## 6 Recent Applications in Metabolic Engineering for Xanthophylls Production  
Microalgae are considered to be ideal model hosts for metabolic engineering since they offer many advantages, such as a simplicity of culture and fast growth rates compared with plants. Furthermore, eukaryotic microalgae present more genetic and physiological similarities with plant cells than bacteria [96]. In addition, many microalgae have an active central terpenoid metabolism, allowing a great enough supply of precursors and a high capacity for xanthophylls storage [96]. Nevertheless, studies into the modification of these pigment pathways in microalgae are infrequent. Mutagenesis, with its various methods, has been applied to wild strains of microalgae to improve their xanthophyll production. In 2001, [PERSON] and collaborators used mutagenesis to increase production of zeaxanthin by _Dunaliella salina_ and ultimately succeeded in generating two overproducing strains of zeaxanthin. One of these zeaxanthin epoxidase mutants was recognized in the study carried out by [PERSON] and [PERSON] in 2003 [106]. Similar mutations have also been conducted in other microalgae strains, such as _Scenedesmus obliquus_ and _Chlamydomonas reinhardtii_[107]. The zeaxanthin content (per cell) is 15-fold higher than the wild type under non-stressed conditions [108]. Zexaanthin has previously been engineered by chemical mutagenesis into _Chlorella zofingiensis_[109]. Its production reached 4 and 7 mg/g DW in mixotrophic and photosynthetic conditions, respectively [109]. Manipulation of microalgae to make them able to produce higher quantities of xanthophylls can also been carried out by the inactivation or the overexpression of own genes or by the expression of genes from other species [16]. This would allow us to obtain genetically modified species of microalgae with ameliorated or decreased enzymatic activities and able to accumulate the desired pigments. Recently, in 2018, [PERSON] et al. introduced the exogenous \(\beta\)-carotene oxygenase gene (CrtR) from the strain _Synechococcus elongatus_ (PCC 7002), to prolong carotenoid pathway toward zeaxanthin production in vivo [110]. Associating the CrtR gene with the insertion of the galactose transporter from _Escherichia coli_ led to respective zeaxanthin yields of 9 mg/g DW and 8 mg/g DW in autotrophic and mixotrophic conditions [110]. ASX is commercially produced, generally for its human health benefits, and is used as an anti-inflammatory compound and as a skin protector. It can also be used as an essential component of aquaculture feed [71; 111]. _Haematococcus pluvialis_, the most common strain of microalgae, used for the production of ASX, accumulates between 9 and 36 mg/g DW of this pigment depending on culture conditions. However, production of ASX by other species is currently under study to bypass the constraints of culture observed with _Haematococcus_ strains [112]. Even if _Synechocystis_ sp., a cyanobacteria, does not naturally express the genes encoding for BKT and CRTR-B, and the enzymes can be engineered using genes from _Haematococcus pluvialis_ under the promoters cpc560 for BKT and psbA2 CrtR-B [113]. These successful gene expressions led to the conversion of ASX precursors echinenone and zeaxanthin, into 4.8 mg/g DW ASX in nitrogen deprivation [113]. Similarly, the _Chlamydomonas reinhardtii_ UVM4 strain mutant has become able to produce ASX after a redesign by codon optimization of the Bkt gene. The latter was a dysfunctional endogenous gene [114] which was targeted at the chloroplast with a psaD transit sequence. Hence, ASX production yields of 1 and 3 mg/L/day in autotrophy and mixotrophy conditions were respectively achieved [114]. Additionally, the endogenous PDS was recently overexpressed in the chloroplast of _Haematococcus pluvialis_ by optimizing codons and using the psbA promoter and UTR [115], demonstrating the capacity of expressing a nuclear gene successfully in _Haematococcus pluvialis_ chloroplast.