2.2 Development of recombinant strains
Primers and plasmids used in this study are listed in Tables S2 and S3. The suicide vector, pEX18Tc, was used (Hoang et al., 1998). Fragments of 500−1000 bp for flanking genes to be deleted were amplified fromZ. mobilis , which were cloned into E. coli DH5α for amplification. The recovered plasmids were fused with enzymatically digested pEX18Tc and confirmed by sequencing. Subsequently, the plasmids were transformed into E. coli JM110 for demethylation for efficient transformation into Z. mobilis ZM4 or ZM401 through electroporation. RM medium supplemented with tetracycline was used to select colonies harboring the target plasmids, and 5% sucrose was added to the RM medium for counter-selection of homologous colonies (Li et al., 2013). The selected mutants were verified using tetracycline sensitivity and Colony PCR. The shuttle vector pHW20a was used to carry target genes (Dong et al., 2011). The overexpressed genes were colonized from Z. mobilis , which, together with the promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (Pgap), were amplified by PCR for fusion with pHW20a. The expression plasmids were demethylated inE. coli JM110 and then transformed into Z. mobilis as previously described (Xia et al., 2018). When engineered with the empty vector pHW20a, no significant difference was observed compared with the wild-type strains ZM4 and ZM401 (Fig. S2). Therefore, ZM4/pHW20a and ZM401/pHW20a were used as controls for the comparative analysis. All the engineered strains were verified by PCR.