腺苷酸脱氨酶在乳酸克鲁维酵母中构建表达

Expression of adenosine monophosphate deaminase in Kluyveromyces lactis

【目的】实现鼠灰链霉菌来源经密码子优化后的腺苷酸脱氨酶基因在乳酸克鲁维酵母(Kluyveromyces lactis GG799)中组成型表达。【方法】以鼠灰链霉菌(Streptomyces murinus)来源的腺苷酸脱氨酶(AMP)基因经密码子优化后作为模板,设计特异性引物,PCR扩增AMP脱氨酶基因opt-AMPD,以p KLAC1为载体构建重组表达质粒p KLAC1-opt-AMPD,经Sac II线性化后电转化法转入K.lactis GG799,筛选得到重组菌株,测定酶活,经His TrapTM HP纯化后得到AMP脱氨酶,并优化重组菌的发酵培养基。【结果】对AMP脱氨酶基因进行了密码子优化后,构建了重组K.lactis GG799/p KLAC1-opt-AMPD,实现组成型表达,密码子优化后AMP脱氨酶酶活提高到586±50 U/m L。SDS-PAGE结果显示,纯化后的AMP脱氨酶为单一条带,蛋白大小约为60 k D。优化的发酵培养基为(g/L):葡萄糖40、蛋白胨20、酵母粉15、Na Cl 8、KCl 10、Mg SO4 2,30°C、200 r/min发酵120 h,酶活达到2 100±60 U/m L。【结论】实现了密码子优化后的腺苷酸脱氨酶基因在乳酸克鲁维酵母GG799内的组成型表达,为实现腺苷酸脱氨酶的重组高效表达和发酵生产进行了有益探索。

[Objective] We constructed a recombinant Kluyveromyces lactis GG799 strain to constitutively produce adenosine monophosphate（AMP） deaminase. [Methods] The codons of AMP deaminase gene derived from Streptomyces murinus were optimized and used as template. We designed primers and amplified the opt-AMPD gene. The opt-AMPD gene was cloned into the expression plasmid p KLAC1. The recombinant expression plasmid was linearized by Sac II and transformed into K. lactis GG799 by electrotransformation. We determined the AMP deaminase activity of positive transformants. The AMP deaminase was purified by His TrapTM HP and we preliminary optimized the fermentation medium of K. lactis GG799. [Results] The codons of AMPD gene were optimized and a recombinant K. lactis GG799/p KLAC1-opt-AMPD was constructed to constitutively produce AMP deaminase. The AMP deaminase activity reached 586±50 U/m L after optimizing codon. The purified AMP deaminase showed asingle band on SDS-PAGE and the molecular weight by SDS-PAGE was about 60 k D. The preliminary optimized medium contained 40 g/L glucose, 20 g/L peptone, 15 g/L yeast extract, 8 g/L Na Cl, 10 g/L KCl, 2 g/L Mg SO4. The activity of AMP deaminase reached 2 100±60 U/m L cultured in flask after 120 h at 30 °C with agitation 200 r/min. [Conclusion] These codons of AMPD gene were optimized and AMP deaminase was constitutively produced in the K. lactis GG799. It is a valuable exploration about high efficiency recombinant expression and production of AMP deaminase.