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 .