High-Level Expression and Secretion of Methyl Parathion Hydrolase in Bacillus subtilis WB800

Abstract

The methyl parathion hydrolase (MPH)-encoding gene mpd was placed under the control of the P43 promoter and Bacillus subtilis nprB signal peptide-encoding sequence. High-level expression and secretion of mature, authentic, and stable MPH were achieved using the protease-deficient strain B. subtilis WB800 as the host.Organophosphate compounds are used extensively in agricultural and domestic pest control and as chemical warfare agents; these compounds inhibit the acetylcholinesterase of animals (8). Mass application of these compounds in the environment causes serious problems. Organophosphate hydrolase (OPH) isolated from soil microorganisms detoxified organophosphates effectively (14,18), and bioremediation of organophosphate compounds in the environment by using bacterial enzymes may provide an efficient, convenient, and economical method for detoxification. We previously isolated Plesiomonas sp. strain M6, which is capable of hydrolyzing methyl parathion at high efficiency (5), and the gene (mpd) encoding a novel methyl parathion hydrolase (MPH) was cloned (5) and expressed in Escherichia coli (9). However, the previous work showed that E. coli could process only a small proportion of the inactive precursor polypeptide, comprising the signal peptide and the mature enzyme, to produce the active MPH (9).Most microorganisms that produce OPH are gram-negative bacteria, and the OPH is located intracellularly or secreted into the periplasm. The outer membrane acts as a permeability barrier and limits interaction between the pesticides and OPH residing within the cells (12). This bottleneck reduces the application efficacy. Bacillus subtilis can serve as an efficient and safe host for recombinant protein secretion (3,4,7,16,17). Secreted proteins usually remain in biologically active forms (13,15), and downstream purification is greatly simplified. In this work, the mpd gene was fused with the nprB signal peptideencoding sequence, and the organophosphate hydrolase was expressed and secreted in B. subtilis under the control of the P43 promoter (20).The methyl parathion hydrolase-encoding gene mpd (GenBank accession no. AF338729) cannot be expressed in B. subtilis by using its own promoter. To achieve expression and secretion of MPH in B. subtilis, the secretion signal peptideencoding sequence of the nprB gene was fused with the mature mpd gene. A typical cleavage site (ASA-A) (19) for signal peptidase I was designed for the release from B. subtilis of MPH with an authentic N terminus (Fig. 1B). The mature mpd gene sequence (9) was cloned using the primer pair P1 (5Ј-G CGCTGCAGCACCGCAGGTG-3Ј) and P2 (5Ј-CGCAAGCT TTCATCATCACTTGGGGTTGACGACCGA-3Ј) from plasmid pMT1 (5). To introduce the PstI site (underlined) upstream, the codon GCC encoding the first amino acid of mature MPH was modified to GCA (Fig. 1B). Two additional stop codons (TGATGA) and the HindIII site (underlined) were introduced with primer P2. Primers P3 (5Ј-CGCGGAT CCTGATAGGTGGTATGTTTTCGC-3Ј) and P4 (5Ј-CTTG GTCAAGTTGCGCATGTGTACATTCCTCTCTT-3Ј) were used to ...

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