Development of highly efficient whole-cell catalysts of cis-epoxysuccinic acid hydrolase by surface display – Bioresources and Bioprocessing

Construction of CESH[L] surface display systems on E. coli cells

To obtain an anchoring motif that is suitable for CESH[L] surface display on E. coli cells, five anchoring motifs including three endogenous motifs (LOA, MipAV₁₄₀, and YiaTR₂₃₂) and two heterogenous INP truncation motifs (InaKN and InaPbN) were chosen to construct the surface display systems. The gene of each anchoring motif and the gene of CESH[L] with codon-optimization for E. coli were sequentially cloned into pET vectors for protein expression. Considering the internal restriction sites, the gene of the anchoring motif for LOA, MipAV₁₄₀, YiaTR₂₃₂, and InaKN was first inserted into the NdeI/NcoI sites of pET22b, and the gene of CESH[L] was then inserted into the NcoI/XhoI sites, while the genes of the anchoring motif InaPbN and CESH[L] were inserted into NcoI/BamHI sites and BamHI/HindIII sites of pET28a sequentially. The five recombinant plasmids were obtained and each plasmid was transformed into E. coli BL21(DE3), forming five CESH[L] surface display systems. Using these systems, the fusion proteins would be displayed on the cell surface after the protein expression is induced by the addition of IPTG.

Determination of surface display efficiency of the five display systems

The surface display efficiency of the five display systems was first assessed by trypsin accessibility assays (Fig. 1a). Trypsin cleaves proteins on the C-terminal side of lysine and arginine residues, so proteins attached to the cell surface with extracellular exposure can be digested by trypsin while the intracellular proteins are inaccessible by trypsin. The trypsin accessibility can be easily determined by SDS-PAGE analysis of trypsin-treated and untreated whole cells, which provides the information of the target protein locations (Maurer et al. 1997). To evaluate whether trypsin accessibility assay is effective for CESH[L], whole cells and cell lysate of the CESH[L] intracellular expression system (Cui et al. 2012) were treated with trypsin. SDS-PAGE analysis showed the same intact CESH[L] bands for the trypsin-untreated and treated cells, but the CESH[L] band of the cell lysate after trypsin treatment almost disappeared (lanes 1–3 in Fig. 1a). This demonstrated that CESH[L] can be digested by trypsin, but trypsin cannot pass through the cell membranes. Therefore, trypsin accessibility assay can be used to ascertain the surface localization of the expressed CESH[L].

CESH[L] surface display efficiencies of different anchoring motifs. a SDS-PAGE analysis of the whole cells or the cell lysate with and without trypsin treatment. Lanes 1–2, whole cells of intracellular CESH[L] expression system without and with trypsin treatment; Lane 3, trypsin-treated cell lysate of the intracellular CESH[L] expression system. Lanes 4–13, each pair of lanes are trypsin-untreated and treated whole cells expressing LOA-CESH[L], MipAV₁₄₀-CESH[L], YiaTR₂₃₂-CESH[L], InaKN-CESH[L], and InaPbN-CESH[L], respectively. Lanes M, standard protein molecular weight markers. b Whole-cell activities of various CESH[L] expression systems incubated at 4 °C for different days. c Comparison of the surface display systems and intracellular expression system. Intra, LOA, MipA, YiaT, InaKN, and InaPbN represent the cells expressing intracellular CESH[L], LOA-CESH[L], MipAV₁₄₀-CESH[L], YiaTR₂₃₂-CESH[L], InaKN-CESH[L], and InaPbN-CESH[L], respectively. Intra-L, Intra-T, and Intra-T-S represent the whole-cell lysate, the Triton X-100 treated cells, and the supernatant after the Triton X-100 treatment, respectively, of the intracellular expression system. The activities of all surface display systems were measured after 2-day incubation at 4 °C, while the activities of the intracellular expression system were measured without incubation. Symbols ** indicate significant differences with p-values less than 0.01

SDS-PAGE analysis of trypsin-treated and untreated whole cells expressing fusion proteins LOA-CESH[L], MipAV₁₄₀-CESH[L], YiaTR₂₃₂-CESH[L], InaKN-CESH[L], and InaPbN-CESH[L] showed that the protein expression levels and the cell surface display levels were different in these systems (Fig. 1a). Trypsin treatment did not digest the LOA-CESH[L] band (lanes 4–5 in Fig. 1a), suggesting a failed display of LOA-CESH[L] in this system. The bands of MipAV₁₄₀-CESH[L], InaKN-CESH[L], and InaPbN-CESH[L] in the trypsin-treated cells were appreciably thinner than those in the untreated cells (lanes 6–7, 10–11, and 12–13 in Fig. 1a). The band of YiaTR₂₃₂-CESH[L] in trypsin-treated cells was completely disappeared, but the amount of expressed YiaTR₂₃₂-CESH[L] protein was much less than the expressed fusion proteins in other systems (lanes 8–9 in Fig. 1a). These results indicated that the fusion proteins MipAV₁₄₀-CESH[L], YiaTR₂₃₂-CESH[L], InaKN-CESH[L], and InaPbN-CESH[L] were successfully displayed onto the E. coli cell surface with varying degrees.

After confirming that CESH[L] can be displayed on the E. coli cell surface, the whole-cell CESH[L] activity of each cell surface display system was examined (Fig. 1b). Surprisingly, the whole-cell activities of all cell surface display systems were very low after the induction and only the YiaTR₂₃₂-CESH[L] system exhibited an activity of 0.34 ± 0.09 U/OD. However, after incubation at 4 °C for a few days, the activities of the InaKN-CESH[L] and InaPbN-CESH[L] systems increased significantly. Particularly, the InaPbN-CESH[L] system exhibited a very high activity of 1.67 ± 0.04 U/OD after the 2-day incubation. Combined with the trypsin accessibility assays, these results indicate that the fusion proteins with different anchoring motifs showed not only different expression and display efficiency, but also different folding levels. The InaPbN-CESH[L] system showed the highest activity after a 2-day incubation at 4 °C and was chosen for the following studies.

As a comparison, the whole-cell and lysate activity of the cells with soluble CESH[L] in an intracellular expression system (Cui et al. 2012) was 0.09 ± 0.02 U/OD and 1.29 ± 0.03 U/OD, respectively (Fig. 1c), indicating that the membrane penetrability of the substrate CES and/or the product TA is very low. Because treatments with surfactants have been used to enhance cell permeability and improve bioconversion efficiency (Rosenberg et al. 1999; Zhang and Qian 2000), the whole cells of the intracellular expression system were treated with the surfactant Triton X-100 to enhance the cell permeability. The activity of the surfactant-treated cell culture increased significantly, reaching 0.62 ± 0.05 U/OD. However, after centrifugation, the supernatant of the surfactant-treated culture retained the activity of 0.26 ± 0.03 U/OD, indicating more than one-third of activities arise from the enzyme leakage instead of the permeability for substrates/products. Furthermore, the InaPbN-CESH[L] system after 2 days of incubation at 4 °C showed an activity higher than the lysate of the intracellular expression system, suggesting an excellent expression and display level of the InaPbN-CESH[L] system without the permeability problem.

Optimization of the InaPbN-CESH[L] surface display system

Since the InaPbN-CESH[L] surface display system showed the highest activity among the five systems, the production conditions of this system were further optimized. To study the effect of the inducer IPTG, different concentrations of IPTG from 0.2 to 1.0 mM were used to induce the expression of InaPbN-CESH[L]. Trypsin accessibility assays showed that both the amount and the surface display efficiency of expressed InaPbN-CESH[L] were almost the same under these IPTG concentrations (Fig. 2a). The effect of cultivation temperature on InaPbN-CESH[L] expression after the induction was also studied at 16 °C, 20 °C, 25 °C, 30 °C, and 37 °C. The InaPbN-CESH[L] expression levels and the display efficiencies were different under these temperatures and the best expression temperature is 30 °C according to the SDS-PAGE analysis (Fig. 2b). However, the cells with protein expression at 30 °C showed low activity and poor stability during the incubation at 4 °C (Fig. 2c). The activity of the cells with protein expression at 16 °C, 20 °C, and 25 °C increased significantly in the first 2 days and the cells with protein expression at 25 °C showed the best activity and stability. Thus, 25 °C was used as the optimum expression temperature for the InaPbN-CESH[L] system in the following studies.

SDS-PAGE analysis of the InaPbN-CESH[L] expression. a The effect of the inducer IPTG concentrations. Lanes 1–12, each pair of lanes are trypsin-untreated and treated whole cells induced by IPTG with the concentrations of 0.0 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, and 1.0 mM. Lane M, standard protein molecular weight markers. b The effect of the expression temperature. Lanes 1–10, each pair of lanes are trypsin-untreated and treated whole cells with the expression temperatures 16 °C, 20 °C, 25 °C, 30 °C, and 37 °C. Lane M, standard protein molecular weight markers. c The whole-cell enzymatic activities of InaPbN-CESH[L] with different expression temperatures. d Fraction analysis of intracellular expressed CESH[L] (lanes 1–3) and displayed InaPbN-CESH[L] (lanes 4–6). W, whole-cell lysate; S, supernatant after centrifugation of the cell lysate; P, pellet after centrifugation of the cell lysate. Lane M, standard protein molecular weight markers

According to Fig. 2b, about half (49.6% estimated by Bio-Rad Image Lab Software) of the total InaPbN-CESH[L] proteins expressed at 25 °C were displayed on the cell surface. To check the form of undisplayed proteins, the cells expressing InaPbN-CESH[L] were lysed by ultrasonication. After centrifugation, all InaPbN-CESH[L] proteins were in the pellet and no InaPbN-CESH[L] protein was detected in supernatants, suggesting no soluble InaPbN-CESH[L] proteins were inside cells (Fig. 2d). Therefore, the un-displayed InaPbN-CESH[L] should be in inclusion bodies or buried in membranes. As a control, intracellular CESH[L] without anchoring motif was found mainly in supernatants, indicating that CESH[L] is largely soluble in E. coli cells with a small amount forming inclusion bodies.

Effects of pH and temperature on enzymatic activity and stability of the InaPbN-CESH[L] system

Whole-cell enzymatic activities of InaPbN-CESH[L] in buffers under different pH were detected. The results showed that the InaPbN-CESH[L] surface display system had the highest enzyme activity in Tris–HCl buffer at pH 8.0 and pH 8.5 (Fig. 3a). However, the system showed higher stability at pH 8.0 than at pH 8.5 (Fig. 3b). The whole-cell enzymatic activity of the cells in Tris–HCl buffer at pH 8.0 showed no significant loss at 4 °C within 7 days. Similar to the phenomena presented in previous sections, the whole-cell enzymatic activity of the cells showed a significant increase (about five times) after one-day incubation and reached the highest value after 2-day incubation. This phenomenon suggested that there was a maturation step of the displayed InaPbN-CESH[L], which was completed in the first 2 days at 4 °C.

Effects of pH and temperature on surface-displayed InaPbN-CESH[L] enzymatic activity and stability. a Relative enzymatic activities in buffers with different pH values. The activities were measured after incubation at 4 °C for 5 h. The highest whole-cell enzymatic activity in Tris–HCl buffer (pH8.5) was set to 100%. b The long-term stability at 4 °C in Tris–HCl buffers with different pH values. c Trypsin accessibility assays of the InaPbN-CESH[L] displayed whole cells after incubation at 4 °C. Lanes 1–8, each pair of lanes are trypsin-untreated and treated whole cells with different lengths of incubation time. Lane M, standard protein molecular weight markers. d The long-term stability of the InaPbN-CESH[L] displayed whole cells incubated at different temperatures

The increased activity observed in the InaPbN-CESH[L] system incubated at 4 °C might originate from the slow folding of displayed proteins or the slow secretion of intracellular proteins in inclusion bodies. To further address the mechanism of the phenomenon, the trypsin accessibility assays of the whole cells during the incubation were performed (Fig. 3c). The results showed that a small fraction of un-displayed proteins was continually further displayed on the cell membrane during the first three days (lanes 2, 4, 6, and 8 in Fig. 3c). Almost all InaPbN-CESH[L] proteins were displayed after 3-day incubation (lane 8 in Fig. 3c) and the total displayed proteins increased slightly during the first three days (lanes 1, 3, 5, and 7 in Fig. 3c). Considering that the activity increased about five times after one-day incubation and increased slightly since then (Fig. 3b), it could be concluded that most of the further displayed proteins in the second and third days did not significantly contribute to the activity increase during the incubation. Therefore, a maturation step, i.e., the gradual folding of the displayed proteins, occurring during the incubation at 4 °C should be the major reason for the observed activity increase.

The stability of the InaPbN-CESH[L] system incubated at different temperatures was also studied. When the cells were incubated at 37 °C, no maturation step was observed and 57.82% of whole-cell enzyme activity was lost only after one day. When incubated at 16 °C and 25 °C, the whole-cell enzyme activities rose slightly in the first few days, and thereafter, the enzyme activity began to decrease. The highest whole-cell enzyme activities were about 0.8 U/OD during the incubation. When incubated at 4 °C, the whole-cell enzyme activity reached the highest activity (1.67 ± 0.02 U/OD) after 2-day incubation. Subsequently, the activity declined slightly but maintained a constant high level. After 15-day incubation, 91.66% (1.53 ± 0.08 U/OD) of the highest activity remained (Fig. 3c). Therefore, the InaPbN-CESH[L] system is quite stable at 4 °C and suitable for long-term storage.

Yield comparison of the surface display system and the intracellular expression system

For the intracellular expression system using the plasmid pET28a-CESH[L], the total enzymatic activity of CESH[L] in the cell lysate from 250 ml cell culture was 17,490 ± 187 U, corresponding to 55.56 ± 0.59 mg CESH[L] according to a standard curve of the activity and the purified enzyme. For the InaPbN-CESH[L] surface-display system after 2-day incubation at 4 °C, the whole-cell enzymatic activity was 1.54 ± 0.01 U for 20 μl cell culture and the total enzymatic activity was 19,193 ± 183 U for 250 ml culture, corresponding to 60.97 ± 0.58 mg active CESH[L] excluding the fusion anchoring motifs. Therefore, the InaPbN-CESH[L] surface display system displayed a higher amount of CESH[L] than the enzymes expressed in the intracellular system. Assuming that the cell number of 1.0 OD is 5 × 10⁸ per ml (Stevenson et al. 2016), the display level of the InaPbN-CESH[L] system was estimated to be about 1.9 × 10⁶ molecules per cell. The final cell density of the intracellular expression system using the plasmid pET28a-CESH[L] was slightly lower than the InaPbN-CESH[L] surface-display system (OD₆₀₀ 4.5 vs. 5.0), and the CESH[L] molecular number of the intracellular expression system was estimated to be 1.9 × 10⁶, indicating the similar amount of CESH[L] molecules were produced in each cell of both systems.

Evaluation of the reusability of the InaPbN-CESH[L] display system

After incubation of the InaPbN-CESH[L] display cells at 4 °C for different days, three rounds of whole-cell enzymatic activity measurements were performed to evaluate the reusability of the system. For the cells with 2-day incubation, three usages of the whole cells remained about 98.44%, 95.39%, and 75.73% of their original activity. Furthermore, the cells with 5- and 8-day incubation also showed good reusability similar to the cells with 2-day incubation. For the incubation time longer than eight days, the reusability of whole cells was impaired, but the system with 14-day incubation still showed about 51.17% of the highest activity in the third usage of the cells (Fig. 4). These results indicated that the InaPbN-CESH[L] display system has good reusability.

Repeated use of the InaPbN-CESH[L] surface display system. The cells after protein expression were incubated at 4 °C for different days and then the whole-cell enzyme activities were repetitively assayed four times. After each enzymatic reaction, the cells were centrifuged and washed with the assay buffer, and then the whole-cell enzyme activities were immediately assayed again as repetitive usage