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\begin{document}

\title{Pyrazines -- valuable flavour \& fragrance compounds: Biocatalytic
synthesis and industrial applications.}



\author[1]{Frederik Mortzfeld}%
\author[2]{Chiam Hashem}%
\author[3]{Kvetka Vrankova}%
\author[4]{Margit Winkler}%
\author[1]{Florian Rudroff}%
\affil[1]{TU Wien Faculty of Technical Chemistry}%
\affil[2]{Graz University of Technology}%
\affil[3]{Axxence Slovakia, s.r.o.}%
\affil[4]{ACIB GmbH}%


\vspace{-1em}



  
  \date{June 11, 2024}


\begingroup
\let\center\flushleft
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\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Substituted alkyl pyrazines - other than being extracted from various
natural sources such as coffee beans, cocoa beans, nuts and vegetables -
can be synthesized by the use of traditional chemical methods or by the
help of certain microorganisms. The importance of pyrazines for food
industry is expected to grow in the upcoming years due to the higher
demand for ready meals, coffee and chocolate drinks; the roasty, nutty
and earthy smell is reminiscent for coffee and cocoa depending on
substitution and concentration of pyrazines. The growing awareness of
people about the ingredients and the origin of their daily food has
strongly influenced the market with labels like 'organic` and 'natural`.
Many flavor ingredients prepared by biotechnology methods have conquered
the market in recent years and are destined to replace and optimize the
ineffective (0.01\% pyrazine/kg biomass) extraction from plants or
animal sources. This overview focuses on the achievements and the
upcoming challenges in pyrazine synthesis. Major parts deal with the
extraction of natural products from sugar molasses, the chemical
synthesis, fermentation by microorganisms and preparative methods by
biocatalysis. The different types of production are decisive for the
declaration and value of the final product and can span from 200-3500
\$/kg for the synthetically produced or the naturally extracted
2,5-dimethylpyrazine, respectively.%
\end{abstract}%



\sloppy


Mini-review / Perspective

\textbf{Title: \emph{Pyrazines: Valuable flavor and fragrance compounds
- Synthesis and industrial applications}}

{[}Frederik B. Mortzfeld{]}\textsuperscript{1},

{[}Chiam Hashem{]}\textsuperscript{2}

{[}Kvetoslava Vrankov\selectlanguage{ngerman}á{]}\textsuperscript{3}

{[}Margit Winkler{]}\textsuperscript{2}

{[}Florian Rudroff{]}\textsuperscript{1,*}

\textsuperscript{1} Institute of Applied Synthetic Chemistry, TU Wien,
Getreidemarkt 9, 1060 Vienna, Austria.

\textsuperscript{2} Institute of Molecular Biotechnology, TU Graz,
Petersgasse 12, 8010 Graz, Austria

\textsuperscript{3} Axxence Slovakia, s.r.o., Mickiewiczova 9, 81107
Bratislava, Slovakia

\textbf{Correspondence:} {[}Prof. Florian Rudroff, Institute of Applied
Synthetic Chemistry, TU Wien, Getreidemarkt 9/163-OC, 1060 Vienna,
Austria{]}.

\textbf{E-mail} : {[}florian.rudroff@tuwien.ac.at{]}

\textbf{Keywords:} Biocatalysis, Biotransformation, Chemo-Enzymatic
Reactions, Fragrance and Flavor Compounds, Pyrazine,

\textbf{Abbreviations:}

\textbf{AmDH} , amine dehydrogenase; \textbf{AS} , Acetolactate
synthase;\textbf{CAGR} , Compound Annual Growth Rate; \textbf{CDH} ,
cellobiose dehydrogenase; \textbf{GRAS} , Generally recognized as
safe;\textbf{ox.,} oxidation; \textbf{PDC} , pyruvate
decarboxylase;\textbf{TDH,} l-threonine-3-dehydrogenase;
\textbf{TDA,}l-threonine deaminase; \emph{t}\textbf{-BuOK} ,
Potassium\emph{tert} -butoxide; \textbf{ThDP} , thiamine
diphosphate;\textbf{SucA} , ThDP-dependent E1 subunit of the
\selectlanguage{greek}α-\selectlanguage{english}ketoglutarate dehydrogenase complex

A\textbf{bstract}

Substituted alkyl pyrazines - other than being extracted from various
natural sources such as coffee beans, cocoa beans, nuts and vegetables -
can be synthesized by the use of traditional chemical methods or by the
help of certain microorganisms. The importance of pyrazines for food
industry is expected to grow in the upcoming years due to the higher
demand for ready meals, coffee and chocolate drinks; the roasty, nutty
and earthy smell is reminiscent for coffee and cocoa depending on
substitution and concentration of pyrazines. The growing awareness of
people about the ingredients and the origin of their daily food has
strongly influenced the market with labels like 'organic` and 'natural`.
Many flavor ingredients prepared by biotechnology methods have conquered
the market in recent years and are destined to replace and optimize the
ineffective (0.01\% pyrazine/kg biomass) extraction from plants or
animal sources. This overview focuses on the achievements and the
upcoming challenges in pyrazine synthesis. Major parts deal with the
extraction of natural products from sugar molasses, the chemical
synthesis, fermentation by microorganisms and preparative methods by
biocatalysis. The different types of production are decisive for the
declaration and value of the final product and can span from 200-3500
\$/kg for the synthetically produced or the naturally extracted
2,5-dimethylpyrazine, respectively.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Graphical abstract.}

\textbf{1 Introduction}

\emph{Short History \& Appearances of Pyrazines, Extraction from
Molasses}

Humankind has always been fascinated by the food it eats. Historically,
herbs and spices were used to improve the taste and preserve the shelf
life of food {[}1{]}. Later, thermal processing such as frying, cooking
and baking marked the beginning of changing the properties and flavors
of foods, indifferent about the occurrence of triggered organic
reactions at high temperatures. Both, the advent of modern organic
chemistry and the increasing demand of food flavors - from strawberry to
vanilla -- are the reasons why chemically synthesized flavors have
appeared in our food. Many of these have been inspired by naturally
occurring fermentation processes with the help of further achievements
in chemistry, microbiology, biochemistry and genetics {[}2{]}. To cover
global demands, further progress is necessary. For example, the world's
strawberry harvest can cover only a small amount for strawberry flavored
products such as strawberry jams, dairy products, ice cream, shampoos,
room fragrances \emph{etc} . and is often enriched in flavour with
chemical or biological synthesized flavorings {[}personal consultations
with Axxence Slovakia s.r.o.{]}.

Today, most flavorings are produced by chemical synthesis or derived
from natural sources. However, market analysis shows a customer trend
craving products that are `natural` or at least containing `natural
flavor` which means that ingredients are coming from natural sources
such as a spice, fruit, or vegetable (see part of the EU regulation in
chapter 2.4 \emph{(EEC No 1334/2008)).} The legislation of the
regulations varies from country to country. Most commercial flavor
compounds do not qualify to be labelled 'natural' despite being the
structural equivalent of natural flavors because they are synthesized
{[}3{]} chemically. Biological preparation is more often hindered due to
high production costs of compatible unit operations or deficiency in
availability of sources. Vanilla flavor is a popular example since only
less than 1\% of its global consumption derives from vanilla orchids
{[}4{]}.\{Kundu, 2017 \#420;Fache, 2016 \#424\} Thousands of tons of
vanillin are produced chemically, although `biovanillin' is emerging
{[}5{]}. Public perception increases the pressure on industrial
processes towards more sustainability. However, extraction from plants
is often limited by low concentrations of the desired compounds and
their appearance in variable and complex mixtures.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 1.} Overview on alkyl pyrazines. The most processed alkyl
pyrazines\textbf{1} -\textbf{7} . Annual volumes of use of pyrazine
derivatives used as flavoring agents in Europe 2004 is stated in
kg/year, whereas the * represents the quantitative natural occurrence of
alkyl pyrazines in foods in Europe in tonnes per year {[}6{]}. Special
attention belongs to compounds \textbf{1} , \textbf{3} , \textbf{4,} and
\textbf{8} which are commercially available as pure substance declared
as natural whereas others are at least partially chemical synthesized.
Short notice: To the best of our knowledge these are the latest official
volumes of the WHO and EFSA {[}7{]}. Non-commercial Synthetic
alkyl-pyrazines\textbf{11} -\textbf{16} .

The consequences are high extraction costs and a product line that
suffers from high risk of change in taste and smell. Therefore,
industrial research is turning towards bio-based and/or biocatalytic
strategies. Nowadays, the use of certain microorganisms {[}8{]} is well
known and declared as natural and safe by food authorities {[}9{]}
(EFSA, FDA \emph{etc.} ), and offers the chance for a better economic
feasibility with mild reaction conditions, high specificity and
simplified downstream processes due to higher purities, better
reproducibility and constant sourcing in contrast e.g. to the natural
feedstock extraction.

Pyrazines are volatile, nitrogen-containing heterocyclic compounds
widely distributed in plants {[}10{]}, insects {[}11{]}, fungi and
bacteria {[}12{]}. They act as an odor signal to repel predators and
effectively prevent vegetative tissue or immature fruit from being eaten
{[}13{]}. For this reason, pyrazines find various applications as
ingredients in pesticides, insecticides, dyes, and pharmaceutical
compounds {[}14{]}. Pyrazines gained attention from the food industry as
important ingredients in raw and roasted foods. Especially alkylated
pyrazines are in the focus, as they have strong olfactory properties
(Figure 1). They can be extracted from natural sources such as potatoes,
nuts and coffee with applications in numerous areas, e.g.
2,3-dimethypyrazine \textbf{6} (coffee, nutty, roasty) is used in
various beverages and sauces, while 2,5-dimethylpyrazine
\textbf{3}(nutty, musty, earthy, roasted cocoa) is used in breakfast
cereals. 2,3,5-Trimethylpyrazine \textbf{1} (chocolate enhancer) and
2,6-dimethylpyrazine \textbf{4} (roasty, nutty, beef) are used in meals
with coffee, cocoa, meat or potatoes as a roasted flavour.
2-Ethyl-3-methylpyrazine \textbf{2} (nutty, musty, corn, raw, earthy,
oily) is used in peanut products, popcorn, and in bread. The perceived
odor depends on the dilution (recommended 0.1\% or less in dipropylene
glycol) of the substance. Methoxylated pyrazines are mainly used in the
perfume industry to improve the odor of cosmetics and toiletries
{[}15{]} (fresh, green, woody, ambery, oriental, musky, minty and
herbaceous). Pyrazine flavors are often added to microwave foods because
roasting odors are not formed in microwaves at temperatures below 200\selectlanguage{ngerman}°C
{[}16{]}.

The growing demand for the `natural` alkyl pyrazines described above
cannot be satisfied by extraction and concentration techniques from
natural sources alone, since the natural content of e.g. 0.01 wt. \%
pyrazines in sugar beet molasses is low {[}14c, 17{]}. Modern
biotechnology, recognized as a sustainable economic process, could cope
with the growing demand for `natural` compounds in the future and may
further increase the amount may fulfil the market demand of selectively
substituted pyrazines.

\textbf{2 Different approaches to produce the same molecule}

Pyrazines are formed by the condensation of two molecules of
\selectlanguage{greek}α-\selectlanguage{english}aminocarbonyls such as amino acids or amino sugars (Maillard reaction)
{[}18{]}. The two aminocarbonyl compounds firstly react to form a
dihydropyrazine and subsequently undergo aromatization by oxidation with
air or elimination of a hydroxyl group to form the side chain. Pyrazines
can be extracted and distilled in low amounts directly from natural
sources such as vegetables, coffee, cocoa and nuts, most of them
deliberately formed at elevated temperature in roasting processes in the
case of coffee and cocoa {[}17, 19{]} (Figure 3, \textbf{C} ) or
unintentionally in refining processes of e.g. white sugar {[}20{]}.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 2.} Retrosynthetic analysis shows different ways leading
to the same molecule. Overview about different strategies: A) Chemical
synthesis via amination of 1,2-diketones and subsequent
dimerization/condensation to pyrazine starting from oxidized chemical
precursors. B) Combination of chemical precursor and enzymatic amination
synthesis using aminotransferase (ATA-113).

An example that has been studied over centuries is
3-isobutyl-2-methoxypyrazine, a characteristic odor in grapes (Figure
3,\textbf{C} ), which is biosynthesized by the plant from leucine and
glycine {[}21{]} or leucinamide and glyoxal {[}10{]}. Efforts have been
made to identify these hypothetical precursors without final
confirmation.

In general, chemical synthesis cannot convert two amino acids to a
pyrazine core without a suitable catalyst. However, it is feasible to
start from 1,2-diketones and diamines or aminoketones and amino
aldehydes or other synthetic equivalents (Figure 2, \textbf{A} ). The
enzymatic synthesis can combine chemical precursors and enzyme catalysis
to obtain the final product (Figure 2, \textbf{B} ). Microbial synthesis
uses the cellular network of naturally pyrazine producing microorganisms
to form selectively substituted pyrazines by feeding the microorganism
the necessary precursors (Figure 3, \textbf{D} ). The advantages and
challenges of chemistry, biocatalysis and biotechnology are summarized
below.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 3.} Retrosynthetic analysis shows different ways leading
to the same molecule. Overview about different strategies: C) Extraction
and distillation from natural sources (vegetables, nuts and beans) with
focus on the biosynthesis of 3-isobutyl-2-methoxypyrazine in grapes from
amino acids leucine and glycine and D) Biobased syntheses by in vivo
biotransformation starting from amino acids and sugars.

\textbf{2.1 Chemical Synthesis}

The most prominent route for traditional pyrazine synthesis is the
reaction of 1,2-diketones in the presence of ammonia to form diamines or
aminoketones {[}22{]}. The list of examples is long, nevertheless an
easy high yielding representative example is the reaction of diketones
with diamines in methanol at room temperature under dry conditions in
the presence of \emph{tert} -BuOK in up to 5 hours, that yields above 70
\% of alkylated pyrazines {[}23{]}.

\textbf{2.2 Enzymatic approach}

In 2018, Turner \emph{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
\selectlanguage{greek}α-\selectlanguage{english}aminoketones that underwent oxidative dimerization to the final
product (Figure 2){[}25{]}. In the case of pyrazines, the chirality 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. Substrates were exhausted after
72 hours and pyrazine was extracted in pure form from the aqueous phase,
but the yield of pyrazine was still moderate at 50-65\% for
symmetric\textbf{11,} deriving from cyclohexane-1,2-dione and \textbf{8}
deriving from diacetyl, and 32\% for the non-symmetric \textbf{12} from
pentane-2,3-dione (Figure 1). The explanation for by-product formation
requires further studies e.g. identification of possible double aminated
by-products in the aqueous phase or extraction problems. The question
whether dimerization to the pyrazine core occurred in the aqueous buffer
or in organic solvent after extraction remains elusive and needs further
investigations.

On the one hand, diketones can be produced by different chemical steps
from various building blocks (Figure 2). On the other hand, there are
also biological options, since the biological route is known to be via
acetoin \textbf{10} (3-hydroxy-butan-2-one, Figure 2). Recent
developments have been made with a 'new' ThDP-dependent lyase, which is
able to synthesize acetoin building blocks from smaller subunits e.g.
pyruvate and activated acetaldehyde enzymatically. Pohl \emph{et
al.}showed with engineered PDC from \emph{Zymomonas mobilis} (\emph{Zm}
PDC) and \emph{Acetobacter pasteurianus} (\emph{Ap} PDC) they can obtain
excellent yields of 61-98\% from the combinations of arylated aldehydes
with 1) 3-oxobutanoic acid and 2) alkyl aldehydes. At the same time, it
was published how to obtain excellent pyrazine yields from either
acetaldehyde with SucA {[}ThDP-dependent E1 subunit of the
\selectlanguage{greek}α-\selectlanguage{english}ketoglutarate dehydrogenase complex from \emph{Escherichia
coli}(\emph{E.coli} )\emph{)} {]}, or from pyruvate (3-oxopropionic
acid) with an cellobiose dehydrogenase (\emph{CDH} ){[}26{]}.

In industrial processes, cost is one of the determining factors. The
price of ATAs (e.g. ATA-113) is much higher than chemical amination with
simple ammonia. The enantiomeric integrity of the amination is of great
importance e.g. for pharmaceuticals; however, for the production of
planar heterocycles the enantioselectivity of the amination is
redundant, whereas high regioselectivity of the condensation is
essential.

In this context, non-symmetric pyrazines could be synthesized
regioselective with ATA-~113 {[}25{]} in an one-pot approach, whereas a
standard chemical synthesis would only allow access to symmetric or a
mixture of non-symmetric pyrazines.

\textbf{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 \emph{Pseudomonas sp.} , \emph{Bacillus sp.}
,\emph{Chondromyces sp.} {[}27{]}\emph{,} and \emph{Streptomyces
sp.}{[}28{]}.

Furthermore, Li et al. (2017) identified the strain \emph{Paenibacillus
aceti} L14\textsuperscript{T} 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-\emph{iso} -butylpyrazine, 2-\emph{iso}
-butyl-3-methylpyrazine and 1-(5-\emph{iso}
-butyl-2-pyrazinyl)-1-propanone (1.6 g/l). For further understanding,
the group sequenced the genome of L14\textsuperscript{T}identifying
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, \emph{Corynebacterium glutamicum} produces small amounts of
trimethylpyrazine \textbf{1} , dimethylpyrazines \textbf{3} , \textbf{4}
,\textbf{6} , and tetramethylpyrazine \textbf{8,} and the biosynthesis
relates to the metabolism of branched-chain amino acids.
Wittmann\emph{et 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 \textbf{10} to aminoketones. The last step is
the condensation of aminoketones and concomitant oxidation to pyrazines
{[}30{]}.

In contrast, a study by Nawrath \emph{et al.} (2010) highlighted a
different route proposal: in \emph{Myxobacteria,} pyrazines are proposed
to arise from branched amino acids such as valine via reduction to
valinal and dimerization of the resulting aminoaldehydes to
\textbf{13}{[}12{]}.

Silva-Junior \emph{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
bacterium\emph{Serratia~marcescens} 3B2 leading to symmetric
2,5-dimethylpyrazine \textbf{3} , but more importantly the non-symmetric
3-ethyl-2,5-dimethylpyrazine\textbf{5a} both starting with l-threonine,
which is converted to aminoacetone. Subsequently, condensation and
dehydration lead to compound \textbf{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 \textbf{5a} {[}31{]}. In comparison,
natural pyrazine\textbf{5a} is only available by extraction from natural
sources in mixtures with its regio-isomer 3-ethyl-2,6-dimethylpyrazine
\textbf{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 \selectlanguage{greek}α-\selectlanguage{english}amino acids
according to A) dimerization of \selectlanguage{greek}α-\selectlanguage{english}amino aldehydes and B) dimerization of
\selectlanguage{greek}α-\selectlanguage{english}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
in\emph{E.~coli} by using a monomodular, non-ribosomal peptide
synthetase HqlA from \emph{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
pyrazines\textbf{15} . 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 \emph{Pseudomonas fluorescens} SBW25 was introduced
into\emph{E.~coli} to produce reactive aminoketone species, which can
immediately dimerize to yield pyrazines. The gene sequence included the
synthesis of the natural \selectlanguage{greek}α-\selectlanguage{english}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 \textbf{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 \textbf{5a/b} attracted the attention of Zhang \emph{et al.} in
2020 after the isolation of a strain of \emph{Bacillus subtilis} . They
proposed the biosynthetic pathway starting from L-threonine and
D-glucose, which were tested by substrate addition and isotope-labelling
experiments. \emph{B. subtilis} is GRAS and there is evidence for the
formation of other valuable pyrazines in this organism {[}34{]}.

\textbf{2.4 Industrial Applications and 'Natural` certification as
driving force of bio-based approaches}

Lately, there has been an increasing call for the sustainability in
industry, especially in food for daily consumption and healthy food. The
global market for the flavor and fragrance industry has tripled since
the beginning of the 21\textsuperscript{st} century. According to a new
market research study, this industry will grow at a CAGR (Compound
Annual Growth Rate) of approximately 3.1\% over the next five years,
reaching US\$~30.5 billion in 2024 (US\$~25.5 billion in 2019). In
addition to the CAGR of 3.1\%, the natural and organic flavors sector is
growing much faster, with an average annual growth rate of 5.7\% to
US\$~11 billion in 2026 from US\$~6.8 billion in 2019 {[}35{]}. This
growth is mainly derived due to the global pressure for the use of
flavors in food production, triggered by customer's demand as well as
the increasing cost efficiency through technological progress and the
concomitant possibility to declare products as natural.

The European regulation on flavors (EEC No 1334/2008) defines in article
3 (2) c): \emph{``Natural flavoring substance shall mean a flavoring
substance obtained by appropriate physical, enzymatic or microbiological
processes from material of vegetable, animal or microbiological origin
either in the raw state or after processing for human consumption by one
or more of the traditional food preparation processes''.}

To the best of our knowledge, no biotechnological syntheses for
industrial production of alkylated pyrazine derivatives, with the
declaration natural, have currently been registered. Thus, extraction
from natural sources is the only access to natural pyrazines to this
end.

\textbf{2.5 Conclusions}

In recent decades, many different bacterial and enzymatic processes for
the synthesis of natural fragrances and flavors have been described in
literature. Few of these processes have made it into industrial
large-scale production, due to high barriers in the strongly cost-driven
beverage and food industry.

In the case of alkyl pyrazines, the increasing demand can no longer be
supplied economically from natural sources.

As discussed above, the consumer can no longer be satisfied by
commercial chemical synthesis and the dependency on fossil oil chemicals
is becoming a crucial negative factor. Production in engineered
microorganisms such as \emph{Pseudomonas} sp., \emph{Bacillus}
sp.,\emph{Chondromyces} sp. and \emph{Streptomyces} sp. opens
possibilities for the future of small \emph{in vivo} factories. Up to
now, pyrazine producing strains were identified and modified by gene
knockouts or transfer of genes into other organisms to produce several
types of pyrazines. However, these systems are far too unspecific and
produce many different products. Cell factories are more difficult to
optimize compared to conventional chemistry, where typically one main
product and few by-products are obtained. Recently, optimized \emph{in
vivo}factories of heterologous genes for pyrazine synthesis in \emph{E.
coli}have been thoroughly described. However, the product range has been
very limited so far and has little to do with the 7 most required
alkylpyrazines. For these 7 compounds, smart routes yet need to be
designed. While industry will continue to improve running processes,
academic developments in technological science will expand the
complementary fields of chemistry and microbiology to deliver
commercially affordable and sustainable products in the next decades.

\textbf{Acknowledgement}

C.H. has been supported by the Austrian Science Fund (FWF):
{[}860262{]}.

\textbf{Conflict of interest}

The authors declare no conflict of interest.

\textbf{References}

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