of the phenotypic changes produced have not yet been unravelled. We investigated the determinants of Cu resistance in a Saccharomyces cerevisiae strain that was evolved to tolerate up to 2.5 g CuSO(4) l(-1) in the culture medium. We found that the content of intracellular Cu and the expression levels of several genes encoding proteins involved in Cu metabolism and oxidative stress response were similar in the Cu-tolerant (evolved) and the Cu-sensitive (non-evolved) strain. The major difference detected in the two strains was the copy number of the gene CUP1, which encodes a metallothionein. In evolved cells, a sevenfold amplification of CUP1 was observed, accounting for its strongly and steadily increased expression. Our results implicate CUP1 in protection of the evolved S. cerevisiae cells against Cu toxicity. In these cells, robustness towards Cu is stably inheritable and can be reproducibly selected by controlling environmental conditions. This finding corroborates the effectiveness of laboratory evolution of whole cells as a tool to develop microbial strains for biotechnological applications.
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| 2. |
- Brocca, Stefania, et al.
(författare)
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Mutants provide evidence of the importance of glycosydic chains in the activation of lipase 1 from Candida rugosa
- 2000
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 9:5, s. 985-990
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Tidskriftsartikel (refereegranskat)abstract
- Sequence analysis of Candida rugosa lipase 1 (LIP1) predicts the presence of three N-linked glycosylation sites at asparagine 291, 314, 351. To investigate the relevance of sugar chains in the activation and stabilization of LIP1, we directed site mutagenesis to replace the above mentioned asparagine with glutamine residues. Comparison of the activity of mutants with that of the wild-type (wt) lipase indicates that both 314 and 351 Asn to Gln substitutions influence, although at a different extent, the enzyme activity both in hydrolysis and esterification reactions, but they do not alter the enzyme water activity profiles in organic solvents or temperature stability. Introduction of Gln to replace Asn35 is likely to disrupt a stabilizing interaction between the sugar chain and residues of the inner side of the lid in the enzyme active conformation. The effect of deglycosylation at position 314 is more difficult to explain and might suggest a more general role of the sugar moiety for the structural stability of lipase 1. Conversely, Asn291Gln substitution does not affect' the lipolytic or the esterase activity of the mutant that behaves essentially as the wt enzyme. This observation supports the hypothesis that changes in activity of Asn314Gln and Asn351Gln mutants are specifically due to deglycosylation.
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