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Design and application of a lactulose biosensor

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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.

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Engineering C2E for elevated lactulose production.(a) The BLGR strains co-expressing LacI-L5 and the C2E random mutagenesis library grown on LB agar supplemented with 10 mM lactose under UV 302 nm. (b) Lactulose productions of the selected variants expressing the C2E mutants at 37 °C, compared with that of strain expressing the wild-type C2E (C2E-WT). (c) Western-blot analysis of mutant C2 expression as compared with the wild-type enzyme. (d) Specific activities of wild-type C2E (C2E-WT) and mutant C2 at 37 °C and 80 °C.
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f4: Engineering C2E for elevated lactulose production.(a) The BLGR strains co-expressing LacI-L5 and the C2E random mutagenesis library grown on LB agar supplemented with 10 mM lactose under UV 302 nm. (b) Lactulose productions of the selected variants expressing the C2E mutants at 37 °C, compared with that of strain expressing the wild-type C2E (C2E-WT). (c) Western-blot analysis of mutant C2 expression as compared with the wild-type enzyme. (d) Specific activities of wild-type C2E (C2E-WT) and mutant C2 at 37 °C and 80 °C.

Mentions: To test the effectiveness of strain BLGR harboring plasmid pLac7-L5 as a whole-cell biosensor of lactulose, it was used as a high-throughput screening tool for engineering C2E from Caldicellulosiruptor saccharolyticus32 for improved lactulose production. The plasmid pN25-C2E harboring the C2E random mutagenesis library was co-transformed with the plasmid pLac7-L5 into strain BLGR. Lactulose endogenously produced by C2E mutants was found to bind to LacI-L5 and activate the expression of GFP inside the cell. Two colonies, expressing mutant enzymes C1 and C2, showing stronger GFP fluorescence than colonies expressing wild-type C2E on LB agar supplemented with lactose, were selected from the library (Fig. 4a). Because LacI-L5 was not responsive to lactose, any fluorescence expression would be induced by the lactulose produced by C2E mutants inside the cells using lactose as the substrate. HPLC analyses verified enhanced lactulose production by these selected variants compared with that of the strain expressing wild-type C2E. The best of the variants, C2(E272D, F339S), displayed ~22-fold increase in lactulose production at 37 °C (Fig. 4b). Western blot analysis revealed that the expression level of mutant C2 was significantly elevated by ~32-fold, compared with that of the wild-type enzyme (Fig. 4c), although no obvious increase in codon usage bias was observed in the mutations in mutant C2. The pN25-C2E plasmid expressing mutant C2 was isolated and retransformed into strain BLGR, and a significantly elevated expression level of the mutant C2E was confirmed.


Design and application of a lactulose biosensor
Engineering C2E for elevated lactulose production.(a) The BLGR strains co-expressing LacI-L5 and the C2E random mutagenesis library grown on LB agar supplemented with 10 mM lactose under UV 302 nm. (b) Lactulose productions of the selected variants expressing the C2E mutants at 37 °C, compared with that of strain expressing the wild-type C2E (C2E-WT). (c) Western-blot analysis of mutant C2 expression as compared with the wild-type enzyme. (d) Specific activities of wild-type C2E (C2E-WT) and mutant C2 at 37 °C and 80 °C.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Engineering C2E for elevated lactulose production.(a) The BLGR strains co-expressing LacI-L5 and the C2E random mutagenesis library grown on LB agar supplemented with 10 mM lactose under UV 302 nm. (b) Lactulose productions of the selected variants expressing the C2E mutants at 37 °C, compared with that of strain expressing the wild-type C2E (C2E-WT). (c) Western-blot analysis of mutant C2 expression as compared with the wild-type enzyme. (d) Specific activities of wild-type C2E (C2E-WT) and mutant C2 at 37 °C and 80 °C.
Mentions: To test the effectiveness of strain BLGR harboring plasmid pLac7-L5 as a whole-cell biosensor of lactulose, it was used as a high-throughput screening tool for engineering C2E from Caldicellulosiruptor saccharolyticus32 for improved lactulose production. The plasmid pN25-C2E harboring the C2E random mutagenesis library was co-transformed with the plasmid pLac7-L5 into strain BLGR. Lactulose endogenously produced by C2E mutants was found to bind to LacI-L5 and activate the expression of GFP inside the cell. Two colonies, expressing mutant enzymes C1 and C2, showing stronger GFP fluorescence than colonies expressing wild-type C2E on LB agar supplemented with lactose, were selected from the library (Fig. 4a). Because LacI-L5 was not responsive to lactose, any fluorescence expression would be induced by the lactulose produced by C2E mutants inside the cells using lactose as the substrate. HPLC analyses verified enhanced lactulose production by these selected variants compared with that of the strain expressing wild-type C2E. The best of the variants, C2(E272D, F339S), displayed ~22-fold increase in lactulose production at 37 °C (Fig. 4b). Western blot analysis revealed that the expression level of mutant C2 was significantly elevated by ~32-fold, compared with that of the wild-type enzyme (Fig. 4c), although no obvious increase in codon usage bias was observed in the mutations in mutant C2. The pN25-C2E plasmid expressing mutant C2 was isolated and retransformed into strain BLGR, and a significantly elevated expression level of the mutant C2E was confirmed.

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.


Related in: MedlinePlus