Engineering oxygen-independent biotin biosynthesis in Saccharomyces cerevisiae

Engineering oxygen-independent biotin biosynthesis in Saccharomyces cerevisiae

Wronska, A.K. (TU Delft BT/Industriele Microbiologie)
van den Broek, M.A. (TU Delft BT/Industriele Microbiologie)
Perli, T. (TU Delft BT/Industriele Microbiologie)
de Hulster, A.F. (TU Delft BT/Industriele Microbiologie)
Pronk, J.T. (TU Delft BT/Biotechnologie)
Daran, J.G. (TU Delft BT/Industriele Microbiologie)

BT/Biotechnologie

2021

An oxygen requirement for de novo biotin synthesis in Saccharomyces cerevisiae precludes the application of biotin-prototrophic strains in anoxic processes that use biotin-free media. To overcome this issue, this study explores introduction of the oxygen-independent Escherichia coli biotin-biosynthesis pathway in S. cerevisiae. Implementation of this pathway required expression of seven E. coli genes involved in fatty-acid synthesis and three E. coli genes essential for the formation of a pimelate thioester, key precursor of biotin synthesis. A yeast strain expressing these genes readily grew in biotin-free medium, irrespective of the presence of oxygen. However, the engineered strain exhibited specific growth rates 25% lower in biotin-free media than in biotin-supplemented media. Following adaptive laboratory evolution in anoxic cultures, evolved cell lines that no longer showed this growth difference in controlled bioreactors, were characterized by genome sequencing and proteome analyses. The evolved isolates exhibited a whole-genome duplication accompanied with an alteration in the relative gene dosages of biosynthetic pathway genes. These alterations resulted in a reduced abundance of the enzymes catalyzing the first three steps of the E. coli biotin pathway. The evolved pathway configuration was reverse engineered in the diploid industrial S. cerevisiae strain Ethanol Red. The resulting strain grew at nearly the same rate in biotin-supplemented and biotin-free media non-controlled batches performed in an anaerobic chamber. This study established an unique genetic engineering strategy to enable biotin-independent anoxic growth of S. cerevisiae and demonstrated its portability in industrial strain backgrounds.

Anoxic growth
Biotin biosynthesis
Gene dosage
Prokaryotic pathway
Vitamin B7

http://resolver.tudelft.nl/uuid:4dbf037d-e1d2-4d96-b9c9-6a9e806e2baa

https://doi.org/10.1016/j.ymben.2021.05.006

1096-7176

Metabolic Engineering, 67, 88-103

Institutional Repository

journal article

© 2021 A.K. Wronska, M.A. van den Broek, T. Perli, A.F. de Hulster, J.T. Pronk, J.G. Daran