4 DISCUSSION
In present work, to efficiently insert epPCR product into the chromosome
of B. subtilis , we first employed a PCR based multimerization
method to fuse epPCR product with flanking region and
AbR; then, the insertion construct was transformed
into supercompetent cells using a modified protocol, generating
3.53×105 transformations/μg DNA. The library could be
created within one day and the library is large enough for the need of
directed evolution (104-105 mutants
are usually screened in directed evolution). Although the library
generated here is smaller than those constructed by multimeric plasmid
method (Li et al. 2017; Zhang and Sathitsuksanoh et al. 2011), our
method solves the problems of plasmid instability and heterozygosity
encountered in
plasmid
based method.
A crucial step in present method is the assembly of the insertion
construct (LF-AbR-GOI-RF). We had attempted to fuse
the three fragments (LF-AbR, GOI and RF) through PCR
extension and Gibson assembly, but both methods resulted in low yield of
insertion construct. On contrary, PCR based multimerization method
(Shafikhani et al. 1997; You et al. 2012) realized efficient assembly of
insertion construct (Figure 2d). Another key step is competent cell
preparation and transformation. Strain SCK6 was grown in YN medium and
induced by 1.5% xylose, which remarkably improves transformation
efficiency as described by Li et al. (2017). We further demonstrated
that increasing the amount of competent cell and the length of flanking
region can enhance the transformation efficiency of the insertion
construct (Figure 2c).
Plasmid or chromosome is used to carry expression cassette when
producing a protein in B. subtilis . Plasmid mediated protein
expression has the limitations of plasmid instability and safety concern
(the use of AbR) (Bron et al. 1991). Chromosomal
integration manner can overcome the limitations of plasmid manner, but
low gene dose usually leads to low protein yield. Therefore,
transcription and translation level of GOI need to be dramatically
enhanced to achieve high protein yield in integration manner. Mutant MT1
harbors two mutations in SPAprE coding region, both of
which could improve the yield of MPH (Figure 4b and 4d). Nijland et al.
(2007) reported that a single amino acid change remarkably increased the
secretion level of β-toxin. Therefore, the random mutagenesis strategy
established here can find its application in improving protein secretion
level in B. subtilis .