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Design and application of a lactulose biosensor

View Article: PubMed Central - PubMed

ABSTRACT

In this study the repressor of Escherichia coli lac operon, LacI, has been engineered for altered effector specificity. A LacI saturation mutagenesis library was subjected to Fluorescence Activated Cell Sorting (FACS) dual screening. Mutant LacI-L5 was selected and it is specifically induced by lactulose but not by other disaccharides tested (lactose, epilactose, maltose, sucrose, cellobiose and melibiose). LacI-L5 has been successfully used to construct a whole-cell lactulose biosensor which was then applied in directed evolution of cellobiose 2-epimerase (C2E) for elevated lactulose production. The mutant C2E enzyme with ~32-fold enhanced expression level was selected, demonstrating the high efficiency of the lactulose biosensor. LacI-L5 can also be used as a novel regulatory tool. This work explores the potential of engineering LacI for customized molecular biosensors which can be applied in practice.

No MeSH data available.


Effects of lactose on wild-type LacI (a) and LacI-L5 (b).
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f3: Effects of lactose on wild-type LacI (a) and LacI-L5 (b).

Mentions: Because lactose, whose binding increases the affinity of the lac repressor for the operator, acts as an anti-inducer of wild-type LacI33, its effects on LacI-L5 was studied. For LacI-L5, the anti-inducing effect of lactose remained similar to that in wild-type LacI, however, the induction of LacI-L5 by lactulose was not inhibited by lactose (Fig. 3).


Design and application of a lactulose biosensor
Effects of lactose on wild-type LacI (a) and LacI-L5 (b).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384092&req=5

f3: Effects of lactose on wild-type LacI (a) and LacI-L5 (b).
Mentions: Because lactose, whose binding increases the affinity of the lac repressor for the operator, acts as an anti-inducer of wild-type LacI33, its effects on LacI-L5 was studied. For LacI-L5, the anti-inducing effect of lactose remained similar to that in wild-type LacI, however, the induction of LacI-L5 by lactulose was not inhibited by lactose (Fig. 3).

View Article: PubMed Central - PubMed

ABSTRACT

In this study the repressor of Escherichia coli lac operon, LacI, has been engineered for altered effector specificity. A LacI saturation mutagenesis library was subjected to Fluorescence Activated Cell Sorting (FACS) dual screening. Mutant LacI-L5 was selected and it is specifically induced by lactulose but not by other disaccharides tested (lactose, epilactose, maltose, sucrose, cellobiose and melibiose). LacI-L5 has been successfully used to construct a whole-cell lactulose biosensor which was then applied in directed evolution of cellobiose 2-epimerase (C2E) for elevated lactulose production. The mutant C2E enzyme with ~32-fold enhanced expression level was selected, demonstrating the high efficiency of the lactulose biosensor. LacI-L5 can also be used as a novel regulatory tool. This work explores the potential of engineering LacI for customized molecular biosensors which can be applied in practice.

No MeSH data available.