中文摘要：
近年来,大肠杆菌作为一种模式生物被广泛应用于各种功能化合物的生产,氨基酸因其具有广泛的药用价值而备受关注。本文利用合成生物学手段,在大肠杆菌中引入L-2,3-二氨基丙酸外源路径,首次实现了其生物合成,具体工作如下:1、设计了三条外源合成路径,将外源基因连接到pETDuet-1载体上,以BL21 (DE3)为外源酶表达宿主。通过镍柱纯化得到三组外源酶,进行体外酶活测试。三组外源酶中,来源于金黄色葡萄球的SbnA,SbnB能够以谷氨酸和磷酸丝氨酸为底物,将其转化为目的产物L-DAP,另外两组酶并无活性。2、将外源基因sbnA,sbnB转入野生型BW25113成功实现的L-DAP的生物合成。通过内源基因的过表达以及支路基因的敲除,进一步提高了目标产物的产量,L-DAP由最初的174.6mg·L-1提高到378.4 mg.L-1。测试了 BL21 (DE3)菌株的发酵能力,并进行菌株优化,最终确定BW25113作为宿主菌。3、将代谢路径分为外源基因与内源基因两个模块,通过调控内源基因与外源基因的表达强弱来进行优化,最终确定外源基因构建在高拷贝的pZE12-luc载体上,内源基因连接在中拷贝的pCS27质粒上为最优组合。4、探究了不同酵母粉浓度下L-DAP对细胞生长的抑制作用,发现添加足量的酵母粉大肠杆菌能够恢复正常生长,说明L-DAP抑制了某些氨基酸的合成,酵母粉的加入弥补了氨基酸合成的不足。最后敲除ygeX基因,并不能解除L-DAP在培养基中浓度的降低,说明ygeX基因在大肠杆菌中表达较弱,对L-DAP降解不明显。5、试图利用反义RNA干扰调控TCA循环,设计了三个不同的靶向序列干扰eno基因,成功构建pCS-SerA-SerC-EnoasRNA载体。产量并未达到预期结果。建立了特定的颜色反应测定L-DAP浓度,确定30 min为颜色反应终止时间。
英文摘要：
In recent years, E.coli has been widely used as a model organism in the production of various functional compounds, and amino acids have attracted much attention because of its wide medicinal value. In this paper,we used synthetic biology method to introduce L-2,3-diaminopropionic acid exogenous pathways into E.coli for the first time to realize its biosynthesis. The specific work is as follows:Three exogenous synthetic pathways were designed. The foreign gene was ligated into pETDuet-1 vector, and BL21 (DE3) was used as host to express exogenous enzyme. Three groups of exogenous enzymes were obtained by purification of nickel column, and the enzyme activity was tested in vitro. Among the three group exogenous enzymes, SbnA and SbnB derived from Staphylococcus aureus could use glutamic acid and phosphoserine as the substrate, and transformed them into the target product L-2,3-Diaminopropionic Acid, but no activity detected in the other enzymes.Transform the exogenous genes sbnA and sbnB into wild-type E. coli BW25113 successfully achieved L-DAP biosynthesis, and then via overexpression of the endogenous gene and knockout of the branch gene to further increase the yield of the target product. The titer of L-DAP was increased from the initial 178.4 mg·L-1 to 378.6 mg-L-1. The fermentation ability of BL21 (DE3) strain was tested and we optimized the strain.Finally, BW25113 was used as host bacteria.The metabolic pathways were divided into two modules, The foreign gene and the endogenous gene module. By module optimization to regulate the expression of endogenous gene and exogenous gene. Finally,The optimal combination is that use the high copy number of plasmid pZE12-luc to express exogenous gene and the pCS27 plasmid to express endogenous gene.The inhibitory effects of L-DAP on cell growth were investigated under different yeast concentrations. It was found that the addition of sufficient amount of yeast could restore normal growth of E.coli,indicating that L-DAP inhibited the synthesis of certain amino acids, and the addition of yeast powder made up for the deficiency of amino acid synthesis. The knockout of the ygeX gene did not relieve the decrease of L-DAP concentration in the culture medium, which indicated that ygeX gene expression in E.coli is weak and had no obvious degradation to L-DAP.We tried to use antisense RNA to regulate TCA cycle, Three different targeting sequences were designed to interfere with the eno gene,and the pCS-SerA-SerC-EnoasRNA vector was successfully constructed,but the production did not meet the expected results. At last a specific color reaction was established to determine the L-DAP concentration, and the reaction time is 30 min.
