Effects Of Inorganic Nitrogen And Short-Chain Carboxylic Acids On Metabolism Of Astaxanthin Biosynthesis By Xanthophyllomyces Dendrorhous

Astaxanthin is a high value-added carotenoid that can be used not only as a feed additive in special aquaculture,but also as a natural antioxidant and free radical scavenger for health foods and high grade cosmetics.In addition,animal tests have demonstrated that astaxanthin plays an important role in enhancing immunity and anticancer.Xanthophyllomyces dendrorhous have the advantages of short growth cycle,small footprint,and easy achieve of high-density cultivation,thus it has become a hot research object for producing natural pigment and high value-added product astaxanthin in recent years.At present,the production of astaxanthin from X.dendrorhous is faced with a situation of insufficient production capacity and low content.It is necessary to further optimize the production conditions and explore the synthesis mechanism of intracellular astaxanthin in X.dendrorhous for enhancing the intracellular astaxanthin content of X.dendrorhous.In this study,X.dendrorhous UV3-721,a laboratory-preserved strain,was used as a research strain.Few studies have studied the effects of inorganic nitrogen on the synthesis of astaxanthin from X.dendrorhous.Therefore,different ammonium salts and nitrates were selected to study their effects on cell growth and astaxanthin accumulation of X.dendrorhous.The regulation mechanism of ammonium nitrogen on the synthesis of astaxanthin was elucidated by studying the changes of the transcription level of the gene that encoding key enzyme in the astaxanthin synthesis pathway o f X.dendrorhous.and the relative abundance of X.dendrorhous intracellular small molecule metabolites.Meanwhile,the effects of four short-chain carboxylic acids related to TCA cycle on the growth and astaxanthin synthesis of X.dendrorhous were also studied.On this basis,the fermentation amplification technique of increasing the yield of astaxanthin by adding ammonium nitrogen and short-chain carboxylic acid was studied,and the cheap nitrogen source for the production of astaxanthin by X.dendrorhous was found.The specific results are as follows:First,the effects of adding different inorganic nitrogen on cell growth and astaxanthin synthesis of X.dendrorhous were investigated.The results show that the addition of ammonium nitrogen can enhance the ability of the cells to synthesize astaxanthin.Subsequently,the effects of adding time of ammonium sulfate and adding concentration on astaxanthin production by X.dendrorhous were investigated,and the optimal adding time of ammonium sulfate was determined as the initial cultivation period and the adding concentration was 1 g/L.The astaxanthin content of the X.dendrorhous reached 9.06 mg/g,which was 64.4%higher than the controlSecondly,the effects of ammonium sulfate on the expression of six key genes in the astaxanthin biosynthesis pathway were investigated.The results showed that the upstream genes ipi and crtE in the astaxanthin synthesis pathway were in the stagnation phase and logarithmic growth phase(0～72 h)shows a higher relative expression,which may be related to the synthesis of related precursors and other carotenoids in the preparation of the upstream pathway.The relative expression of the downstream genes crtYB,crtI,crtS and crtR was higher after the stable growth period(72～144 h),indicating that the upstream precursor substances and other carotenoids accumulated in the early stage were effectively flowed to the end products synthesis of astaxanthin.Thirdly,the changes of the relative abundance of intracellular small molecule metabolites in X.dendrorhous was studied.What changes obviously is the significant weakening of fatty acid metabolism in the ammonium sulfate experimental group,which led to the influx of more acetyl-CoA flow into the mevalonate synthesis upstream of the carotenoid synthesis pathway,thereby promoting the accumulation of arotenoids such as astaxanthin.Moreover,the accumulation of intracellular acidic substances in the cells causes the internal environment to be acidic.which is also beneficial to the accumulation of astaxanthin in the cells.Fourthly,the effects of four short-chain carboxylic acids(acetate,pyruvate,succinic acid and malic acid)related to the TCA cycle on the growth and astaxanthin synthesis of X.dendrorhous was studied.The results showed that adding 30 mM sodium acetate at 24 h could significantly increase the biomass of X.dendrorhous,and the biomass reached 4.66 g/L,which was about 38.2%higher than the control group.The adding of 40 mM pyruvate at 24 h promoted the X.dendrorhous growth and astaxanthin accumulation of X.dendrorhous to some extent.The strategy of adding pyruvate and ammonium sulfate to some extent alleviated the inhibition of ammonium sulfate on the growth of the cells.The yield and content of astaxanthin reached 33.24 mg/L and 8.20 mg/g,respectively,which is higher than the control group 33.3%and 36.4%,respectively.Fifthly,the fermentation amplification technique for improving the yield of astaxanthin by combined pyruvate and ammonium sulfate in a 5 L fermentor was investigated.The results showed that the biomass and astaxanthin content were significantly improved compared with the control group.After 6 days of fermentation.the maximum biomass reached 13.40 g/L,the total carotenoid and astaxanthin yields are 270.96 mg/L and 99.66 mg/L,and the astaxanthin content is 7.65 mg/g,40%,83.7%,and 54.5%higher than the control group,respectively.