管钊，吴明科，陈吴方，陈美丽，马江锋，姜岷

(南京工业大学生物与制药工程学院材料化学工程国家重点实验室，江苏南京210009)

摘要：利用大肠杆菌厌氧制备丁二酸过程中，采用氨水作为pH调节剂不仅可以中和酸性产物还可提供无机氮，被菌体利用，然而高浓度的积累会抑制菌体生长及代谢产酸的能力。为增强大肠杆菌对高浓度的耐受性，以(NH4)2HPO4为供体，通过在连续培养装置中不断提高(NH4)2HPO4浓度，以获得可耐受的产丁二酸大肠杆菌。结果表明：突变株在胁迫下，摇瓶厌氧发酵72 h，细胞干质量浓度(DCW)可达1.82 g/L，丁二酸产量为11.72 g/L，分别比出发菌株提高了1.6和4.6倍。进一步地，在5 L发酵罐上考察其利用氨水调节pH生产丁二酸的能力，厌氧发酵90 h，丁二酸质量浓度达到27.32 g/L，生产强度为0.30 g/(L·h)，比出发菌株分别提高88.1%和87.5%。

关键词：进化代谢；耐铵；丁二酸；大肠杆菌

doi:10.3969/j.issn.1672-3678.2015.06.010

收稿日期：2015-03-19

基金项目：国家重点基础研究发展计划(973计划)(2013CB733901)；江苏省高校优势学科建设工程

作者简介：管钊(1989—)，男，江苏南通人，硕士研究生，研究方向：生物工程；姜岷(联系人)，教授，E-mail：jiangmin@njtech.edu.cn

中图分类号：TQ921

文献标志码：A

文章编号：1672-3678(2015)06-0055-06

Abstract：During the process of succinic acid production by Escherichia coli,ammonia was adopted as pH regulator, not only could neutralize organic acid but also as nitrogen source for growth. would seriously inhibit the growth and metabolic capability of the strain.To improve the tolerance of E. gradually which was provided from(NH4)2HPO4. was obtained. in flask,dry cell weight of the mutant reached 1.82 g/L and succinic acid concentration reached 11.72 g/L within 72 h fermentation,which was 1.6- and 4.6-fold compared with the parent strain respectively.Furthermore,the ability of the mutant using ammonia as pH regulator for succinic acid production was investigated in a 5-L fermentor.After 90 h fermentation,concentration and productivity of succinic acid reached 27.32 g/L and 0.30 g/(L·h),which were increased by 88.1% and 87.5% compared with the parent strain.

Keywords：metabolic evolution; ammonium-tolerant; succinic acid; Escherichia coli

Isolation of ammonium-tolerant mutant of Escherichia coli for succinic acid production by metabolic evolution

GUAN Zhao,WU Mingke,CHEN Wufang,CHEN Meili,MA Jiangfeng,JIANG Min

(State Key Laboratory of Materials-Oriented Chemical Engineering,College of Biotechnology

and Pharmaceutical Engineering,Nanjing Tech University,Nanjing 210009,China)

丁二酸(琥珀酸)作为一种重要的C4平台化合物，是制备可降解生物材料的主要原料，被广泛应用于食品、农业、医药等行业，具有广阔的市场前景。目前，丁二酸的生产方法主要有化学合成和生物合成法。与化学合成法相比，生物合成法具有对环境污染小、反应条件温和、能耗低等优点，因此受到越来越多研究人员的关注[2-3]。丁二酸生产的优……