Plasmids and polymerase chain reaction (PCR) primers used in this study are listed in Table 1. Common procedures, including genomic DNA preparation, restriction digestions, transformations, and other standard molecular biological techniques were performed as previously described . All restriction enzymes and T4 DNA ligase were purchased from New England Biolabs (Ipswich, USA). PCR was performed following the manufacturer’s instructions, using a high fidelity KOD-Plus-Neo polymerase (Toyobo, Osaka, Japan). Plasmid preparation and gel extraction kits were obtained from Promega (Madison, USA) and oligonucleotides were synthesized by Bioneer (Daejeon, Korea). The MrBBS gene (GenBank accession number: KJ020282) was optimized to E. coli codons and synthesized by Bioneer (Additional file 1: Figure S1). We deposited the nucleotide sequence data of E. coli codon-optimized MrBBS in the GenBank database (accession number: KU680479). The synthesized MrBBS was amplified by PCR with Bis-IF and Bis-IR primers (Table 1) and the plasmid backbone was amplified with the Bis-VF and Bis-VR primers from pTrc99A (GE Healthcare). The two PCR-amplicons were assembled via the Gibson Assembly Method  using Gibson Assembly Master Mix (New England Biolabs), to produce the pTSN-MrBBS plasmid. The entire polycistronic MVA pathway genes were inserted into pTSN-MrBBS through XbaI restriction site digestion of both the pTSN-MrBBS and the pSNA plasmids containing all the mevalonate pathway genes , followed by ligation with T4 DNA ligase. The resultant plasmid was designated pSNA-MrBBS. To overexpress FPP synthase, the ispA gene (GenBank accession number: AAC73524) was PCR-amplified from E. coli MG1655 genomic DNA, using IspAI-F and IspAI-R primers. To avoid proofreading errors introduced during PCR, the amplified ispA gene was first inserted into the XbaI restriction site of pTSN-MrBBS (instead of pSNA-MrBBS). The pTSN-MrBBS-IspA and pSNA plasmids were digested with XbaI followed by ligation with T4 DNA ligase to create pSNA-MrBBS-IspA. The direction of the MVA pathway genes was verified by Sanger sequencing.