The Improvement Of Succinate Production By Regulating ATP Generation And NADH/NAD~+ Ratio In Escherichia Coli

Succinic acid(SA)has been recognized as one of the most important bio-based building block chemicals due to its numerous potential applications.A lot of strategies were reported to improve succinic acid production in Escherichia coli,such as blocking competitive pathway,activating glyoxylic acid pathway,overexpressing key succinate-production enzymes,and improving the supply of energy and reducing equivalents.In the previous study,we constructed a series of succinate-producing strains.In this paper,we regulate the generation of ATP and the ratio of NADH/NAD+ to improve succinate production by the adjustment of the ratio of substrate,the modification of respiratory chain and the application of ATP futile cycling.Glucose and xylose are the main components of cellulosic hydrolyzate,we previously engineered Escherichia coli YL104H to efficiently produce succinate from glucose.Furthermore,the present study proved that YL104H could also co-utilize xylose and glucose for succinate production.However,anaerobic succinate accumulation using xylose as the sole carbon source failed,probably because of an insufficient supply of energy.By analyzing the ATP generation under anaerobic conditions in the presence of glucose or xylose,we indicated that succinate production was affected by the intracellular ATP level,which can be simply regulated by the substrate ratio of xylose to glucose.The improved succinate production was achieved by increased ATP supply in previous studies,we for the first time regulated the ATP generation by optimizing the sugar ratios to improved succinate production.Different proportions of mixed sugars were tried for succinate fermentation,and the highest titer was achieved with a glucose-to-xylose ratio of 1:2.This finding was confirmed by succinate production using an artificial mixture containing different xylose to glucose ratios.Using xylose mother liquor,a waste containing both glucose and xylose derived from xylitol production,a final succinate titer of 61.66 g/L with an overall productivity of 0.95 g/L/h was achieved.We previously engineered Escherichia coli YL104 to efficiently produce succinate from glucose,but the overall yield of YL104 remains to be improved.In this study,we investigated the relationships between the NADH/NAD+ ratio,ATP level,and overall yield of succinate production by using glucose as the carbon source in YL104.First,the use of sole NADH dehydrogenases increased the overall yield of succinate by 7%and substantially decreased the NADH/NAD+ ratio.Second,the soluble fumarate reductase from Saccharomyces cerevisiae was overexpressed to manipulate the anaerobic NADH/NAD+ ratio and ATP level.Third,another strategy for reducing the ATP level was applied by introducing ATP futile cycling for improving succinate production.Finally,a combination of these methods exerted a synergistic effect on improving the overall yield of succinate,which was 39%higher than that of the previously engineered strain YL104.Increasing the supply of energy and reducing equivalents is a common method to improve succinate production.In this paper,it was found that the optimized NADH/NAD+ ratio and ATP level are more beneficial for succinate synthesis.Moreover,these strategies can be extended to other biochemicals production processes.