Rolling circle amplification of synthetic DNA accelerates biocatalytic determination of enzyme activity relative to conventional methods

Timin Hadi,Joel O Melby,Douglas E Fuerst,Nicole Nozzi,Erik Kvam,Wei Gao

doi:10.1038/s41598-020-67307-9

Timin Hadi, Joel O Melby + Show 4 more

Open Access

https://doi.org/10.1038/s41598-020-67307-9

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Abstract

The ability to quickly and easily assess the activity of large collections of enzymes for a desired substrate holds great promise in the field of biocatalysis. Cell-free synthesis, although not practically amenable for large-scale enzyme production, provides a way to accelerate the timeline for screening enzyme candidates using small-scale reactions. However, because cell-free enzyme synthesis requires a considerable amount of template DNA, the preparation of high-quality DNA “parts” in large quantities represents a costly and rate-limiting prerequisite for high throughput screening. Based on time-cost analysis and comparative activity data, a cell-free workflow using synthetic DNA minicircles and rolling circle amplification enables comparable biocatalytic activity to cell-based workflows in almost half the time. We demonstrate this capability using a panel of sequences from the carbon-nitrogen hydrolase superfamily that represent possible green catalysts for synthesizing small molecules with less waste compared to traditional industrial chemistry. This method provides a new alternative to more cumbersome plasmid- or PCR-based protein expression workflows and should be amenable to automation for accelerating enzyme screening in industrial applications.

Highlights

The ability to quickly and assess the activity of large collections of enzymes for a desired substrate holds great promise in the field of biocatalysis
Cell-free protein synthesis (CFPS) is one technique that has been explored to accelerate the process of identifying enzymes for industrial applications[4]
While protein may be rapidly expressed in less than 24 hours via cell-free protein synthesis, the DNA template encoding the protein-of-interest often takes many days to convert from a digital sequence into physical DNA before protein expression can occur

Summary

Introduction

The ability to quickly and assess the activity of large collections of enzymes for a desired substrate holds great promise in the field of biocatalysis. Based on time-cost analysis and comparative activity data, a cell-free workflow using synthetic DNA minicircles and rolling circle amplification enables comparable biocatalytic activity to cell-based workflows in almost half the time We demonstrate this capability using a panel of sequences from the carbon-nitrogen hydrolase superfamily that represent possible green catalysts for synthesizing small molecules with less waste compared to traditional industrial chemistry. Screening panels are typically 96-well collections of recombinant enzymes produced in a convenient expression host (usually E. coli) This form of cell-based production is adapted to automated workflows, but new strategies for speeding progress and reducing costs of promising “hits” are increasingly desired[1,3]. PCR amplicons are unstable in nuclease-containing cell extracts unless subsequently converted into closed-circular DNA10,11, or synthesized with long protective 5′/3′ extensions[12], or coordinated with exonuclease inhibitors[13]

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