A selection assay for haloalkane dehalogenase activity based on toxic substrates

Abstract

Based on natural selection and the survival of the fittest by evolutionary adaption, a smart high-throughput system was developed to select active haloalkane dehalogenase variants from a large mutant library. Only active enzyme variants can hydrolyse toxic halogenated alkanes to promote growth, whereas inactive mutants starve or die due to the toxic compound. With this powerful tool, huge enzyme mutant libraries can be screened within a few days. The selection is done without any artificial substrates that are hard to synthesize and they also resemble typical ones for haloalkane dehalogenases. Three saturation libraries, with a size of more than 10(6) cells, based on inactive variants of the haloalkane dehalogenases DhaA or DhlA were successfully screened to retrieve active enzymes. The enrichment of the active wild-type enzyme in contrast to the inactive variants was about 340-fold. In addition, this selection approach can be applied for continuous directed evolution experiments for the enrichment of cells expressing adapted haloalkane dehalogenases.

Fully automatized high‐throughput enzyme library screening using a robotic platform

“…The successful accumulation of revertants in a continuous selection process (Fig. 7) was exemplified for a knock-out library (Fibinger et al, 2015). The statistical portion of wild-type revertants-containing the re-introduced residues in the active site-in the libraries was 0.26-3.125%.…”

“…After screening 166 MTPs of the selection-based evolutionary process a strong accumulation of revertants was found by the automatized phenol red assay. Further details have been already reported elsewhere (Fibinger et al, 2015).…”

“…Fifty microliter of the whole cell suspension were added to 150 mL of the assay solution (140 mL HEPES, 1 mM, pH 8.2, 20 mg/mL phenol red supplemented with 10 mL of a mixture of 100 mM haloalkanes 1,2-dibromoethane, 1-iodopropane, 1-bromobutane, 1-iodobutane, and 4-bromobutyronitrile in acetonitrile). All five substrates are known to be accepted by the HLD and using a mixture increases the chance to find desired active variants (Fibinger et al, 2015). The absorption at 430 and 560 nm was measured at the beginning and after 24 h of incubation at 37 C. The phenol red indicator color change to yellow, indicated by a change in the ratio of both absorption values, was used for signaling the release of protons and subsequent catalytic conversion of the haloalkanes to the corresponding alcohols.…”

“…Additionally, haloalkanes are common pollutants that can be degraded to harmless alcohols resulting in the generation and/or release of growth substrates. To find appropriate enzyme variants for this purpose, a selection methodology was established for the selective accumulation of E. coli cells expressing active HLDs (Fibinger et al, 2015). In this study the activity of DhaA (Newman et al, 1999) and DhlA (Janssen et al, 1989) were destroyed by substituting catalytically necessary residues.…”

Strategies for directed and adapted evolution as part of microbial strain engineering

“…In addition, some excrete enzymes (such as cellulase, amylase, lipase and polygalacturonase) can generate a hydrolytic zone around a colony that may be used to screen high activity strains. Likewise, if the evolved enzyme or pathway can enable conversion of a toxic substance into biomass growth, colony size screening can be used.…”