## 3 Biotechnology-Based Improvement of Astaxanthin Production in _X. dendrohous_
This fact has enabled the cloning process of all genes involved in phytoene synthesis, phytoene desaturation, and cyclisation and formation of 3-hydroxy and 4-keto. Thus, knowledge of the molecular biology of _X. dendorhous_ allows researchers to direct the genetic modifications and increase the metabolic precursors flow to the carotenoid biosynthetic pathway [48; 59; 79; 80]. In order to divert metabolite flow from the sterol pathway towards carotenoid biosynthesis, _X. dendorhous_ was transformed with the _crtE_ cDNA (geranylgeranyl pyrophosphate synthase). Transformants were obtained with higher carotenoid levels including astaxanthin due to increased levels of geranylgeranyl pyrophosphate synthase [74]. The genetic improvement of _Xanthophyllomyces_ strains by metabolic engineering has been achieved by upregulating phytoene synthase and lycopene cyclase leading to an increase in \(\beta\)-carotene and echinenone titres. These results suggested that the oxygenation reactions might be rate limiting [48]. The combination of chemical mutagenesis and genetic engineering by targeting limiting reactions (including overexpression of _crtYB_ and _crtS_ genes) led to the generation of an overproducing strain of astaxanthin (9.7 mg per gram of dry weight) [75]. These authors described the construction of new transformation plasmids for the stepwise expression of the bottleneck genes in the carotenoid biosynthetic pathway. As a result, titres were comparable to those provided by _H. pluvialis_, the leading commercial producer of natural astaxanthin [22]. Enhancement of _crtS_ gene transcriptional levels led to an increase in the transcription levels of related genes (_crtE_, _crtYB_, _crtI_) in the astaxanthin biosynthetic pathway. A scheme of carotenoid biosynthesis in _X. dendorhous_ involving alternative bicyclic and monocyclic pathways was proposed by [PERSON] and co-workers in 2015 [81]. \begin{table}
\begin{tabular}{c c c c} \hline
**Targets** & **Approach** & **Result** & **Ref.** \\ \hline _crtYB_ gene & Deactivation & No carotenoids & [48] \\ \hline _crtYB_ gene & Overexpression & Accumulation of \(\beta\)-carotene and echinenone & [48] \\ \hline _crtl_ gene & Overexpression & Increase torulene and HDCO and decrease echinenone, \(\iota\)-carotene and astaxanthin & [48] \\ \hline _crtR_ gene & Description of its role & Required together with the _crtS_ gene for the conversion of \(\beta\)-carotene to astaxanthin. & [59] \\ \hline double _cyp61_ genes & Deletion & Enhanced astaxanthin production by 1,4-fold compared with the parental strain & [77] \\ \hline _crtE_ gene under Padh4r & Evaluation of promotors & Increase in intracellular astaxanthin by 1.7-fold compared with parental & [78] \\ \hline _acT, lmgS_ and _lmgR_ genes & Triple overexpression & Enhanced volumetric astaxanthin production by 1.4-fold compared with that of the control strain & [23] \\ \hline _acT/lmgS/lmgR/crtE/crtS_ genes & Combined overexpression & Enhanced volumetric astaxanthin production by 2.1-fold higher compared with the control strain & [23] \\ \hline Combination of \(\beta\)-conventional mutagenesis and _crtYB_ gene expression & Combined overexpression & 22 times higher astaxanthin specific production than for the wild type & [75] \\ \hline \end{tabular}
\end{table} 
Table 1: Examples summary of _X. dendorhous_ (_P. rhodozyma_) genetic engineering. The limiting step in the carotenoid pathway is phytoene synthase. In order to increase the _crtYB_ gene copy number, integration plasmids were constructed. Thus, the transformants with higher copy number accumulated carotenoid intermediates missed in the parental strain. Some of them are substrates and intermediates of astaxanthin synthase, which can be transformed to astaxanthin by the addition of the astaxanthin synthase gene [82]. Recently, a system to completely delete target diploid genes in _X. dendrorho_ was developed. Diploid _CYP61_ genes involved in the synthesis of ergosterol that inhibits the pathway for mevalonate (substrate for isoprenoid biosynthesis), were deleted.