Screening And Characterizing High Astaxanthin Content Haematococcus Pluvialis And Metabolimic Profiling Of Astaxanthin Accumulation Process

Astaxanthin is one of caroteoids that have strong antioxidative capacity,it's a high value-added health food.Currently,Haematococcus pluvialis and yeast can be used to produce astaxanthin,the latter one is difficult for industrial production due to its poor production efficiency.Thus,industrial production of astaxanthin always uses H.pluvialis.For producing astaxanthin efficiently,we use ethyl methane sulfonate as mutagenic agent mutagenize H.pluvialis,and in order to research the reason of increased astaxanthin yield,we measured the content of intracellular fatty acid and transcriptional level of key genes in astaxanthin synthesis pathway.Meanwhile,we also applied metabonomics to study molecular mechanism of H.pluvialis in growth cycle and astaxanthin accumulation process.Results are as follows:(1)After preliminary screening,we obtained 997 strains and through secondary screening,51 strains were left.After further screening,we got seven mutants of H.pluvialis with higher growth rate,and astaxanthin yield were increased in six of them.The cell wall of mutants all had concaves while cell wall of wild-type was relatively smooth;but after hight light stress,wild-type and mutants all showed some concaves.(2)Octadecenoic acid is one of intracellular fatty acid that were increased in all 7 mutants.PSY gene and BKT gene are main restriction gene of astaxanthin biosynthesis pathway,the both transcription levels of mutants were increased.So,we infered that increased content of octadecenoic acid and increased transcription level of PSY gene and BKT gene were the main reasons of increased astaxanthin yield.(3)When H.pluvialis exposured to high light,the growth of H.pluvialis was divided into three phases,i.e.green phase,yellow phase and red phase based on the astaxanthin and chlorophyll contents.A total of 81 metabolites were analyzed by principal component analysis(PCA),partial least squares analysis(PLS)and hierarchical clustering analysis(HCA).The results suggested that three samples had a significantly different clustering,and cells exposured to high light might have led to systematic changes in cell metabolism pathways.Further research found that in yellow phase,the content of some cytoprotective metabolites such as sucrose,proline and glutamic acid,were higher than green phase.Their metabolic precursors glyceraldehyde 3-phosphate,pyruvic acid and a-ketoglutarate are the intermediates of the Calvin cycle and the TCA cycle,it's may implied those two cycles activity were enhanced.In conclusion,we infered that the metabolic regulation mode of H.pluvialis,i.e.high light enhanced the activity of Calvin cycle and TCA cycle,then promoted the accumulation of intracellular cytoprotective metabolites.In red phase,with the accumulation of astaxanthin,the content of intracellular metabolites were decreased.