Inducible Promoter Replacement in Z. mobilis

Replacement of the native pdc promoter by the IPTG-inducible promoter P_T7A1 [22 (link)] in the Z. mobilis chromosome was achieved by homologous recombination using the suicide vector pZP950. Homology arms adjacent to the P_pdc on the chromosome were chosen to be 800 bp long and were flanking a spectinomycin resistance cassette as well as an expression cassette for lacI and the LacI-regulated promoter P_T7A1 [22 (link)]. Z. mobilis ZM4 was transformed with pZP950 via electroporation as previously described [9 (link)] and selection was carried out using 200 µg/ml spectinomycin on ZCM plates in the presence of 1 mM IPTG. Resulting colonies were tested on ZCM plates with and without 1 mM IPTG and colonies that only grew in the presence of IPTG were tested by colony PCR (see Additional File 1 for primer sequences) using OneTaq Quick-Load 2X Master Mix (New England Biolabs Inc., USA). The resulting homogenous strain with P_pdc replaced by P_T7A1 was named sGB027 (ZM4 ΔP_pd :: spR-lacI-P_T7A1).
Plasmids for either lactate dehydrogenase or alanine dehydrogenase expression were introduced into sGB027, resulting in strains sGB029 and sGB038, respectively.