A Preliminary Study On The CDNA Cloning Of Wakame ?-carotene Desaturase And Its Function

Fucoxanthin contributes more than 10% of the total carotenoids in seaweed and exhibits various bioactivities.However,several steps in its biosynthesis pathway are still unclear and rare genes coding key enzymes in this pathway in brown algae has been studied.In this study,the cDNA encoding?-carotene desaturase(ZDS),a key enzyme which catalyzes the formation of lycopene,was obtained by RT-PCR and 5'/3' RACE method from brown seaweed U.pinnatifida Suringar(UpZDS).UpZDS gene contained an open reading frame(ORF)of 1821 bp in length which encoded 606 amino acid residues.The results of bioinformatics analysis of UpZDS gene coding protein showed the presence of a chloroplast signal peptide composed of 51 amino acid residues at its N-terminus,a consensus Gly-rich sequence and a NAD/FAD binding domain.Homologous analysis indicated that UpZDS protein was highly conservative to ZDS from other plants.According to the results of a genetic complementation experiment in engineered Escherichia coli,the function of UpZDS gene was verified as engineered E.coli could produce lycopene in the presence of UpZDS,crtE,crtB and crtP.In this study,the engineering bacteria co-transformed with crtE,crtB,crtP and UpZDS genes were constructed.The production of three kinds of carotenoids(lycopene,neurosporene and ?-carotene)in engineering bacteria under different conditions of three single factors was investigated.The three single factors were different inoculation volumes,different IPTG concentrations and different induction temperatures.The results showed that the production of ?-carotene was the highest,followed by neurosporene,and the production of lycopene was the least under the tested culture conditions.In the detection of effect of the inoculation volumes on production of carotenoids,lycopene reached the highest production of 0.637 ?g/mL at the inoculation volume of 1%.The neurosporene and?-carotene reached the highest contents at 3% inoculation volume,with 2.182 ?g/mL and 2.820?g/mL,respectively.In detection of effect of IPTG concentrations on production of carotenoids,lycopene reached a maximum content of 0.691 ?g/mL at the IPTG concentration of 0.5 mmol/L.Both neurosporene and ?-carotene reached a maximum content at the IPTG concentration of 0.7 mmol/L,which was 2.156 ?g/mL and 2.392 ?g/mL,respectively.In with the examination of effect of temperatures on production of carotenoids,lycopene,neurosporene and ?-carotene reached amaximum content of 0.727 ?g/mL,2.301 ?g/mL and 3.744 ?g/mL,respectively,at temperature of20?.These findings provided powerful evidence that UpZDS protein reported in this study might have similar functions to the corresponding ZDS from other plants.This study would lay the foundation for the elucidation of biosynthesis pathway of fucoxanthin in U.pinnatifida Suringar.