Both of the phosphoenolpyruvate carboxylase (PPC) and phosphoenolpyruvate carboxykinase (PCK) could catalyze the reaction from phosphoenolpyruvate (PEP) to oxaloacetic acid (OAA) in the pathways of anaerobic mixed acid fermentation for E.coli.In addition,the reaction catalyzed by PCK generates ATP,which is more beneficial to the growth of the strain and the succinic acid production theoretically.In this study,we constructed a ppc defective strain using λ-Red homologous recombination system with the E.coli W3110 (△pfl,△ldh) as the parent strain.Based on that,Bacillus subtilis pck was overexpressed.The preliminary anaerobic fermentation experiments showed that both strains partially recovered the ability to consume glucose through the overexpression of pck.Besides,the ppc defective strain showed the most excellent performance,the rate of glucose consumption and succinate production were 4.2 and 15.3 folds as much as those of the parent strain,respectively.

The fermentation characteristics of recombinant E.coli JM001(△ppc)/pTrc99a-pck for succinate production was investigated.The results showed that its glucose consumption and succinate production rates were 4.2 and 15.3 folds as much as those of the parent strain E.coli JM001,respectively.The mass yield of succinate to glucose was 54%,besides the acetic acid concentration was rather low.It could be concluded that the reaction from phosphoenolpyruvate (PEP) to oxaloacetic acid (OAA) catalyzed by PEP carboxykinase (PCK) was more beneficial to the cell growth and succinate production compared to PEP carboxylase (PPC).Further investigation was carried out to optimize the fermentation conditions.The results showed that bacterial sludge was much better than 10% (v/v) broth for the inoculation from aerobic phase to the anaerobic one,succinate mass yield increased from 55.2% to 65.1% and the entire time to consume 20g/L glucose reduced to 48h.The results of glucose tolerance experiment showed that between 20g/L and 60g/L initial glucose,cell growth and succinate production were both satisfactory,succinate mass yield was from 64.18% to 69.10% with the increase of glucose concentration.But when the glucose was higher than 60g/L,cell growth and succinate production were clearly repressed.The higher the glucose concentration was,the more obvious the inhibition effects were.For 7L fermentor research,the initial glucose of the anaerobic phase was 40.25g/L,after 96 h it was completely consumed.The succinate mass yield was 67.75%.During the anaerobic phase,the glucose consumption rate was 0.42g/(L·h) and succinate productivity was 0.28g/(L·h).

Under anaerobic conditions and in the absence of exogenous electron acceptors,Escherichia coli ferments glucose to a mixture of products consisting of acetate,formate and ethanol,as well as a small amounts of lactate and succinate.In order to reduce the co-products lactate and formate, a ldh pfl double mutant has been constructed based on the RED recombination system and the site-specific recombinant technology.The fermentation results indicated that deficient in both of the two enzymes showed significant growth defects because of pyruvate accumulation which would negatively affect the phosphoenolpyruvate-dependent sugar transport.Since E.coli lacks the anaplerotic enzyme pyruvate carboxylase,pyruvate accumulation and succinate formation in this strain could be altered by directing pyruvate to the succinate branch with this enzyme.Hence,a recombinant plasmid pTrc99a-pyc was constructed and transformed to JM1307.Results showed:with 8 g/L initial glucose,the cell density of JM1307(pTrc99a-pyc)enhanced to OD600 2.5,and the main product was succinate(2.8 g/L)with little acetic acid,and no formic acid and lactic acid were produced.

We studied the effect of aerobic glucose-feeding strategies,which controlled the cell growth on the anaerobic fermentation.The optimum transition time was the one when aerobically grown at special growth rate of 0.07 h-1,at OD600 = 30.E.coli AFP111 showed that the optimal fermenta-tion capability with a final succinic acid concentration of 94.2 g/L,a mass yield of 105% and productivity of 1.76 g/(L.h) during the anaerobic stage.

ABSTRACT Aerobic growth conditions significantly influenced anaerobic succinate production in two-stage fermentation by Escherichia coli AFP111 with knockouts in rpoS, pflAB, ldhA, and ptsG genes. At a low cell growth rate limited by glucose, enzymes involved in the reductive arm of the tricarboxylic acid cycle and the glyoxylate shunt showed elevated activities, providing AFP111 with intracellular redox balance and increased succinic acid yield and productivity.

Succinic acid is regarded as C4 platform chemical, which has a huge potential market with wide applications in biopolymer, food, medicine, and so on. Production of succinic acid by microorganism fer-mentation has attracted much attention in recent times. By fermentation, succinic acid could be produced from renewable resources in place of petroleum with low cost and low pollution; especially CO2 could be fixed by microorganism to form the product, which is a promising way to deal with the green gas. Some E. coli have been chosen as good succinic acid producing strains because of their clear genetic backgrounds, convenience to be modified and controlled, and good growth properties with low nutrients requirements. The progress of genetic engineering strategy and metabolic engineering technology for construction of succinic aicd producing recombinant E. coli has been introduced in this paper with the discussion of the future re-search in this area.

The techniques of saccharifying liquid preparation from cornhusks by acid hydrolysis were stud- ied.The optimum parameters for the hydrolysis based on the yield of total sugar were determined by means of orthogonal experiments and regulative experiments.The best conditions of hydrolysis were as follows:hy- drolysis temperature 110℃,vitriol concentration 1.0%,particle diameter 20～40 meshes,reaction time 90min.The yield of total sugar was 90.37% and the concentration of total sugar was 85 g/L.The saccharif- ying liquid from cornhusks was decolorized by active carbon.Yield of decolorization was 92.27%.Losing rate of total sugar was below 5% and the concentration of furfurol was 0.236 g/L.The shaking flasks exper- iment for the production of succinic acid was carried out and the results showed that the saccharifying liquid could be used as the carbon source substitute for microbe fermentation of succinic acid.