Interplay of metagenomics and in vitro compartmentalization

Abstract

In recent years, the application of approaches for harvesting DNA from the environment, the so-called, 'metagenomic approaches' has proven to be highly successful for the identification, isolation and generation of novel enzymes. Functional screening for the desired catalytic activity is one of the key steps in mining metagenomic libraries, as it does not rely on sequence homology. In this mini-review, we survey high-throughput screening tools, originally developed for directed evolution experiments, which can be readily adapted for the screening of large libraries. In particular, we focus on the use of in vitro compartmentalization (IVC) approaches to address potential advantages and problems the merger of culture-independent and IVC techniques might bring on the mining of enzyme activities in microbial communities.

Figure 1

Flow chart illustrating the identification, isolation and further engineering of novel enzymes using metagenomic and directed evolution approaches. Newly identified enzymes from metagenomic libraries can serve as an ideal starting point for the directed evolution of enzymes with improved properties. High‐throughput screening for enzymatic activity can be used, both for screening large metagenomic libraries and subsequently, for directed evolution experiments.

Figure 2

Mining genomes and metagenomes for novel enzymes. A gene library is created from environmental samples (Step 1–3) and used to screen for novel genes (Step 4) cloned into bacteria which can be sequenced (Step 5a). The encoded proteins expressed in appropriate host are then subjected to structure‐function analyses (central panel). Alternatively, large‐scale sequencing of bulk DNA is used for archiving and sequence homology screening purposes to capture the largest amount of the available genetic resources present in environmental samples (Step 5b).

Figure 3

Selections by FACS sorting of double emulsion droplets. A gene library is transformed into bacteria, and the encoded proteins are expressed in the cytoplasm, the periplasm, or on the surface of the cells (Step 1). The bacteria are dispersed to form a water‐in‐oil (w/o) emulsion, with typically one cell per aqueous microdroplet. Alternatively, an in vitro transcription/translation reaction mixture containing a library of genes is dispersed to form a w/o emulsion with typically one gene per aqueous microdroplet. The genes are transcribed and translated within the microdroplets (Step 2). Proteins with enzymatic activity convert the non‐fluorescent substrate into a fluorescent product and the w/o emulsion is converted into a water‐in‐oil‐in‐water (w/o/w) emulsion (Step 3). Fluorescent microdroplets are separated from non‐fluorescent microdroplets using a fluorescence activated cell sorter (FACS) (Step 4). Bacteria or genes from fluorescent microdroplets which encode active enzymes are recovered and the bacteria are propagated or the DNA is amplified using the polymerase chain reaction. These bacteria or genes can then be re‐compartmentalized for further rounds of selection.