We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.
Cellulase enzymes (endoglucanases, cellobiohydrolases, and β-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12.
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (“halos”) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness.
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.
1. Screening plate preparation
2. Endoglucanase library generation and screen
3. Representative Results:
An example of the readout for this high throughput screen is shown in Figure 3. Clones can be identified as inactive, weakly active, and highly active based on the size of the degradation zones. This is especially useful if an enzyme of known activity is included in the library as a reference. As shown here, the identification of active enzymes is robust and reproducible. However, please be aware that adequate labeling of the screening plates is extremely important. The researcher must be able to identify active variants after washing the colonies away, in order to pick them from the master plate stored at -80°C for further characterization. Finally, it is difficult to assess a priori the relationship between activities on artificial vs natural substrates. Therefore, the researcher must test all positive clones on industrially relevant material to determine protein fitness.
Figure 1. Schematic outline of the screening procedure. Cells are transformed with an endoglucanase library and plated selectively for single colonies. Clones picked and grown overnight in 96 well deep well plates for (1) storage at -80°C and (2) replica plating on plates containing CMC and IPTG to induce endoglucanase expression. Active clones are picked from the master plate stored at -80°C.
Figure 2. Cloning and expression of fungal catalytic domains in E. coli. (A) Endoglucanase expression vector. Genes were cloned into pET22b(+) (Novagen) under control of the T7 promoter. (B) Right panel: endoglucanase-expressing BL21 (DE3) colonies. Left panel: CMC degradation halos after washing and development with Congo Red.
Figure 3. Sample results from CMC/Congo Red screen. Pictures taken of screening plates after washing and Congo Red development. All colonies on screening plates were of similar size.
The protocol described here enables rapid and high throughput identification of active enzymes, with minimal manipulation. Activity detection is fairly sensitive, and qualitatively reflects the amount of activity within the cell. Its ease of use makes this method suitable for a wide range of enzyme libraries, only limited by functional expression in E. coli. Furthermore, activity screening of this kind is not restricted to fungal cellulases, but can be adapted for any endoglucanase activity, or any cellulase activity for which there is a soluble substrate (like CMC).
The authors have nothing to disclose.
This work was funded by the Gordon and Betty Moore Foundation, and by the UNCF/Merck Science Initiative.
Reagent | Company | Product number |
---|---|---|
IPTG | Sigma | I1284 |
Congo Red | Sigma | C6277 |
Carboxymethyl Cellulose | Sigma | 360384 |
NaCl | Sigma | S1679 |
Bacto-agar | BD Biosciences | 214030 |
Bioassay plates | Thermo Scientific | 240845 |