l-malate is one of the most important platform chemicals widely used in food, metal cleaning, textile finishing, pharmaceuticals, and synthesis of various fine chemicals. Recently, the development of biotechnological routes to produce l-malate from renewable resources has attracted significant attention. A potential l-malate producing strain E. coli BA040 was obtained by inactivating the genes of fumB, frdABCD, ldhA and pflB. After co-overexpression of mdh and pck, BA063 achieved 18 g/L glucose consumption, leading to an increase in l-malate titer and yield of 13.14 g/L and 0.73 g/g, respectively. Meantime, NADH/NAD+ ratio decreased to 0.72 with the total NAD(H) of 38.85 µmol/g DCW, and ATP concentration reached 715.79 nmol/g DCW. During fermentation in 5L fermentor with BA063, 41.50 g/L glucose was consumed within 67 h with the final l-malate concentration and yield of 28.50 g/L, 0.69 g/g when heterologous CO2 source was supplied. The availability of NAD(H) was correlated positively with the glucose utilization rate and cellular metabolism capacities, and lower NADH/NAD+ ratio was beneficial for the accumulation of l-malate under anaerobic conditions. Enhanced ATP level could significantly enlarge the intracellular NAD(H) pool under anaerobic condition. Moreover, there might be an inflection point, that is, the increase of NAD(H) pool before the inflection point is followed by the improvement of metabolic performance, while the increase of NAD(H) pool after the inflection point has no significant impacts and NADH/NAD+ ratio would dominate the metabolic flux. This study is a typical case of anaerobic organic acid fermentation, and demonstrated that ATP level, NAD(H) pool and NADH/NAD+ ratio are three important regulatory parameters during the anaerobic production of l-malate.

中文翻译：

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大肠杆菌的代谢工程，厌氧生产L-苹果酸。

l-苹果酸是最重要的平台化学品之一，广泛用于食品，金属清洁，纺织品整理，药物以及各种精细化学品的合成中。近来，从可再生资源生产l-苹果酸的生物技术路线的发展引起了极大的关注。通过灭活fumB，frdABCD，ldhA和pflB的基因，获得了潜在的产苹果酸的菌株大肠杆菌BA040。在mdh和pck共同过量表达后，BA063的葡萄糖消耗量为18 g / L，导致l-苹果酸滴度的增加，产量分别为13.14 g / L和0.73 g / g。同时，NADH / NAD +比降低至0.72，总NAD（H）为38.85 µmol / g DCW，ATP浓度达到715.79 nmol / g DCW。在带有BA063、41的5L发酵罐中发酵。在67小时内消耗了50 g / L葡萄糖，最终的l-苹果酸浓度和产量为28.50 g / L，当提供异源CO2来源时为0.69 g / g。NAD（H）的有效性与葡萄糖利用率和细胞代谢能力呈正相关，而较低的NADH / NAD +比值有利于厌氧条件下l-苹果酸的积累。增强的ATP水平可以在厌氧条件下显着扩大细胞内NAD（H）库。此外，可能存在拐点，即拐点之前NAD（H）库的增加随后代谢性能的改善，而拐点之后NAD（H）库的增加没有明显影响NADH / NAD +比值将主导代谢通量。