食品科学

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密码子优化及透明颤菌血红蛋白共表达提高耐热脂肪酶在毕赤酵母的表达

王建荣,刘丹妮,夏 雨,杨 玲,刘金山,唐 业,陈丽芝,黄佳乐,李阳源*   

  1. 广东溢多利生物科技股份有限公司,广东 珠海 519060
  • 出版日期:2016-10-15 发布日期:2016-12-01

Enhanced Production of Thermomyces lanuginosus Lipase in Pichia pastoris by Codon Optimization and Co-expression of Vitreoscilla Hemoglobin

WANG Jianrong, LIU Danni, XIA Yu, YANG Ling, LIU Jinshan, TANG Ye, CHEN Lizhi, HUANG Jiale, LI Yangyuan*   

  1. Guangdong VTR Bio-Tech Co. Ltd., Zhuhai 519060, China
  • Online:2016-10-15 Published:2016-12-01

摘要:

根据毕赤酵母密码子的偏好性,通过在线软件对Thermomyces lanuginosus脂肪酶基因(tll)和透明颤菌血红蛋白基因(vhb)进行密码子优化。优化后的脂肪酶基因(tll-opt)和透明颤菌血红蛋白基因(vhb-opt)中碱基GC含量分别由原来的46%提高到49%和42%提高到51%,碱基A、T、C、G均匀分布,减少了AT和GC富集区,利于tll-opt和vhb-opt基因在毕赤酵母X33中的表达。将tll和tll-opt连接到表达载体pPICZαA并转入毕赤酵母X33中。摇瓶培养条件下,含有tll和tll-opt重组工程菌的最大酶活力分别为7 U/mL和16 U/mL。50 L发酵罐培养条件下,含有tll和tll-opt重组工程菌的最大酶活力分别为201 U/mL和430 U/mL。为了进一步提高含tll-opt重组工程菌的表达酶活性,将vhb-opt转入该重组工程菌得到工程菌VHb+(含有tll-opt和vhb-opt)。重组工程菌VHb+在摇瓶培养条件下最大酶活力为23 U/mL,分别是优化后基因和原始基因最大表达酶活力的1.39 倍和3.28 倍。重组工程菌VHb+在50 L发酵罐瓶培养条件下最大酶活力为610 U/mL,分别是优化后基因和原始基因最大表达酶活力的1.41 倍和3.03 倍。

关键词: 密码子优化, 脂肪酶, 透明颤菌血红蛋白, 高密度发酵

Abstract:

The codon usage of Thermomyces lanuginosus lipase (tll) and Vitreoscilla hemoglobin (vhb) gene were optimized
by online software according to P. pastoris preferred codon usage. The G + C contents of the codon-optimized genes tll-opt
and vhb-opt were increased from 46% to 49% and 42% to 51%, respectively. The nucleotides A, T, C and G dispersed evenly
in the optimized genes, thereby eliminating AT- or GC-rich motifs. The tll and tll-opt genes were ligated into pPICZαA and
transformed into P. pastoris X33, respectively. The recombinant strains containing tll and tll-opt were cultivated in shaking
flask and 50 L bioreactor, respectively. In shake-flask level, the maximum lipase activities of X33/tll and X33/tll-opt were
7 and 16 U/mL, respectively. In 50 L bioreactor, the maximum lipase activities produced by X33/tll and X33/tll-opt were
201 U/mL and 430 U/mL. To further increase the expression of tll-opt, the vhb-opt gene was transformed into the
recombinant strain containing tll-opt to form recombinant strain VHb+. In shake-flask level, the maximum lipase activity
of VHb+ was 23 U/mL, which was 139% and 328% higher than that from the tll-opt gene and the wild-type gene (tll),
respectively. The maximum lipase activity of VHb+ in 50 L bioreactor was 610 U/mL, which was 141% and 303% higher
than that from the tll-opt gene and the wild-type gene (tll).

Key words: codon optimization, lipase, Vitreoscilla hemoglobin, high cell density fermentation

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