Expanding metabolism for total biosynthesis of the nonnatural amino acid L-homoalanine

Kechun Zhang, Han Li, Kwang Myung Cho, James C. Liao

Research output: Contribution to journalArticlepeer-review

132 Scopus citations


The dramatic increase in healthcare cost has become a significant burden to the world. Many patients are denied the accessibility of medication because of the high price of drugs. Total biosynthesis of chiral drug intermediates is an environmentally friendly approach that helps provide more affordable pharmaceuticals. Here we have expanded the natural metabolic capability to biosynthesize a non-natural amino acid L-homoalanine, which is a chiral precursor of levetiracetam, brivaracetam, and ethambutol. We developed a selection strategy and altered the substrate specificity of ammoniumassimilating enzyme glutamate dehydrogenase. The specificity constant kcat/K m of the best mutant towards 2-ketobutyrate is 50-fold higher than that towards the natural substrate 2-ketoglutarate. Compared to transaminase IlvE and NADH-dependent valine dehydrogenases, the evolved glutamate dehydrogenase increased the conversion yield of 2-ketobutyrate to L-homoalanine by over 300% in aerobic condition. As a result of overexpressing the mutant glutamate dehydrogenase and Bacillus subtilis threonine dehydratase in a modified threonine-hyperproducing Escherichia coli strain (ATCC98082, △rhtA), 5.4 g/L L-homoalanine was produced from 30 g/L glucose (0.18 g/g glucose yield, 26% of the theoretical maximum). This work opens the possibility of total biosynthesis of other nonnatural chiral compounds that could be useful pharmaceutical intermediates.

Original languageEnglish (US)
Pages (from-to)6234-6239
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number14
StatePublished - Apr 6 2010


  • Chiral chemicals
  • Directed evolution
  • Metabolic engineering
  • Synthetic biology


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