Genomics | Microbial Evolution & Genomics | Biocatalysis | Directed Molecular Evolution | Small Molecule Chemistry
A constitutive expression system for high-throughput screening
Dirk Aerts*, Tom Verhaeghe, Marjan De Mey, Tom Desmet, Wim Soetaert
*Corresponding author: Dirk Aerts
Centre of Expertise for Industrial Biotechnology and Biocatalysis, Ghent University, Ghent, Belgium
F1000Posters 2011, 2: 632 (poster) [ENGLISH]
Poster [1.29 MB]
9th Carbohydrate Bioengineering Meeting 2011, 15 - 18 May 2011, P1
The high-throughput screening of mutant libraries is typically perceived as the bottleneck in directed evolution. To that end, assays are usually performed in multi-well plates. Before a library can be screened, soluble expression of the enzymes has to be achieved.
To reduce the amount of consumables and number of pipetting steps in high-throughput screening, a constitutive expression system was developed that comprises four different promoters of varying strength. The system was validated by the expression of different sucrose phosphorylase enzymes from Leuconostoc mesenteroides, Lactobacillus acidophilus and Bifidobacterium adolescentis in 96-deep- and low-well plates at three temperatures.
Drastically improved soluble expression in mini-cultures was observed for the enzymes from L. mesenteroides strains by reducing the promoter strength from strong to intermediate and by expressing the proteins at lower temperatures. In contrast, the enzymes from B. adolescentis and L. acidophilus were expressed most efficiently with a strong promoter.
The constitutive expression of sucrose phosphorylases in low-well plates resulted in a level of activity that is equal or even better than what was achieved by inducible expression. Therefore, our plasmid set with varying constitutive promoters will be an indispensable tool to optimize enzyme expression for high-throughput screening.
No relevant conflicts of interest declared.
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