大肠杆菌琥珀酸和乙酸合成途径的删除及其重组菌株的D-乳酸发酵

Elimination of succinate and acetate synthesis in recombinant Escherichia coli for D-lactate production

菌株CICIM B0013-030(B0013,ack-pta,pps,pflB)可积累D-乳酸作为主要发酵产物,然而副产物琥珀酸和乙酸的含量分别高达乳酸的11.9%和7.1%。为构建副产物含量低的产D-乳酸重组大肠杆菌菌株,本研究删除了菌株B0013-030的琥珀酸(frdA)和乙酸(tdcDE)合成途径,并考察了重组菌株在摇瓶和发酵罐中经两阶段发酵(好氧生长菌体和厌氧发酵产酸)利用葡萄糖发酵D-乳酸的性能。结果表明,分别构建含有frdA::difGm和tdcDE::difGm突变盒的重组质粒,并利用Red重组系统将突变盒整合于染色体上的目的基因,再利用Xer重组系统去除抗生素抗性基因,依次获得了重组菌株B0013-040B(B0013-030,frdA)和B0013-050B(B0013-040B,tdcDE)。摇瓶发酵结果表明,frdA基因的删除使得菌株B0013-040B副产物琥珀酸的含量降低了80.8%;在7 L发酵罐中进行乳酸发酵,菌株B0013-040B的D-乳酸产量达114.5 g/L,光学纯度大于99.9%,但仍积累1.0 g/L琥珀酸和5.4 g/L乙酸。进一步删除了tdcD和tdcE基因的菌株B0013-050B,在7 L发酵罐中生产111.9 g/L D-乳酸,乙酸和琥珀酸的合成量分别降低为0.4 g/L,其他副产物含量也维持较低水平,表明该菌株具有较优良的D-乳酸发酵性能。

When Eshcerichia 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 light of the lower byproduct levels and high lactate production,strain B0013-050B may prove useful for D-lactate production.