Identification of Homophenylalanine Biosynthetic Genes from the Cyanobacterium Nostoc punctiforme PCC73102 and Application to Its Microbial Production by Escherichia coli

摘要: L-Homophenylalanine (L-Hph) is a useful chiral building block for synthesis of several drugs, including angiotensin-converting enzyme inhibitors and the novel proteasome inhibitor carfilzomib. While chemoenzymatic route fully developed, we investigated microbial production L-Hph to explore possibility more efficient sustainable approach production. We hypothesized that synthesized from L-Phe via mechanism homologous 3-methyl-2-oxobutanoic acid conversion 4-methyl-2-oxopentanoic during leucine biosynthesis. Based on bioinformatics analysis, found three putative homophenylalanine biosynthesis genes, hphA (Npun_F2464), hphB (Npun_F2457), hphCD (Npun_F2458), in cyanobacterium Nostoc punctiforme PCC73102, located around gene cluster responsible anabaenopeptin constructed Escherichia coli strains harboring hphABCD-expressing plasmids achieved fermentative L-Phe. To our knowledge, this first identification genes any organism. Furthermore, improve low efficiency initial strain, optimized expression hphA, hphB, hphCD, which increased yield ∼630 mg/liter. The L-Leu E. were also compared. This analysis revealed HphB has comparatively relaxed substrate specificity can perform function LeuB, but HphA HphCD show tight cannot complement LeuA LeuC/LeuD functions, vice versa. Finally, range tolerance L-Hph-producing strain was examined, showed m-fluorophenylalanine, o-fluorophenylalanine, L-tyrosine accepted as substrates corresponding homoamino acids generated.