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Applied and Environmental Microbiology, June 2007, p. 4011-4019, Vol. 73, No. 12
0099-2240/07/$08.00+0     doi:10.1128/AEM.00730-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Desensitization of Feedback Inhibition of the Saccharomyces cerevisiae {gamma}-Glutamyl Kinase Enhances Proline Accumulation and Freezing Tolerance{triangledown}

Tomoko Sekine,1,{dagger} Akari Kawaguchi,1,{dagger} Yoshimitsu Hamano,1 and Hiroshi Takagi2*

Department of Bioscience, Fukui Prefectural University, 4-1-1 Kenjojima, Matsuoka-cho, Fukui 910-1195, Japan,1 Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan2

Received 1 April 2007/ Accepted 10 April 2007

In response to osmotic stress, proline is accumulated in many bacterial and plant cells as an osmoprotectant. The yeast Saccharomyces cerevisiae induces trehalose or glycerol synthesis but does not increase intracellular proline levels during various stresses. Using a proline-accumulating mutant, we previously found that proline protects yeast cells from damage by freezing, oxidative, or ethanol stress. This mutant was recently shown to carry an allele of PRO1 which encodes the Asp154Asn mutant {gamma}-glutamyl kinase (GK), the first enzyme of the proline biosynthetic pathway. Here, enzymatic analysis of recombinant proteins revealed that the GK activity of S. cerevisiae is subject to feedback inhibition by proline. The Asp154Asn mutant was less sensitive to feedback inhibition than wild-type GK, leading to proline accumulation. To improve the enzymatic properties of GK, PCR random mutagenesis in PRO1 was employed. The mutagenized plasmid library was introduced into an S. cerevisiae non-proline-utilizing strain, and proline-overproducing mutants were selected on minimal medium containing the toxic proline analogue azetidine-2-carboxylic acid. We successfully isolated several mutant GKs that, due to extreme desensitization to inhibition, enhanced the ability to synthesize proline better than the Asp154Asn mutant. The amino acid changes were localized at the region between positions 142 and 154, probably on the molecular surface, suggesting that this region is involved in allosteric regulation. Furthermore, we found that yeast cells expressing Ile150Thr and Asn142Asp/Ile166Val mutant GKs were more tolerant to freezing stress than cells expressing the Asp154Asn mutant.

* Corresponding author. Mailing address: Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan. Phone: 81-743-72-5420. Fax: 81-743-72-5429. E-mail: hiro{at}bs.naist.jp

{triangledown} Published ahead of print on 20 April 2007.

{dagger} T.S. and A.K. contributed equally to this work.

Applied and Environmental Microbiology, June 2007, p. 4011-4019, Vol. 73, No. 12
0099-2240/07/$08.00+0     doi:10.1128/AEM.00730-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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  • Kaino, T., Tateiwa, T., Mizukami-Murata, S., Shima, J., Takagi, H. (2008). Self-Cloning Baker's Yeasts That Accumulate Proline Enhance Freeze Tolerance in Doughs. Appl. Environ. Microbiol. 74: 5845-5849 [Abstract] [Full Text]  
  • Nevoigt, E. (2008). Progress in Metabolic Engineering of Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 72: 379-412 [Abstract] [Full Text]  


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