2.3 Bio-Based Synthesis
The production of pyrazines is abundant in plants, but so far only a few bacteria have been reported to be able to synthesize pyrazines, including Pseudomonas sp. , Bacillus sp. ,Chondromyces sp. [27], and Streptomyces sp.[28].
Furthermore, Li et al. (2017) identified the strain Paenibacillus aceti L14T as a highly efficient pyrazine producer by genome analysis. The strain was isolated from a solid-state acetic acid fermentation culture and is qualified for the production of 1.6 g/l pyrazines including 2,3-di-iso -butylpyrazine, 2-iso -butyl-3-methylpyrazine and 1-(5-iso -butyl-2-pyrazinyl)-1-propanone (1.6 g/l). For further understanding, the group sequenced the genome of L14Tidentifying genetic structures suitable for pyrazine biosynthesis based on precursor amino acids such as valine, leucine and isoleucine as the crucial intermediates [29]. Genome analysis will form the basis for further studies on increasing selectivity and productivity using biotransformation instruments.
In nature, Corynebacterium glutamicum produces small amounts of trimethylpyrazine 1 , dimethylpyrazines 3 , 4 ,6 , and tetramethylpyrazine 8, and the biosynthesis relates to the metabolism of branched-chain amino acids. Wittmannet al. (2010) have shown through a combination of gene knockout and feeding experiments that the biosynthetic pathway to the Top 7 (Figure 1) alkylated pyrazines, starting from pyruvate, requires the activity of acetolactate synthase (AS) and proceeds via acetolactate, followed by a sequence of transamination and oxidation steps transforming acetoin 10 to aminoketones. The last step is the condensation of aminoketones and concomitant oxidation to pyrazines [30].
In contrast, a study by Nawrath et al. (2010) highlighted a different route proposal: in Myxobacteria, pyrazines are proposed to arise from branched amino acids such as valine via reduction to valinal and dimerization of the resulting aminoaldehydes to 13[12].
Silva-Junior et al. recently studied a leaf-cutter ant-associated bacterium which is producing pyrazines used as trail pheromones in ant species. The study proposed the biosynthetic pathway in the bacteriumSerratia marcescens 3B2 leading to symmetric 2,5-dimethylpyrazine 3 , but more importantly the non-symmetric 3-ethyl-2,5-dimethylpyrazine5a both starting with l-threonine, which is converted to aminoacetone. Subsequently, condensation and dehydration lead to compound 1 . In the presence of acetate, the dehydration takes place with simultaneous addition of acetate to C3, resulting in a carbonyl group, which is subsequently reduced and eliminated to obtain exclusively 5a [31]. In comparison, natural pyrazine5a is only available by extraction from natural sources in mixtures with its regio-isomer 3-ethyl-2,6-dimethylpyrazine 5b , which is emphasizing the importance of this selective route.
The following two examples could provide some new strategies for the enzymatic synthesis of pyrazine derivatives from natural α-amino acids according to A) dimerization of α-amino aldehydes and B) dimerization of α-aminoketones in living cells.
An attempt to establish a pyrazine producing cell factory that uses the pathway via the dimerization of aldehydes was carried out in 2018 inE. coli by using a monomodular, non-ribosomal peptide synthetase HqlA from Penicillium herquei [32]. The enzyme reduces amino acids, in this case l-tyrosine, to the corresponding amino-aldehyde, which is then dimerized to the substituted pyrazines15 . The binding of the acid to HqlA consumes ATP and the actual reduction requires NADPH.
Another approach was recently published (2020), in which an enzymatic cascade from Pseudomonas fluorescens SBW25 was introduced intoE. coli to produce reactive aminoketone species, which can immediately dimerize to yield pyrazines. 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].
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-labelling experiments. B. subtilis is GRAS and there is evidence for the formation of other valuable pyrazines in this organism [34].