Efficient synthesis of l-tert-leucine through reductive amination using leucine dehydrogenase and formate dehydrogenase coexpressed in recombinant E. coli

• l-tert-Leucine was synthesized through reductive amination of trimethylpyruvate.
• LeuDH was coexpressed with FDH in recombinant Escherichia coli for NADH regeneration.
• Substrate feeding strategy was adopted to improve the process efficiency.
• 1.5 M of trimethylpyruvate was stoichiometrically converted to l-Tle with >99% ee.
• The continuous procedure reached a higher space-time yield of 786 g L−1 d−1.

Enantiopure l-tert-leucine (l-Tle) was synthesized through reductive amination of trimethylpyruvate catalyzed by cell-free extracts of recombinant Escherichia coli coexpressing leucine dehydrogenase (LeuDH) and formate dehydrogenase (FDH). The leudh gene from Lysinibacillus sphaericus CGMCC 1.1677 encoding LeuDH was cloned and coexpressed with NAD+-dependent FDH from Candida boidinii for NADH regeneration. The batch reaction conditions for the synthesis of l-Tle were systematically optimized. Two substrate feeding modes (intermittent and continuous) were addressed to alleviate substrate inhibition and thus improve the space-time yield. The continuous feeding process was conveniently performed in water at an overall substrate concentration up to 1.5 M, with both conversion and ee of >99% and space-time yield of 786 g L−1 d−1, respectively. Furthermore, the preparation was successfully scaled up to a 1 L scale, demonstrating the developed procedure showed a great industrial potential for the production of enantiopure l-Tle.

An efficient preparation of l-Tle was developed through reductive amination of trimethylpyruvate catalyzed by cell-free extracts of recombinant E. coli coexpressing leucine dehydrogenase and NAD+-dependent formate dehydrogenase.Figure optionsDownload as PowerPoint slide