The SREBP (Sterol Regulatory Element-Binding Protein) pathway: a regulatory bridge between carotenogenesis and sterol biosynthesis in the carotenogenic yeast Xanthophyllomyces dendrorhous

Xanthophyllomyces dendrorhous is a basidiomycete yeast that naturally produces the red–orange carotenoid astaxanthin, which has remarkable antioxidant properties. The biosynthesis of carotenoids and sterols share some common elements that have been studied in X. dendrorhous. For example, their synthesis requires metabolites derived from the mevalonate pathway and in both specific pathways, cytochrome P450 enzymes are involved that share a single cytochrome P450 reductase, CrtR, which is essential for astaxanthin biosynthesis, but is replaceable for ergosterol biosynthesis. Research on the regulation of carotenoid biosynthesis is still limited in X. dendrorhous; however, it is known that the Sterol Regulatory Element-Binding Protein (SREBP) pathway, which is a conserved regulatory pathway involved in the control of lipid metabolism, also regulates carotenoid production in X. dendrorhous. This review addresses the similarities and differences that have been observed between mammal and fungal SREBP pathways and what it is known about this pathway regarding the regulation of the production of carotenoids and sterols in X. dendrorhous.

Rice Straw Hydrolysis and Cultivation of Xanthophyllomyces dendrorhous for Astaxanthin Production and Antioxidant Properties

Astaxanthin is a natural pigment with strong antioxidant activity and is widely supplied as dietary supplement. The red yeast Xanthophyllomyces dendrorhous is one of the potential sources for astaxanthin production. Rice straw was considered for utilization on cultivation and astaxanthin production. Rice straw was treated by autoclave-assisted alkaline pretreatment. and hydrolyzed by cellulase hydrolysis. X. dendrorhous TISTR5730 was cultured and accumulated as the astaxanthin on rice straw hydrolysate. The highest reducing sugar concentration of rice straw hydrolysate was 89.82±0.39g/L (0.71±0.01g/g) when using rice straw (3.3%) and cellulase loading (3 mL). High cell density X. dendrohous TISTR5730 cultivation on rice straw hydrolysate with 20 and 40 g/L of initial reducing sugar was investigated. 40 g/L was appropriate for biomass production while 20 g/L was suitable for astaxanthin accumulation. The highest astaxanthin content was 417.28±50.89 μg/g cell basis at 192 h. Astaxanthin productivity and yield coefficient were 0.01±0.00 mg/L/h and 0.11±0.01 mg/g sugar consumed. The antioxidant activities of astaxanthin were determined by DPPH and ABTS scavenging and FRAP reducing power. The produced astaxanthin represented the high antioxidant activities with IC50 of 9.30 and 1.67 μg/mL of DPPH and ABTS scavenging respectively and FRAP reducing power of 5.31±0.07 μg TEAC/mL. This research indicated that rice straw hydrolysate could be an alternative medium for astaxanthin production. Antioxidant activity of astaxanthin was proved and was feasible for further applications.

Metabolic engineering for high yield synthesis of astaxanthin in Xanthophyllomyces dendrorhous

Astaxanthin is a carotenoid with a number of assets useful for the food, cosmetic and pharmaceutical industries. Nowadays, it is mainly produced by chemical synthesis. However, the process leads to an enantiomeric mixture where the biologically assimilable forms (3R, 3′R or 3S, 3′S) are a minority. Microbial production of (3R, 3′R) astaxanthin by Xanthophyllomyces dendrorhous is an appealing alternative due to its fast growth rate and easy large-scale production. In order to increase X. dendrorhous astaxanthin yields, random mutant strains able to produce from 6 to 10 mg/g dry mass have been generated; nevertheless, they often are unstable. On the other hand, site-directed mutant strains have also been obtained, but they increase only the yield of non-astaxanthin carotenoids. In this review, we insightfully analyze the metabolic carbon flow converging in astaxanthin biosynthesis and, by integrating the biological features of X. dendrorhous with available metabolic, genomic, transcriptomic, and proteomic data, as well as the knowledge gained with random and site-directed mutants that lead to increased carotenoids yield, we propose new metabolic engineering targets to increase astaxanthin biosynthesis.

The cause of on-target point mutations generated by CRISPR-Cas9 treatment in the yeast Xanthophyllomyces dendrorhous

ABSTRACTRecognizing outcomes of DNA repair induced by CRISPR-Cas9 cutting is vital for precise genome editing. Reported DNA repair outcomes after Cas9 cutting include deletions/insertions and low frequency of genomic rearrangements and nucleotide substitutions. Thus far, substitution mutations caused by CRISPR-Cas9 has not attracted much attention. Here, we identified on-target point mutations induced by CRISPR-Cas9 treatment in the yeast Xanthophyllomyces dendrorhous by Sanger and Illumina sequencing. Different from previous studies, our findings suggested that the on-target mutations are not random and they cannot render the gRNA effective. Moreover, these point mutations showed strong sequence dependence that is not consistent with the observations in Hela cells, in which CRISPR-mediated substitutions were considered lacking sequence dependence and conversion preferences. Furthermore, this study demonstrated that the NHEJ components Ku70, Ku80, Mre11, or RAD50, and the overlapping roles of non-essential DNA polymerases were necessary for the emergence of point mutations, increasing the knowledge on CRISPR-Cas9 mediated DNA repair.

Improvement of a Specific Culture Medium Based on Industrial Glucose for Carotenoid Production by Xanthophyllomyces dendrorhous

In this study, a low-cost chemically defined (CD) culture medium was proposed and evaluated with the aim of replacing culture media such as yeast mold (YM) and yeast peptone dextrose (YPD), commonly used for growth and carotenoid production by Xanthophyllomyces dendrorhous. Initially, the CD culture medium was compared to the YM and YPD. The growth in optical density (OD) and carotenoid production (mg/L) of the cultures reached 4.88, 6.76, 5.79, and 0.67, 0.92, and 0.69, respectively. The CD culture served as the basis of an improved specific culture medium containing industrial glucose. Additionally, in this new formulation, vitamins, glutamate, and other compounds were evaluated. Industrial glucose more than doubled carotenoid production; however, the addition of vitamins was not essential for X. dendrorhous cultivation. Moreover, glutamate and Na2HPO4 proved to be highly significant factors (p-value < 0.05), increasing carotenoid biosynthesis from 0.67 to 1.33 mg/L. The specific culture was successfully used in a bioreactor at 2 L and 110 L pilot-scale levels, increasing carotenoid production up to 2 mg/L. It was demonstrated that the CD-specific culture medium is an efficient alternative to conventional culture media to carry out carotenoid production at the laboratory and pilot levels, with promising potential for industrial scaling.