Genetic Modification And Structural Analysis Of D-carbamoylase Provide Insights On Recombinant Protein Solubility

One of the greatest bottlenecks in producing recombinant proteins in Escherichia coli is that the target proteins, when expressed in a high amount, are often present in an insoluble form without detectable activities. This affects research on characterization of the target proteins and their application. D-carbamoylase (DCase), catalyzing a rate-limiting step in a two-step reaction system for producing D-p-hydroxyphenylglycine (D-HPG), is an important enzyme in semi-synthesis ofβ-lactam antibiotics in industry. We used a co-expression system with molecular chaperone and directed evolution techniques to improve soluble expression of DCase in Escherichia coli, followed by investigatng the relationship between structure and function of the enzyme.A DCase gene was cloned from Burkholderia pickettii, and over-expressed in E. coli as a recombinant protein. The resultant DCase activity was, however, very low because nearly 80% of recombinant DCase was partitioned into insoluble aggregates. To facilitate the expression of soluble and active DCase, three different of molecular chaperones, DnaK-DnaJ-GrpE (DnaKJE), GroEL-GroES (GroELS) and trigger factor (TF), were co-expressed with the DCase, respectively. The target protein aggregate from DCase overproduction were alleviated with the aid of GroELS, resulting in a three-fold increase in DCase enzyme activity compared to the wild-type strain. However, owing to problems related to application of expression inducer in above strategy, alternative method is used for directed evolution of the protein. Directed evolution incorporates Darwinian principles of mutation and selection into experimental strategies for improving biocatalyst or enzyme properties in the laboratory. In this study, error-prone PCR and DNA shuffling techniques are applied to randomly mutate its encoding sequence, followed by an efficient screening based on structural complementation. Several mutants of DCase with reduced aggregation are isolated. Solubility tests of these mutants and several other mutants generated by site-directed mutagenesis indicate that three amino acid residues of DCase (A18, Y30 and K34) are related to the DCase protein solubility in DCase. In the structure model of the DCase-M3 homotetramer and the DCase-M3 monomer, the amino acid of the 18th position is situated at a turn betweenβ-sheet andα-helix, and amino acids of both the 30th and 34th positions were are located inα-helix. The three residues are distributed on the surface of the target protein, and are located far from the catalytic sites (Glu⁴⁷, Lys¹²⁷ and Cys¹⁷²). In silico structural modeling analyses further suggested that hydrophilicity and/or negative charge at these three residues may be responsible for the increased solubility of DCase proteins produced in E. coli. Substitutions of A¹⁸ and Y³⁰ with selected hydrophilic amino acids revealed that solubility of A18T and Y30N muteins was clearly improved. Additional replacement at the K³⁴ position showed that increased negative charge of K34E mutein led to improvement of solubility of the mutated proteins. Based on the information, multiple engineering-designed mutants were constructed by site-directed mutagenesis; among them, a triple mutant A18T/Y30N/K34E (named as DCase-M3) was successfully over-expressed in E. coli with up to 80% of DCase-M3 proteins in soluble form. These results indicate that more soluble proteins can be obtained by combination of mutations. DCase-M3 was purified to homogeneity and a comparative analysis with WT DCase showed that DCase-M3 enzyme is similar to the native DCase in its biochemical properties, including kinetic and thermodynamic parameters, optimal temperature, optimal pH, and thermo-stability at 60℃and 65℃.Based on the data obtained from laboratory studies, the WT and the mutant DCase were fermented and the DCase activity was dertermined for industrial use. Firstly, we fermented the WT DCase and a single-point mutant DCase (K34E), using three different culture media (LB, TB and improved industrial culture medium) and three inducing temperatures (22℃,30℃ and 37℃), respectively. The results showed that K34E mutein had a higher activity than that of WT Dcase in each culture conditions. Considering that two enzymes (D-hydantoinase and D-carbamoylase) are involved in D-HPG production, it was interesting to see their combinational effect after mutation, DHase and mutant DCase (K34E) genes were cloned in Escherichia coli in rank to form E. coli BL21 (DE3)/pCHWT (DHase/ DCase) and E. coli BL21 (DE3) /pCHMU (DHase/ K34E). The fermentation results showed that D-HPG production efficiency of E. coli BL21(DE3)/pCHMU was twice as much as that of E. coli BL21(DE3)/pCHWT. In addition, we enlarged the fermentation scale to 5L for WT DCase and DCase-M3 constructs. It showed that activity of DCase-M3 mutein was increased by seven-fold incomparison with that of WT DCase, indicating that DCase-M3 mutein has a great potential in industrial application.