# Challenges and Potential in Increasing Lutein Content in Microalgae
## 2 Lutein Sequestration Versus Structural Function  
Sequestration capacity is critical for lutein accumulation because the structural form of this pigment, with an essantial role in fulfilling photosynthetic functions, only has a minimal content.  
The accumulation of lutein is the ultimate result of three major processes: biosynthesis, degradation and sequestration. The biosynthesis of lutein has been extensively studied and reviewed. This includes the metabolic pathway, transcript regulation and post transcript regulation [2; 43; 44; 45]. The degradation of lutein and other carotenoids by carotenoid cleavage dioxygenases (CCDs) has also garnered much attention [46; 47]. Moreover, carotenoid sequestration has been recognized as an essential factor for carotenoid accumulation in  
\begin{table}
\begin{tabular}{c c c c} \hline
**Microalgal Strains** & **Strategy** & **Lutein Yield (mg/g)** & **References** \\ \hline _C. sorokiniana_ MB-1-M12 & Random mutagenesis & 7.52 & [24] \\ _C. sorokiniana_ mutant & Random mutagenesis & 7.0 & [16] \\ DMR-56\&8 & Dysfunction of _BKT1_ by chemical & \\ _C. zofingiensis_ mutant CZ-bkt1 & mutation & 13.81 & [28] \\ _Chlamydomonas reinhardtii_ & Overexpression _PSY_ gene from _Dunaliella salina_ & 2.2-fold increase of lutein content & [29] \\ _C. reinhardtii_ & Overexpression _PSY_ gene from _C. zofingiensis_ & 2.2-fold increase of lutein content & [30] \\ _C. zofingiensis_ & Overexpressing _PDS_ gene & Total carotenoid content increased & \\ _C. reinhardtii_ & Overexpression _OR_ gene from _A. thaliana_ & 1.9-fold increase of lutein content & [32] \\ \hline \end{tabular}
\end{table}
Table 2: Examples of mutagenesis and metabolic engineering strategies for altering lutein content in microalgae strains.  
plants [45; 48; 49]. In contrast, the limitation of lutein sequestration in algae has been neglected. This work thus focuses on surveying current findings and knowledge related to lutein sequestration in microalgae.  
As illustrated in Figure 1, the biosynthesis of lutein occurs on the inner chloroplast membrane of plants and algae [50]. Due to the lipophilic characteristic of the unsaturated polyene chain and the hydrophobic hydroxyl groups on the rings, it is difficult for lutein to float freely in the hydrophilic environment of the chloroplast and attaches tightly to the membrane. Thus, once lutein is synthesized, lutein accumulates on the inner chloroplast membrane. Without proper sequestration, this leads to feedback inhibition on lutein synthesis or degradation by ROS or CCDs. Therefore, lutein sequestration is essential for lutein accumulation, and the storage capacity of lutein might determine the upper limit of lutein content in the organisms.  
Based on current knowledge, lutein could bond to LHC-associated proteins as a structural and functional pigment involved in light-harvesting and nonphotochemical quenching (NPQ) in plant green tissues and microalgae [1; 51]. Lutein could also be stored in carotenoid-lipoprotein substructures and the plastoglobulin, an oil environment, within the chromoplasts of flower petals such as marigold. It was found that lutein could be massively accumulated in the plastoglobulin of the petals as an \"oil form\" [52; 53; 54]. In contrast, lutein content is usually low in the leaves as a \"structural form\" in marigold. This difference in the two lutein sequestration methods might lead to the variance in the level of lutein accumulation. Therefore, exploring the storage capacity of lutein under the two methods can provide a rough estimation of the level of content in lutein producing organisms.  
A standard is required to estimate the storage capacity of lutein in LHCs and lipid droplets, but there is not a widely accepted, accurate standard to address this problem. Based on current knowledge, the mass ratio between the accumulated lutein and the lutein storage medium can possibly be used to estimate the capacity of lutein sequestration.