# Challenges and Potential in Increasing Lutein Content in Microalgae
## 1 Introduction
Maximum lutein content of 11.8 mg/g, with a lutein productivity of 25.0 mg/L/d, was achieved by exposing the strain to 1000 umol m\({}^{-2}\) s\({}^{-1}\) of light intensity [22]. Efforts have also been made on strain improvements for increasing lutein content. Random mutagenesis has been used as a useful technique to improve strains and isolate lutein-rich algal mutants [25]. Chemical mutagens and UV-irradiance were attempted to generate mutant strains. High lutein yield algal mutants were isolated by resistance to inhibitors of the carotenogenic pathway. In this way, several algal mutants with high lutein content have been isolated (Table 2). For example, [PERSON] et al. obtained several high lutein-yield _C. sorokiniana_ mutants through random mutagenesis with N-methyl-N\({}^{\prime}\)-nitroosoguanidine (MNNG). The highest lutein content, 7.0 mg/g, was achieved by the mutant DMR5 and DMR-8 [26]. A 7.52 mg/g lutein content was achieved with the mutant strain _C. sorokiniana_ MB-1-M12 under mixotrophic conditions [24]. _Chromochloris zofingiensis_ (also named as _Chlorella zofingiensis_) could accumulate lutein, zeaxanthin, \(\beta\)-carotene and aastaxanthin under different cultivation conditions [27]. After mutagenesis by MNNG, [PERSON] et al. isolated a _C. zofingiensis_ mutant with a dysfunctional _BKT1_ gene. In this mutant, the content of lutein, zeaxanthin and \(\beta\)-carotene reached 13.81 \(\pm\) 1.23, 7.00 \(\pm\) 0.82, and 7.18 \(\pm\) 0.72 mg/g, separately [28]. \begin{table}
\begin{tabular}{c c c c}
**Microalgal Strains** & **Lutein Content (mg/g)** & **Lutein Productivity (mg/L/d)** & **References** \\ \hline C. _sorokiniana_ FZU60 & 9.57 & 11.57 & [20] \\ _Parachlorella_ sp. JD-076 & 11.8 & 25 & [22] \\ _Desmodesmuts_ sp. F51 & 5.56 & 5.22 & [23] \\ _Scenedesmus obliquus_ FSP-3 & 4.52 & 4.15 & [17] \\ _C. sorokiniana_ MB-1 & 5.86 & 2.39 & [24] \\ \end{tabular}
\end{table} 
Table 1: Lutein content and productivity of microalgae strains under desirable cultivation conditions. Metabolic and genetic engineering are also used as effective tools to manipulate the rate-limiting step of the metabolic pathway for increasing lutein content and productivity in algal strains [33]. Although genetic engineering of algae is still in the initial stages, the use of this technique to enhance lutein content in microalgae has made progress in recent years. For example, new molecular technologies such as CRISPR/Cas9 genome engineering technology and genome sequencing have improved understanding of lutein metabolism regulation in algae species [34; 35; 36; 37]. Phytoene synthase (PSY) and phytoene desaturase (PDS) are essential enzymes that catalyze rate-limiting carotenoid biosynthesis steps in plants and algae. Overexpression of these genes may increase lutein content in algal cells. For example, the heterologous _PSY_ gene from _D. salina_ and _C. zofingiensis_ has been overexpressed in _C. reinhardtii_[29; 30]. The _PSY_ transformants exhibited a 1.6-fold and 2.0-fold increase of lutein content in transgenic cells compared with the wild type. The endogenous _PDS_ gene was overexpressed in _C. zofingiensis_ and resulted in a 32.1% increase in the total carotenoid content [31]. Another approach taken is to enhance the enzymatic activity of PSY. The orange (_OR_) gene encodes a plastid-associated protein, which triggers the chloroplast to chromoplast differentiation in plants. Many studies have focused on the OR protein's positive effect on the post-transcriptional regulation of carotenoids by stabilizing the PSY protein and maintaining the PSY protein level [38; 39; 40]. Recently, an _OR_ gene from arabidopsis (_AtOR_) was expressed in the alga _C. reinhardtii_, resulting in a 1.9-fold and 1.7-fold increase in lutein \(\beta\)-carotene, respectively [32]. In sum, research on the production of microalgae-based lutein has made remarkable progress. Recent work has approached the commercialization threshold of microalgal-based lutein production.