Tailored carbon partitioning for phototrophic production of (E)-α-bisabolene from the green microalga Chlamydomonas reinhardtii.

Photosynthetic microbial hosts such as cyanobacteria and eukaryotic microalgae have recently emerged as alternative engineering platforms for the sustainable light-driven bio-production of terpenoids. Many desirable compounds with numerous applications can be produced in microorganisms by heterologous expression of terpene synthases. However, success of green microbial systems has been hampered by issues such as insufficient enzyme expression titers and low flux to desired terpenoid products from carbon fixed during photosynthesis. This work demonstrates how the green microalga Chlamydomonas reinhardtii can be engineered to produce the sesquiterpene biodiesel precursor (E)-α-bisabolene. Through strategic genetic engineering, substantial enhancements of productivity were achieved by coordinated tuning of the isoprenoid metabolism, combining serial enzyme loading for terpene synthase overexpression and amiRNA-based repression of competing pathways. Up to 10.3 ± 0.7mg bisabolene·g-1 cell dry weight could be produced in five days, which represents more than a 15-fold increase over single synthase expression strains. Investigation of strain performance in scale-up cultivations determined overall bisabolene productivity benefits from light:dark cycles. Mixotrophic cultivation can yield up to 11.0 ± 0.5mg bisabolene per liter in seven days in these conditions, and phototrophic production of 3.9 ± 0.2mg per liter was feasible. These achievements represent an important milestone in the engineering of C. reinhardtii towards the goal of designing sustainable, light-driven, green-cell algal bio-factories.