Enzymatic Cascade in Pseudomonas that Produces Pyrazine from α‐Amino Acids

Abstract

Pyrazines are widespread chemical compounds that include pheromones and odors. Herein, a novel mechanism used by Pseudomonas fluorescens SBW25 to biosynthesize monocyclic pyrazines is reported. Heterologous expression of the papABC genes that synthesize the natural α‐amino acid 4‐aminophenylalanine (4APhe), together with three adjacent papDEF genes of unknown function, in Escherichia coli resulted in the production of 2,5‐dimethyl‐3,6‐bis(4‐aminobenzyl)pyrazine (DMBAP), which comprised two symmetrical aminobenzyl moieties derived from 4APhe. It is found that PapD is a novel amino acid C‐acetyltransferase, which decarboxylates and transfers acetyl residues to 4APhe, to generate an α‐aminoketone, which spontaneously dehydrates and condenses to give dihydro DMBAP. PapF is a novel oxidase in the amine oxidase superfamily that oxidizes dihydro DMBAP to yield the pyrazine ring of DMBAP. These two enzymes constitute a unique mechanism for synthesizing monocyclic pyrazines and might serve as a novel strategy for the enzymatic synthesis of pyrazine derivatives from natural α‐amino acids.

Pyrazines: Synthesis and Industrial Application of these Valuable Flavor and Fragrance Compounds

“…In 2018, Turner et al reported a chemo-enzymatic synthesis of substituted pyrazines using an amino transaminase (S-selective, ATA-113, Codexis [24] ) in the presence of a suitable amine donor, which mediated the key amination of the 1,2-diketone precursor to -aminoketones that underwent oxidative dimerization to the final product (Figure 4). [25] In the case of pyrazines, the chirality S. marcescens 3B2 l-threonine → aminoacetone 2,5-dimethylpyrazine 3 3-ethyl-2,5-dimethylpyrazine 5a [31] HqlA NRPS from P. herquei in E. coli l-thyrosine 4,4′-(pyrazine-2,5-diyl-bis(methylene))diphenol 15 [32] ATRed NRPS from X. indica l-phenylalanine 4,4′-(pyrazine-2,5-diyl-bis(methylene))diphenyl [33] SBW25 from P. fluorescens in E. coli 4-aminophenyl alanine 2,5-dimethyl-3,6-bis(4-aminobenzyl)pyrazine 16 [34] B. subtilis l-threonine, d-glucose 2-ethyl-3,5(3,6)-dimethyl pyrazine 5a/b [35] of the amine group is irrelevant for the synthesis of the aromatic heterocycle core. All reactions were carried out at room temperature with isopropyl amine as the amine donor.…”

“…The novel biosynthetic gene cluster comprised of 4-aminophenylalanine (4APhe) C-acetyltransferase, dihydropyrazine oxidase, and a methyltransferase. [34] A potential industrial strain candidate for 2-ethyl-3,5(3,6)dimethyl pyrazine 5a/b attracted the attention of Zhang et al in 2020 after the isolation of a strain of Bacillus subtilis. They proposed the biosynthetic pathway starting from l-threonine and d-glucose, which were tested by substrate addition and isotope-labeling experiments.…”

“…The novel biosynthetic gene cluster comprised of 4‐aminophenylalanine (4APhe) C‐acetyltransferase, dihydropyrazine oxidase, and a methyltransferase. [ 34 ]…”

Quantitative Analysis of Pyrazines and Their Perceptual Interactions in Soy Sauce Aroma Type Baijiu

“…Pyrazines are six-membered heterocyclic compounds with two nitrogen atoms at positions 1 and 4 [14]. Pyrazines are responsible for universally desirable and enjoyable aromas and have been identified in many foods and beverages, particularly as significant volatile components of various nuts, such as peanuts [15], almonds [16], hazelnuts [17] and pecans [18].…”

Pyrazines – valuable flavour & fragrance compounds: Biocatalytic synthesis and industrial applications.

“…The gene sequence included the synthesis of the natural α-amino acid 4-aminophenyl alanine and several other genes for the transformation to non-natural symmetric 2,5-dimethyl-3,6-bis(4-aminobenzyl)pyrazine 16 . The novel biosynthetic gene cluster comprised of 4-aminophenylalanine (4APhe) C-acetyltransferase, dihydropyrazine oxidase, and a methyltransferase [33].…”