# Caroenoid Production from Microalgae: Biosynthesis, Salinity Responses and Novel Biotechnologies
## 5 Future Prospects  
### Genetic Modifications of Microalgae for Salt Tolerance and Carotenoid Accumulation  
Microalgae are widespread and large in number, and only a small part of them have been studied. Therefore, there are still numerous novel microalgae species that may be able to tolerate salinity and accumulate carotenoids. Here, we mainly focus on three methods to get expected microalgae strains: (1) adaptive evolution, (2) random mutagenesis and (3) targeted genetic engineering. Different to strain isolation, adaptive evolution can obtain an expected strain with a target phenotype. Adaptive evolution has been widely utilized for strain improvement to stress tolerance in various microalgae species, such as _C. reinhardtii_ for 200 mM salt tolerance [108] and _Chlorella sp._ for high phenol concentration [109]. It was performed for improving the tolerance of a freshwater strain _Chlorella sp._ AE10 to 30 g/L salt for 138 days (46 cycles). The genes of the resulting strain _Chlorella sp._ S30 related to Calvin-Benson cycle, C4-dicarboxylic acid cycle and crassulacean acid metabolism (CAM) pathways were upregulated, which was beneficial for CO\({}_{2}\) fixation under salt stress [85]. However, salinity acclimation of microalgae involves different functional genes and numerous pathways, which makes the exploitation of specific genes in salt tolerance more difficult. Chemical mutagens, such as N-methyl-N\({}^{\prime}\)-nitro-N-nitrosoguanidine (MNNG) and ethyl methane sulfonate (EMS), are effective in mutant generation and may lead to mutants with enhanced carotenoid accumulation [110]. When genes in the carotenoid synthesis pathway (shown in Figure 1) are mutated, carotenoid profiles in microalgae may change. By chemical mutagenesis with EMS, carotenoid content in _Coelastrum sp._ C1-G1 was increased about 2-fold over its mother strain [39]. The Astaxanthin-overproduction strain _H. pluvialis_ MT 2877 is a mutant by MNNG, which produced 4-fold astaxanthin over the WT strain [111]. _C. zofingiensis bkt1_, a chemical mutagen by MNNG, can accumulate high amounts of three essential carotenoids, e.g., zeaxanthin (7.00 \(\pm\) 0.82 mg/g), lutein (13.81 \(\pm\) 1.23 mg/g) and \(\beta\)-carotene (7.18 \(\pm\) 0.72 mg/g) under different cultivation conditions [33], which can serve as a competent option for large-scale cultivation. _Chlorella sp._ was irradiated by \({}^{137}\)Se- \(\gamma\) ray and domesticated with a seawater culture medium (salinity 3% wt.) under 15% CO\({}_{2}\) stress. The biomass yield of the mutant was increased by 25% with 54.9% DW of lipids [112]. Although random mutagenesis could lead to novel traits, unpredictable traits may also show up, and the mutated genes still need further verification, such as gene sequencing, knockout and retro-complementation. Targeted genetic engineering could generate specific insertions, deletions or substitutions in the host genome while avoiding random results [113]. Up until now, a lot of genes related to salt responses have been recognized, such as _nhaP_ encoding a Na\({}^{+}\)/H\({}^{+}\) antiporter, _codA_ encoding choline oxidase to synthesize compatible solutes and _Dps_ encoding DNA-binding proteins (Dps). Their introduction or overexpression strains have shown enhanced salt-tolerant abilities and potentials for seawater cultivation, which have been comprehensively reviewed [114]. Genetic engineering of microalgae for carotenoid hyperaccumulation is now a mature technology [115]. Genetic engineering of the green algae _C. zofingiensis_ with a modified norfluxazon-resistant endogenous _pds_ gene resulted in up to 54.3% higher astaxanthin [116]. Transformation and expression of _dxs_ and _psy_ genes in _P. tricornutum_ can increase fucoxanthin content by 2.4-fold and 1.8-fold, respectively [117]. _H. pluvialis_ can also act as a gene donor. Transgenic _D. salina_ with _bkt_ and _chyb_ genes from _H. pluvialis_ was capable of astaxanthin biosynthesis with a better tolerance to high light [118].