ABSTRACT
Hyper-osmotic stress is one of the key factors that decrease the efficiency of biological succinic acid production. To increase the osmotic stress tolerance of succinate-producing Escherichia coli, we studied the influence of IrrE, an exogenous global regulator, on cell osmotic stress resistance. Fermentation results showed that cell growth and succinic acid production by the recombinant increased under different Na+ concentrations. Meanwhile, the maximum dry cell mass, glucose consumption and succinic acid concentration increased 15.6%, 22% and 23%, respectively, when fermented in a 5-L bioreactor. Expressing IrrE improved cell resistance to hyper-osmotic stress. Further comparison of intracellular osmoprotectants (trehalose and glycerol) concentrations showed that trehalose and glycerol concentrations in the recombinant increased. This suggested that introduction of IrrE could enhance intracellular osmoprotectants accumulation which conferred cell with improved resistance to osmotic stress.
ABSTRACT
Sugarcane molasses containing large amounts of sucrose is an economical substrate for succinic acid production. However, Escherichia coli AFP111 cannot metabolize sucrose although it is a promising candidate for succinic acid production. To achieve sucrose utilizing ability, we cloned and expressed cscBKA genes encoding sucrose permease, fructokinase and invertase of non-PTS sucrose-utilization system from E. coli W in E. coli AFP111 to generate a recombinant strain AFP111/pMD19T-cscBKA. After 72 h of anaerobic fermentation of the recombinant in serum bottles, 20 g/L sucrose was consumed and 12 g/L succinic acid was produced. During dual-phase fermentation comprised of initial aerobic growth phase followed by anaerobic fermentation phase, the concentration of succinic acid from sucrose and sugarcane molasses was 34 g/L and 30 g/L, respectively, at 30 h of anaerobic phase in a 3 L fermentor. The results show that the introduction of non-PTS sucrose-utilization system has sucrose-metabolizing capability for cell growth and succinic acid production, and can use cheap sugarcane molasses to produce succinic acid.
Subject(s)
Bioreactors , Escherichia coli , Genetics , Metabolism , Escherichia coli Proteins , Genetics , Fermentation , Membrane Transport Proteins , Genetics , Metabolic Engineering , Molasses , Saccharum , Chemistry , Succinic Acid , Chemistry , Sucrose , ChemistryABSTRACT
Succinic acid is one of the key intermediates in the tricarboxylic acid cycle (TCA)and has huge potentials in biopolymer, food, medicine applications. This article reviews recent research progress in the production of succinic acid by microbial fermentation, including discovery and screening of the succinic-acid-producing microbes, the progress of genetic engineering strategy and metabolic engineering technology for construction of succinic acid-producing strains, and fermentation process control and optimization. Finally, we discussed the limitation of current progress and proposed the future research needs for microbial production of succinic acid.
Subject(s)
Actinobacillus , Genetics , Metabolism , Anaerobiospirillum , Genetics , Metabolism , Fermentation , Industrial Microbiology , Methods , Metabolic Engineering , Methods , Succinic Acid , MetabolismABSTRACT
During the anaerobic fermentation by Escherichia coli AFP111 for succinic acid production, the viable cell concentration and productivity were decreased with the raising of succinic acid concentration. In order to restore cellular succinic acid productivity and prolong fermentation time, we collected strains and refreshed medium for repetitive succinic acid production. However, productivity is lower than that in the anaerobic fermentation before reusing strains. To enhance the productivity, strains were aerobically cultivated for 3 h in pure water before anaerobic fermentation. The activities of key enzymes were enhanced for better performance in producing succinic acid at anaerobic stage. After three rounds of repetitive fermentations, succinic acid concentration and yield reached to 56.50 g/L and 90% respectively. The succinic acid productivity was 0.81 g/(L x h), which was 13% higher than the repetitive fermentations without aerobic activation of the strains.
Subject(s)
Aerobiosis , Anaerobiosis , Culture Media , Escherichia coli , Genetics , Metabolism , Fermentation , Genetic Engineering , Glucose , Metabolism , Industrial Microbiology , Succinic Acid , MetabolismABSTRACT
Escherichia coli NZN111 is a promising strain with ldhA and pflB genes inactivated for the production of succinic acid. However, with these mutations, NAD+ could not be regenerated from NADH, and an unbalanced NADH/NAD+ ratio eliminated cell growth and glucose utilization under anaerobic conditions. Nicotinic acid mononucleotide adenylyltransferase (NAMNAT), encoded by the nadD gene, catalyzes the reaction from nicotinic acid mononucleotide (NaMN) to nicotinic acid adenine dinucleotide (NaAD) during the synthetic pathway of NAD(H). Overexpression of the nadD gene could enhance the concentration of NAD(H) and maintain a suitable NADH/NAD+ ratio. In this study, we constructed a recombinant strain E. coli NZN111/pTrc99a-nadD, and overexpressed NAMNAT with 1.0 mmol/L of IPTG under anaerobic conditions in sealed bottles. Compared to E. coli NZN111, the concentrations of NAD+ and NADH in the recombinant strain increased by 3.21-fold and 1.67-fold, respectively. The total concentration of NAD(H) was increased by 2.63-fold, and the ratio of NADH/NAD+ decreased from 0.64 to 0.42. The recombinant strain restored the cell growth and glucose utilization under anaerobic conditions. After 72 h, the recombinant strain could consume 14.0 g/L of glucose to produce 6.23 g/L of succinic acid, and the concentration of succinic acid was 19-fold higher than in E. coli NZN111.