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.
作者
余子辰
王锐麒
张震
徐庆阳
陈宁
余义发
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)
出处
《食品与发酵工业》
CAS
CSCD
北大核心
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)。
