Engineering All-Round Cellulase for Bioethanol Production

ACS Synthetic Biology, vol. 12, pp. 2187–2197

Abstract

One strategy to decrease both the consumption of crude oil and environmental damage is through the production of bioethanol from biomass. Cellulolytic enzyme stability and enzymatic hydrolysis play important roles in the bioethanol process. However, the gradually increased ethanol concentration often reduces enzyme activity and leads to inactivation, thereby limiting the final ethanol yield. Herein, we employed an optimized Two-Gene Recombination Process (2GenReP) approach to evolve the exemplary cellulase CBHI for practical bioethanol fermentation. Two all-round CBHI variants (named as R2 and R4) were obtained with simultaneously improved ethanol resistance, organic solvent inhibitor tolerance, and enzymolysis stability in simultaneous saccharification and fermentation (SSF). Notably, CBHI R4 had a 7.0- to 34.5-fold enhanced catalytic efficiency ( k cat / K M ) in the presence/absence of ethanol. Employing the evolved CBHI R2 and R4 in the 1G bioethanol process resulted in up to 10.27% (6.7 g/L) improved ethanol yield (ethanol concentration) than non-cellulase, which was far more beyond than other optimization strategies. Besides bioenergy fields, this transferable protein engineering routine holds the potential to generate all-round enzymes that meet the requirement in biotransformation and bioenergy fields.

Authors 8

1. Minghui Wang

   Nanjing Normal University

   Affiliation as printed

   School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, No. 2 Xuelin Road, Nanjing 210097, China

2. Haiyang Cui Aachen Institute of Biotechnology

   RWTH Aachen University

   Affiliation as printed

   Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany

3. Anni Li

   Nanjing Normal University

   Affiliation as printed

   School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, No. 2 Xuelin Road, Nanjing 210097, China

4. Ulrich Schwaneberg Aachen DWI - Leibniz Institute for Interactive Materials Institute of Biotechnology

   DWI – Leibniz Institute for Interactive Materials · RWTH Aachen University

   Affiliation as printed

   DWI-Leibniz Institute for Interactive Materials, Forckenbeckstraße 50, 52074 Aachen, Germany

   Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany

5. Lihui Zhang

   Nanjing Normal University

   Affiliation as printed

   School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, No. 2 Xuelin Road, Nanjing 210097, China

6. Junnan Wei corresponding

   Nanjing Normal University

   Affiliation as printed

   School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, No. 2 Xuelin Road, Nanjing 210097, China

7. Xiujuan Li corresponding

   Nanjing Normal University

   Affiliation as printed

   School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, No. 2 Xuelin Road, Nanjing 210097, China

8. He Huang

   Nanjing Tech University · Nanjing Normal University

   Affiliation as printed

   School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, No. 2 Xuelin Road, Nanjing 210097, China

   School of Pharmaceutical Sciences, Nanjing Tech University, No. 30 South Puzhu Road, Nanjing 211816, China