提高产反式-4-羟脯氨酸工程菌产量的发酵策略

Strategies on Improving thetrans4-Hydroxyproline Production from the Engineering StrainE. coli

DOI：10.3969/j.issn.1673-1689.2019.11.006

中文关键词: 反式-4-羟脯氨酸 大肠杆菌工程菌 缺陷型 透明颤菌血红蛋白 发酵优化

英文关键词: trans-4-Hydroxyproline,engineering strainE. coli,defective strain,vitreoscilla hemoglobin,fermentation optimization

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中文摘要:

为了实现羟脯氨酸的高产，首先以缺陷型菌株E. coliJM109ΔargB/pUC19-BH作为实验菌，通过单因素优化及正交试验，优化得到新的培养基配方及培养条件。其次通过基因工程手段引入透明颤菌血红蛋白VHB基因，构建菌株E. coliJM109ΔargB/pUC19-BHV，进行高密度发酵。结果显示，发酵培养基为：麦芽糖10 g/L，甘油5 g/L，胰蛋白胨22 g/L，K₂HPO₄4 g/L，FeSO₄（Fe^{2+∶VC=1∶1） 5 mmol/L，MgSO₄·7H2O 1.7 g/L，NaCl 3 g/L，CaC l₂0.005 g/L；培养条件为：初始pH 8.5，温度30 ℃，接种体积分数4%，转速220 r/min。此条件下，菌株E. coliJM109ΔargB/pUC19-BH羟脯氨酸产量约为1 602 mg/L，约为优化前的1.4倍. 在7 L罐发酵培养，菌株E. coliJM109ΔargB/pUC19-BHV的羟脯氨酸产量约为18.3 g/L，与JM109ΔargB/pUC19-BH相比，提高了13.2%，说明VHB 基因的引入有利于羟脯氨酸产量提高.}

英文摘要:

To enhance the yield oftrans-4-Hydroxyproline from the engineering strainE. coliJM109ΔargB/pUC19-BH，optimization of fermentation medium and culture condition was carried out with a single-factor and orthogonal design experiment. Further，a recombinant E. coli JM109ΔargB/pUC19-BHV，containing the vitrescilla hemoglobin gene VHB was constructed to be desirable for high-density fermentation. Results showed that the fermentation medium was optimized as follows：maltose 10 g/L，glycerol 5 g/L，trypsin 22 g/L，K₂HPO₄4 g/L，FeSO₄：VC 5 mmol/L，MgSO₄1.7 g/L，NaCl 3 g/L and CaCl2 0.005 g/L. The optimal fermentation conditions were as below：inoculum size 4%（v/v），initial pH 8.5 and culture temperature 30 ℃. At 24 h of the fermentation，the yield of P4H reached 1 602 mg/L，which was about 1.4 times of that before the optimization. In a 7 L fermenter，the recombinantE. coliJM109ΔargB/pUC19-BHV yielded 18.3 g/L of P4H，13.2% higher than that of the parental strain，indicating that the introduction of VHB gene was beneficial for the high-density production of trans-4-Hydroxyproline.

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