Study On The Regulation Mechanism Of Exogenous Additives On Astaxanthin Synthesis By Xanthophyllomyces Dendrorhous

Astaxanthin is an important ketocarotenoid that is widely used in the food,aquaculture,nutraceutical and pharmaceutical industries because of its strong antioxidant activity and numerous biological functions.The basidiomycetous red yeast Xanthophyllomyces dendrorhous was regarded as one of the best resources for natural astaxanthin due to its ability to accumulate large amounts of astaxanthin through high-density fermentation and easy cultivation.However,there are some problems in application of X.dendrorhous,such as low yield and high cost.These are due to the fact that the fermentation strategies have not been well optimized,and also the metabolic regulation mechanism is still not clear.Therefore,in this study,the effects of exogenous additives on the cell growth and astaxanthin synthesis of X.dendrorhous was studied and their metabolic mechanisms were analyzed with comparative metabonomics and transcriptome.The aim of this study was to provide a better understanding of the mechanism of cell growth and astaxanthin biosynthesis in X.dendrorhous.In this study,the effects of phytohormone on cell growth and astaxanthin synthesis of X.dendrorhous were investigated.The results showed that,the optimal phytohormone was determined as 6-benzylaminopurine(6-BAP)and its optimized adding concentration and time were 0.25 mg/L at 24 h.The highest biomass of 3.33 g/L,the highest astaxanthin yield of 15.57 mg/L and the maximum astaxanthin content of 4.67 mg/g DCW were achieved,which were 22.0%,34.1%and 24.2%higher than those of the control group,respectively.It indicated that it was beneficial to improve astaxanthin accumulation by feeding phytohormone and present a new strategy to improve astaxanthin production in X.dendrorhous.To further understand the metabolic response of X.dendrorhous to 6-BAP,time-course metabolomics and gene expression levels of X.dendrorhous cultures with and without 6-BAP feeding were investigated.In total,39 metabolites were identified by GC-MS,and most of these metabolites were primarily associated with tricarboxylic acid cycle(TCA cycle),glycolysis,carbohydrate,amino acids and fatty acids.The metabolic analysis revealed that 6-BAP facilitated glucose consumption,promoted the metabolic flux from glycolysis,suppressed the TCA cycle,drove carbon flux into fatty acid and mevalonate biosynthesis,and finally facilitated the formation of astaxanthin.ROS analysis suggested that the anti-stress mechanism in X.dendrorhous might be induced by 6-BAP.Additionally,the process of 6-BAP significantly upregulated the expression of six key genes(hmgR,idi,crtE,crtYB,crtI and crtS)involved in pathways related to astaxanthin biosynthesis.This research demonstrates the metabolomic mechanism of phytohormone stimulation of astaxanthin production in X.dendrorhous,which established the fundamental for large-scale production.Finally,the effect of Na-citrate on cell growth and astaxanthin synthesis of X.dendrorhous was studied.The results show that,its optimized adding concentration and time were 3 g/L at 24 h.The highest biomass and astaxanthin yield were 6.04 g/L and 22.53 mg/L,which were 1.8 and 2.0 times higher than those of the control group,respectively.It indicated that it was beneficial to improve astaxanthin accumulation by feeding Na-citrate.Furthermore,transcriptomics and metabolomics analysis was conducted to study the metabolic pathways of X.dendrorhous in response to Na-citrate.The metabolic analysis revealed that Na-citrate facilitated glucose consumption,promoted the metabolic flux from glycolysis,regulated the TCA cycle,thereby providing more energy and materials for the synthesis of astaxanthin.The inhibition of protein synthesis also provides sufficient acetyl-CoA,ATP,and NADPH for the synthesis of astaxanthin.The gene analysis revealed that the addition of Na-citrate can promote the up-regulated expression of glycolytic genes,sugar uptake-related genes on cell membrane,and key genes of mevalonate.It also can promote the up-regulation of cellular oxidative stress-related genes,which may be important genes in astaxanthin synthesis.Based on the above research,this work firstly reports the effect of 6-BAP and Na-citrate regulation on astaxanthin synthesis in X.dendrorhous and combining metabolomics and transcriptomics analysis,the cell metabolic mechanism of astaxanthin synthesis by X.dendrorhous was explored,which provides an effective fermentation strategy and a new perspective for regulating the production of astaxanthin by X.dendrorhous...