Bielefeld University, Faculty of Biology, Center for Biotechnology (CeBiTec), Universitätsstrasse 27, 33615 Bielefeld, Germany.
Bielefeld University, Faculty of Biology, Center for Biotechnology (CeBiTec), Universitätsstrasse 27, 33615 Bielefeld, Germany.
Metab Eng. 2018 Jan;45:211-222. doi: 10.1016/j.ymben.2017.12.010. Epub 2017 Dec 16.
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 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.
光合微生物宿主，如蓝藻和真核微藻，最近已成为替代工程平台，用于可持续的光驱动萜类化合物的生物生产。许多具有多种应用的理想化合物可以通过萜烯合酶的异源表达在微生物中产生。然而，绿色微生物系统的成功受到许多问题的阻碍，例如酶表达滴度不足和从光合作用固定的碳到所需萜烯产物的通量低。这项工作展示了如何对莱茵衣藻进行工程改造，以生产倍半萜生物柴油前体（E）-α-法尼烯。通过战略性的遗传工程，通过协调调节异戊二烯代谢，结合串联酶加载以过表达萜烯合酶和 amiRNA 抑制竞争途径，实现了生产力的大幅提高。在五天内可生产 10.3 ± 0.7mg 法尼烯·g 细胞干重，比单酶表达菌株提高了 15 倍以上。在扩大培养中研究菌株性能确定了光暗循环对法尼烯总体生产力的益处。在这些条件下，混合营养培养可在七天内产生高达 11.0 ± 0.5mg/L 的法尼烯，而光营养生产 3.9 ± 0.2mg/L 是可行的。这些成就代表了向设计可持续、光驱动、绿色细胞藻类生物工厂的目标对莱茵衣藻进行工程改造的重要里程碑。
