Plasmid Construction for Mevalonate Pathway

pBb plasmids were prepared as the BglBrick standard expression vectors25 (link). The top portion of the mevalonate pathway contains genes for the conversion of acetyl CoA to mevalonate: acetoacetyl-CoA synthase from E. coli (atoB), HMG-CoA synthase from S. cerevisiae (HMGS), and an amino-terminal truncated version of HMG-CoA reductase from S. cerevisiae (HMGR). The bottom portion of the mevalonate pathway contains genes for the conversion of mevalonate toFPP: mevalonate kinase from S. cerevisiae (MK), phosphomevalonate kinase from S. cerevisiae (PMK), phosphomevalonate decarboxylase from S. cerevisiae (PMD), IPP isomerase from E. coli (idi), and farnesyl diphosphate synthase from E. coli (ispA). pJBEI-2704 (pBbA5c-MevT-MBIS): The construction of this plasmids has been previously reported12 (link). pJBEI-2997 (pBbA5c-MevT(CO)-MBIS(CO)): pBbA5c-MevT(CO) was constructed by ligating pBbA5c vector (BamHI/XhoI) and BglBrick compatible codon optimized HMGS and HMGR inserts (BglII/XhoI), using BglBrick cloning strategy with compatible BglII and BamHI restriction. Individual genes were PCR-amplified from pAM45 (ref. 16 (link)) with primers contain EcoRI and BglII sites at 5′-end and BamHI and XhoI sites at 3′-end of each gene. The BglBrick restriction sites found in each gene were removed by site-specific mutagenesis. pBbA5c-MevT(CO)-MBIS(CO) was prepared by the ligation of pBbA5c-MevT(CO) vector (BamHI/XhoI) and MBIS with E. coli codon optimized MK and PMK insert (BglII/XhoI). pJBEI-2999 (pBbA5c-MevT(CO)-P_trc-MBIS(CO)): The plasmid was prepared by two-step ligation of pBbA5c-MevT(CO) vector (BamHI/XhoI) and a double terminator-Ptrc gene part (BglII/XhoI) and MBIS(CO) insert (BglII/XhoI) by standard BglBrick cloning strategy.