High-Throughput Screening of Higher Α-Amylase Producing Bacillus Licheniformis by Fluorescence-Activated Droplet Sorting

Abstract

Backgroundα-Amylases is one of the most important starch degrading enzymes and has the widest range of industrial applications. Bacillus licheniformis has been widely used as a cell factory for industrial production of amylase. However, difficulties in genetic modification of B. licheniformis have limited its widespread use. Directed evolution, based on the combination of random mutagenesis and high throughput screening (HTS), has been proven an effective strategy in strain improvement for increasing the productivity, but it requires a suitable HTS system to screen the desired mutants. Droplet-based microfluidics has emerged as a powerful tool for single-cell screening with ultra-high throughput, however, the accessibility of a droplet microfluidic HTS platform to users having no background in microfluidics is still an issue. ResultsHere, we first developed a microfluidic HTS platform based on fluorescence-activated droplet sorting (FADS) technology. This platform allowed (i) encapsulation of single cells in monodisperse water-in-oil droplets; (ii) cell growth and protein production in droplets; (iii) sorting of droplets based on their fluorescence intensities. To validate the platform, a model selection experiment of a binary mixture of Bacillus strains was performed and a 45.6-fold enrichment was achieved at a sorting rate of 300 droplets per second. Furthermore, we used the platform for the selection of higher α-amylase-producing strains from a library of B. licheniformis strains (a strain already used at industrial-scale). The B. licheniformis mutant library was generated by atmospheric and room temperature plasma (ARTP) mutagenesis. The clones displaying over 50% improvement in α-amylases productivity compared to the wild-type were isolated.ConclusionsWe established an efficient droplet microfluidic platform which consisted of droplet generation, droplet incubation, and sorting of droplets with a throughput of up to 1 × 106 droplets per h. The screening platform was demonstrated by successfully identifying B. licheniformis clones with improved α-amylase production. We believe that the droplet platform could be extended to the development of other industrially valuable strains.