法夫酵母β-胡萝卜素转化酶(Asy)基因的克隆及可溶性表达研究

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

摘要:

【目的】从高产虾青素的法夫酵母(Phaffia rhodozyma) 7B12菌株中克隆β-胡萝卜素转化酶基因(β-Carotene converting enzyme gene, asy), 并将该基因在大肠杆菌(Escherichia coli)中进行可溶性表达, 为深入研究该酶的性质及应用提供基础。【方法】采用cDNA末端快速扩增技术, 克隆得到asy基因全长cDNA序列, 将其克隆到表达载体pET32a中, 通过优化温度和IPTG浓度提高其可溶表达量; 进一步在E. coli BL21(DE3)中共表达携带asy基因的重组质粒和pACCAR16Δcrtx质粒(携带由乙酰辅酶A合成β-胡萝卜素的基因链), 用液相色谱分析共转化菌株中类胡萝卜素种类的变化, 鉴定可溶性表达的Asy酶活性。【结果】法夫酵母7B12菌株asy基因的cDNA序列与GenBank能检索到的唯一一条asy基因mRNA序列(Accession No. DQ002007.1)一致性达到97%, 该序列总长1 971 bp, 最大开放阅读框为1 614 bp, 编码538个氨基酸, 在E. coli BL21(DE3)中表达的Asy融合蛋白分子量约为70 kD; 条件优化后转化asy基因的重组菌在26 °C、0.5?mmol/L IPTG的条件下诱导时, 可溶性融合蛋白比例达85%。与pACCAR16Δcrtx单质粒转化相比, 共转化pACCAR16Δcrtx及asy基因的菌株类胡萝卜素产物的成分发生了明显的变化, 其中α-胡萝卜素的含量明显减少, 伴随出现了3种新的色素, 根据出峰时间判断其中一种为β-胡萝卜素和虾青素的中间产物——β-隐黄质。【结论】从法夫酵母中克隆得到asy基因, 通过优化诱导温度及IPTG浓度等条件提高了该基因在E. coli BL21(DE3)中的可溶性表达量, 经鉴定可溶性的Asy融合蛋白具有转化β-胡萝卜素的活性。

关键词:法夫酵母, β-胡萝卜素转化酶, 克隆, 可溶性表达, 活性分析

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.

Key words:Phaffia rhodozyma, β-Carotene converting enzyme, Clone, Soluble expression, Activity analysis