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.