Engineering heterologous molybdenum-cofactor-biosynthesis and nitrate-assimilation pathways enables nitrate utilization by Saccharomyces cerevisiae

Engineering heterologous molybdenum-cofactor-biosynthesis and nitrate-assimilation pathways enables nitrate utilization by Saccharomyces cerevisiae

Perli, T. (TU Delft BT/Industriele Microbiologie)
van der Vorm, Daan N.A. (Student TU Delft)
Wassink, Mats (Student TU Delft)
van den Broek, M.A. (TU Delft BT/Industriele Microbiologie)
Pronk, J.T. (TU Delft BT/Biotechnologie)
Daran, J.G. (TU Delft BT/Industriele Microbiologie)

BT/Biotechnologie

2021

Metabolic capabilities of cells are not only defined by their repertoire of enzymes and metabolites, but also by availability of enzyme cofactors. The molybdenum cofactor (Moco) is widespread among eukaryotes but absent from the industrial yeast Saccharomyces cerevisiae. No less than 50 Moco-dependent enzymes covering over 30 catalytic activities have been described to date, introduction of a functional Moco synthesis pathway offers interesting options to further broaden the biocatalytic repertoire of S. cerevisiae. In this study, we identified seven Moco biosynthesis genes in the non-conventional yeast Ogataea parapolymorpha by SpyCas9-mediated mutational analysis and expressed them in S. cerevisiae. Functionality of the heterologously expressed Moco biosynthesis pathway in S. cerevisiae was assessed by co-expressing O. parapolymorpha nitrate-assimilation enzymes, including the Moco-dependent nitrate reductase. Following two-weeks of incubation, growth of the engineered S. cerevisiae strain was observed on nitrate as sole nitrogen source. Relative to the rationally engineered strain, the evolved derivatives showed increased copy numbers of the heterologous genes, increased levels of the encoded proteins and a 5-fold higher nitrate-reductase activity in cell extracts. Growth at nM molybdate concentrations was enabled by co-expression of a Chlamydomonas reinhardtii high-affinity molybdate transporter. In serial batch cultures on nitrate-containing medium, a non-engineered S. cerevisiae strain was rapidly outcompeted by the spoilage yeast Brettanomyces bruxellensis. In contrast, an engineered and evolved nitrate-assimilating S. cerevisiae strain persisted during 35 generations of co-cultivation. This result indicates that the ability of engineered strains to use nitrate may be applicable to improve competitiveness of baker's yeast in industrial processes upon contamination with spoilage yeasts.

Metabolic engineering
Molybdenum cofactor
Nitrate assimilation
Nitrate reductase
Saccharomyces cerevisiae

http://resolver.tudelft.nl/uuid:6b2bdd67-154a-4e58-b739-fd704c9d57d9

https://doi.org/10.1016/j.ymben.2021.02.004

1096-7176

Metabolic Engineering, 65, 11-29

Institutional Repository

journal article

© 2021 T. Perli, Daan N.A. van der Vorm, Mats Wassink, M.A. van den Broek, J.T. Pronk, J.G. Daran