3. Construction of L-tert-Leucine Producing Strain by Expressing Heterologous Leucine Dehydrogenase and Formate Dehydrogenase in Escherichia coli

L-tert-Leucine is an unnatural amino acid that is a key intermediate for the synthesis of several important drugs. The L-tert-Leucine synthesis can be performed continuously by the collaboration of leucine dehydrogenase and formate dehydrogenase. In this study, recombinant strains of Escherichia coli expressing leucine dehydrogenase (LeuDH) and formate dehydrogenase (FDH), respectively, and the strain co-expressing the two enzymes were constructed. The activity for the two enzymes of the cell extraction from different recombinant strains was determined. L-tert-Leucine was successfully synthesized by the recombinant strains, and the yield in different conditions was compared. The production of L-tert-Leucine was the highest when cell extraction of strains containing pLeuDH and pFDH, 1 mL cells extract could produce 4.5 mg L-tert-Leucine, while 1 mL whole cells could only produce 1.05 mg L-tert-Leucine. The yield of L-tert-Leucine was 3.375 mg/mL cell extraction of the strain containing pLeuDHFDH when NAD was added, while the yield fell to 1.635 mg/mL when the whole cell was used.