共表达磷酸烯醇式丙酮酸羧激酶和烟酸转磷酸核糖激酶提高重组大肠杆菌发酵木糖产丁二酸

Enhancing succinate production from xylose by co-expression of phosphoenolpyruvate carboxykinase and nicotinic-acid phosphonbosyltransferase in recombinant Escherichia coli

野生型E.coli K12能够在厌氧条件下代谢木糖生长,但是丁二酸不是其主要的代谢终产物。而在E.coli BA203(Δldh A,Δpfl B,Δppc)中,通过过量表达磷酸烯醇式丙酮酸羧化激酶(PCK),即E.coli BA204,使其能够在厌氧条件下利用木糖发酵生产丁二酸。为了进一步提高生物量及丁二酸的产量,通过过量表达烟酸转磷酸核糖激酶(NAPRTase)提高NAD(H)的生成,从而提高木糖代谢速率。因此采用2种方法构建了共表达烟酸转磷酸核糖激酶和磷酸烯醇式丙酮酸羧化激酶的基因工程菌,即E.coli BA208(BA203/p Trc99a-pnc B-pck)和E.coli BA209(BA203/p Trc99a-pck-trc-pnc B)。通过实验发现:厌氧发酵72 h,BA209消耗16.7 g/L木糖,生成15.8 g/L丁二酸,乙酸含量有所降低,而丙酮酸的量几乎不变。BA209中NAD(H)总量和ATP含量较BA208和BA204都有明显的提高。这为考察NAD(H)和ATP 2种辅因子对重组大肠杆菌利用木糖合成丁二酸的影响提供了研究平台。

Wild Escherichia coli K12 can use xylose to grow under anaerobic conditions, but succinate is not the dominant fermentation product. We overexpressed phosphoenolpyruvate carboxykinase （PCK） in E. coli BA203, a IdhA ,pflB, and ppc deletion strain, named E. coli BA204. The resultant strain could use xylose to produce succinate under anaerobic conditions. To further improve the biomass and succinate production,the NAD （H） pool size was increased by overexpression of nicotinic acid phosphoribosyhransferase （NAPRTase）. As a result, xylose metabolic rate was accelerated. In this case, two methods were used to construct the recombinant strains, E. coli BA208 （BA203/pTrc99a-pncB-pck） and E. coli BA209 （ BA203/ pTrc99a-pck-trc-pncB） by co-expression of PCK and NAPRTase. After fermentation for 72 h, 16. 7 g/L ofxylose was consumed in BA209, producing 15.8 g,/L succinate. Moreover, lower amount of acetic acid was detected,pyruvic acid was almost the same as that of the parent strain. The total amount of NAD （H） and ATP in BA209 were both increased significantly than those in BA208 and BA204. Our study provides the possibility to consider the two factors of NAD （H） and ATP to improve succinate production from xylose.