## 3 Biotechnology-Based Improvement of Astaxanthin Production in _X. dendrohous_  
Chemical synthesis is relatively complex even for simple carotenoids such as \(\beta\)-carotene. Thus, several companies and academic laboratories have investigated biological sources of astaxanthin. Among these sources, _P. rhodozyma/X. dendrohous_ and the microalga _H. pluvialis_ stand out because of their ability to synthesise astaxanthin. However, biological production of astaxanthin in natural isolates presents a well-known drawback; the very low specific production. Thus, the specific astaxanthin production of wild type strains of _X. dendrohous_ is 200-400 \(\mu\)g/g of yeast dry weight [61; 62]. Besides, the thickness of yeast cell walls and capsule, which apparently hinders astaxanthin uptake, seems to be also relevant for production and downstream processing. In order to obtain a competitive natural source of this xanthophyll it has been necessary to increase this production by a factor of 10-50. Classical random mutagenesis methods by using physical or chemicals agents have been successfully applied [63; 64; 34; 43; 65]. Some of these mutants obtained by random mutagenesis and screening with relevant dry cell weight productivity rates [_P. rhodozyma_ E5042 (2.5 mg/g); _X. dendrohous_ VKPM Y2476 (4.1 mg/g); _P. rhodozyma_ JMU-MVP14 (6.01 mg/g)] [66; 67; 68]. Nevertheless, one of the drawbacks of this approach is the introduction of secondary mutations that may affect the physiology, viability, or metabolic capacity of the cell. Besides, genetic instability of the mutants, reduction in the production of biomass, and accumulation of undesired intermediaries have frequently been detected [64; 69]. The selection of astaxanthin super-producer mutants in solid medium conditioned the detection of strains due to the low levels of aeration, which inhibit the production of oxygenated carotenoids. As a result, different selection procedures have been developed. On the one hand, positive selection by means of inhibitors of carotenogenesis (\(\beta\)-ionone, diphenylamine) has been developed [70]. On the other hand, negative selection, such as the increased sensitivity of carotenoid-producing strains to antimycin A (a respiratory inhibitor), has been another option [71; 72]. The yield improvement of microbial products has been traditionally faced up by means of: (i) treatment with mutagenic agents (e.g., nitrogen mustards, ultraviolet irradiation, X-ray); (ii) increasing the biosynthetic genes; or (iii) redirecting the metabolic precursors [73]. As a consequence, a promising strategy is metabolic engineering (see Table 1), which includes: (i) the overexpression of carotenoid biosynthetic genes (e.g., \(crtYB\) gene); (ii) the metabolic flow increase towards the synthesis of specific pathway precursor molecules (e.g., geranylgeranyl-pyrophosphate synthase encoding gene (\(crtE\))), or (iii) the supplementation with carotenogenesis precursors (e.g., mevalonate) [20; 47; 62; 74; 75]. However, this metabolic redirection, which can be included in the new trends of synthetic biology, has needed several methodological updates prior to validating the final effects along the astaxanthin production improvement history. As an example, transformation methods for _Xanthophyllomyces_ have been developed and optimised using (i) constitutive promoters from the yeast itself [e.g., actin, glyceraldehyde-3-phosphate dehydrogenase (\(gpd\)) or NADP-dependent glutamate dehydrogenase (\(gdhA\)) genes]; (ii) stable integration in the genome by means of the ribosomal DNA of _X. dendrohous_ introduced into the transformation vectors; and (iii) optimised resistance genes (e.g., kanamycin resistance gene of transposon Tn5 or hygromycin resistance gene (_lph_)) [21; 76]. Under optimum conditions, the transformation efficiency obtained was 1 \(\times\) 10\({}^{3}\) transformants per microgram of plasmid DNA [76]. The carotenoid pathway of _X. dendorhous_ is well established (Figure 1).