Abstract
Backgroundl-2-aminobutyric acid (l-ABA) is an unnatural amino acid that is a key intermediate for the synthesis of several important pharmaceuticals. To make the biosynthesis of l-ABA environmental friendly and more suitable for the industrial-scale production. We expand the nature metabolic network of Escherichia coli using metabolic engineering approach for the production of l-ABA.ResultsIn this study, Escherichia coli THR strain with a modified pathway for threonine-hyperproduction was engineered via deletion of the rhtA gene from the chromosome. To redirect carbon flux from 2-ketobutyrate (2-KB) to l-ABA, the ilvIH gene was deleted to block the l-isoleucine pathway. Furthermore, the ilvA gene from Escherichia coli W3110 and the leuDH gene from Thermoactinomyces intermedius were amplified and co-overexpressed. The promoter was altered to regulate the expression strength of ilvA* and leuDH. The final engineered strain E. coli THR ΔrhtAΔilvIH/Gap-ilvA*-Pbs-leuDH was able to produce 9.33 g/L of l-ABA with a yield of 0.19 g/L/h by fed-batch fermentation in a 5 L bioreactor.ConclusionsThis novel metabolically tailored strain offers a promising approach to fulfill industrial requirements for production of l-ABA.
Highlights
Construction of engineered E. coli strain for l‐threonine production To expand metabolism for producing l-2-aminobutyric acid (l-ABA), we designed a unique metabolic pathway in E. coli, where l-threonine was taken as a precursor for l-ABA synthesis (Fig. 1)
ΔrhtAΔilvIH/Gap-ilvA*-Pbs-leuDH without any obvious reduction of cell growth when promoters of different strengths including Pbs [30] and Gap from E. coli BL21 (DE3) were employed to regulate the expression of ilvA* and leuDH. These results indicated that the disruption of ilvIH drived more carbon flux to l-ABA and proper adjustments of attenuating the expression of ilvA* by a relatively weak promoter and enhancing the expression of leuDH by a strong promoter were beneficial for the biosynthesis of l-ABA in this strain
In this study, a novel strain E. coli THR ΔrhtAΔilvIH/ Gap-ilvA*-Pbs-leuDH for l-ABA production was constructed through metabolic engineering
Summary
TPM medium was used to monitor the production of l-ABA, during the growth of cells at 35 °C. TPM medium contains per liter: glucose, 50 g; yeast extract, 6 g; MgSO4·7H2O, 2 g; K H2PO4, 4 g; (NH4)2SO4, 14 g; betaine, 1 g; l-methionine, 0.149 g; l-lysine, 0.164 g; trace metal solution, 5 mL and C aCO3, 30 g. The fermentation supernatants were filtered through a 0.22 μm syringe filter (Nylon; Jinteng, Tianjin, China) and used for determination of residual glucose, amino acids and organic acids. The residual concentration of glucose in the media was measured using a glucose analyzer (YSI model 2300, Xylem Inc., Rye Brook, NY, USA) [37] and the amino acids were determined using an amino acid analyzer (SYKAMS-433D, SYKAM, Munich, Germany). Amino acid standards were purchased from Sykam (Amino Acid Calibration Solution H, Sykam, Germany)
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