提高产反式-4-羟脯氨酸工程菌产量的发酵策略

Strategies on Improving the trans-4-Hydroxyproline Production from the Engineering Strain E. coli

为了实现羟脯氨酸的高产,首先以缺陷型菌株E.coli JM109ΔargB/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.coli JM109ΔargB/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(Fe2+∶VC=1∶1)5 mmol/L,MgSO4·7H2O 1.7 g/L,Nacl 3 g/L,Cacl20.005 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.coli JM109ΔargB/pUC19-BH羟脯氨酸产量约为1602 mg/L,约为优化前的1.4倍.在7 L罐发酵培养,菌株E.coli JM109ΔargB/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.

To enhance the yield of trans-4-Hydroxyproline from the engineering strain E.coli JM109ΔargB/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 CaCl20.005 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 1602 mg/L,which was about 1.4 times of that before the optimization.In a 7 L fermenter,the recombinant E.coli JM109ΔargB/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.