进化代谢选育高渗透压耐受型产琥珀酸大肠杆菌

Isolation of high osmotic-tolerant mutants of Escherichia coli for succinic acid production by metabolic evolution

摘要:

在以碳酸钠为酸中和剂的大肠杆菌两阶段发酵产琥珀酸的过程中，由于Na+的积累造成发酵体系中渗透压的提高，严重抑制了琥珀酸的产物浓度。为了增强大肠杆菌对渗透压的耐受性，考察了利用进化代谢方法筛选高渗透压耐受型高产琥珀酸大肠杆菌菌株的可行性。进化代谢系统作为一种菌株突变装置，可以使菌体在连续培养条件下以最大的生长速率生长。以NaCl为渗透压调节剂，通过在连续培养装置中逐步提高NaCl浓度使菌体在高渗透压条件下快速生长，最终得到了一株高渗透压耐受型琥珀酸生产菌株Escherichia coli XB4。以碳酸钠为酸中和剂，在7 L发酵罐中利用Escherichia coli XB4进行两阶段发酵，厌氧培养60 h后，琥珀酸产量达到了69.5 g/L，琥珀酸生产速率达到了1.81 g/(L·h)，分别比出发菌株提高了18.6％和20％。

关键词:琥珀酸，大肠杆菌，渗透压，进化代谢

Abstract:

Succinic acid production was inhibited by high osmotic pressure caused by the accumulation of sodium ions in the process of two-stage fermentation by Escherichia coli using Na2CO3 as the pH regulator. To enhance the resistance of this strain to osmotic stress, the possibility to isolate high NaCl-tolerant mutant strain of Escherichia coli for succinic acid production by metabolic evolution was investigated. The metabolic evolution system was used as a mutant-generating system, allowing the cells to be continuously cultured at the maximum specific growth rate. The mutant strain can grow at maximum rate in the condition of high osmotic by gradually improving the concentration of NaCl in a continuous culture. Then the high osmotic-tolerant mutant strain of E. coli XB4 was selected with NaCl as the osmo-regulator. When using Na2CO3 as the pH regulator, E. coli XB4 was used in a 7.0 L fermenter during two-stage fermentation. After 60 h anaerobic fermentation, the mutant strain XB4 produced 69.5 g/L succinic acid with a productivity of 1.18 g/(L·h), which were increased by 18.6% and 20% compared with that of the parent strain.

Key words:succinic acid, Escherichia coli, osmotic, metabolic evolution