Overproduction of the Escherichia coli Chaperones GroEL-GroES in Rhodococcus ruber Improves the Activity and Stability of Cell Catalysts Harboring a Nitrile Hydratase

Abstract

Three combinations of molecular chaperones from Escherichia coli (i.e., DnaK-DnaJ-GrpEGroEL- GroES, GroEL-GroES, and DnaK-DnaJ-GrpE) were overproduced in E. coli BL21, and their in vitro stabilizing effects on a nitrile hydratase (NHase) were assessed. The optimal gene combination, E. coli groEL-groES (ecgroEL-ES), was introduced into Rhodococcus ruber TH3. A novel engineered strain, R. ruber TH3G was constructed with the native NHase gene on its chromosome and the heterologous ecgroEL-ES genes in a shuttle plasmid. In R. ruber TH3G, NHase activity was enhanced 37.3% compared with the control, TH3. The in vivo stabilizing effect of ecGroEL-ES on the NHase was assessed using both acrylamide immersion and heat shock experiments. The inactivation behavior of the in vivo NHase after immersion in a solution of dynamically increased concentrations of acrylamide was particularly evident. When the acrylamide concentration was increased to 500 g/l (50%), the remaining NHase activity in TH3G was 38%, but in TH3, activity was reduced to 10%. Reactivation of the in vivo NHases after varying degrees of inactivation was further assessed. The activity of the reactivated NHase was more than 2-fold greater in TH3G than in TH3. The hydration synthesis of acrylamide catalyzed by the in vivo NHase was performed with continuous acrylonitrile feeding. The final concentration of acrylamide was 640 g/l when catalyzed by TH3G, compared with 490 g/l acrylamide by TH3. This study is the first to show that the chaperones ecGroEL-ES work well in Rhodococcus and simultaneously possess protein-folding assistance functions and the ability to stabilize and reactivate the native NHases.

The less conserved metal-binding site in human CRISP1 remains sensitive to zinc ions to permit protein oligomerization

“…The human CRISP1 constructs that were transformed into different E. coli strains are summarized in Supplementary Figure 2A. The E. coli pG-KJE8/ BL21 strain possesses two chaperone systems that enhance the solubility of proteins [29][30][31] and the Origami 2(DE3) pLysS strain improves disulfide bond formation in the cytoplasm of E. coli 32 . Initially, the human CRISP1 coding sequence was optimized according to the codon bias of E.coli 33 and fused with the N-terminal His-tag.…”

Recent Advances and Promises in Nitrile Hydratase: From Mechanism to Industrial Applications

“…To overcome the obstacle that there might be a low expression level of heterologous protein, as well as to avoid the formation of inactive and insoluble protein, tremendous efforts have been made including ribosome binding site engineering, molecular chaperone co-expression, and tag fusion strategy. Tian et al (2016) introduced a shuttle plasmid which carries ecgroEL-ES, a molecular chaperone gene, to R. ruber TH3G strain hosting an NHase gene. The chaperones assist protein-folding and even reactivate the native NHases (Tian et al, 2016).…”

“…Tian et al (2016) introduced a shuttle plasmid which carries ecgroEL-ES, a molecular chaperone gene, to R. ruber TH3G strain hosting an NHase gene. The chaperones assist protein-folding and even reactivate the native NHases (Tian et al, 2016). Moreover, in E. coli, the soluble expression level of recombinant NHase could be improved with the help of molecular chaperones and thus low inducing temperature was avoided.…”

Novel Chaperones RrGroEL and RrGroES for Activity and Stability Enhancement of Nitrilase in Escherichia coli and Rhodococcus ruber

“…For example, with EcGroEL-EcGroES overexpression, the stability of Cryj2 significantly increased compared with the wild-type protein [16]. The activity of nitrile hydratase (NHase) remained at 229 U/L with EcGroEL-EcGroES protection, while that of the control enzyme without chaperones dropped to zero after heat shock at 50 • C for 10 min [17]. Besides, overexpression of EcGroEL-EcGroES in R. ruber could stabilize NHase after heat shock [17].…”