Metabolic engineering of Escherichia coli for L-malate production anaerobically

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Keywords: ATP; Anaerobic fermentation; Escherichia coli; L-malate; NAD(H).

Fig. 1

Engineered metabolic network for L-malate production from glucose in E. coli anaerobically. The undesired genes deleted in this study are marked in the pathways are deleted as marked with yellow oblique bar. The over-expressed and heterologous introduction pathways were marked with red bold lines. Not all enzyme-catalyzed steps and intermediates are shown. Pyr, pyruvate; Fum, Fumarate; Suc, succinate; CIT, citrate; ICIT, isocitrate; PEP, phosphoenolpyruvate; OAA, oxaloacetate; ACoA, acetyl-coenzyme A; ACP, acetyl phosphate; NA, nicotinic acid; NaMN, nicotinic acid mononucleotide; NAD, nicotinamide adenine dinucleotide; NaAD, desamido NAD. The genes involved in the metabolic pathways: ldhA, lactate dehydrogenase; pflB, pyruvate-formate lyase; frdABCD, fumarate reductase; fumB, fumarase; mdh, malate dehydrogenase; ppc, phosphoenolpyruvate carboxylase; pck, phosphoenolpyruvate carboxykinase; pykF, pykA, pyruvate kinase; pta, phosphate acetyltransferase; ackA, acetate kinase; adhE, alcohol dehydrogenase; ptsG, phosphotransferase system; pncB, NA phosphoribosyltransferase; nadD, NaMN adenylyltransferase; nadE, NAD synthetase

Fig. 2

Anaerobic phase of recombinant strain BA063 with CO₂ (a) and N₂ (b) to maintain the anaerobic condition in 5-L fermenter. The experiments were performed in triplicate. DCW, Dry cell weight