# Marine Microalgae for Potential Lutein Production
## 4 Engineering of Biosynthetic Pathways in Microalgae for Lutein Production  
Microalgae as biomolecule production platforms have long been explored through engineering of their biosynthetic pathways by chemical mutagenesis as well as targeted genetic engineering of specific genes. Chemical mutagens such as \(N\)-methyl-\(N^{\prime}\)-nitro-\(N\)-nitroosquanidine (MNNG) and Ethyl Methane Sulfonate (EMS) have been successfully used for generating microalgal mutants with high contents of carotenoids including lutein [21; 28; 29; 30]. In a recent study, a lutein-deficient _Chlorella vulgaris_ (CvLD) strain was generated by chemical random mutagenesis and the strain was identified as an enhanced producer of violaxanthin [21]. Sequence analysis of the lycopene \(\varepsilon\)-cyclase gene (_lcy-e_) of the mutant strain CvLD revealed a single mutation (A336V), which might have resulted in a conformational change of the conserved region of CvLCYE (lycopene \(\varepsilon\)-cyclase) with reduced biochemical activity. For genetic engineering of microalgae, robust genetic transformation protocols for the nuclear, chloroplast, or mitochondrial genomes are available [31]. The key methods for delivering DNA to microalgae are electroporation, shaking of cells with glass beads, and the biolistic (particle gun bombardment) method [32]. Most of the enzymes of secondary metabolism are encoded within the nuclear DNA; however, some of these enzymes are actually targeted to function in the chloroplast [33]. Since most of the enzymes of the carotenogenic pathway are found in the microalgal chloroplast, it was suggested that nuclear and/or chloroplast transformation can be used for the metabolic engineering of the carotenoid biosynthetic pathway [34]. Genetic engineering of microalgae through chloroplast transformation was mostly achieved by the biolistic method [35]. Although the low expression level of the target gene is the main drawback of nuclear transformation, it is the method of choice when appropriate post translational modifications of the target protein are essential [31]. The phytoene synthase gene (_psy_) from _Dunaliella salina_[28] and _Chlorella zofingiensis_[36] was nuclear transformed to the model microalga _Chlamydomonas reinhardtii_, and the derivative strains, respectively, produced 2.6- and 2.2-fold higher amounts of lutein compared to the wild type strain. Another study with the microalga _Chlamydomonas reinhardtii_ involved a point mutation of the native gene for phytoene desaturase (_pds_) which was then nuclear transformed. The derivative strain, consisting of the mutant enzyme with increased desaturase activity biosynthesis, increased the amount of lutein, \(\beta\)-carotene, zeaxanthin, and violaxanthin [37]. In a very similar approach, [PERSON] et al. [38] nuclear-transformed _Chlorella zofingiensis_ with a mutant version of the native _pds_ gene. The derivative strain, consisting of the mutant phytoene desaturase enzyme with increased desaturase activity, accumulated a higher amount of total carotenoids (32%) and astaxanthin (54%) compared to the parent strain. In a recent metabolic engineering study with _Chlamydomonas reinhardtii_, the gene for phytoene-\(\beta\)-carotene synthase (_pbs_) from the red yeast _Xanthophyllomyces dendorrhoea_ was cloned into pMS188 vector and nuclear transformed [39]. This derivative strain possesses the bifunctional enzyme with both phytoene synthase (_psy_) and lycopene cyclisation (_lcy-b_) activities. This is the first heterologous expression system for carotenoids biosynthesis, which resulted in a simultaneous increase in lutein (60%) and \(\beta\)-carotene (38%) under low light conditions. In the lutein biosynthetic pathway, the lycopene \(\varepsilon\)-cyclase gene (_lcy-e_) acts as a key regulator of the \(\alpha\)-branch, while lycopene \(\beta\)-cyclase (_lcy-b_) acts in both the \(\alpha\)-branch and \(\beta\)-branch.