Engineering Escherichia coli for high-yielding 2,5-Dimethylpyrazine synthesis from L-Threonine by reconstructing metabolic pathways and enhancing cofactors regeneration.

2,5-Dimethylpyrazine (2,5-DMP) is important pharmaceutical raw material and food flavoring agent. Recently, engineering microbes to produce 2,5-DMP has become an attractive alternative to chemical synthesis approach. In this study, metabolic engineering strategies were used to optimize the modified Escherichia coli BL21 (DE3) strain for efficient synthesis of 2,5-DMP using L-threonine dehydrogenase (EcTDH) from Escherichia coli BL21, NADH oxidase (EhNOX) from Enterococcus hirae, aminoacetone oxidase (ScAAO) from Streptococcus cristatus and L-threonine transporter protein (EcSstT) from Escherichia coli BL21, respectively. We further optimized the reaction conditions for synthesizing 2,5-DMP. In optimized conditions, the modified strain can convert L-threonine to obtain 2,5-DMP with a yield of 2897.30 mg/L. Therefore, the strategies used in this study contribute to the development of high-level cell factories for 2,5-DMP.

Expression of Novel L-Leucine Dehydrogenase and High-Level Production of L-Tert-Leucine Catalyzed by Engineered Escherichia coli.

Leucine dehydrogenase (LDH) is a NAD+-dependent oxidoreductase, which can selectively catalyze α-keto acids to obtain α-amino acids and their derivatives. It plays a key role in the biosynthesis of L-tert-leucine (L-Tle). As a non-naturally chiral amino acid, L-Tle can be used as an animal feed additive, nutrition fortifier, which is a perspective and important building block in the pharmaceutical, cosmetic, and food additive industry. In this study, four hypothetical leucine dehydrogenases were discovered by using genome mining technology, using the highly active leucine dehydrogenase LsLeuDH as a probe. These four leucine dehydrogenases were expressed in Escherichia coli BL21(DE3), respectively, and purified to homogeneity and characterized. Compared with the other enzymes, the specific activity of PfLeuDH also shows stronger advantage. In addition, the highly selective biosynthesis of L-Tle from trimethylpyruvic acid (TMP) was successfully carried out by whole-cell catalysis using engineered E. coli cells as biocatalyst, which can efficiently coexpress leucine dehydrogenase and formate dehydrogenase. One hundred-millimolar TMP was catalyzed for 25 h, and the yield and space-time yield of L-Tle reached 87.38% (e.e. >99.99%) and 10.90 g L-1 day-1. In short, this research has initially achieved the biosynthesis of L-Tle, laying a solid foundation for the realization of low-cost and large-scale biosynthesis of L-Tle.