Introduction
The worldwide market for flavors and fragrances generates a 30 billion
US-$ revenue volume per year, and grows at a 5 % annual rate. Natural
flavor production is of increasing importance, predominantly in Europe
and North America. The U.S. Food & Drug Administration defines natural
flavors as those deriving their chemicals from animal or plant sources,
as opposed to artificial flavors that use synthetic chemicals in the
production process.
Industrial biotechnology can fulfill naturality prerequisites through
novel, sustainable processes. At the same time, its potential to replace
current chemical synthesis routes remains controversial across different
fields of study such as the production of specialty and fine chemicals.
Here, a promising approach to the synthesis of flavors and fragrances is
the development of in vitro multi-enzyme cascade reactions. These
combine the benefits of enzymatically catalyzed reactions (e.g. high
selectivity, mild reaction conditions) with the concept of process
integration. In literature, cascade reactions with up to ten reaction
steps and eight enzymes have been successfully established, albeit
usually limited to the application in an aqueous phase. Few examples
show the application in both aqueous and organic phases: they, however,
make use of process integration (extraction steps) solely in order to
enhance reaction turnover, without implementing additional reaction
steps in the organic phase. In previous papers, our research group
conclusively proved the applicability of multi-enzyme cascade reactions
in a multiphase system for the production of specialty chemicals through
the example of cinnamyl cinnamate with integrated cofactor regeneration
and integrated intermediate extraction. As a consequent step towards
production-scale process development, within this line of research we
successfully implemented a continuous production process for the
flavoring agent cinnamyl cinnamate in a three liter miniplant reactor
setup.
Cinnamyl cinnamate is extracted as a natural component from Balsam of
Peru, and used as a flavoring agent in cosmetic products and perfumes.
Currently, two main chemical production processes are in use: styrene
oxidative carbonylation with carbon monoxide, oxygen and aliphatic
alcohols in the presence of palladium and sodium propionate, or cinnamic
aldehyde synthesis in absolute ether with aluminum ethylate. Contrary to
the conventional chemical approach, in this study we investigate an
innovative production process for cinnamyl cinnamate that fits the
criteria of both the U.S. Department of Health and Human Services and
the European Union for labelling the product as a “natural flavor”.
An important requirement to scale new production processes up for
industrial application from an economic and/or ecological point of view
is the possibility of a mathematical description of said process for
continuous process control and development. However, due to the use of
various enzymes and multiple phases, the mathematical description of
complex biotechnological processes such as multi-enzyme cascade
reactions constitutes an obstacle to biotechnological process
development. Therefore, this study takes on the challenge of introducing
a mathematical model to describe a complex biotechnological production
process implemented in a miniplant. Our model is implemented in Aspen
Custom Modeler® V8.8 (Aspen Technology, Inc., U.S.A.),
and validated using experimental data obtained from the miniplant. In
this study, we present the cinnamyl cinnamate-producing multi-enzyme
cascade reaction sequence and discuss our mathematical model as well as
experimental results for the miniplant. Furthermore, a mathematical
optimization tool is used to perform simulation runs with the validated
model, while simultaneously varying the process conditions to find
optimal process operating windows, thereby proving the benefits of
computer-aided process development in biotechnology.