摘要
为了获得高产反式-4-羟脯氨酸的菌株,基于大肠杆菌的代谢网络模型的指导,以大肠杆菌E.coli BL21(DE3)Δput A为出发菌株,通过基因敲除技术成功敲除arg B基因,阻断L-脯氨酸合成的前体物L-谷氨酸的分支代谢途径,增加L-脯氨酸合成的代谢流,构建了精氨酸缺陷型菌株E.coli BL21(DE3)Δput AΔarg B。同时转入表达质粒p UC19-pro B2A-Ptrp2-hyp,该质粒含有突变基因pro B2,该突变基因所编码的谷氨酸激酶受L-脯氨酸的反馈抑制作用显著降低。摇瓶发酵结果表明,在外源添加600 mg/L L-精氨酸时,该重组菌株产反式-4-羟脯氨酸的量达到312.67 mg/L,较菌株E.coli BL21(DE3)Δput A/p UC19-pro B2A-Ptrp2-hyp提高了25.29%。
In order to obtain high-yield strains of trans-4-hydroxyproline, based on the guidance of E. coli meta- bolic network model and using E. eoli BL21 （DE3）AputA as the original strain, gene argB was knocked out success- fully, which blocked metabolic pathways branch of L-glutamic acid, L-proline synthetic precursors and increased syn- thesis of L-proline metabolic flux. The constructed arginine-deficient strain E. eoli BL21 （ DE3 ） AputAAargB was transformed with the expression plasmid pUC19-proB2A-Ptrp2-hyp. The expression plasmid contains the mutant gene proB2 encoding glutamate kinase that significantly reduced L-proline feedback inhibition. Fermentation results indica- ted thatthe trans-4-hydroxyproline production of recombinant strain reached 312.67 mg/L exogenous arginine, which increased by 25 Ptrp2-hyp. 29% compared with E. eoli BL21 （DE3 er addition of 600 mg/L AputA/ pUC19-proB2A-ptrp2-hyp.
出处
《食品与发酵工业》
CAS
CSCD
北大核心
2017年第1期24-30,共7页
Food and Fermentation Industries
基金
国家自然科学基金(30970058)
江苏省自然科学基金(BK2012554)
工业生物技术教育部重点实验室(江南大学)开放课题基金(KLIB-ZR200801)
