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Functional expression of a heterologous nickel-dependent, ATP-independent urease in Saccharomyces cerevisiae.
- Source :
-
Metabolic engineering [Metab Eng] 2015 Jul; Vol. 30, pp. 130-140. Date of Electronic Publication: 2015 May 30. - Publication Year :
- 2015
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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 Ni(2+)-dependent enzyme and Saccharomyces cerevisiae does not express native Ni(2+)-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±0.01 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 Ni(2+) 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 Ni(2+)-dependent enzymes can be functionally expressed in S. cerevisiae.<br /> (Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)
- Subjects :
- Cation Transport Proteins genetics
Schizosaccharomyces genetics
Schizosaccharomyces metabolism
Schizosaccharomyces pombe Proteins genetics
Urease genetics
Cation Transport Proteins biosynthesis
Gene Expression
Nickel metabolism
Saccharomyces cerevisiae genetics
Saccharomyces cerevisiae metabolism
Schizosaccharomyces pombe Proteins biosynthesis
Urease biosynthesis
Subjects
Details
- Language :
- English
- ISSN :
- 1096-7184
- Volume :
- 30
- Database :
- MEDLINE
- Journal :
- Metabolic engineering
- Publication Type :
- Academic Journal
- Accession number :
- 26037463
- Full Text :
- https://doi.org/10.1016/j.ymben.2015.05.003