共表达N-乙酰转移酶和磷脂酶重组菌的构建及发酵优化

Construction of Co-Expressed N-Acetyltransferase and Phospholipase Gene in Recombinant Strain and Optimization of Fermentation Conditions

DOI：10.3969/j.issn.1673-1689.2018.08.011

中文关键词: N-乙酰转移酶 毕赤酵母 尖孢镰刀菌 磷脂酶 高密度发酵

英文关键词: N-acetyl transferase,P. pastoris,Fusarium oxysporum,phospholipase,high density fermentation

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中文摘要:

为了提高磷脂酶在毕赤酵母中的表达量，构建了N-乙酰转移酶（Mpr1）和磷脂酶共表达的重组毕赤酵母。Mpr1是一类能催化乙酰基团在乙酰辅酶A和胺之间转移的胞内酶，能够降低酵母细胞内的ROS水平，具有抗活性氧（ROS）氧化胁迫生理功能。来源于尖孢镰刀菌的磷脂酶（phospholipase）是一类可以将磷脂水解为小分子脂肪酸、甘二酯和磷酸胆碱的胞外酶。研究了Mpr1对重组菌的生长和磷脂酶表达量的影响。为了获得在3.6 L发酵罐中表达磷脂酶的最佳发酵条件，分别从诱导温度、起始诱导菌体浓度和甲醇诱导浓度3个方面研究了重组菌的高密度发酵，以恒溶氧反馈调节补加甘油和甲醇流加仪在线监测流加甲醇。结果表明：当诱导温度、起始诱导菌体质量浓度和甲醇诱导体积分数分别为28 ℃，40 g/L和1.0%，诱导120 h后磷脂酶活达到最高，为8 100 U/mL，总蛋白含量为8.3 mg/mL，为前期工作中磷脂酶单独表达时的1.33倍。

英文摘要:

To increase the production of phospholipase，a strain ofP. pastorisKM71 containing the gene co-expressing the N-cetyltransferase（Mpr1） and phospholipase was constructed. Mpr1 is an intracellular enzyme that catalyzes the transfer of acetyl groups between acetyl CoA and amines. Mpr1 has significant physiological functions in ROS oxidative stress resistance ability. Phospholipase，which come from Fusarium oxysporum is a group of hydrolases that hydrolyze phospholipids，releasing a variety of products，such as lysophospholipids，free fatty acids. Constructing the recombinantP. pastorisKM71/pPIC9K-phospholipase/pPICZA-Mpr1，the impact of Mpr1 on yeast cell growth and production of phospholipase from Fusarium oxysporum was investigated. In order to obtain the optimal condition of phospholipase fermentation，we observed the influence of temperature，initial induction cell density and methanol concentration on the high density fermentation process in 3.6 L fermentor ofP. pastorisKM71/pPIC9K-phospholipase/ pPICZA-MPR1. By using glycerol feeding strategy with DO control in high-density fermentation and maintained the residual methanol concentration constantly with a methanol sensor. The result showed that the optimum condition were listed as follows：the temperature was 28 ℃，the initial induction cell density was 40 g/L，the methanol concentration was 1.0%. Under the condition，the phospho- lipase activity reached 8 100 U/mL and the protein concentration（8.3 mg/mL） at cultivation of 120 h，which was 1.33 times than before.

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