Fig. 1
Modular and metabolic engineering of S. elongatus PCC 7942 for photosynthetic amorpha-4,11-diene and squalene productions from CO2. a Heterologous expression of key enzymes derived from E. coli were introduced to direct the carbon flux to the native 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway as a module of OverMEP. The second module (TPS terpene synthase) is an enzymatic conversion of farnesyl diphosphate (FPP) to either heterologous sesqui- or triterpene as a final carbon sink. Based on the OverMEP strains, heterologous amorphadiene synthase (ADS) and squalene synthase (SQS) were expressed, respectively. A molecule of FPP was converted to amorphadiene catalyzed by ADS, or a reductive dimerization of two molecules of FPP was catalyzed by SQS to produce squalene. b Schematic diagrams of the construction of S. elongatus PCC 7942 strains that produce either amorphadiene or squalene. The heterologous genes dxs, dxs-idi, dxs-dxr-idi, dxr-idi-ispA, dxs-idi-ispA, dxs-dxr-idi-ispA were introduced to a neutral site I (NSI) of S. elongatus genomic DNA, respectively. Subsequently, the ADS or SQS genes were introduced to neutral site II (NSII). Each strain was verified by PCR using a pair of oligonucleotides shown as red arrows and their sequences are listed in Additional file 1: Table S1. The corresponding DNA fragments (1, 2 and 3) are shown in gel images. Also, the DNA sequences were verified correctly. Abbreviations of the corresponding gene are dxs (Ec) 1-deoxy-d-xylulose-5-phosphate synthase of E. coli, idi (Ec) isopentenyl diphosphate isomerase of E. coli, dxr (Ec) 1-deoxy-d-xylulose 5-phosphate reductase of E. coli, ispA (Ec) farnesyl diphosphate synthase (IspA) of E. coli, ADS (Aa) amorpha-4,11-dien synthase of A. annua, SQS (Sc), squalene synthase of S. cerevisiae, G3P glyceraldehyde 3-phosphate, DXP 1-deoxy-d- xylulose-5-phosphate, MEP 2-C-methyl-d-erythritol-4-phosphate, CDP-ME 4-diphosphocytidyl-2-C-methyl-d-erythritol; CDP-MEP 4-diphosphocytidyl-2C-methyl-d-erythritol-2-phosphate, MEcPP 2C-methyl-d-erythritol-2,4-cyclodiphosphate, HMBPP (E)-4-hydroxy-3-methylbut-2-enyl-diphosphate, IPP isopentenyl diphosphate, DMAPP dimethylallyl diphosphate, FPP farnesyl diphosphate