Selection-Based Molecular Breeding of Non-Mevalonate Pathway for Efficient Production of Terpenes

Terpenoids are one of the most diverse families of natural compounds that include valuable chemicals as pharmaceuticals, flavors, and fragrances. Dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP) are common building blocks for prenyl pyrophosphates, the precursors of all the terpenoids. To increase the production level of terpenoids in heterologous microbial hosts, numbers of gene targets that affect terpenoid synthesis have been identified using the color screening based on the carotenoid pigmentation. With the growth-coupled selection systems, throughput of the search for those targets could be much improved, enabling the rapid access to even more diverse set of gene targets that improves the supply capacity of the prenyl-pyrophosphates of the host.

 It has been recognized that the overexpression of heterologous terpene synthases often exhibit the negative impact on the cell growth¹, due to the reduction of the biosynthesis of ubiquinones and lipid carriers. On the course of an attempt to hyper-produce geraniol in Escherichia coli, we accidentally found that the overexpression of the mutant of Ocimum basilicum geraniol synthase (GESM53), the variant previously obtained by the screening for improved precursor consumption², deprive some strains of E. coli almost entirely of their colony-forming capacities. Interestingly, the cell growth was completely restored by the additional overexpression of Idi, the known gene targets that elevate prenyl-pyrophosphate production upon overexpression. Therefore, we concluded that the depletion of prenyl pyrophosphates via GPP, not the product toxicity of geraniol, seemed to be responsible for the lethality of overexpression of GESM53. This also implies that, we could identify the genes having “booster” effect on precursor supply for terpernes, simply by searching for the clones that makes host cell grow in the presence of GESM53 overexpressed.

 We developed an arabinose-inducible-GESM53-expression plasmid. Upon induction of GESM53 with 0.2% arabinose, the colony forming unit (c.f.u) of the cell harboring this plasmid dropped by 10⁴-fold. We also tested that the additional overexpression of each and all genes involved in the endogenous MEP pathway and found that Idi and FPPS fully recovered the c.f.u. With this selection system set up, we conducted the directed evolution of IspH, which catalyze the final step of MEP pathway. While the wildtype IspH failed to restored the cell growth, four mutants of IspH were found to confer growth capacity where GESM53 being expressed. We found that the co-expression of these ispH mutants alone significantly elevate the production level of carotenoids and monoterpenes.

(1) Wang, C. et al., Metab. Eng. 13, 648–655 (2011)

(2) Furubayashi, M. et al., PLoS One 9, e93317 (2014)