Functional expression of a heterologous nickel-dependent, ATP-independent urease in Saccharomyces cerevisiae

Functional expression of a heterologous nickel-dependent, ATP-independent urease in Saccharomyces cerevisiae

Milne, N.
Luttik, M.A.H.
Cueto Rojas, H.F.
Wahl, A.
Van Maris, A.J.A.
Pronk, J.T.
Daran, J.G.

Applied Sciences

BT/Biotechnology

2015-05-30

In microbial processes for production of proteins, biomass and nitrogen-containing commodity chemicals, ATP requirements for nitrogen assimilation affect product yields on the energy producing substrate. In Saccharomyces cerevisiae, a current host for heterologous protein production and potential platform for production of nitrogen-containing chemicals, uptake and assimilation of ammonium requires 1 ATP per incorporated NH3. Urea assimilation by this yeast is more energy efficient but still requires 0.5 ATP per NH3 produced. To decrease ATP costs for nitrogen assimilation, the S. cerevisiae gene encoding ATP-dependent urease (DUR1,2) was replaced by a Schizosaccharomyces pombe gene encoding ATP-independent urease (ure2), along with its accessory genes ureD, ureF and ureG. Since S. pombe ure2 is a Ni2+-dependent enzyme and Saccharomyces cerevisiae does not express native Ni2+-dependent enzymes, the S. pombe high-affinity nickel-transporter gene (nic1) was also expressed. Expression of the S. pombe genes into dur1,2? S. cerevisiae yielded an in vitro ATP-independent urease activity of 0.44±0.01 µmol min?1 mg protein?1 and restored growth on urea as sole nitrogen source. Functional expression of the Nic1 transporter was essential for growth on urea at low Ni2+ concentrations. The maximum specific growth rates of the engineered strain on urea and ammonium were lower than those of a DUR1,2 reference strain. In glucose-limited chemostat cultures with urea as nitrogen source, the engineered strain exhibited an increased release of ammonia and reduced nitrogen content of the biomass. Our results indicate a new strategy for improving yeast-based production of nitrogen-containing chemicals and demonstrate that Ni2+-dependent enzymes can be functionally expressed in S. cerevisiae.

Saccharomyces cerevisiae
ATP-independent urease
Ni-dependent enzyme
ATP conservation
nitrogen metabolism
physiology

http://resolver.tudelft.nl/uuid:7fc8cea1-e81d-4608-9f5f-9f20289c3cad

Elsevier

1096-7176

https://doi.org/10.1016/j.ymben.2015.05.003

Metabolic Engineering, 30, 2015

Institutional Repository

journal article

© 2015 The Authors
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)