Ptrchis2b

As mentioned above, E. coli BL21(DE3) has its native MEP pathway to form IPP and DMAPP. Therefore, the MEP pathway for sabinene synthesis was constructed by harboring the plasmid pHB1 to introduce the exogenous sabinene synthase. Furthermore, to enhance the metabolic flux into GPP by catalyzing the conversion of DMAPP and IPP, the GPP synthase (IspA or GPPS2) was overexpressed or introduced.
E. coli BL21(DE3) harboring pHB7 and pTrcLower was constructed to form the MVA pathway for sabinene synthesis. The mvaE (Genbank: AF290092) was amplified with the primer mvaE-F and mvaE-R from genomic DNA of Enterococcus faecalis (ATCC 700802D-5) and then cloned into pHB5 and with restriction enzymes Nco I and Bam HI, creating pHB6. The mvaS (Genbank: AF290092) was amplified from genomic DNA of E. faecalis (ATCC 700802D-5) with the primer mvaS-F and mvaS-R and cloned into pHB6 and with restriction enzymes Sac I and Pst I, creating pHB7. The ERG12, ERG8, ERG19 and IDI1 genes from S. cerevisiae (ATCC 204508) were cloned into pTrcHis2B (Invitrogen, Carlsbad, CA) using a method of successive hybridization to yield pTrcLower
[42 (link)].

The P. aeruginosa gene PA2428 encoding PPK2 was codon-optimized for expression in E. coli and purchased from Thermo Fisher Scientific GeneArt. It was received in the expression plasmid pET100/D-TOPO and named pAU005. The gene is expressed under control of T7 promoter with an N-terminal His-tag, X-press epitope and EK-recognition site for tag cleavage. The glk gene encoding E. coli glucokinase (EGlk) was PCR amplified from genomic DNA of E. coli (strain K12) using the oligonucleotides EGlk-Forward (5′-AT ACCATGGGTACAAAGTATGCATTAGTCGG-3′) and EGlk-Reverse (5′-AGACCTTAGGTCACATTCTGGGTTCTGGACAT CATCACCATCATCACTAAGAATTCATA-3′). The forward primer introduced a NcoI site and an extra codon for glycine at the 5′-end of the coding sequence of EGlk. The reverse primer appended a Gly-Ser-Gly linker and a 6x His tag, stop codon and EcoRI restriction site near the 3′-end. The purified PCR fragment was cloned into NcoI and EcoRI sites of vector pTrcHis2B (Invitrogen) generating plasmid pAU007. EGlk is expressed under control of Trc promoter. The construct was verified by DNA sequencing. Plasmids were transformed into E. coli BL21 (DE3) strain and transformants were selected from LB agar plates containing 100 mg/L of ampicillin.