Cooperation of phytoene synthase, pyridoxal kinase and carbonic anhydrase for enhancing carotenoids biosynthesis in genetic Chlamydomonas reinhardtii

Background: Microalgae, a group of photosynthesis microorganisms, are one of natural producer of carotenoids effectively. Among all, Chlamydomonas reinhardtii is a potential host which has high growth rate and complete genomic information, thus it is easy for gene manipulation. Herein, CC-400 as a cell-wall deficient strain, was chosen for overexpression of genes to improve carotenoid production under the optimization of medium and cultural conditions. Results: At first, phytoene synthase (PSY) was cloned and transformed into CC-400, leading to 38% increment in beta-carotene and 14% in lutein compared to wild type which produced 8.6 mg/L beta-carotene and 2.7 mg/L lutein via TAP medium. By stepwise integration of pyridoxal kinase (PdxY) and carbonic anhydrase (SyCA) to generate SPPY strain, the biomass and carotenoid titer were significantly increased when using Two-layer photoreactor. Medium with doubling acetic acid, additional γ-aminobutyric acid (GABA), or 5-aminolevulinic acid (5-ALA) were tested, thus treated with 5-ALA increased 3.8 folds for lutein and 2.6 folds for beta-carotene when compared to CC-400, with final titer of 10.83 mg/L and 22.82 mg/L respectively at 7 days. Conclusions: Phytoene synthase as the enzyme for rate-determining step in carotenoids synthesis where geranylgeranyl pyrophosphate converted into phytoene was over-expressed and the high-level of beta-carotene and lutein in SPPY were achieved. Gene pdxY induced the biomass and carotenoid production with improve on co-factor PLP regeneration while carbonic anhydrase strengthened the CO₂ uptake. The best combination of genetic engineering and culture condition make C. reinhardtii CC-400 more competitive for carotenoid biosynthesis.