Carbohydrate-Active Enzyme (CAZyme) Engineering

Carbohydrate-active enzymes (or CAZymes) are a broad category of enzymes and functional proteins/domains that can synthesize, degrade, modify and/or recognize glycans and glycoconjugates (1). CAZymes like cellulases and hemicellulases are responsible for the deconstruction of cellulosic biomass into soluble sugars. Cellulases are predominantly glycosyl hydrolases (GHs) that comprise of a carbohydrate-binding module (CBM) that binds to specific substrates and a catalytic module that hydrolyzes the glycan polymer. Fungi (Trichoderma reesei) and bacteria (Clostridium thermocellum) can degrade plant biomass by secreting a complex synergistic cocktail of CAZymes (2-3) that have become cornerstone biocatalysts for cellulosic biorefineries.

We are currently interested in increasing CAZyme activity for biomass conversion to reduce cost of biofuel production. CAZyme activity can be increased by addition of synergistic enzymes (4-5), reducing non-productive protein binding to biomass (6-7), and rational/directed-evolutionary engineering approaches to increase CAZyme specific activity. We are currently funded by the NSF to engineer cellulases with reduced enzyme binding to lignin and cellulose. Also, with development of novel single-molecule (SM) techniques to characterize cellulase motility bound to cellulose surfaces (8), we are engineering and characterizing processive cellulases using complementary bulk/SM assays to better understand the rate-limiting steps for cellulose bioconversion to sugars. We also collaborate with researchers at the Great Lakes Bioenergy Research Center (9) to develop novel enzymes to hydrolyze unnatural cellulosic allomorphs.

References

  1. CAZyme database (http://www.cazy.org)
  2. Chundawat SPS, Lipton MS, Purvine SO, Uppugundla N, Gao D, Balan V, Dale BE: Proteomics based compositional analysis of complex cellulase-hemicellulase mixtures. J Proteome Res 2011, 10:4365–4372.
  3. Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS: Microbial Cellulose Utilization: Fundamentals and Biotechnology. Microbiol Mol Biol Rev 2002, 66:506–577.
  4. Gao D, Chundawat SPS, Krishnan C, Balan V, Dale BE: Mixture optimization of six core glycosyl hydrolases for maximizing saccharification of ammonia fiber expansion (AFEX) pretreated corn stover. Bioresour Technol 2010, 101:2770–2781.
  5. Chundawat SPS, Bellesia G, Uppugundla N, Sousa L, Gao D, Cheh A, Agarwal U, Bianchetti C, Phillips G, Langan P, Balan V, Gnanakaran S, Dale BE: Restructuring the crystalline cellulose hydrogen bond network enhances its depolymerization rate. J Am Chem Soc 2011, 133:11163–11174.
  6. Gao D, Chundawat SPS, Uppugundla N, Balan V, Dale BE: Binding Characteristics of Trichoderma reesei Cellulases on Untreated, Ammonia Fiber Expansion and Dilute-acid Pretreated Lignocellulosic Biomass. Biotech Bioeng 2011, 108:1788–1800.
  7. Gao D, Chundawat SPS, Sethi A, Balan V, Gnanakaran S, Dale BE: Increased enzyme binding to substrate is not necessary for more efficient cellulose hydrolysis. Proc Natl Acad Sci 2013, 110:10922–10927.
  8. Brady SK, Sreelatha S, Feng F, Chundawat SPS, Lang MJ: Cellobiohydrolase 1 from Trichoderma reesei degrades cellulose in single cellobiose steps. Nature Communications, 2015, 6, 10149.
  9. Lim S, Chundawat SPS, Fox BG: Expression, Purification and Characterization of a Functional Carbohydrate-Binding Module from Streptomyces sp. SirexAA-E. Protein Expr Purif 2014, 98:1–9.

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