Increased NADPH concentration obtained by metabolic engineering of the pentose phosphate pathway in Aspergillus niger
Increased NADPH concentration obtained by metabolic engineering of the pentose phosphate pathway in Aspergillus niger
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DOI：
10.1111/j.1742-4658.2005.04554.x
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发表时间：
2005-03-01
期刊：
影响因子：
5.4
通讯作者：
Ruijter, GJG
中科院分区：
文献类型：
--
作者：
Poulsen, BR;Nohr, J;Ruijter, GJG
许多生物合成反应和生物转化受到NADPH可用性低的限制。为了增加NADPH浓度和/或通过尼日尔曲霉中戊糖磷酸途径的通量，克隆编码葡萄糖6-磷酸脱氢酶（gsdA）、6-磷酸葡萄糖酸脱氢酶（gndA）和转酮醇酶（tktA）的基因并在单独的菌株中过表达。细胞内NADPH浓度增加2至9倍，作为6-磷酸葡萄糖酸脱氢酶的13倍过量生产的结果。虽然葡萄糖6-磷酸脱氢酶和转酮醇酶的过量产生改变了几种代谢产物的浓度，但并没有导致NADPH浓度增加。为了建立这三种基因的过表达的影响，在含有基本培养基的生物反应器培养物的指数期和稳定期中详细表征野生型和过表达菌株，所述基本培养基以葡萄糖作为碳源，以铵或硝酸盐作为氮源和最终细胞密度限制底物。酶，中间代谢产物，多元醇池（内和细胞外），有机酸，生长速率和速率常数诱导的酸生产在postexponential阶段进行了测量。没有一个修饰的菌株具有改变的生长速率。偏最小二乘回归显示了NADPH和多达40个其他变量（酶和代谢物的浓度）之间的相关性，并且可以从相对容易获得的数据（酶，多元醇和草酸盐的浓度）预测细胞内NADPH浓度。该预测可用于筛选工程菌株或其他生物突变体中的高NADPH水平。
Many biosynthetic reactions and bioconversions are limited by low availability of NADPH. With the purpose of increasing the NADPH concentration and/or the flux through the pentose phosphate pathway in Aspergillus niger, the genes encoding glucose 6-phosphate dehydrogenase (gsdA), 6-phosphogluconate dehydrogenase (gndA) and transketolase (tktA) were cloned and overexpressed in separate strains. Intracellular NADPH concentration was increased two- to ninefold as a result of 13-fold overproduction of 6-phosphogluconate dehydrogenase. Although overproduction of glucose 6-phosphate dehydrogenase and transketolase changed the concentration of several metabolites it did not result in increased NADPH concentration. To establish the effects of overexpression of the three genes, wild-type and overexpressing strains were characterized in detail in exponential and stationary phase of bioreactor cultures containing minimal media, with glucose as the carbon source and ammonium or nitrate as the nitrogen source and final cell density limiting substrate. Enzymes, intermediary metabolites, polyol pools (intra- and extracellular), organic acids, growth rates and rate constant of induction of acid production in postexponential phase were measured. None of the modified strains had a changed growth rate. Partial least square regressions showed the correlations between NADPH and up to 40 other variables (concentration of enzymes and metabolites) and it was possible to predict the intracellular NADPH concentration from relatively easily obtainable data (the concentration of enzymes, polyols and oxalate). This prediction might be used in screening for high NADPH levels in engineered strains or mutants of other organisms.