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].