Glycoside hydrolases from a targeted compost metagenome, activity-screening and functional characterization

Abstract

Background: Metagenomics approaches provide access to environmental genetic diversity for biotechnology applications, enabling the discovery of new enzymes and pathways for numerous catalytic processes. Discovery of new glycoside hydrolases with improved biocatalytic properties for the efficient conversion of lignocellulosic material to biofuels is a critical challenge in the development of economically viable routes from biomass to fuels and chemicals.

Results: Twenty-two putative ORFs (open reading frames) were identified from a switchgrass-adapted compost community based on sequence homology to related gene families. These ORFs were expressed in E. coli and assayed for predicted activities. Seven of the ORFs were demonstrated to encode active enzymes, encompassing five classes of hemicellulases. Four enzymes were over expressed in vivo, purified to homogeneity and subjected to detailed biochemical characterization. Their pH optima ranged between 5.5 - 7.5 and they exhibit moderate thermostability up to ~60-70°C.

Conclusions: Seven active enzymes were identified from this set of ORFs comprising five different hemicellulose activities. These enzymes have been shown to have useful properties, such as moderate thermal stability and broad pH optima, and may serve as the starting points for future protein engineering towards the goal of developing efficient enzyme cocktails for biomass degradation under diverse process conditions.

Figure 1

Diverse CAZy-family genes members were identified in the metagenome from switchgrass compost. The putative reconstructed genes are predicted to fall into 12 different CAZy families. The most abundant families are GH10, GH43, and GH51. Homology-based protein structure predictions were generated using AS2TS (proteinmodel.org) [10]. All members were expressed in E coli and screened for expression and activity. Colored structures were expressed and characterized biochemically while the black and white structures were not.

Figure 2

All of the codon-optimized genes are highly expressed in E. coli. Twenty-two genes in each of three expression vectors were expressed in E. coli. pET57 expression lysates (panel A), pET60 expression lysates (panel B), and pVP16 expression lysates (panel C) were analyzed with a LabChip GXII Protein Assay (Caliper Life Sciences, Hopkinton, MA). Ordering of samples for each panel is alphanumeric for each vector backbone. Expression of all genes was confirmed in each of the three vectors.

Figure 3

Purified JMC25406 is active at neutral pH and is moderately thermostable.A – JMC25406 has a pH optimum between 5.5 and 7.5 for both α-arabinofuranosidase and β-xylosidase activities. B – JMC25406 enzymatic activities are stable to heating up to 60°C for 20 min. C – DSF of purified JMC25406 gives an estimated T_M of 65.6°C. A representative plot from triplicate experiments is shown for purified JMC25406 and a buffer control.

Figure 4

JMC01245 and JMC37744 have broad pH optima and moderate thermal stability.A – Both JMC01245 and JMC37744 have pH optima for endoxylanase activity around 6–7, but JMC37744 maintains significant activity up to pH 10. B – JMC37744 endoxylanase activity is resistant to heating up to 60°C, while JMC01245 activity is stable up to 70°C.

Figure 5

JMC09349 has a broad pH optimum and moderate thermal stability.A – JMC09349 has a pH optimum of 6.5 and maintains significant α-fucosidase activity between pH 3–8.5. B – JMC09349 enzymatic activity is thermo-tolerant up to 50°C for 20 min. C – DSF of purified JMC09349 gives an estimated T_M of 50.3°C. A representative plot from triplicate experiments is shown for purified JMC09349 and a buffer control.