Engineering cyanobacteria and the key enzyme CrtE for terpenoid production

Abstract

Cyanobacteria are photosynthetic prokaryotes that perform oxygenic photosynthesis. Due to their ability to use the photon energy of sunlight to fix carbon dioxide into biomass, cyanobacteria are promising hosts for the sustainable production of terpenoids, also known as isoprenoids, a diverse class of natural metabolites with potential as advanced biofuels and high-value chemicals. However, the cyanobacterial enzymes involved in the biosynthesis of the terpene precursors needed to make more complicated terpenoids are poorly characterized. Here we show the crystal structures of geranylgeranyl pyrophosphate synthase from a model cyanobacterium Synechococcus sp. PCC 7002, which is able to synthesize C20-geranylgeranyl pyrophosphate from C5-isopentenyl pyrophosphate and C5-dimethylallyl pyrophosphate. Using site-directed mutagenesis, we identified the amino acid residues Met-87 and Ser-88 play important roles in controlling product chain elongation, and Lys-53 contributes to substrate binding. Crystallisation of putative solanesyl pyrophosphate synthase from Synechococcus sp. PCC 7002 and geranyl pyrophosphate synthase from Picea abies were performed in this study. The predicted structure of Syn7002 putative SPPS showed high similarity to that of the SPPS3 from Arabidopsis thaliana (PDB: 3APZ); it is worth noting that the homology study indicates that both enzymes might use a similar mechanism to control the chain-length of the product. Phylogenetic analysis was performed with all the function determined trans-prenyltransferases, providing additional information to investigate their evolutionary relationships. In this work, I tested different inducible systems for recombinant gene expression in cyanobacteria. An engineered Synechococcus sp. PCC 7002 expressing geraniol synthase and GPPS showed a dramatic decrease in carotenoids and chlorophylls upon gene induction, consistent with the possibility that the carbon flux was diverted from pigment synthesis to monoterpene synthesis. Overall, our results provide new structural and functional information on the cyanobacterial PTSs, that could lead to the development of improved cyanobacterial platforms for terpenoid production.