利用谷氨酸棒状杆菌高效表达枯草芽孢杆菌的谷氨酰胺合成酶发酵生产L-谷氨酰胺

Glutamine Synthetase of Bacillus subtilis is Applied to Corynebacterium glutamicum Fermentation System to Produce L-Glutamine

谷氨酰胺合成酶(GS)是用于合成L-谷氨酰胺(L-Gln)过程中的关键酶,然而GS的酶活受到多种因素的影响,导致其酶活不强,产物L-Gln偏低.本研究以GS的基因为研究对象,构建谷氨酸棒状杆菌(Corynebacterium glutamicum)的高效表达系统.利用C.glutamicum中的两种强启动子:tac启动子(Ptac)和麦芽糖启动子(Pmal),比较不同启动子的作用下GS的酶活力;其次,为了避免腺苷酰化对C.glutamicum的GS的抑制作用,将GS的腺苷酰化位点突变成苯丙氨酸(Tyr405Phe);同时由于枯草芽孢杆菌(Bacillus subtilis)的GS不受腺苷酰化作用的影响,故将Bacillus subtilis的GS基因导入到C.glutamicum的表达系统中,比较两种基因来源不同GS的酶活高低,以期达到高产L-Gln的目的.本研究第一次应用Bacillus subtilis来源的GS在C.glutamicum的系统中表达.最终的结果表明,在C.glutamicum的表达系统中,Ptac比Pmal的效果更好,来自Bacillus subtilis的GS比来自C.glutamicum的GS酶活力更高.重组菌BJ2有最高的酶活力和产量,L-Gln的最终产量达到32.5 g/L.

Glutamine synthetase（ GS） is the key enzyme responsible for L-glutamine（ L-Gln） synthesis. However,enzymatic activity of GS is influenced by many factors,leading to the low enzyme activity and the low production of L-Gln. In this study,in order to improve the yield of L-Gln,a high-effective expression system was constructed in Corynebacterium glutamicum with gln A gene. We tested two different strong promoters,promoter tac（ Ptac）,and promoter mal（ Pmal）,for a comparison of the activity of GS. After then,for avoiding the adenylylation in C. glutamicum,we constructed plasmids containing the point mutation of gln A,in which Tyr405 is replaced by a phenylalanine residue.Since the glutamine synthetase in Bacillus subtilis is not regulated via adenylylation,we also compared C. glutamicum GS activity with Bacillus subtilis＇ s to detect the enzyme activity in the given system. Light point of this study is to express GS of Bacillus subtilis in C. glutamicum expression system for the first time. The results reveal that promoter tac was more capable for producing L-Gln in C. glutamicum system. GS enzyme from Bacillus subtilis had the higher activity than C. glutamicum＇s. The recombinant strain BJ2 has the highest GS activity and produce 32.5 g / L L-glutamine.