Fermentative production of the unnatural amino acid L-2-aminobutyric acid based on metabolic engineering.

BACKGROUND: L-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. RESULTS: In 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. CONCLUSIONS: This novel metabolically tailored strain offers a promising approach to fulfill industrial requirements for production of L-ABA.

Increased productivity of L-2-aminobutyric acid and total turnover number of NAD+/NADH in a one-pot system through enhanced thermostability of L-threonine deaminase.

OBJECTIVE: To strengthen NADH regeneration in the biosynthesis of L-2-aminobutyric acid (L-ABA). RESULTS: L-Threonine deaminase (L-TD) from Escherichia coli K12 was modified by directed evolution and rational design to improve its endurance to heat treatment. The half-life of mutant G323D/F510L/T344A at 42 °C increased from 10 to 210 min, a 20-fold increase compared to the wild-type L-TD, and the temperature at which the activity of the enzyme decreased by 50% in 15 min increased from 39 to 53 °C. The mutant together with thermostable L-leucine dehydrogenase from Bacillus sphaericus DSM730 and formate dehydrogenase from Candida boidinii constituted a one-pot system for L-ABA biosynthesis. Employing preheat treatment in the one-pot system, the biosynthesis of L-ABA and total turnover number of NAD+/NADH were 0.993 M and 16,469, in contrast to 0.635 M and 10,531 with wild-type L-TD, respectively. CONCLUSIONS: By using the engineered L-TD during endured preheat treatment, the one-pot system has achieved a higher productivity of L-ABA and total turnover number of coenzyme.