Calcium Carbonate Addition Improves L-Methionine Biosynthesis by Metabolically Engineered Escherichia coli W3110-BL.

L-Methionine (L-Met) is a sulfur-containing amino acid, which is one of the eight essential amino acids to human body. In this work, the fermentative production of L-Met with genetically engineered Escherichia coli W3110-BL in a 5-L fermentor was enhanced through supplement of Ca2+ into the fermentation medium. With the addition of 30 g/L calcium carbonate (CaCO3), the titer of L-Met and yield against glucose reached 1.48 g/L and 0.09 mol/mol glucose, 57.45% higher than those of the control, respectively. The flux balance analysis (FBA) revealed that addition of CaCO3 strengthened the tricarboxylic acid cycle and increased the intracellular ATP concentration by 39.28%. The re-distribution of carbon, ATP, and cofactors flux may collaborate to improve L-Met biosynthesis with E. coli W3110-BL. The regulation of citrate synthase and oxidative phosphorylation pathway was proposed to be important for overproduction of L-Met. These foundations provide helpful reference in the following metabolic modification or fermentation control for further improvement of L-Met biosynthesis.

Effect of dissolved oxygen on L-methionine production from glycerol by Escherichia coli W3110BL using metabolic flux analysis method.

L-Methionine is an essential amino acid in humans, which plays an important role in the synthesis of some important amino acids and proteins. In this work, metabolic flux of batch fermentation of L-methionine with recombinant Escherichia coli W3110BL was analyzed using the flux balance analysis method, which estimated the intracellular flux distributions under different dissolved oxygen conditions. The results revealed the producing L-methionine flux of 4.8 mmol/(g cell·h) [based on the glycerol uptake flux of 100 mmol/(g cell·h)] was obtained at 30% dissolved oxygen level which was higher than that of other dissolved oxygen levels. The carbon fluxes for synthesizing L-methionine were mainly obtained from the pathway of phosphoenolpyruvate to oxaloacetic acid [15.6 mmol/(g cell·h)] but not from the TCA cycle. Hence, increasing the flow from phosphoenolpyruvate to oxaloacetic acid by enhancing the enzyme activity of phosphoenolpyruvate carboxylase might be conducive to the production of L-methionine. Additionally, pentose phosphate pathway could provide a large amount of reducing power NADPH for the synthesis of amino acids and the flux could increase from 41 mmol/(g cell·h) to 51 mmol/(g cell·h) when changing the dissolved oxygen levels, thus meeting the requirement of NADPH for L-methionine production and biomass synthesis. Therefore, the following modification of the strains should based on the improvement of the key pathway and the NAD(P)/NAD(P)H metabolism.

Enhanced L-methionine production by genetically engineered Escherichia coli through fermentation optimization.

Microbial fermentation for L-methionine (L-Met) production based on natural renewable resources is attractive and challenging. In this work, the effects of medium composition and fermentation conditions were investigated to improve L-Met production by genetically engineered Escherichia coli MET-3. Statistical optimization techniques including Plackett-Burman (PB) design and Box-Behnken design (BBD) were adopted first to optimize the culture medium. Results of PB-designed experiments indicated that the culture medium components including glucose, yeast extract, KH2PO4, and MgSO4.7H2O had significant effects on L-Met biosynthesis. With their best-predicted concentration established by BBD (glucose 37.43 g/L, yeast extract 0.95 g/L, KH2PO4 1.82 g/L, and MgSO4.7H2O 4.51 g/L), L-Met titer was increased to 3.04 g/L from less than 2.0 g/L. For further enhancement of L-Met biosynthesis, the fermentation conditions of batch cultivation carried out in a 5-L fermentor were optimized, and the optimum results were obtained at an agitation rate of 300 rpm, medium pH of 7.0, and induction temperature of 28 °C. Based on the optimization parameters, fed-batch fermentation with the modified medium was conducted. As a result, great improvement of L-Met titer (12.80 g/L) and yield (0.13 mol/mol) were achieved, with an increase of 38.53% and 30.0% compared with those of the basal medium, respectively. Furthermore, higher L-Met productivity of 0.261 g/L/h was obtained, representing 2.13-fold higher in comparison to the original medium. The results may provide a helpful reference for further study on strain improvement and fermentation control.