Metabolic Engineering of Ketocarotenoid Biosynthetic Pathway into the model organism Chlamydomonas reinhardtii

• Main Author: Tran, Nam Trung
• Format: Others
• Language: en
• Published: 2019
• Online Access: https://tuprints.ulb.tu-darmstadt.de/8965/1/01082019_Dissertation_NT_Tran.pdf; Tran, Nam Trung <http://tuprints.ulb.tu-darmstadt.de/view/person/Tran=3ANam_Trung=3A=3A.html> (2019): Metabolic Engineering of Ketocarotenoid Biosynthetic Pathway into the model organism Chlamydomonas reinhardtii.Darmstadt, Technische Universität, [Ph.D. Thesis]

Ketocarotenoids represent a special group of carotenoids characterized by presence of one or several carbonyl (C=O) groups in their ionone rings. Due to their excellent anti-oxidative characteristics, several ketocarotenoids such as canthaxanthin and astaxanthin are valuable pigments highly sought by feed, cosmetical and nutraceutical industries. In this work I aimed to introduce ketocarotenoid biosynthetic pathway into the model organism Chlamydomonas reinhardtii via overexpression of its native key enzyme ß-carotene ketolase (CrBKT). High transgene expression and transgene stability were attained with help of the bicistronic Ble2A system in which, the selection marker - zeocin-resistance conferring ble gene - is directly linked to gene-of-interest via the so-called self-cleavage foot-and-mounth-disease-virus (FMDV) 2A sequence. Functionality of Ble2A system was proven by successful production of the fluorescence protein mCherry in Chlamydomonas. CrBKT’s enzymatic activity was also successfully confirmed by heterologous production in carotenoid-producing E. coli, indicated by the almost complete conversion of ß-carotene to canthaxanthin and of zeaxanthin to astaxanthin. Two strains of Chlamydomonas were chosen for metabolic engineering: strain UVM-4 which boasted improved transgene expression and strain CC-4102 in which only ß-carotene and zeaxanthin are accumulated due to mutations in beta-carotene biosynthetic pathway and xanthophyll cycle. Transformation of both strains with CrBKT overepxression vectors yielded hundreds of zeocin-resistant colonies but only in ~10% of them could the integration of CrBKT be confirmed via PCR. Ketocarotenoid production was not detected in any PCR-positive lines either. Success was only achieved when transformation conditions were changed, namely algal cells were transformed and recovered in dark on growth medium supplemented with yeast extract and tryptone. Under these conditions, I was able to isolate four CrBKT-overproducing transformants. All four lines were characterized by their pale green color as well as their drastically reduced chlorophyll contents. Canthaxanthin production was also detected in two lines, whose concentration stood at about 10% of total cellular carotenoids (~0.1 pg/cell). An intriguing phenomenon was also observed with these pale green canthaxanthin-producing transformants. When cultivated in light, they promptly reverted back to the normal dark green color. Canthaxanthin could no longer be detected and zeocin resistance was seeminly impaired as well. Though the cause of this phenomenon could not be pinpointed with certainty, it did help explain the failure of my earlier experiments. Taken together, these results formed the foundation for future projects of ketocarotenoid metabolic engineering in microalgae. Several potential pitfalls that might be encountered were identified and strategies to overcome them were also suggested.