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Appl Environ Microbiol. 1988 April; 54(4): 996-1002
Copyright © 1988, American Society for Microbiology. All Rights Reserved.

Strain Improvement of Rhodotorula graminis for Production of a Novel L-Phenylalanine Ammonia-Lyase

Steve A. Orndorff^,*, Nina Costantino, David Stewart and Don R. Durham

Protein Engineering Department, Genex Corporation, Gaithersburg, Maryland 20877

ABSTRACT

L-Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) from Rhodotorula rubra has been used in the commercial manufacture of L-phenylalanine from trans-cinnamic acid. In this study, R. graminis PAL was investigated. Mutant strain GX6000 was isolated after ethyl methanesulfonate mutagenesis of wild-type R. graminis GX5007 by selecting for resistance to phenylpropiolic acid, an analog of trans-cinnamic acid. Mutant strain GX6000 produced inducible PAL at levels four- to fivefold higher than had wild-type R. graminis. Furthermore, this strain had several other physiological traits that make it more commercially useful than R. rubra. For example, during fermentation, the PAL half-life was three- to fivefold longer, PAL specific activity was six to seven times higher, and PAL synthesis was significantly less inhibited by temperatures above 30°C. Induction of PAL in strain GX6000 appeared to be less tightly regulated; L-leucine acted synergistically with L-phenylalanine, the physiological inducer, to increase the PAL specific activity and titer to 165 U/g (dry weight) and 3,000 U/liter, respectively, a 40% increase over the effect of L-phenylalanine alone. Strain GX6000 PAL showed significantly greater stability in bioreactors for the synthesis of L-phenylalanine, a finding that is consistent with the stability properties observed during fermentation.

^* Corresponding author.

Present address: The NutraSweet Co., Box 2387, Augusta, GA 30903.

Present address: National Cancer Institute, Frederick Cancer Research Facility, Frederick, MD 21701.

Present address: W. R. Grace & Co., Washington Research Center, Columbia, MD 21044.

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