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Cel7A Engineering and Expression

  • Author(s): Dana, Craig Matthew
  • Advisor(s): Clark, Douglas S
  • Blanch, Harvey W
  • et al.
Abstract

Renewable fuels produced from biomass-derived sugars are receiving increasing attention. Lignocellulose-degrading enzymes derived from fungi are attractive for saccharification of biomass because they can be produced at higher titers and at significantly less cost than those produced by bacteria or archaea. However, their properties can be suboptimal; for example, they are subject to product inhibition and are sensitive to small changes in pH. Furthermore, increased thermostability would be advantageous for saccharification as increased temperature may reduce the risk of microbial contamination. Therefore, there is a need for a generalized platform that can be applied to the engineering of these enzymes. Commercially available lignocellulose-degrading enzymes are produced using a hypersecreting strain of the filamentous fungus Trichoderma reesei. Among the enzymes secreted by this organism, the cellulase Cel7A is present in the highest concentration and is the only enzyme responsible for non-reducing end directed exo-acting cellulolytic activity. Additionally, the enzyme's presence is critical for growth of T. reesei on cellulosic substrates. Here, a general mutagenesis platform that employed the budding yeast Saccharomyces cerevisiae was developed to improve the properties of Cel7A. Secretion of Cel7A at titers of 26 mg/L with limited hyperglycosylation was achieved using an S. cerevisiae strain with upregulated protein disulfide isomerase, an engineered α-factor prepro leader, and the deletion of a plasma membrane ATPase. Because cellulase activities are difficult to screen in high-throughput, a DNA shuffling based library generation technique that results in a high percentage of active clones was developed called Biased Clique Shuffling (BCS). BCS allows for the control of DNA diversity during library generation. Applying this technique to 11 homologous Cel7A genes, we generated several libraries that were rich in activity and identified chimeras with improved thermostability, thermal activity, and product inhibition. The libraries generated using the BCS technique were far superior as a source of active and stable chimeras compared to an equimolar library prepared from the same 11 genes (as is classically prepared using DNA shuffling). Finally, we found that Cel7A expressed in the filamentous fungus N. crassa had twice the specific activity at 65°C and a 10°C higher Tm relative to Cel7A expressed in S. cerevisiae. Through a study of the three known post-translational modifications, namely glycosylation, disulfide bond formation, and N-terminal glutamine cyclization, we revealed that S. cerevisiae expressed Cel7A with an unmodified N-terminus, unlike native Cel7A which has an N-terminal pyroglutamate. Furthermore cyclizing the unmodified N-terminal glutamine in the Cel7A expressed in S. cerevisiae to form pryoglutamate in-vitro with glutaminyl cyclase increased the enzyme's specific activity and thermostability to match those Cel7A expressed in N. crassa. This unprecedented result demonstrates the importance of the hydrophobic pyroglutamate in the N-terminal position of Cel7A.

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