磷酸盐和pH值协同调控大肠杆菌发酵合成5-羟基色氨酸  

Phosphate and pH synergistically modulate the synthesis of 5-hydroxytryptophan by fermentation in Escherichia coli

作　　者：余子辰 王锐麒 张震 徐庆阳[1,2,3] 陈宁 余义发[4] YU Zichen;WANG Ruiqi;ZHANG Zhen;XU Qingyang;CHEN Ning;YU Yifa(College of Biotechnology,Tianjin University of Science and Technology,Tianjin 300457,China;Tianjin Engineering Lab of Efficient and Green Amino Acid Manufacture,Tianjin 300457,China;National and Local United Engineering Lab of Metabolic Control Fermentation Technology,Tianjin 300457,China;Nanning Hanhe Biotechnology Co.Ltd.,Nanning 530000,China)

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]天津市氨基酸高效绿色制造工程实验室,天津300457 [3]代谢控制发酵技术国家地方联合工程实验室,天津300457 [4]南宁汉和生物科技股份有限公司,广西南宁530000

出　　处：《食品与发酵工业》2023年第10期114-121,共8页Food and Fermentation Industries

基　　金：天津市合成生物技术创新能力提升行动计划项目(TSBICIP-KJGG-005-08);天津市科技计划项目(21ZYQCSY00050)。

摘　　要：为了提高大肠杆菌合成5-羟基色氨酸能力,该研究设计构建了pSTV28-TPH150过表达质粒,增强了羟化酶基因TPH150的表达,以HTP10为出发菌株(代谢工程实验室保藏,专利号:CN202110714155.1),电转化pSTV28-TPH150质粒得到菌株HTP10-1,发酵结果显示,存在副产物L-色氨酸积累过多,质粒不稳定等问题,因此在初始发酵工艺的基础上,通过设计4个pH梯度进行发酵对比试验和4种不同磷酸二氢钾流加策略来探究最适发酵工艺。实验结果表明,在菌株HTP10-1发酵pH值为6.7,并于发酵培养基中添加1 g/L磷酸二氢钾,葡萄糖补料瓶中添加3 g/L磷酸二氢钾时,菌体比生长速率达到最优,发酵结束后5-羟基色氨酸积累量最高达到5.46 g/L,副产物L-色氨酸积累量减少到1.37 g/L,质粒丢失率较对照组减少32%,极大地提高了菌株HTP10-1的质粒稳定性,验证了磷酸盐与发酵pH调控菌体生长的可行性,对质粒工程菌的发酵控制具有重要参考意义。In order to improve the ability of Escherichia coli to synthesize 5-hydroxytryptophan,the overexpression plasmid pSTV28-TPH150 was designed and constructed to enhance the expression of hydroxylase gene TPH150.Using HTP10 as the original strain and electrotransforming it with the pSTV28-TPH150 plasmid to obtain the strain HTP10-1.The fermentation results showed that the by-product L-tryptophan accumulated too much and the plasmid was unstable.Therefore,on the basis of the initial fermentation process,four pH gradients were designed to carry out fermentation contrast experiments and four different KH_(2)PO_(4) flow addition strategies to explore the optimal fermentation process.The experimental results showed that the optimal growth rate of the bacteria was achieved when the fermentation pH of strain HTP10-1 was 6.7 and 1 g/L KH_(2)PO_(4) was added to the fermentation medium and 3 g/L KH_(2)PO_(4) was added to the glucose replenishment bottle.After the fermentation,the accumulation of 5-hydroxytryptophan reached the highest level of 5.46 g/L.The accumulation of L-tryptophan,a by-product,was reduced to 1.37 g/L,and the plasmid loss rate was reduced by 32%compared with the control group,which greatly improved the plasmid stability of strain HTP10-1.The feasibility of regulating the growth of bacteria by phosphate and fermentation pH was verified,which has important reference significance for the fermentation control of plasmid engineering bacteria.

关 键 词：大肠杆菌 5-羟基色氨酸 质粒 PH值 磷酸二氢钾 比生长速率 

分 类 号：TQ922[轻工技术与工程—发酵工程]