In this study, culture conditions, including dissolved oxygen (DO) content, presence of osmoprotectants, residual glucose concentration, and ammonium sulfate-feeding strategies, were investigated for decreasing the inhibition effects of acetic acid, ammonium, and osmotic stress on L-lysine fermentation by Escherichia coli. The results revealed that higher DO content and lower residual glucose concentration could decrease acetic acid accumulation, betaine supplementation could enhance osmotic stress tolerance, and variable speed ammonium sulfate-feeding strategy could decrease ammonium inhibition. Thus, with 25 % DO content, 0-5.0 g/L of residual glucose concentration, and 1.5 g/L of betaine supplementation, 134.9 g/L of L-lysine was obtained after 72 h of culture, with L-lysine yield and productivity of 45.4 % and 1.9 g/(L · h), respectively.

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Efficient biocatalytic process construction by relieving substrate and product inhibitions via identification and engineering of enzyme conformational dynamics.

BackgroundThe six-carbon circular non-proteinogenic compound l-pipecolic acid is an important chiral drug intermediate with many applications in the pharmaceutical industry. In the present study, we developed a metabolically engineered strain of Escherichia coli for the overproduction of l-pipecolic acid from glucose.ResultsThe metabolic pathway from l-lysine to l-pipecolic acid was constructed initially by introducing lysine cyclodeaminase (LCD). Next, l-lysine metabolic flux from glucose was amplified by the plasmid-based overexpression of dapA, lysC, and lysA under the control of the strong trc promoter to increase the biosynthetic pool of the precursor l-lysine. Additionally, since the catalytic efficiency of the key enzyme LCD is limited by the cofactor NAD+, the intracellular pyridine nucleotide concentration was rebalanced by expressing the pntAB gene encoding the transhydrogenase, which elevated the proportion of LCD with bound NAD+ and enhanced l-pipecolic acid production significantly. Further, optimization of Fe2+ and surfactant in the fermentation process resulted in 5.33 g/L l-pipecolic acid, with a yield of 0.13 g/g of glucose via fed-batch cultivation.ConclusionsWe expanded the metabolic pathway for the synthesis of the chiral pharmaceutical intermediate l-pipecolic acid in E. coli. Using the engineered E. coli, a fast and efficient fermentative production of l-pipecolic acid was achieved. This strategy could be applied to the biosynthesis of other commercially and industrially important chiral compounds containing piperidine rings.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0666-0) contains supplementary material, which is available to authorized users.

Bio-production of cadaverine from cheap carbon sources for synthesizing bio-based polyamides is becoming more common. Here, a novel fermentation process for cadaverine bio-production from glucose was implemented by using a microbial consortium of two engineered Escherichia coli strains to relieve the toxic effect of cadaverine on fermentation efficiency. To achieve controllable growth of strains in the microbial consortium, two engineered E. coli strains grown separately on different carbon sources were first constructed. The strains were, an L-lysine-producing E. coli NT1004 with glucose as carbon source, and a cadaverine-producing E. coli CAD03 with glucose metabolism deficiency generated by modifying the PTSGlc system with CRISPR-Cas9 technology and inactivating cadaverine degradation pathways. Co-culturing these two engineered E. coli strains with a mixture of glucose and glycerol led to successful production of cadaverine. After optimizing cultivation conditions, a cadaverine titer of 28.5 g/L was achieved with a multi-stage constant-speed feeding strategy.