Polymer characterization and optimization of conditions for the enhanced bioproduction of benzaldehyde by Pichia pastoris in a two- ;phase partitioning bioreactor

Benzaldehyde, with its apricot and almond‐like aroma, is the second most abundantly used molecule in the flavor industry, and is most commonly produced via chemical routes, such as by the oxidation of toluene. Biologically produced benzaldehyde, whether by extraction of plant material or via microbial biotransformation, commands a substantial price advantage, and greater consumer acceptance. Methylotrophic yeast, such as Pichia pastoris, contain the enzyme alcohol oxidase (AOX), which, in the presence of alcohols other than methanol, are able to yield aldehydes as dead‐end products, for example, benzaldehyde from benzyl alcohol. In this work, we have determined that benzaldehyde, and not benzyl alcohol, is inhibitory to the transformation reaction by P. pastoris, prompting the development of a selection strategy for identifying sequestering polymers for use in a partitioning bioreactor that was based on the ratio of partition coefficients (PCs) for the two target molecules. Additionally, we have now confirmed for the first time, that the mechanism of solute uptake by amorphous polymers is via absorption, not adsorption. Finally, we have adopted a common strategy used for the production of heterologous proteins by P. pastoris, namely the use of a mixed methanol/glycerol feed for inducing the required AOX enzyme, while reducing the time required for high density biomass generation. All of these components were combined in a final experiment in which 10% of the polymer Kraton D1102K, whose PC ratio of benzaldehyde to benzyl alcohol was 14.9, was used to detoxify the biotransformation in a 5 L partitioning bioreactor, resulting in a 3.4‐fold increase in benzaldehyde produced (14.4 g vs. 4.2 g) relative to single phase operation, at more than double the volumetric productivity (97 mg L−1 h−1 vs. 41 mg L−1 h−1). Biotechnol. Bioeng. 2013; 110: 1098–1105. © 2012 Wiley Periodicals, Inc. Pichia pastoris has high alcohol oxidase activity and, although often directed at methanol metabolism, can non‐specifically oxidize other alcohols, here benzyl alcohol to benzaldehyde, an important fragrance/flavour compound. Benzaldehyde strongly inhibited the biotransformation, however, and a Two‐Phase Partitioning Bioreactor (TPPB) was employed using amorphous polymer beads to sequester the produced benzaldehyde. The uptake mechanism was confirmed to be by absorption, not adsorption, as is seen in non‐selective materials such as activated carbon. The benzaldehyde volumetric produ