| Summary: | 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 <i>Rr</i>GroEL and <i>Rr</i>GroES from <i>Rhodococcus ruber</i>, 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, <i>E. coli</i> and <i>R. ruber</i>. In the <i>E. coli</i> host, fusion expression of nitrilase with either <i>Rr</i>GroES or <i>Rr</i>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 <i>R. ruber</i> via either a plasmid-only or genome-plus-plasmid method. Through integration of the nitrilase gene into the <i>R. ruber</i> genome at the site of nitrile hydratase (NHase) gene via CRISPR/Cas9 technology and overexpression of <i>Rr</i>GroES or <i>Rr</i>GroEL with a plasmid, the engineered strains <i>R. ruber</i> TH3 dNHase::<i>Rr</i>Nit (pNV18.1-P<i>ami</i>-<i>Rr</i>Nit-P<i>ami</i>-<i>Rr</i>GroES) and TH3 dNHase::<i>Rr</i>Nit (pNV18.1-P<i>ami</i>-<i>Rr</i>Nit-P<i>ami</i>-<i>Rr</i>GroEL) were constructed and showed remarkably enhanced nitrilase activity and thermal stability. In particular, the <i>Rr</i>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<i>ami</i>-<i>Rr</i>Nit), respectively. These findings are of great value for production of diverse chemicals using free bacterial cells as biocatalysts.
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