Prokaryotic Expression Of β-carotene Converting Enzyme (Asy) Gene And Analysis Of Its Differential Expression In Phaffia Rhodozyma

Astaxanthin is an interesting carotenoid with ultra-antioxidant performance, which has a lotof biological functions including pigmentation, antitumor, enhancing immunity, and is widelyused in aquaculture, cosmetics, health food and so on. Phaffia rhodozyma is an important sourceof natural astaxanthin. β-carotene converting enzyme (Asy) is a sole enzyme which takes part inthe oxidation of β-carotone to astaxanthin, few studies about which are reported at present.Therefore, this paper has carried out the following research on the asy gene of Phaffiarhodozyma:1. We cloned the cDNA of asy gene from Phaffia rhodozyma7B12by Rapid Amplificationof cDNA Ends technique. The obtained cDNA was1971bp and the longest open reading framewas1614bp which could encode538amino acids. In the end, the homology between newcloned cDNA sequence of asy gene (Accession NO.HM204708.1) and the only reported asymRNA sequence (Accession NO.DQ002007.1) was97%.2. The asy gene was inserted into the expression vector pET32a and the recombinantplasmid pET32-asy was constructed. The recombinant plasmid pET32-asy was then transformedinto E. coli BL21(DE3), and a fusion protein with the molecular weight about70kDa wasexpressed. Under the optimizing condition (induced by0.5mmol/L IPTG, at26C,5h),85%fusion protein expressed by recombinant pET32-asy was soluble. Compared with thecomponents of pigment in the E.coli strain only transformed with pACCAR16Δcrtx and thestrain co-transformed with pACCAR16Δcrtx and pET32-asy, we found some changes ofcarotenoid components. The peak presented α-carotene was disappeared and three new peakswere shown. The peak time suggested it was β-cryptoxanthin, a metabolic intermediate in thepathway of astaxanthin sythesis from β-carotene.3. The relationship between different carotenoids production and asy gene transcription infour different kinds of Phaffia rhodozyma strains was preliminary analyzed in this thesis. Theresults showed that JMU-LY322and JMU-15spw were unable to synthesize astaxanthin, but theasy gene could be transcripted in the two strains. JMU-MVP14and JMU-VDL668couldproduce astaxanthin. Compared with JMU-VDL668, the asy gene transcription amount washigher in JMU-MVP14at48h. The yields of carotenoids, β-carotene, and astaxanthin of JMU-MVP14were significantly higher than JMU-VDL668. These results indicatied that thetranscription of high-level asy gene might be one of the reasons for astaxanthin overproducing.4. The results of adding β-carotene at different culture time showed that the totalcarotenoid production of JMU-VDL668was maintained between300μg/g and400μg/g.JMU-VDL668could take in β-carotene from extracellular environment when the total amount ofcarotenoid was near or less than300μg/g. This showed that there might be a threshold of thetotal amount of carotenoids in JMU-VDL668, JMU-VDL668could take in β-carotene fromextracellular environment when total amout of carotenoids was below the threshold.5. The relationship between asy gene expression and production of carotenoids, β-carotene,astaxanthin from JMU-MVP14and JMU-VDL668was studied in the thesis. The result showedthat when two srtains were in growth phase, the transcription level of asy gene was high, and themaximum transcription amount of asy gene was detected at36h. It was low when strains were instable phase, and minimum transcription amount was detected at96h. So in the periodmentioned before, the level of asy gene expression was closely related to cell growth.Furthermore, the transcription level of asy gene increased rapidly after the secondary growth ofJMU-VDL668, then the β-carotene from extracellular environment could be converted intoastaxanthin by the strain. This indicated that the secondary growth of JMU-VDL668couldsignificantly promote the level of asy gene transcription and caused the increasing its ability ofastaxanthin synthsis. In addition, the astaxanthin yields of JMU-MVP14increased following bythe decreasing of β-carotene yields during108-120h and at the same time, the transcription levelof asy gene was still low. Therefore, this results indicated that including regulating the amount ofenzyme, some other regulational mechanisms existed to regulate the conversion from β-caroteneto astaxanthin in the yeast.