2.3 Generation of MPH mutant library
The right flanking region (RF) of amyE locus was amplified from strain BPC1 using primer pair P1/P5. Another fragment containing left flanking region (LF), ZeoR andPcry3A was amplified from strain BPC1 using primer pair P6/P2. These two fragments were fused by overlap PCR (Shevchuk et al. 2004) using primer pair P1/P2, resulting in fragment RF-LF-ZeoR-Pcry3A . The cleavage site Sac I was introduced between RF and LF.
The coding region of SPaprE and MPH was amplified by error-prone PCR with primer pair P3/P4 using the Starmut Random Mutagenesis Kit (GenStar, Beijing, China). The PCR reaction solution with a total volume of 50 μL contained 0.02 ng/μL template, 25 μL 2×buffer, 3 μL StarMut Enhancer, 0.4 mM primer pair P3/P4. The PCR was conducted as follows: initial denaturation at 95 °C for 2 min and subsequent steps of 94 °C for 30 s, annealing at 56 °C for 1 min, and extension at 72 °C for 1 min for 30 cycles total. The overlap of 47 bp between the left end of the epPCR product and the right end of RF-LF-ZeoR-Pcry3A was introduced through primers P4 and P1, and the overlap of 45 bp between the right end of the epPCR product and the left end of RF-LF-ZeoR-Pcry3A was introduced through primers P3 and P2.
To generate the multimer of fragment LF-ZeoR-Pcry3A -mpd -RF, each 50 μL reaction system contained 0.2 mM dNTP, equal molar amount of RF-LF-ZeoR-Pcry3A and epPCR product (~30 pmol for each fragment), and 0.04 U/μL Phusion polymerase without the addition of primers. The prolonged overlap extension PCR was conducted as follows: denaturation at 98 °C for 30 s; 30 cycles of denaturation at 98 °C for 15 s, annealing at 60 °C for 15 s, and extension at 72 °C at a rate of 2 kb/min; followed by 72 °C extension for 10 min. The multimer product was digested with Sac I to release the monomer of fragment LF-ZeoR-Pcry3A -mpd -RF which was transformed into the competent cells of strain SCK6 according to following procedure.