Jiang Min, Chen Xu, Liang Liya, Liu Rongming, Wan Qing, Wu Mingke, Zhang Hanwen, Ma Jiangfeng, Chen Kequan, Ouyang Pingkai
State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 211816, Jiangsu, China.
State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 211816, Jiangsu, China.
Enzyme Microb Technol. 2014 Mar 5;56:8-14. doi: 10.1016/j.enzmictec.2013.12.011. Epub 2013 Dec 19.
Succinate is not the dominant fermentation product from xylose in wild-type Escherichia coli K12. E. coli BA 203 is a lactate dehydrogenase (ldhA), pyruvate formate lyase (pflB), and phosphoenolpyruvate (PEP)-carboxylase (ppc) deletion strain. To increase succinate accumulation and reduce byproduct formation, engineered E. coli BA204, in which ATP-forming PEP-carboxykinase (PEPCK) is overexpressed in BA203, was constructed and produced 2.17-fold higher succinate yield. To further improve the biomass and the consumption rate of xylose, nicotinic acid phosphoribosyltransferase (NAPRTase), a rate limiting enzyme in the synthesis of NAD(H), was also overexpressed. Thus, co-expression of PEPCK and NAPRTase in recombinant E. coli BA209 was investigated. In BA209, the pck gene and the pncB gene each have a trc promoter, hence, both genes are well expressed. During a 72-h anaerobic fermentation in sealed bottles, the total concentration of NAD(H) in BA209 was 1.25-fold higher than that in BA204, and the NADH/NAD+ ratio decreased from 0.28 to 0.11. During the exclusively anaerobic fermentation in a 3-L bioreactor, BA209 consumed 17.1 g L⁻¹ xylose and produced 15.5 g L⁻¹ succinate. Furthermore, anaerobic fermentation of corn stalk hydrolysate contained 30.1 g L⁻¹ xylose, 2.1 g L⁻¹ glucose and 1.5 g L⁻¹ arabinose, it produced a final succinate concentration of 17.2 g L⁻¹ with a yield of 0.94 g g⁻¹ total sugars.
琥珀酸并非野生型大肠杆菌K12利用木糖发酵产生的主要产物。大肠杆菌BA 203是乳酸脱氢酶（ldhA）、丙酮酸甲酸裂解酶（pflB）和磷酸烯醇丙酮酸（PEP）羧化酶（ppc）缺失菌株。为了增加琥珀酸积累并减少副产物生成，构建了工程大肠杆菌BA204，其中在BA203中过表达了生成ATP的PEP羧激酶（PEPCK），其琥珀酸产量提高了2.17倍。为了进一步提高生物量和木糖消耗率，还过表达了烟酰胺磷酸核糖转移酶（NAPRTase），它是NAD（H）合成中的限速酶。因此，研究了PEPCK和NAPRTase在重组大肠杆菌BA209中的共表达。在BA209中，pck基因和pncB基因均有trc启动子，因此，两个基因均能良好表达。在密封瓶中进行72小时厌氧发酵期间，BA209中NAD（H）的总浓度比BA204高1.25倍，NADH/NAD⁺比值从0.28降至0.11。在3-L生物反应器中进行纯厌氧发酵时，BA209消耗了17.1 g L⁻¹木糖，产生了15.5 g L⁻¹琥珀酸。此外，对含有30.1 g L⁻¹木糖、2.1 g L⁻¹葡萄糖和1.5 g L⁻¹阿拉伯糖的玉米秸秆水解液进行厌氧发酵，最终琥珀酸浓度为17.2 g L⁻¹，产率为0.94 g g⁻¹总糖。
