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

Abstract

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 nonnatural 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 ammonium-assimilating enzyme glutamate dehydrogenase. The specificity constant k(cat)/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, DeltarhtA), 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.

Fig. 1.

Biosynthesis of L-homoalanine and its pharmaceutical applications. (A) Chemical synthetic routes of antiepileptic and antituberculosis drugs from the chiral intermediate L-homoalanine. (B) Constructing a nonnatural metabolic pathway for L-homoalanine fermentation. Engineered E. coli can overproduce the natural amino acid threonine from glucose. Threonine is converted to 2-ketobutyrate by threonine dehydratase. Then L-homoalanine is synthesized from 2-ketobutyrate by amination.

Fig. 2.

Comparison of different amination enzymes on the production of L-homoalanine. E. coli cultures were inoculated in M9 medium with addition of 10 g/L 2-ketobutyrate and incubated at 37 °C for 24 h. (A) Wild-type E. coli BW25113. Overexpression of (B) transaminase IlvE (C) valine dehydrogenase from Streptomyces avermitilis (D) valine dehydrogenase from Streptomyces coelicolor (E) valine dehydrogenase from Streptomyces fradiae (F) evolved glutamate dehydrogenase GDH1 (G) evolved glutamate dehydrogenase GDH2. Error bars: standard deviations from three independent experiments.

Fig. 3.

Selection strategy to evolve glutamate dehydrogenase (GDH) for amination of 2-ketobutyrate. (A) Wild-type GDH assimilates ammonia directly into glutamate. (B) Knocking out transaminase genes avtA and ilvE from the chromosome makes wild-type E. coli valine auxotrophic, which can be complemented by a mutant GDH active on aminating 2-ketoisovalerate. (C) 2-ketobutyrate is chemically similar to the valine precursor, 2-ketoisovalerate. A mutant GDH active on 2-ketoisovalerate is likely to be active on 2-ketobutyrate.

Fig. 4.

Construction of GDH library for evolution. (A) Binding pocket of Clostridium symbiosum glutamate dehydrogenase (PDB: 1BGV) complexed with its natural substrate glutamate. Residues K89, T193, V377, and S380 are within a radius of 6 Å of the γ-carbon of glutamate. (B) Sequence alignment of C. symbiosum and E. coli GDH. The binding pocket is conserved, and the corresponding residues of E. coli GDH are K92, T195, V377, and S380. These residues were subjected to site-saturation mutagenesis with randomized NNK codon. A library size of 2 million members was transformed into valine auxotrophic E. coli and selected for mutants growing in M9 minimal medium. (C) Growth curve of E. coli (ΔavtA, ΔilvE) transformants in minimal medium. −Val means absence of valine. +Val means presence of valine. Or cells are transformed with GDH1 (K92L, T195A, V377A, and S380C mutations) or GDH2 (K92V and T195S mutations) mutant. Cells did not grow up in absence of valine in the minimal medium, while GDH mutants could rescue cell growth without valine addition.

Fig. 5.

Production of L-homoalanine with different combinations of E. coli strains and threonine dehydratases. (A) (1) BW25113 (2) BW25113 with overexpression of GDH2, (3) ATCC98082 with overexpression of TdcB & GDH2, (4) ATCC98082 with overexpression of IlvA_EC & GDH2, (5) ATCC98082 with overexpression of IlvA_BS & GDH2, (6) ATCC98082 (ΔrhtA) with overexpression of IlvA_BS and GDH2. (B) The yield of L-homolalanine biosynthesis from glucose. Error bars: standard deviations from three independent experiments.