Manipulating ATP supply improves in situ CO2 recycling by reductive TCA cycle in engineered Escherichia coli
Manipulating ATP supply improves in situ CO2 recycling by reductive TCA cycle in engineered Escherichia coli
复制标题
DOI：
10.1007/s13205-020-2116-7
复制
发表时间：
2020-02-19
期刊：
影响因子：
2.8
通讯作者：
Li, Si-Yu
中科院分区：
文献类型：
--
作者：
Chen, Ching-Hsun;Tseng, I-Ting;Li, Si-Yu
在大肠杆菌中引入pGETS118KAFS，将KOR(编码α-酮戊二酸：铁氧还蛋白氧化还原酶)、ACL(编码依赖于ATP的柠檬酸裂解酶)、FrD(编码富马酸还原酶)和SDH(编码琥珀酸脱氢酶)与枯草芽孢杆菌的有序基因组装在一起，重建了还原三羧酸(RTCA)循环。以E.coliMZLF(E.coliBL21(DE3)Delta zwf，Delta LDH，Delta frd)为研究对象，以C-2/C-1[(乙醇+乙酸盐)/(甲酸盐+CO2)]为指标，考察重组RTCA对CO2就地循环利用的有效性。研究表明，通过能量泵(EP)提供ATP，甲酸盐向硝酸盐提供电子形成ATP，使C-2/C-1比从1.03+/-0.00提高到1.49+/-0.02。同样，当通过引入异源乙醇产生途径(PLOI295)来增加ATP产量时，C-2/C-1比率进一步增加到1.79+/-0.02。综上所述，ATP供应是重组RTCA循环原位回收二氧化碳的限速步骤。C-1的降幅很大，但这些回收的C-1的目的地尚不确定。
The reductive tricarboxylic acid (rTCA) cycle was reconstructed in Escherichia coli by introducing pGETS118KAFS, where kor (encodes alpha-ketoglutarate:ferredoxin oxidoreductase), acl (encodes ATP-dependent citrate lyase), frd (encodes fumarate reductase), and sdh (encodes succinate dehydrogenase) were tandemly conjugated by the ordered gene assembly in Bacillus subtilis (OGAB). E. coli MZLF (E. coli BL21(DE3) Delta zwf, Delta ldh, Delta frd) was employed so that the C-2/C-1 [(ethanol + acetate)/(formate + CO2)] ratio can be used to investigate the effectiveness of the recombinant rTCA for in situ CO2 recycling. It has been shown that supplying ATP through the energy pump (the EP), where formate donates electron to nitrate to form ATP, elevates the C-2/C-1 ratio from 1.03 +/- 0.00 to 1.49 +/- 0.02. Similarly, when ATP production is increased by the introduction of the heterologous ethanol production pathway (pLOI295), the C-2/C-1 ratio further increased to 1.79 +/- 0.02. In summary, the ATP supply is a rate-limiting step for in situ CO2 recycling by the recombinant rTCA cycle. The decrease in C-1 is significant, but the destination of those recycled C-1 is yet to be determined.