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E. Zukic, A. Glieder, M. Geier, F. Rudroff, M.D. Mihovilovic:
"Orange Peel Application - Artificial Enzymatic Cascade Expressed in Pichia Pastoris";
Poster: 9^th International Congress on Biocatalysis, Hamburg, Deutschland; 26.08.2018 - 30.08.2018.

R-(+)-Limonene (1) is industrially isolated from orange peels, a side product of the orange juice production and
therefore available in large quantities1. The Mihovilovic group found a way to transform enzymatically a waste
product into the highly valuable product carvolactone (2). This transformation requires a multi-step synthesis
which is still a challenge in the classical chemistry. The enzymatic approach managed to handle this multi-step
synthesis as a one-pot reaction, without isolating the intermediates, which is usually required in the classical
chemistry. The hydroxylation reaction of limonene was done by CumDo, expressed in Pseudomonas putida S12
(P. putida) which was then connected with the well-established mini-pathway in Escherichia coli (E. coli) using
the hydroxylated limonene as the starting material. The following reactions were done by an alcohol
dehydrogenase (RR-ADH), an enoate reductase (XenB) and a Baeyer-Villiger monooxygenase (CHMO Acineto ). The
whole cascade was introduced through a mixed-culture approach to synthesize a chiral carvolactone (2).
Carvolactone is an interesting building block which can serve as a monomer for polymer production through a
ring opening polymerization. Their olefinic side chains can be easily functionalized and crosslinked. 1,2
Several attempts have been performed to express CumDo in E. coli, but due to the size and the complexity of
the enzyme, P. putida turned out to be the best expression host. Recently, a new approach was designed in
order to carry out the synthetic pathway in a single host. The well-known methylotropic yeast Pichia pastoris
(P. pastoris) was selected for this aim due to its ability of heterologous expression of several proteins in high
quantities. In addition, it is suitable for large scale growth and culture for bioreactors. 3 In the present study, the
sequences of the enzymes, which were used for the previous approach, were optimized for P. Pastoris and
introduced through a Gibson Assembly into the pBSYAOX vector. The results of this strategy are herein
presented.

pichia, limonene, cascade