Random mutagenesis by insertion of error-prone PCR products to the
chromosome of Bacillus subtilis
Bacillus subtilis is an attractive host for directed evolution of the
enzymes whose substrates cannot be transported across the cell membrane.
However, generation of mutant library in B. subtilis still suffers
problems of small library size, plasmid instability and heterozygosity.
Here, large library of random mutant was created through inserting
error-prone PCR (epPCR) product to the chromosome of B. subtilis.
Specifically, epPCR product was fused with flanking regions and
antibiotic resistant marker using a PCR-based multimerization method,
generating insertion construct. epPCR product was integrated into
chromosome via homologous recombination after insertion construct was
transformed into the supercompetent cells of B. subtilis strain SCK6.
The transformation efficiency of insertion construct was improved though
increasing the number of competent cell and the length of flanking
regions. A library containing 3.5×105 random mutant was construction
using per μg insertion construct, which is sufficient for directed
evolution. Moreover, the library generation process could be
accomplished within one day. The effectiveness of this method was
confirmed by improving the activity of Methyl Parathion Hydrolase (MPH)
toward chlorpyrifos and to enhance the secretion level of MPH in B.
subtilis. Taken together, present work provides a fast and efficient
method to integrate epPCR product into the chromosome of B. subtilis,
facilitating directed evolution and expression optimization of target