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

Milne N, Luttik MAH, Rojas HFC, Van Maris AJA, Pronk JT, Daran JM, Wahl SA (2015)

Publication Type: Journal article

Publication year: 2015

Journal

Metabolic Engineering Elsevier

Book Volume: 30

Pages Range: 130-140

DOI: 10.1016/j.ymben.2015.05.003

Abstract

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 dur1,2δ S. cerevisiae yielded an in vitro ATP-independent urease activity of 0.44±0.01μmolmin^-1mg 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.

Authors with CRIS profile

Sebastian Aljoscha Wahl

Involved external institutions

Delft University of Technology (TU Delft)

Netherlands (NL)

How to cite

APA:

Milne, N., Luttik, M.A.H., Rojas, H.F.C., Van Maris, A.J.A., Pronk, J.T., Daran, J.M., & Wahl, S.A. (2015). Functional expression of a heterologous nickel-dependent, ATP-independent urease in Saccharomyces cerevisiae. Metabolic Engineering, 30, 130-140. https://dx.doi.org/10.1016/j.ymben.2015.05.003

MLA:

Milne, Nicholas, et al. "Functional expression of a heterologous nickel-dependent, ATP-independent urease in Saccharomyces cerevisiae." Metabolic Engineering 30 (2015): 130-140.

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