Strategies For Improving Astaxanthin Synthesis And Studies On Regulation Of Metabolic Pathway In Phaffia Rhodozyma

Astaxanthin is a kind of xanthophyll possessing multiple biological activities, and it has great commercial potential used for the aquaculture, food, pharmaceutical and cosmetics industries. Phaffia rhodozyma is an astaxanthin-producing yeast used as feed additive. However, suppression of astaxanthin synthesis by high temperature and high concentration of glucose become application bottlenecks in large-scale astaxanthin production by P. rhodozyma. A moderate-temperature, glucose-tolerance, hyperprodution mutant strain of P. rhodozyma, termed MK19, was selected by mutagenesis in previous studies. The new strategies were presented for farther improving astaxanthin production, and the characteristic of carotenogenic metabolic flux was analyzed in terms of metabolic regulation in this study.It was indicated that oxidative stress created by H2O2 and salinity could stimulate the increase of astaxanthin yield in MK19 by 300% and 63% respectively. H2O2 feeding at early culture stage and 0.1% initial NaCl concentration were conductive to promoting astaxanthin accumulation. Based on the results of carotenoid profile, transcriptional pattern of carotenogenic genes, the level of catalase (CAT) and H2O2, major astaxanthin-stimulating regulation mechanism were included:1) more isoprenoid precursor was transferred into astaxanthin biosynthesis flux; 2) β-carotene and the unknown carotenoid were converted to astaxanthin; 3) expression of crtI and crtE gene was enhanced by oxidative stress imposed by H2O2 and salinity; 4) compared with CAT, astaxanthin was prioritized for quenching reactive oxygen species (ROS).An accumulated unknown carotenoid was found in MK19, and it was determined as 3-hydroxy-3’, 4’-didehydro-β,Ψ-carotene-4-one (HDCO) by HPLC, mass spectrometry and NMR spectroscopy. HDCO is the major product of the monocyclic carotenoid biosynthesis pathway in P. rhodozyma. A CrtS (astaxanthin synthase)-overexpressing strain (CSR19) was constructed successfully, whose astaxanthin yield was 33.5% higher than MK19 (25.3 mg/L), and the proportion of astaxanthin as a percentage of total carotenoids was 84%. The results of q-PCR analysis showed that enhancement of crtS transcriptional level increased transcriptional levels of related genes(crtE, crtYB and crtI) in the astaxanthin synthesis pathway and carotenogenic genes were co-regulated. CrtS was found to be a bifunctional enzyme that helped convert HDCO to astaxanthin. A scheme of carotenoid biosynthesis in P. rhodozyma involving alternative bicyclic and monocyclic pathways was firstly proposed. The present findings help clarify the regulatory mechanisms that underlie metabolic fluxes in P. rhodozyma carotenoid biosynthesis pathways and to provide feasible strategies for improving the astaxanthin yield.Astaxanthin synthesis of MK19 could tolerate 110 g/L initial glucose concentration, which is the highest glucose tolerance level reported up to now. Analysis at the metabolic and transcript levels illustrates that deregulation of crtYB gene was the main course of de-inhibition of astaxanthin synthesis by glucose. Through 5’RACE and promoter truncation experiments, the functional promoter region of crtYB gene was defined. CREA was found for the first time as a glucose metabolic repressor regulating crtYB expression in P. rhodozyma after homology comparison through Genbank and functional verification. Compared with wild-type strain, in MK19 the expression level of creA was lower and most of CREA was in inactive state. Thus resulting in deregulation of carotenogenic gene transcription by glucose. The present results not only explained the reason for promotion of glucose tolerance in MK19, but also provided important clues for further improving astaxanthin production capacity.This study explored strategies for increasing astaxanthin yield by regulating oxidative stress and carbon source. Stable, secure astaxanthin-hyperproducing strains were constructed. By means of metabolite tracking, gene transcriptional level and bioinformatics analysis, the present findings revealed the two astaxanthin-synthesis pathways and its regulating characteristics in P. rhodozyma.