Identification of homophenylalanine biosynthetic genes from the cyanobacterium Nostoc punctiforme PCC73102 and application to its microbial production by Escherichia coli

Abstract

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

Fig 1

Locations of l-Hph biosynthesis genes in the anabaenopeptin synthetase cluster of N. punctiforme PCC73102 (A) and the proposed l-Hph biosynthetic pathway mediated by HphA, HphCD, HphB, and aromatic aminotransferase (ATase) in a similar route to leucine biosynthesis (B). The proposed Hph biosynthetic pathway is as follows. l-Phe is converted into phenylpyruvic acid via a transamination reaction mediated by an aminotransferase, such as tyrosine aminotransferase (TyrB). Phenylpyruvic acid is condensed with acetyl-coenzyme A (CoA) catalyzed by HphA, and the resulting thioester is spontaneously hydrolyzed, leading to 2-BMA. 2-BMA is converted into 3-BMA (3-benzylmalic acid) via isomerization of a hydroxyl group mediated by HphCD. The hydroxyl moiety of 3-BMA is oxidized by HphB, followed by spontaneous decarboxylation, producing 2-oxo-phenylbutanoic acid (2-OPB). Finally, 2-OPB is converted into l-Hph via a transamination reaction mediated by an aminotransferase, such as TyrB.

Fig 2

HPLC chromatogram of supernatants obtained from fermentative broths of E. coli W3110 expressing the corresponding Hph proteins. Line 1, HphCD expression only; line 2, HphA and HphCD expression; line 3, HphA, HphCD, and HphB expression; line 4, l-Hph standard (1 g/liter). l-Hph and l-Phe elute at 8.1 and 5.2 min, respectively. The peak at 16.1 min is 2-BMA.

Fig 3

Improvement of l-Hph production by E. coli W3110 strains harboring the various plasmids listed in Table 2. The error bars represent standard deviations (SD) of the mean of three independent experiments.

Fig 4

Complementation experiments with Hph and Leu biosynthesis genes. (A) Growth (OD₆₆₀) of leu gene-deficient mutants and mutants complemented with the corresponding hph gene, as listed in Table 3, in M9 medium. The error bars represent SD of the mean of three independent experiments. (B) l-Hph production by strains containing hph and leu gene combinations as listed in Table 3. The yields of l-Hph are calculated as the average of two independent experiments, the values of which are within 10% error.

Fig 5

Classification of substrates used to examine the substrate specificity of the l-Hph-producing system. dl-m-Fluoro-Phe, dl-o-fluoro-Phe, and l-Tyr are converted into the corresponding homoamino acids. β-Methyl-Phe, l-phenylglycine, l-p-iodo-Phe, l-p-nitro-Phe, and l-Trp could not produce the homoamino acids or the biosynthetic intermediates.