Zhimin Li

Improved succinic acid production in the anaerobic culture of an Escherichia coli pflB ldhA double mutant as a result of enhanced anaplerotic activities in the preceding aerobic culture.

ABSTRACT Escherichia coli NZN111 is a pflB ldhA double mutant which loses its ability to ferment glucose anaerobically due to redox imbalance. In this study, two-stage culture of NZN111 was carried out for succinic acid production. It was found that when NZN111 was aerobically cultured on acetate, it regained the ability to ferment glucose with succinic acid as the major product in subsequent anaerobic culture. In two-stage culture carried out in flasks, succinic acid was produced at a level of 11.26 g/liter from 13.4 g/liter of glucose with a succinic acid yield of 1.28 mol/mol glucose and a productivity of 1.13 g/liter·h in the anaerobic stage. Analyses of key enzyme activities revealed that the activities of isocitrate lyase, malate dehydrogenase, malic enzyme, and phosphoenolpyruvate (PEP) carboxykinase were greatly enhanced while those of pyruvate kinase and PEP carboxylase were reduced in the acetate-grown cells. The two-stage culture was also performed in a 5-liter fermentor without separating the acetate-grown NZN111 cells from spent medium. The overall yield and concentration of succinic acid reached 1.13 mol/mol glucose and 28.2 g/liter, respectively, but the productivity of succinic acid in the anaerobic stage dropped to 0.7 g/liter·h due to cell autolysis and reduced anaplerotic activities. The results indicate the great potential to take advantage of cellular regulation mechanisms for improvement of succinic acid production by a metabolically engineered E. coli strain.

Development of succinic acid production from corncob hydrolysate by Actinobacillus succinogenes

Succinic acid is one of the most important platform chemicals since it has great potential in industrial applications. In this study, corncob hydrolysate was used for succinic acid production. After diluted acid treatment, xylose was released from hemicellulose as the predominant monosaccharide in the hydrolysate, whereas glucose was released very little and most was retained as cellulose in the raw material. Without any detoxification, corncob hydrolysate was used directly as the carbon source in the fermentation. Actinobacillus succinogenes could utilize the sugars in the hydrolysate to produce succinic acid efficiently. Through medium optimization, yeast extract was selected as the nitrogen source and MgCO3 was used to control pH. A total of 23.64xa0g/l of succinic acid was produced with a yield of 0.58xa0g/g based on consumed sugar, indicating that the waste corncob residue can be used to produce value-added chemicals practically.

Enhanced 1,3-propanediol production by supply of organic acids and repeated fed-batch culture

In fed-batch culture of Klebsiella pneumoniae, 1,3-propanediol production was growth associated, while the by-products, including lactic acid and ethanol, increased sharply as the cells grew slowly. When the fed-batch culture was supplied with a mixture of organic acids including citrate, fumarate and succinate, cell growth and 1,3-propanediol production increased significantly, whereas the by-products, especially lactic acid and ethanol, decreased sharply. High concentrations of PDO and acetate inhibited cell growth and PDO production. To improve the PDO production, repeated fed-batch culture with addition of the organic acid mixture was performed in a 5-l reactor. The fed-batch culture was repeated five times, and the 1,3-propanediol yield and concentration reached above 0.61xa0molxa0mol−1 and 66xa0gxa0l−1, respectively, in 20xa0h for each cycle. Furthermore, the PDO productivity reached above 3.30xa0gxa0l−1xa0h−1 in each cycle, which was much higher than that of the original fed-batch culture.

Production of glutathione using a bifunctional enzyme encoded by gshF from Streptococcus thermophilus expressed in Escherichia coli

Glutathione (GSH) is one of the most ubiquitous non-protein thiols that is involved in numerous cellular activities. The gene coding for a novel bifunctional enzyme catalyzing the reaction for glutathione synthesis, gshF, was cloned from Streptococcus thermophilus SIIM B218 and expressed in Escherichia coli JM109. In the presence of the precursor amino acids and ATP, the induced cells of E. coli JM109 (pTrc99A-gshF) could accumulate 10.3 mM GSH in 5 h. The S. thermophilus GshF was insensitive to feedback inhibition caused by GSH even at 20 mM. At elevated concentrations of the precursor amino acids and ATP, E. coli JM109 (pTrc99A-gshF) produced 36 mM GSH with a molar yield of 0.9 mol/mol based on added cysteine and of 0.45 mol/mol based on added ATP. When ATP was replaced with glucose, E. coli JM109 (pTrc99A-gshF) produced 7 mM in 3h. Saccharomyces cerevisiae was used to generate ATP for GSH production. In the presence of glucose and the pmr1 mutant of S. cerevisiae BY4742, JM109 (pTrc99A-gshF) produced 33.9 mM GSH in 12h with a yield of 0.85 mol/mol based on added l-cysteine. It is shown that the S. thermophilus GshF can be successfully used for GSH production.

Enzymatic synthesis of glutathione using yeast cells in two-stage reaction

In the present study, permeated yeast cells were used as the catalyst to synthesize glutathione. When waste cells of brewer’s yeast were incubated with the three precursor amino acids and glucose for 36xa0h, 899xa0mg/L of glutathione were produced. To release the feedback inhibition of γ-glutamylcysteine synthetase caused by glutathione, two-stage reaction was adopted. In the first stage, glycine was omitted from the reaction mixture and only γ-glutamylcysteine was formed. Glycine was then added in the second stage, and 1,569xa0mg/L of glutathione were produced. The conditions of the two-stage reaction were optimized using Plackett–Burman design and response surface methodology. Under the optimized condition, commercially available baker’s yeast produced 3,440xa0mg/L of glutathione in 30xa0h, and most of the produced glutathione was in the medium. The two-stage reaction could effectively reduce the feedback inhibition caused by glutathione, but degradation of glutathione was significant.

Process development of succinic acid production by Escherichia coli NZN111 using acetate as an aerobic carbon source.

Escherichia coli strain NZN111 could convert glucose to succinic acid efficiently in anaerobic conditions after the induction of gluconeogenic carbon sources in aerobic conditions. Acetate shows a strong effect on both yield and productivity of succinic acid. In this study, the fed-batch process of succinic acid production by NZN111 using acetate in a chemically defined medium in the aerobic stage was investigated and developed. Increasing cell density could increase succinic acid with a productivity of 3.97 g/(Lh) in the first 8h of the anaerobic phase with an overall yield of 1.42 mol/mol glucose in a 5L fermentor. However, there was strong repression from succinic acid in the later anaerobic stage. When succinic acid exceeded 30 g/L, the glucose consumption rate began to drop sharply along with the succinic acid production rate. Supplementation with glucose from 30 to 70 g/L in the anaerobic stage showed little effect on succinic acid production. Acetic acid and pyruvic acid accumulated had no effect on succinic acid formation because of their low concentration. With acetate as the sole carbon source for aerobic cultivation in the following scale-up, 60.09 g/L of succinic acid was produced with a yield of 1.37 mol/mol in a 50 L bioreactor.

One-pot synthesis of glutathione by a two-enzyme cascade using a thermophilic ATP regeneration system

In vitro cascade catalysis using enzyme-based system is becoming a promising biomanufacturing platform for biofuels and biochemicals production. Glutathione is a pivotal non-protein thiol compound and widely applied in food and pharmaceutical industries. In this study, glutathione was synthesized by a bifunctional glutathione synthetase together with a thermophilic ATP regeneration system through a two-enzyme cascade in vitro. Four bifunctional glutathione synthetases from Streptococcus sanguinis, S. gordonii, S. uberis and Bacillus cereus were applied for glutathione synthesis. The bifunctional glutathione synthetase from S. sanguinis was selected and coupled with the polyphosphate kinase from Thermosynechococcus elongatus BP-1 for regenerating ATP to produce glutathione in one pot. In the optimized system, 28.5mM glutathione was produced within 5h due to efficient ATP regeneration from low-cost polyphosphate. The yield based on added l-cysteine reached 81.4% and the productivity of glutathione achieved 5.7mM/h. The one-pot system indicated a potential biotransformation platform for industrial production of glutathione.

L-Cysteine Production in Escherichia coli Based on Rational Metabolic Engineering and Modular Strategy

L-cysteine is an amino acid with important physiological functions and has a wide range of applications in medicine, food, animal feed, and cosmetics industry. In this study, the L-cysteine synthesis in Escherichia coliEscherichia coli is divided into four modules: the transport module, sulfur module, precursor module, and degradation module. The engineered strain LH03 (overexpression of the feedback-insensitive cysE and the exporter ydeD in JM109) accumulated 45.8u2009mgu2009L-1 of L-cysteine in 48u2009hr with yield of 0.4% g/g glucose. Further modifications of strains and culture conditions which based on the rational metabolic engineering and modular strategy improved the L-cysteine biosynthesis significantly. The engineered strain LH06 (with additional overexpression of serA, serC, and serB and double mutant of tnaA and sdaA in LH03) produced 620.9u2009mgu2009L-1 of L-cysteine with yield of 6.0% g/g glucose, which increased the production by 12 times and the yield by 14 times more than those of LH03 in the original condition. In fed-batch fermentation performed in a 5-L reactor, the concentration of L-cysteine achieved 5.1u2009gu2009L-1 in 32u2009hr. This work demonstrates that the combination of rational metabolic engineering and module strategy is a promising approach for increasing the L-cysteine production in E. coli.

Continuous culture and proteomic analysis of Escherichia coli DH5α and its acetate-tolerant mutant DA19 under conditions of nitrogen source limitation

Escherichia coli DH5α grows poorly in a chemically defined medium, but its acetate-tolerant mutant, DA19, grows fast and produces less acetate. To investigate the differences in metabolism between the two strains, the samples of continuous cultures at steady state were analyzed by two-dimensional SDS-PAGE (2-D SDS-PAGE) and peptide mass fingerprinting (PMF). The results indicated that in DA19, expression of gnd, yddS, and purH (except for adenine supplement) was up-regulated, and expression of atpA, glnK, and ompR, which encode membrane proteins, was significantly up-regulated. Expression of ackA and yahK in DA19 was down-regulated. In the presence of acetate, hchA and ydcW in DH5α were significantly down-regulated. This study shed light on the differences in metabolic properties between DH5α and DA19, and provided some ideas for improving strains through metabolic engineering.

Central pathway engineering for enhanced succinate biosynthesis from acetate in Escherichia coli

Acetate, a non‐food based substrate obtained from multiple biological and chemical ways, is now being paid great attention in bio‐manufacturing and have a strong potential to compete with sugar‐based carbon source. In this study, acetate can be efficiently converted to succinate by engineered Escherichia coli strains via the combination of several metabolic engineering strategies, including reducing OAA decarboxylation, engineering TCA cycle, enhancement of acetate assimilation pathway and increasing aerobic ATP supply through cofactor engineering. The engineered strain HB03(pTrc99a‐gltA, pBAD33‐Trc‐fdh) accumulated 30.9u2009mM of succinate in 72u2009hr and the yield reached the maximum theoretical yield (∼0.50u2009mol/mol). In the resting‐cell experiments, the yield of succinate in HB03(pTrc99a‐gltA) and HB03(pTrc99a‐gltA, pBAD33‐Trc‐fdh) dropped dramatically, although the productivity of succinate increased due to the high cell density. Further deletion of icdA, formed HB04(pTrc99a‐gltA) and HB04(pTrc99a‐gltA, pBAD33‐Trc‐fdh), increased the yield of succinate in the resting‐cell experiments. The highest concentration of succinate achieved 194u2009mM and the yield reached 0.44u2009mol/mol in 16u2009hr by HB04(pTrc99a‐gltA, pBAD33‐Trc‐fdh). The results showed the metabolically engineered E. coli strains have great potential to produce succinate from acetate.