The use of environmentally damaging petrochemical feedstocks can be displaced by fermentation processes based on engineered microbial chassis that recycle biomass-derived carbon into chemicals and fuels. The stable retention of introduced genes, designed to extend product range and/or increase productivity, is essential. Accordingly, we have created multiply marked auxotrophic strains ofClostridium acetobutylicumthat provide distinct loci (pyrE,argH,purD,pheA) at which heterologous genes can be rapidly integrated using allele-coupled exchange (ACE). For each locus, ACE-mediated insertion is conveniently selected on the basis of the restoration of prototrophy on minimal media. TheClostridioides difficilegene (tcdR) encoding an orthogonal sigma factor (TcdR) was integrated at thepyrElocus under the control of the lactose-inducible,bgaR::PbgaLpromoter to allow the simultaneous control of genes/operons inserted at other disparate loci (purDandpheA) that had been placed under the control of the PtcdBpromoter. In control experiments, dose-dependent expression of acatPreporter gene was observed with increasing lactose concentration. At the highest doses tested (10 mM) the level of expression was over 10-fold higher than ifcatPwas placed directly under the control ofbgaR::PbgaLand over 2-fold greater than achieved using the strong Pfdxpromoter of theClostridium sporogenesferredoxin gene. The utility of the system was demonstrated in the production of isopropanol by theC. acetobutylicumstrain carrying an integrated copy oftcdRfollowing the insertion of a synthetic acetone operon (ctfA/B, adc) at thepurDlocus and a gene (sadh) encoding a secondary dehydrogenase atpheA. Lactose induction (10 mM) resulted in the production of 4.4 g/L isopropanol and 19.8 g/L Isopropanol-Butanol-Ethanol mixture.