Zhou Li, Tian Kangming, Zuo Zhirui, Chen Xianzhong, Shi Guiyang, Singh Suren, Wang Zhengxiang
The Key Laboratory of Industrial Biotechnology of Ministry of Education, Center of Bioresource & Bioenergy, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
Sheng Wu Gong Cheng Xue Bao. 2011 Jan;27(1):31-40.
When Escherichia coli CICIM B0013-030 (B0013, ack-pta, pps, pflB) was used for D-lactate production, succinate and acetate were the main byproducts (as much as 11.9 and 7.1% the amount of lactate respectively). In order to decrease the byproduct levels, we inactivated succinate and acetate synthesis in B0013-030. Two recombinant plasmids containing mutation cassettes of frdA::difGm and tdcDE::difGm respectively were constructed first. The mutation cassettes were used to delete the target genes on the chromosomal by Red recombination. Subsequently, the antibiotic resistance gene was excised from the chromosomal by Xer recombination. Thereby, mutants B0013-040B (B0013-030, frdA) and B0013-050B (B0013-040B, tdcDE) were produced. D-lactate producing abilities of the engineered strains were tested both in shake flasks and in bioreactors using two-phase fermentation (aerobic growth and anaerobic fermentation) with glucose as the sole carbon source. When fermentation was carried out in shake flasks, inactivation of frdA in B0013-030 to produce B0013-040B reduced succinate accumulation by 80.8%. When tested in a 7-liter bioreactor, B0013-040B accumulated 114.5 g/L D-lactate of over 99.9% optical purity. However, 1.0 g/L succinate and 5.4 g/L acetate still remained in the broth. Further inactivation of tdcD and tdcE genes in B0013-040B to produce B0013-050B decreased acetate and succinate accumulation to 0.4 g/L and 0.4 g/L respectively, and lactate titer was as much as 111.9 g/L (tested in the 7-liter bioreactor). In lightof the lower byproduct levels and high lactate production, strain B00 13-050B may prove useful for D-lactate production.
当使用大肠杆菌CICIM B0013-030（B0013，ack-pta，pps，pflB）生产D-乳酸时，琥珀酸和乙酸是主要副产物（分别高达乳酸产量的11.9%和7.1%）。为了降低副产物水平，我们使B0013-030中的琥珀酸和乙酸合成失活。首先构建了两个分别含有frdA::difGm和tdcDE::difGm突变盒的重组质粒。通过Red重组，利用突变盒删除染色体上的目标基因。随后，通过Xer重组从染色体上切除抗生素抗性基因。由此，产生了突变体B0013-040B（B0013-030，frdA）和B0013-050B（B0013-040B，tdcDE）。以葡萄糖为唯一碳源，采用两相发酵（好氧生长和厌氧发酵），在摇瓶和生物反应器中测试了工程菌株的D-乳酸生产能力。在摇瓶中进行发酵时，B0013-030中frdA失活产生B0013-040B，使琥珀酸积累减少了80.8%。在7升生物反应器中测试时，B0013-040B积累了114.5 g/L光学纯度超过99.9%的D-乳酸。然而，发酵液中仍残留1.0 g/L琥珀酸和5.4 g/L乙酸。B0013-040B中tdcD和tdcE基因进一步失活产生B0013-050B，使乙酸和琥珀酸积累分别降至0.4 g/L和0.4 g/L，乳酸产量高达111.9 g/L（在7升生物反应器中测试）。鉴于副产物水平较低且乳酸产量较高，菌株B0013-050B可能对D-乳酸生产有用。
