Cloning and soluble expression of β-carotene converting enzyme (Asy) gene from Phaffia rhodozyma

Abstract:

[Objective] We cloned the cDNA of β-carotene converting enzyme gene (asy) from Phaffia rhodozyma 7B12, a high astaxanthin-producing strain, and expressed the recombinant pET32-asy in E. coli BL21(DE3). Our work could lead to an important use for the study of Asy properties and further applications in vitro. [Methods] Using RACE method, we cloned the asy cDNA from Phaffia rhodozyma 7B12, and constructed recombinant plasmid pET32-asy. After optimizing the temperature and IPTG concentration, the soluble expression of Asy was achieved in E. coli BL21(DE3). pET32-asy and pACCAR16Δcrtx which carried the genes chain on the synthesis of beta-carotene by acetyl CoA were co-transformed into E.?coli BL21(DE3), and the changes of carotenoid species were analyzed by HPLC to detect the activity of Asy. [Results] The homology between new cloned cDNA sequence of asy gene (accession No. HM204708.1) and the only reported asy mRNA sequence (accession No. DQ002007.1) was 97%. The obtained cDNA was 1 971 bp in length, the longest open reading frame was 1 614 bp encoding 538 amino acids, and therefore the fusion protein expressed in E.?coli BL21(DE3) was about 70 kD. At the optimizing condition (induced by 0.5 mmol/L IPTG, at 26 °C, 5 h), 85% fusion protein expressed by recombinant pET32-asy was soluble. Compared the components of pigment in the E. coli strain only transformed with pACCAR16Δcrtx with the strain co-transformed with pACCAR16Δcrtx and pET32-asy, we found some changes of carotenoid components. The peak presented α-carotene was disappeared and three new peaks were shown, suggested that β-cryptoxanthin which is one of themetabolic intermediates between β-carotene and astaxanthin were produced because of Asy expression. [Conclusion] Astaxanthin synthase cDNA was cloned from Phaffia rhodozyma 7B12 and the soluble expression of astaxanthin synthase in E. coli BL21(DE3) was obtained. The fusion protein had a certain activity to transform β-carotene.