FUNCTION- OR ACTIVITY-BASED EVALUATION OF THE ENVIRONMENT
Besides these sequence-based approaches, metagenomic libraries can be screened for various functions (Fig. 12.6). Functional approaches include expression screening with various genetic traps and phage biopanning. Even stable isotope probing (see earlier discussion) could be categorized as a function-based approach if the labeled substrate is a specific metabolite that enriches the culture based on metabolic function. Screening a metagenomic library by sequencing has its limits. The function of many genes from exotic organisms cannot be identified by their sequence. In addition, using known genes to screen for new members of a gene family might miss an entire novel class of genes. For example, if a researcher were trying to find enzymes similar to bacteriorhodopsin, primers similar to known bacteriorhodopsins would be used to screen the library. Some genes would be identified, but others would be missed if their sequences were too divergent. Screening this library for light-driven proton pumping would identify any enzyme that carried out this function, regardless of its sequence.
Expression screening depends on the choice of an expression vector. Proteins are expressed from the metagenomic DNA fragments when they are cloned into a vector with transcriptional and translational start and stop sequences. Then an easy assay for the target function must be devised. For example, the library clones might be plated on a particular toxic pollutant. If any library inserts encoded an enzyme that metabolized the pollutant, that particular library clone would grow. The DNA insert is then isolated and identified by sequencing.
Another functional screen involves fusing the metagenomic DNA in frame with a promoterless gene for green fluorescent protein (GFP). The genes for many enzymes are turned on by their own substrates. Therefore, if this type of gene is cloned in front of promoterless GFP, the GFP gene will be regulated by the same substrate. If the substrate of interest is included in the growth medium for the library, any clone with genes that are activated by the substrate will also produce GFP. These cells will fluoresce green. FACS provides a quick and easy way to isolate the fluorescent clones.
The main barrier to function-based analyses is successful gene expression. Getting a library host such as E. coli to express foreign genes is hit or miss, because some may be toxic to the host, some may require other factors for expression, and others may have very low activity. Another problem is simply volume. The number of potential clones that have any chosen gene of interest is usually low, and excessive numbers of clones must be screened in order to identify just one or two genes. For example, the lipases identified from German soil (see Table 12.1) were only found in 1 of 730,000 different metagenomic clones. In another example, only two novel Na+/H+ antiporters were found after screening 1,480,000 clones. This is why culture enrichment strategies are an important aspect of creating metagenomic libraries (see earlier discussion).
Metagenomic phage biopanning uses basically the same method as phage display. Cloned DNA inserts are expressed as fusion proteins with a phage coat protein and displayed on the phage surface. Because the displayed proteins carry only segments of foreign protein, the problems associated with heterologous expression are lessened. The cloned DNA is from any organisms found within the environmental sample. Thus the expressed protein segments could be any part of an enzyme, a membrane protein, etc.; therefore, the success with a phage display library relies on the screening method. For example, phage biopanning can identify binding partners for a particular pollutant, metabolite, or even another protein. If the target molecule is immobilized on a bead, then any phage carrying a protein segment that binds the target will stick to the bead. The phage can then be isolated, and the DNA insert sequenced to identify the sequence responsible.