High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Summary

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.

Abstract

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 cellulases^3-8, 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. jecorina^3,9, 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 chrysosporium^11-12.

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.

Protocol

1. Screening plate preparation

1. Add 25g LB growth medium, 15g agar, and 1.5g carboxymethyl cellulose (CMC) substrate to 1L distilled water, and autoclave to sterilize.
2. After autoclaving, allow medium to cool and add appropriate antibiotics, and IPTG to a final concentration of 100μM.
3. Pour 200mL medium each into 5 large square petri dishes/bioassay trays (240 x 240 x 20mm or larger). Allow plates to dry completely.

2. Endoglucanase library generation and screen

1. Generate a library of endoglucanase catalytic domain(s). This can be accomplished in many ways (e.g., site-directed or random muta....

Discussion

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 act.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
IPTG	Sigma-Aldrich	I1284
Congo Red	Sigma-Aldrich	C6277
Carboxymethyl Cellulose	Sigma-Aldrich	360384
NaCl	Sigma-Aldrich	S1679
Bacto-agar	BD Biosciences	214030
Bioassay plates	Thermo Fisher Scientific, Inc.	240845