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-GrpE-GroEL-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.

Introduction

With the development of biotechnology, it is common to engineer bacterial strains to produce bioproducts, including pharmaceutical intermediates [25,31] and bulk/fine chemicals [4]. Acrylamide (AM) is a key chemical in many industries, particularly for the production of its polymer product, polyacrylamide (PAM). PAM is of great importance in the treatment of sewage and enhanced oil recovery. PAM can also function as an additive in many chemical processes, such as a coagulator or soil conditioner. Therefore, the production of AM has been a central topic of interest for both chemists and biologists, and diverse approaches, including Cu catalysis and enzyme catalysis, have been developed [20,22,26]. Recently, the enzymatic synthesis of AM, the core step of which is the acrylonitrile hydration conducted by the biocatalyst nitrile hydratase (NHase), has received increasing attention because of its high productivity, mild reaction conditions, and environmentally friendly characteristics [23]. Some strains of Rhodococcus [14], Pseudomonas [33], Nocardia [28], and Bacillus [13] containing nitrile hydratases have already been well studied and successfully applied in the industrial production of acrylamide. Comparing all these candidate strains, Rhodococcus species are quite outstanding, and many strains have been reported to be superior cellular catalysts for the hydration synthesis of acrylamide from acrylonitrile, such as Rhodococcus sp. N774 [10], Rhodococcus rhodochrous J-1 [21,35], Rhodococcus sp. M8 [1], and Rhodococcus ruber TH [18]. However, all these strains share a common problem: NHase is quite inactivation-prone during the strong exothermic hydration reaction and is substantially inhibited by its product (AM). Therefore, enhanced thermal stability and acrylamide tolerance for in vivo NHases are urgently required. Various efforts have attempted to improve the resistance of nitrile hydratases to thermal stress or acrylamide, such as sigma factor mutation and active plasmid partitioning [19], the construction of an amidase-negative engineered strain [18], and the introduction of structural salt bridges [6].

Chaperones are a large family of proteins that aid in protein folding in organisms. These proteins are classified into different families according to their molecular weights, such as the heat shock protein (HSP) 90 family and the HSP 60 family. Recently, chaperones have been discovered to be highly relevant for certain focused medical issues. For example, some proteins in the HSP 90 family were found to be vital for cancer detection and treatment [32]. Among all the chaperones, GroEL from Escherichia coli, a member of the HSP 60 family, is probably the best-characterized example. The molecular structures of GroEL and its cochaperone GroES have been clearly delineated, and the formation of the GroEL-GroES complex with ATP is also well characterized [17]. To date, many teams have succeeded in stabilizing different types of enzymes in vitro by adopting strategies relating to molecular chaperones in E. coli. For example, the E. coli chaperone systems can reactivate heat-treated RNA polymerase [36]. It has also been reported that co-expression with genes of GroEL-GroES significantly enhances soluble and functional production of recombinant human interferon-gamma [34]. One study designed an enzyme-chaperone chimera as a new approach to stabilize enzymes [3]. Given such successful examples, some researchers have attempted to use chaperones to stabilize NHase in vitro or to increase NHase activity in E. coli [24,29,30].

For the first time, based on the in vitro verification of the optimal molecular chaperones from E. coli (GroEL-GroES) for stabilizing NHase, we demonstrate a new method of stabilizing NHases in vivo in Rhodococcus ruber TH3, by heterologously overexpressing E. coli groEL-groES. Moreover, the resulting engineered R. ruber strain exhibited superior performance with respect to acrylonitrile hydration and high-titer AM biosynthesis.

 

Materials and Methods

Strains and Plasmids

The bacterial strains and plasmids used in this study are listed in Table 1. R. ruber TH3 is an amiE-negative mutant constructed by knocking out the amidase gene (which converts acrylamide to the byproduct acrylic acid) in R. ruber TH [18]. E. coli TOP10 was used as a host for gene cloning. E. coli BL21(DE3) [pET-NHM] was used for the preparation of in vitro NHase [28]. E. coli BL21 was the host of three plasmids (pG-KJE8, pGro7, and pKJE7) expressing different E. coli chaperones. Antibiotics were supplemented as needed at the following concentrations: ampicillin, 100 μg/ml; tetracycline, 13.5 μg/ml; and kanamycin, 30 μg/ml. Plasmid and genomic DNA isolation, agarose gel electrophoresis, restriction enzyme digestion, DNA ligation, DNA transformation, and chaperone expression were performed using standard procedures [28] or following the manufacturer’s protocols. Restriction enzymes and Taq DNA polymerase were purchased from Takara (Dalian, China) and New England BioLabs (Beijing, China). PCR purification kits and Biospin gel extraction kits were purchased from Stratagene (San Jose, CA, USA) and Bioer Technology (Hangzhou, China), respectively.

Table 1.Bacterial strains and plasmids used in this study.

Construction of R. ruber TH3G

The full-length Pa2 promoter [16] and the groEL-groES genes were PCR amplified from plasmid pGro7. The primers Pa2-sense-HindIII (5’-CCCAAGCTTTGCGGAGGCGGATAC-3’) and Pa2-anti-XbaI (5’-GCTCTAGACTCCTTAGTGACTCGCC-3’) were used to amplify the Pa2 promoter, and the primers Hsp60-sense-XbaI (5’-GCTCTAGAATGAATATTCGTCCATTGCATGAT-3’) and Hsp60-anti-EcoRI (5’-CGGAATTCTTACATCATGCCGCCCATG-3’) were used to amplify the groEL-groES chaperone genes. The PCR conditions included an initial 10 min denaturing step at 95℃; 30 cycles of 95℃ for 30 sec, 58℃ for 30 sec, and 72℃ for 1 min; and a final 10 min extension at 72℃. The PCR products were then ligated into the pNV18.1 shuttle vector [8]. The presence of the correct insert was verified using colony PCR. Subsequent gene sequencing was performed by the SinoCompany. Sequence assembly was performed using DNAMAN and the Basic Local Alignment Search Tool (BLAST) software on the NCBI Website [10].

Cell Culture and Nitrile Hydratase Activity Assay Using Gas Chromatography (GC)

R. ruber TH3 and TH3G were grown at 28℃ in a medium containing 20 g of glucose, 1 g of yeast extract, 7 g of tryptone, 0.5 g of K2HPO4, 0.5 g of KH2PO4, and 0.5 g of MgSO4·7H2O per liter.

E. coli TOP10, E. coli BL21 [pG-KJE8], E. coli BL21 [pGro7], and E. coli BL21 [pKJE7] were routinely grown at 37℃ in Luria-Bertani (LB) medium [27].

The enzyme activity of both in vivo and isolated NHases was measured according to the activity assay described by Ma et al. [18]. Isolated NHases were obtained from E. coli cells by ultrasonication on ice for 30 min (5 sec of ultrasonication followed by a 5 sec pause) at 143 W (in a 950 W Scientz-D ultrasonic cell pulverizer; NingBo Scientz Biotechnology Co., Ltd., China) and centrifugation at 12,000 ×g for 10 min at 4℃. The NHase activity was assayed at 28℃ in a 5 ml reaction mixture containing 100 μl of acrylonitrile and 0.25 g/l free cells or isolated enzymes in 50 mM PBS buffer. After 5 min, the reaction was terminated by the addition of 200 μl of 2.5 M HCl. After a 10 min centrifugation at 12,000 ×g, the supernatant was analyzed using GC as previously described [18].

One unit of NHase activity (U) was defined as the amount of enzyme required to catalyze the formation of 1 μmol acrylamide per minute. The starting nitrile hydratase activity before the addition of acrylamide was defined as 100%.

Thermal Stability Evaluation of NHase

Free cells of R. ruber TH3 and the engineered strain TH3G were centrifuged (12,000 ×g, 3 min) and resuspended in deionized water to OD460nm = 1.2. Then, the free cells were heat shocked at 55℃ for 10 min, followed by a 30 min recovery at 28℃. The enzyme activity of the free cells after such treatment was assayed as described above.

NHase Acrylamide Tolerance Evaluation in R. ruber

Free cells of R. ruber TH3 and the engineered strain TH3G were centrifuged (12,000 ×g, 3 min) and resuspended in deionized water to OD460nm = 1.2. Then, 500 μl of the cells was added to 9.5 ml of a 30% acrylamide solution (mass fraction) to obtain a final volume of 10 ml. After a 30 min immersion, the cells were immediately centrifuged (12,000 ×g, 3 min) and washed twice with deionized water to remove any acrylamide in the liquid. After another 30 min recovery, NHase enzyme activity was measured immediately.

The tolerance of the cells to dynamically enhanced concentrations of acrylamide was assayed as follows: First, a 60% (mass fraction) acrylamide solution was prepared. Then, 20 ml of free R. ruber TH3 cells or engineered TH3G cells (at OD460nm = 1.2) was added to a conical flask subjected to electromagnetic stirring. The 60% AM solution was pumped into the flask via an injection pump at a constant speed of 1.7 ml/min. Samples were removed every 10 min for immediate NHase activity measurement.

Laboratory-Scale Acrylonitrile Hydration and Acrylamide Biosynthesis

Laboratory-scale acrylonitrile hydration was performed in a reaction mixture containing cell catalysts with 50.3 U/ml NHase activity in 100 ml of deionized water (pH 7.0) in a 300 ml conical flask. Agitation was set through the neck. Substrate acrylonitrile was added at a constant rate of 1 ml/min by an injection pump and was immediately transformed into acrylamide via NHase catalysis in the cells. The reaction temperature was measured with a thermometer and maintained at 10-18℃ in an ice-water bath. The catalytic ability of the cells was reduced as the concentration of the AM product increased. When the NHase in the cells was completely inactivated and substrate acrylonitrile accumulated, the reaction was stopped, and the final concentration of AM was measured using GC, as described in the activity assay section above, after the removal of cells by centrifugation. If the AM concentration was very high to generate crystals at the reaction temperature, the solution was heated to dissolve the crystals before loading for GC analysis.

 

Results and Discussion

Preparation and Self-Thermal Stability of Different Molecular Chaperones

As shown in Fig. 1A, three combinations of different E. coli molecular chaperones, namely, DnaK-DnaJ-GrpEGroEL-GroES (the HSP 70 and 60 families), GroEL-GroES (the HSP 60 family), and DnaK-DnaJ-GrpE (the HSP 70 family), were overproduced in E. coli BL21 [pG-KJE8], BL21 [pGro7], and BL21 [pKJE7], respectively. SDS-PAGE results confirmed the successful overproduction after induction, as illustrated in Fig. 1B, in which chaperones DnaK, DnaJ, GrpE, GroEL, and GroES are separately indicated with arrows.

Fig. 1.Expression of different chaperone combinations in engineered E. coli strains and an in vitro evaluation of their self-thermal stability. (A) Maps of the three strains expressing different chaperone combinations. (B) SDS-PAGE analysis of the total cellular proteins of E. coli BL21 [pG-KJE8], BL21 [pGro7], and BL21 [pKJE7]. Lane M, molecular mass markers; lane 1, a blank control of BL21 [pG-KJE8] without induction; lane 2, total cellular protein of BL21 [pG-KJE8] induced with 4 mg/ml L-arabinose and 10 ng/ml tetracycline for 8 h; lanes 3-4, total cellular proteins of BL21 [pGro7] and BL21 [pKJE7], respectively, induced with 4 mg/ml L-arabinose for 8 h. The arrows indicate protein bands sized 70, 57, 40, 22, and 10 kDa that correspond to DnaK, GroEL, DnaJ, GrpE, and GroES, respectively. (C), SDS-PAGE results of the self-thermal stability of the chaperones DnaK, GroEL, DnaJ, GrpE, and GroES in cell lysates after being incubated at 25℃, 50℃, 55℃, or 60℃ for 10 min.

The in vitro self-thermal stability of each chaperone in the cell lysate was assessed by raising the temperature from 25℃ to 50℃, 55℃, or 60℃ for 10 min. The SDS-PAGE results at different temperatures are summarized in Fig. 1C. The chaperones GroEL, GroES, DnaK, and GrpE maintained thermal stability up to 60℃, but the chaperone DnaJ began to precipitate at 55℃. Therefore, subsequent experiments on the protection effect of chaperones on NHase in vitro were performed at 50℃.

Stabilizing Effect of Different Chaperone Combinations on NHase In Vitro

The overproduction of NHase was achieved using the engineered E. coli strain BL21(DE3) [pET-NHM]. The NHase contained two subunits of molecular mass 26 kDa (α-subunit) and 29 kDa (β-subunit), as shown in Fig. 2A.

Fig. 2.In vitro stabilization of NHase by different molecular chaperone combinations. (A) SDS-PAGE of total cellular proteins of E. coli BL21(DE3) [pET-NHM]. Lane M, molecular weight markers; lane 1, overexpressed NHase with two subunits in the cell lysate of E. coli BL21(DE3) [pET-NHM]. Arrows indicate the α-subunit and β-subunit of NHase. (B) The GC-quantified activity of NHase after heat shock (10 min, 50℃) when mixed with different chaperones. C0, original NHase activity before heat shock; C1, control NHase activity after heat shock without the addition of chaperones; LSKJE, chaperone combination GroEL-GroES-DnaK-DnaJ-GrpE; LS, chaperone combination GroEL-GroES; KJE, chaperone combination DnaK-DnaJ-GrpE. The experiments were performed in triplicates.

Cell lysates containing NHase were aliquoted and mixed with equal amounts of the chaperone combinations GroEL-GroES-DnaK-DnaJ-GrpE, GroEL-GroES, or DnaK-DnaJ-GrpE. After a 10 min heat shock at 50℃, soluble proteins remaining in the supernatant after centrifugation were analyzed via SDS-PAGE and GC. According to the SDS-PAGE results, almost all NHase in the control mixture (cell lysate without chaperones) was fully denatured, and the two bands corresponding to the two subunits nearly disappeared. However, by mixing the cell lysate with the molecular chaperones, NHase remained partially active after heat shock, and the NHase bands of the samples mixed with chaperones were still visible. Similarly, the GC quantification results indicated that the remaining activity of the control NHase mixture without chaperones dropped directly to zero. However, for the sample protected by GroEL-GroES, the NHase activity remained at 229 U/l after heat shock, which was higher than that displayed by NHase protected by the other chaperone combinations (GroEL-GroES-DnaK-DnaJ-GrpE and DnaK-DnaJ-GrpE), as shown in Fig. 2B. Therefore, the chaperones GroEL-GroES exhibited a superior stabilizing effect on NHase with respect to thermal stress resistance.

Enhancement of NHase Activity by E. coli GroEL-GroES in R. ruber TH3

By cloning the genes of E. coli GroEL-GroES (ecGroEL-ES) from plasmid pGro7 and inserting them into the E. coli-Rhodococcus shuttle plasmid pNV18.1, the recombinant plasmid pNV18-groEL-ES was obtained, in which a mutant Pa2 promoter was used to transcribe the exogenous genes (as shown in Fig. 3A). After electroporation of this plasmid into R. ruber TH3, the resulting engineered strain R. ruber TH3 [pNV18-groEL-ES] was obtained and the name simplified as TH3G. It contained the native gene of NHase in its chromosome and the heterologous genes of E. coli chaperones ecGroEL-ES on the plasmid, as illustrated in Fig. 3B.

Fig. 3.Construction of R. ruber TH3G and the overproduction of NHase and ecGroEL-ES. (A) Map of shuttle plasmid pNV-GroEL-ES expressing ecgroEL-ES with the Pa2 promoter. (B) Map of R. ruber TH3G containing the NHase gene in its chromosome and the heterologous chaperones ecgroEL-ES gene in a plasmid. (C) SDS-PAGE confirmation of the overexpression of the chaperone genes ecgroEL-ES in R. ruber TH3G. Lane M, protein size marker; lane 1, whole-cell electrophoresis of the control strain R. ruber TH3; lane 2, whole-cell electrophoresis of the engineered strain R. ruber TH3G. The dashed grey arrow pointed to an unknown protein, whose production level was increased by the chaperones GroEL-ES. (D) NHase activity as measured using GC. The experiments were performed in triplicates.

Parallel cell cultures of R. ruber TH3 and TH3G were performed in shake flasks for the overproduction of NHase and the chaperones ecGroEL-ES. Protein production was monitored using SDS-PAGE. In the TH3 control, NHase was natively produced, and the two bands corresponding to the β and α subunits were remarkably strong, as shown in lane 1 of Fig. 3C. In the engineered TH3G strain, the chaperones GroEL and GroES were successfully produced besides NHase; and also, overexpression of the heterologous ecgroEL-ES genes raised the level of some unknown native proteins, such as the one between the β- and α-NHases (lane 2 of Fig. 3C). The GC quantification results indicate that the activity of NHase in the engineered TH3G strain was 4,342 U/l, which was 37.3% higher than in the control TH3 (as shown in Fig. 3D).

In Vivo Stabilizing Effect of ecGroEL-ES on NHase in R. ruber TH3

The stabilizing effect of ecGroEL-ES on NHase in R. ruber TH3 was assessed using both acrylamide immersion and heat shock experiments. When TH3 and TH3G cells were simultaneously immersed in a 300 g/l acrylamide solution for 30 min, the loss of NHase activity was measured and compared, as shown in Fig. 4. Approximately 81.5% of NHase activity remained in the cells of TH3G, but this value dropped to 57.6% in TH3, which lacks the overproduced chaperones (Fig. 4A). Similarly, after a 10 min heat shock at 55℃, 69.4% of NHase activity remained in TH3G, but this value dropped to 61.3% in TH3 (Fig. 4B).

Fig. 4.In vivo stabilizing effect assessments of ecGroEL-ES on NHase in R. ruber TH3. (A) Tolerance of NHase to 300 g/l acrylamide (30%) for 30 min. (B) Thermal stability of NHase after a 10 min heat shock at 55℃. NHase activity was quantified using GC. The experiments were performed in triplicates. (C) Deactivation curve of NHase in cells of R. ruber TH3G and TH3 with or without chaperones ecGroEL-ES, respectively, when immersed in dynamically increased concentrations of AM. The mass fraction of the acrylamide in the solution increased from 0 to 50% (500 g/l) within 60 min. The experiments were performed in triplicates. Arrows pointed to the left-side or right-side vertical coordinate that the curve used.

To further mimic the actual acrylamide accumulation process during the hydration reaction, the inactivation behavior of in vivo NHase immersed in a solution with dynamically increasing concentrations of acrylamide was carefully evaluated, as shown in Fig. 4C.

In TH3G, the initial NHase activity loss was only 24%, but in TH3, the activity loss (50%) was significantly greater under the same conditions (0-10 min). Finally, when the AM concentration was increased to 500 g/l, the remaining NHase activity in TH3G was still 38%, which was 28% higher than that in TH3.

We further harvested the in vivo NHases under varying degrees of inactivation by AM immersion or heat shock and re-immersed them in deionized water with or without the presence of the chaperones ecGroEL-ES to assess their reactivation characteristics with respect to NHase activity after 1 h. The results are illustrated in Fig. 5. At first, the NHase activity remaining in TH3G after each treatment was higher than that of TH3. Furthermore, the reactivated NHase activity in TH3G with overproduced ecGroEL-ES was more than 2-fold greater than that in TH3, indicating that the chaperones ecGroEL-ES helped to stabilize and reactivate NHase in Rhodococcus.

Fig. 5.Activity reactivation assessment of in vivo NHase with or without the chaperones ecGroEL-ES. Two different batches of assessments were performed in which the deactivation of NHase was caused by acrylamide immersion (30% AM, 20 min) or heat shock (60℃, 10 min). The reactivation of NHase was performed by water immersion at 28℃ for 60 min. After each inactivation and reactivation treatment, the cells were washed twice with water and harvested by centrifugation, and the resulting NHase activity was measured using GC. E0 indicates the initial NHase activity before inactivation. E1 and E3 indicate the NHase activity remaining after acrylamide immersion and heat shock, respectively. E2 and E4 indicate the reactivated NHase activity after water immersion corresponding with E1 and E3, respectively. The experiments were repeated two times.

Hydration Synthesis of AM Catalyzed by Engineered R. ruber TH3G

The laboratory-scale hydration synthesis of AM was performed as shown in Fig. 6A. The substrate, acrylonitrile, was continuously fed into a cell solution to initiate the hydration reaction. After 1 h, the reaction was stopped, and abundant acrylamide crystals were observed in the flask catalyzed by TH3G (Fig. 6B). However, for TH3, only a small amount of crystals were observed in the flask (Fig. 6C). Because high concentrations of AM are oversaturated at low temperatures (0-18℃), larger amounts of acrylamide crystals indicate higher concentrations of the acrylamide product. The final AM concentration was measured using GC after the heating and solubilization of all crystals, and those results are summarized in Fig. 6D. For the engineered TH3G strain, the final concentration of AM achieved was as high as 640 g/l (64%), which was markedly greater than that of the TH3 control (490 g/l).

Fig. 6.Hydration reaction of acrylonitrile to acrylamide catalyzed by the engineered R. ruber TH3G strain versus the control, TH3. (A) The experimental setup for the hydration reaction was assembled with a substrate feeding pump, a reactor, and a water bath. A total of 120 ml of acrylonitrile was continuously pumped into an aqueous solution of R. ruber cells (100 ml) in the reactor at a rate of 2 ml/min. (B) Photograph of the hydration reaction solution catalyzed by R. ruber TH3G strain with a large amount of acrylamide crystals. (C) Photograph of the hydration reaction solution catalyzed by R. ruber TH3 strain. (D) The final AM product concentrations after the hydration reactions.

Acrylamide is an important chemical with both scientific research and industrial applications. Compared with the sulfuric acid or copper catalysts previously used for AM synthesis, microbial cells containing NHases as biocatalysts have several advantages. For example, the reaction is performed at ambient temperature and pressure, the rate of bioconversion from substrate to product is quite rapid, and the reaction process is more environmentally friendly. Because of the significant costs of the condensing and purifying processes, a high concentration of the acrylamide product is the objective of bioconversion. Therefore, the activity and stability of overexpressed NHases in Rhodococcus cells are always of great importance for the development of AM bioproduction techniques, with a particular emphasis on acrylamide tolerance and thermal resistance.

Diverse investigations have been performed to enhance the activity and stability of in vivo NHases, including both NHase modification and transcriptional regulation in cells. For example, to improve NHase activity, Shi et al. introduced an atg mutation into the start codon of the NHase α-subunit [28]. To improve the stability of NHase, Chen et al. introduced salt-bridges into a sensitive region of a thermophilic, industrial NHase [7] together with additional site-directed mutagenesis [6]. Ma et al. regulated the transcription spectra of thousands of genes by mutating the sigma factor of Rhodococcus RNA polymerase [19].

It is well acknowledged that under extreme conditions, such as high temperature, condensed salt solutions, and high or low pH levels, a sudden leap in chaperone expression can be observed [9]. Therefore, many studies have focused on the mechanisms of molecular chaperone interactions with other proteins, including human interferon-gamma [34], D-phenylglycine aminotransferase [11], and mammalian mitochondrial malate dehydrogenase. To summarize, two accepted functions of molecular chaperones in a living cell are to help properly fold other functional proteins [17,24,34] and to stabilize, and even reactivate, proteins that have become denatured [3,36]. In light of these functions, it was expected that we could obtain both enhanced native enzyme activity and stability by overproducing molecular chaperones. Some researchers have also highlighted the use of heterologous chaperones cloned from thermophilic bacteria, such as Pyrococcus furiosus [5,15], Methanococcus jannaschii [12], and Synechocystis [2], and their overexpression in E. coli via different approaches, such as co-expression or fusion with the target enzymes.

However, so far, no report has detailed the effects of E. coli chaperones on industrial enzymes natively produced in the cells of Rhodococcus strains. Using the NHase producer and model strain R. ruber TH3 as a cellular catalyst for AM production, we cloned and overproduced the superior E. coli chaperones ecGroEL-ES in Rhodococcus cells via a shuttle plasmid. We specifically focused on changes to the activity and stability of natively expressed NHase in R. ruber TH3. Compared with the control, NHase activity increased 37% in R. ruber overexpressing ecgroEL-ES, which confirmed that the protein-folding function of ecGroEL-ES works well in Rhodococcus. The stabilizing effect of ecGroEL-ES on NHase in the cells of R. ruber TH3 was assessed in terms of both thermal tolerance and acrylamide tolerance. The results indicate that with overproduction of ecGroEL-ES, NHase tolerance to harsh stress, particularly high concentrations of acrylamide, was increased 23%, proving again that ecGroEL-ES exerts a strong stabilizing effect on NHase in R. ruber cells. More interestingly, we further confirmed that NHase deactivated by acrylamide immersion can be markedly reactivated with the assistance of ecGroEL-E, which indicated that the reactivation function of ecGroEL-ES toward denatured NHase also works well in Rhodococcus. Using the engineered TH3G strain as a cellular catalyst for AM production, final acrylamide concentrations reached as high as 640 g/l within 1 h by feeding the substrate acrylonitrile into a small hydration reactor.

This work highlights a new but efficient method of improving both the activity and stability of one or more natively expressed enzymes in uncommon cellular catalysts such as Rhodococcus or other actinomycetes. The result is of great value for the development of cellular catalysts and their subsequent industrial applications.