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

•Carotenoid content is governing by the PSY expression in Chlamydomonas reinhardtii.•PdxY serves as a critical gene for biomass enhancer in genetic PPY strain.•Cooperate PdxY, PSY and SyCA to form SPPY, improving carotenoid titer.•Biomass is increased by extra acetic acid, but carotenoids are boosted by 5-ALA.•Using Two-layer photoreactor to increase biomass and carotenoid production in SPPY. 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. 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. 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.