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

Chunmeng Xu,Huimin Yu,Youxiang Liang,Lingjun Tang,Hui Luo,Song Jiao

doi:10.3390/molecules25041002

Chunmeng Xu, Huimin Yu + Show 4 more

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https://doi.org/10.3390/molecules25041002

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Abstract

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 RrGroEL and RrGroES 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 RrGroES or RrGroEL 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 RrGroES or RrGroEL with a plasmid, the engineered strains R. ruber TH3 dNHase::RrNit (pNV18.1-Pami-RrNit-Pami-RrGroES) and TH3 dNHase::RrNit (pNV18.1-Pami-RrNit-Pami-RrGroEL) were constructed and showed remarkably enhanced nitrilase activity and thermal stability. In particular, the RrGroEL 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-Pami-RrNit), respectively. These findings are of great value for production of diverse chemicals using free bacterial cells as biocatalysts.

Highlights

Nitrilases are enzymes that can convert nitriles to the corresponding acid and ammonia [1,2,3,4].Nitrilases have attracted the attention of many researchers because of their benefits as catalysts, such as mild reaction conditions, environmental friendliness, high specificity and selectivity compared with traditional chemical approaches [4,5]
After urea-induction, the transcription level of RrgroES, RrgroEL, and RrgroEL2 increased by 25, 10, and 32-fold, respectively (Figure S1a); after heat shock, the transcription level of the three chaperone genes was enhanced by 100%, 100%, and 3-fold, respectively (Figure S1b) [14]
These results indicate that RrGroES and RrGroEL, together with RrGroEL2, may play dominant roles in overexpression and thermal stability of intracellular enzymes in R. ruber TH

Summary

Introduction

Nitrilases are enzymes that can convert nitriles to the corresponding acid and ammonia [1,2,3,4]. Nitrilases have attracted the attention of many researchers because of their benefits as catalysts, such as mild reaction conditions, environmental friendliness, high specificity and selectivity compared with traditional chemical approaches [4,5]. Nitrilases are important industrial enzymes due to their wide applications in production of valuable fine or chiral chemicals, such as acrylic acid, which is used to Molecules 2020, 25, 1002; doi:10.3390/molecules25041002 www.mdpi.com/journal/molecules. Better thermal stability of nitrilase is required to maintain high reaction rates [9]. The low thermal stability of nitrilase has limited its industrial application [8]

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