High Through-put Screening And Evaluation Of Fucoxanthin Overproducing Mutant Of Phaeodactylum Tricornutum

Fucoxanthin belonging to allenic carotenoids is widely found in nature and consists of a polyene backbone.It is mainly originated from invertebrate cells and algae,including phaeophyceae,diatomaceae and chrysophyceae.It has been reported that fucoxanthin can be used as a safe and effective dietary supplement.Because of the unique structure of fucoxanthin,it has anti-inflammatory,anti-obesity,anti-tumor,anti-diabetes,anti-malarial effects and other physiological activities.Although fucoxanthin is a pigment with high utilization prospects,it's application is limited due to the low extraction efficiency from seaweeds,difficulties to synthesize chemically and low level of fucoxanthin in brown algae.Phaeodactylum tricornutum is a model species of diatoms.Fucoxanthin content of P.tricornutum ranges from15.42~16.51 mg·g-1,which is a suitable level for fucoxanthin production.In order to improve the production yield of fucoxanthin,physical and chemical mutagenesis could be applied to generate mutants with better performance.However,a rapid method to screen for mutants with higher fucoxanthin content is needed to accelerate the discovery of high-content strains.This paper focuses on the following three aspects:(1)In our previous work,heavy ion irradiation was used for mutagenesis and preparing mutant pools.More than 800 mutant strains were selected and analyzed for OD value,fucoxanthin contents and parameters of the photosynthetic system.We also combined with Rose Bengal stress to screen the mutants.According to the results,12 mutant strains have better phenotypic character than the wild type under Rose Bengal stress.There are 6 strains having significantly higher fucoxanthin productivity than wild type.The fucoxanthin content in the mutant strains ranged between 50.01 mg·L-1 and 62.3mg·L-1 compared to the fucoxanthin content of the wild type which was 35.9 mg·L-1.This method proved to be a suitable for screening mutants with high fucoxanthin content.(2)At present,the concentration of pigments is mainly determined by high performance liquid chromatography(HPLC),which needs at least 3 hours to analyze one sample.In this study,we show that the concentration of fucoxanthin can be assessed using a spectrophotometer measuring values at 663 nm,445 nm,and 750 nm.This method could analyze one sample in 5~10 minutes.This spectrophotometric method is easier and faster than HPLC and the standard error was less than 5% when compared tothe HPLC results.Also,this method can be applied to other diatoms,with standard errors of 3~14.6%.This high-throughput method could improve the efficiency of the mutant screening process.(3)Flow cytometry(FCM),a widely used technology,is convenient for cell pigment analysis and has high sensitivity and accuracy.In previous work,the method of screening fucoxanthin-producing microalgae by FCM has not been reported yet.According to the spectral information of fucoxanthin,we analyzed the correlation between mean fluorescence intensity(MFI)and fucoxanthin content of the cells.The results demonstrated that the Per CP-Cy5-5 channel is better for cells sorting.Based on the results,1×105 positive and negative mutation cells were selected,respectively.Compared to wild type,the positive group achieved 25.5% increase of fucoxanthin content;the negative group decreased 12.6%.Four single clones were randomly selected and cultured under high temperature condition,it was shown that 3 of the 4 mutant strains had higher fucoxanthin productivity than the wild type.This method provides a rapid and efficient way to screen mutant and monitor cellular pigments.