Directed evolution and structural analysis of N-carbamoyl-D-amino acid amidohydrolase provide insights into recombinant protein solubility in Escherichia coli

Abstract

One of the greatest bottlenecks in producing recombinant proteins in Escherichia coli is that over-expressed target proteins are mostly present in an insoluble form without any biological activity. DCase (N-carbamoyl-D-amino acid amidohydrolase) is an important enzyme involved in semi-synthesis of beta-lactam antibiotics in industry. In the present study, in order to determine the amino acid sites responsible for solubility of DCase, error-prone PCR and DNA shuffling techniques were applied to randomly mutate its coding sequence, followed by an efficient screening based on structural complementation. Several mutants of DCase with reduced aggregation were isolated. Solubility tests of these and several other mutants generated by site-directed mutagenesis indicated that three amino acid residues of DCase (Ala18, Tyr30 and Lys34) are involved in its protein solubility. In silico structural modelling analyses suggest further that hydrophilicity and/or negative charge at these three residues may be responsible for the increased solubility of DCase proteins in E. coli. Based on this information, multiple engineering designated mutants were constructed by site-directed mutagenesis, among them a triple mutant A18T/Y30N/K34E (named DCase-M3) could be overexpressed in E. coli and up to 80% of it was soluble. DCase-M3 was purified to homogeneity and a comparative analysis with wild-type DCase demonstrated that DCase-M3 enzyme was similar to the native DCase in terms of its kinetic and thermodynamic properties. The present study provides new insights into recombinant protein solubility in E. coli.

Figure 1. A high-throughput colorimetric screening procedure

(A) Fusion expression construct with DCase–α-fragment in the pMAL-c2x vector. The linker between DCase and the α-fragment of β-gal is Gly-Ser-Ala-Gly-Ser-Ala-Ala-Gly-Ser-Gly-Ala-Ser. (B) Photograph of E. coli colonies expressing genes encoding the WT and evolved DCases in liquid medium. (C) Assay of β-gal activity using the substrate ONPG in vitro. A unit of β-gal activity is defined as the amount of enzyme required to hydrolyse 1 μmol of ONPG to O-nitrophenol and D-galactose per min. (D) Native PAGE of soluble fractions of the WT and evolved DCases expressed with α-fragment fusion tag at 37 °C. MU1–MU4, DCase mutants 1–4 as indicated in the main text.

Figure 2. Sequence alignment of six homologous amidohydrolases

1, DCase from B. pickettii; 2, DCase from Agrobacterium sp. KNK712; 3, DCase from Pseudomonas sp. KNK003A; 4, nitrilase from Polaromonas sp. JS666; 5, aliphatic amidase from Saccharopolyspora spinosa; 6, β-alanine synthase from Brevibacillus agri. The Figure was generated using the ClustalW program and drawn with ESPript. The secondary structure elements of DCase are shown above the sequences. Identical residues are boxed in black. Glu⁴⁷, Lys¹²⁷ and Cys¹⁷², the catalytically important residues, are indicated by stars. The three mutations in DCase, A18T, Y30N and K34E, are indicated by closed circles (●).

Figure 3. Solubility analysis of target proteins that were expressed using the pET expression system at 22 °C

The mutations are listed on the right-hand side of the panels. (A) Solubility analysis of WT DCase and evolved recombinant DCases. (B) Expression test of a single mutant (Y30N), three double mutants (A18T/Y30N; A18T/K34E; and Y30N/K34E) and a triple mutant (A18T/Y30N/K34E). tot, total cell; ppt, precipitant fraction; sup, supernatant fraction.

Figure 4. Equilibrium denaturation curves of WT and Dcase-M3

(A) SDS/PAGE of purified WT DCase and DCase-M3. (B) Urea-induced equilibrium transition curves for the unfolding of WT DCase (▲) and DCase-M3 (●). Curves of urea-induced denaturation were monitored by the fluorescence of tryptophan at 340 nm.

Figure 5. Model of DCase based on its similarity with DCase of Agrobacterium sp. KNK712

(A) The three-dimensional structure of the DCase-M3 homotetramer and monomer. In the DCase-M3 homotetramer (left-hand panel), the three replacements are coloured: A18T (cyan), Y30N (red) and K34E (blue). In the DCase-M3 monomer (right-hand panel), the three mutations in DCase-M3 are indicated as space-filling models including A18T, Y30N and K34E, which are located on the surface of the protein. The catalytically important residues (Glu⁴⁷, Lys¹²⁷ and Cys¹⁷²) are displayed as ball and stick model, and the N- and C-termini are also labelled in the structure. The Figure was generated using RasMol. (B) Charge distribution of the region near the amino acid at position 34 on the molecular surface of modelled WT and evolved DCases (K34E and K34D). The electrostatic potential is coloured: uncharged amino acids (white), negative amino acids (red) and positive amino acids (blue). All Figures were generated using GRASP.