Optimization Of Autotrophic Cultivation Conditions And Related Regulation Mechanism Of Lutein Accumulation In Marine Chlamydomonas

Lutein is an important photosynthetic pigment,which is used extensively as feed additive and effective functional nutrient due to its bright color and the function of protecting cells against photo-oxidative damage.Comparing with marigold,using marine microalgae as the lutein feedstock has many advantages such as high yield,producing several value-added bioproduct and mitigating the shortage of fresh water resources.In this paper,the lutein productivity of Chlamydomonas sp.JSC4 was improved by optimizing the culture conditions.Moreover,the regulation mechanism of lutein accumulation in strain JSC4 was explored by analyzing the transcriptome and expression levels of related genes.The main results are as follows:The different nutritional conditions were individually manipulated to improve the cell growth and lutein production of strain JSC4.The results show that the optimal cell growth and lutein accumulation were obtained in Modified Bold Basal 3N medium with a nitrate-N concentration of 1000 mg/L and a sea salt concentration of 2%.Then,an innovative salinity-gradient operation strategy was used to dramatically improve the lutein productivity of strain JSC4 to 1.92 mg/L/d,which simulated the evaroparation of water and the increase of salinity in the outdoor culture process,providing favorable support for the large-scale lutein production.The light wavelength and temperature were individually operated to improve the cell growth and lutein production of strain JSC4.The results show that the optimal cell growth was obtained under white light and a temperature of 35?,while the optimal lutein content was obtained under blue light and a lower temperature of 20-25?.Moreover,the temperature-gradient strategy and two-stage strategy combining light quality and temperature were used to further improve lutein production.Among them,the two-stage strategy could significantly improve the lutein content from 2.52 to 4.24mg/g,resulting in the highest lutein productivity of 3.25 mg/L/d.The transcriptome sequencing of strain JSC4 was investigated using the high-throughput sequencing technology of Illumina HISeq^TM 2000.The obtained unigenes were compared with databases of NR?NT?KO?Swiss Prot?PFAM?GO?KOG.The carotenoid biosynthesis pathway of strain JSC4 was further elucidated based on the results of transcriptome functional annotation.The effect of light intensity on lutein accumulation of strain JSC4 was investigated.The results show that high lutein productivity(5.08 mg/L/d)was achieved under high light irradiation of 625?mol/m²/s,which was higher than the results obtained in most related studies using phototrophic batch cultivation.It was worth noting that high light condition was conducive to improving cell growth rate but led to a decrease in the lutein content.In order to further explore the regulatory mechanism of lutein accumulation in microalgal cells under different light intensities,the transcription levels of key genes in the lutein synthesis pathway were analyzed.It was found that the transcription level of lut1 gene was significantly down-regulated under high light condition,which was consistent with the change trend of lutein content.The effect of temperature on cell growth and the profiles of cell components in strain JSC4 was studied.The results show that the low temperature(20?)condition could enhance lutein and unsaturated fatty acids accumulation in strain JSC4,while the highest contents of carbohydrate,protein and lipid were obtained at 40?,30? and20?,respectively.It was also found that the transcription levels of the psy,pds,crt L-b,crt Z,zep,and lut1 genes involved in carotenoid synthesis pathway,as well as the bccp,bc,?ct and?ct genes involved in lipid synthesis pathway,were upregulated obviously at low temperature(20?)condition.