| Autor: |
Xu C; Key Laboratory of Industrial Biocatalysis, Ministry of Education, Beijing 100084, China.; Department of Chemical Engineering, Tsinghua University, Beijing 100084, China., Tang L; Key Laboratory of Industrial Biocatalysis, Ministry of Education, Beijing 100084, China.; Department of Chemical Engineering, Tsinghua University, Beijing 100084, China., Liang Y; Key Laboratory of Industrial Biocatalysis, Ministry of Education, Beijing 100084, China.; Department of Chemical Engineering, Tsinghua University, Beijing 100084, China., Jiao S; Key Laboratory of Industrial Biocatalysis, Ministry of Education, Beijing 100084, China.; Department of Chemical Engineering, Tsinghua University, Beijing 100084, China., Yu H; Key Laboratory of Industrial Biocatalysis, Ministry of Education, Beijing 100084, China.; Department of Chemical Engineering, Tsinghua University, Beijing 100084, China.; Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084, China., Luo H; Department of Biological Science and Engineering, University of Science and Technology Beijing, Beijing 100083, China. |
| Abstrakt: |
For large-scale bioproduction, thermal stability is a crucial property for most industrial enzymes. A new method to improve both the thermal stability and activity of enzymes is of great significance. In this work, the novel chaperones Rr GroEL and Rr GroES from Rhodococcus ruber , a nontypical actinomycete with high organic solvent tolerance, were evaluated and applied for thermal stability and activity enhancement of a model enzyme, nitrilase. Two expression strategies, namely, fusion expression and co-expression, were compared in two different hosts, E. coli and R. ruber . In the E. coli host, fusion expression of nitrilase with either Rr GroES or Rr GroEL significantly enhanced nitrilase thermal stability (4.8-fold and 10.6-fold, respectively) but at the expense of enzyme activity (32-47% reduction). The co-expression strategy was applied in R. ruber via either a plasmid-only or genome-plus-plasmid method. Through integration of the nitrilase gene into the R. ruber genome at the site of nitrile hydratase (NHase) gene via CRISPR/Cas9 technology and overexpression of Rr GroES or Rr GroEL with a plasmid, the engineered strains R. ruber TH3 dNHase:: Rr Nit (pNV18.1-P ami - Rr Nit-P ami - Rr GroES) and TH3 dNHase:: Rr Nit (pNV18.1-P ami - Rr Nit-P ami - Rr GroEL) were constructed and showed remarkably enhanced nitrilase activity and thermal stability. In particular, the Rr GroEL and nitrilase co-expressing mutant showed the best performance, with nitrilase activity and thermal stability 1.3- and 8.4-fold greater than that of the control TH3 (pNV18.1-P ami - Rr Nit), respectively. These findings are of great value for production of diverse chemicals using free bacterial cells as biocatalysts. |
