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Applied and Environmental Microbiology, July 2000, p. 2811-2816, Vol. 66, No. 7
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Phosphorylation of Nucleosides by the Mutated Acid Phosphatase from Morganella morganii

Yasuhiro Mihara,1,* Takashi Utagawa,1 Hideaki Yamada,2 and Yasuhisa Asano2

Applied Microbiology Laboratory, Fermentation and Biotechnology Laboratories, Ajinomoto Co., Inc., Kawasaki-ku, Kawasaki-shi 210-8681,1 and Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa, Kosugi, Toyama 939-0398,2 Japan

Received 8 February 2000/Accepted 24 April 2000

A novel nucleoside phosphorylation process using the food additive pyrophosphate as the phosphate source was investigated. The Morganella morganii gene encoding a selective nucleoside pyrophosphate phosphotransferase was cloned. It was identical to the M. morganii PhoC acid phosphatase gene. Sequential in vitro random mutagenesis was performed on the gene by error-prone PCR to construct a mutant library. The mutant library was introduced into Escherichia coli, and the transformants were screened for the production of 5'-IMP. One mutated acid phosphatase with an increased phosphotransferase reaction yield was obtained. With E. coli overproducing the mutated acid phosphatase, 101 g of 5'-IMP per liter (192 mM) was synthesized from inosine in an 88% molar yield. This improvement was achieved with two mutations, Gly to Asp at position 92 and Ile to Thr at position 171. A decreased Km value for inosine was responsible for the increased productivity.

* Corresponding author. Mailing address: Applied Microbiology Laboratory, Fermentation and Biotechnology Laboratories, Ajinomoto Co., Inc., 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki-shi 210-8681, Japan. Phone: 81-44-244-7138. Fax: 81-44-244-4757. E-mail: yasuhiro_mihara{at}ajinomoto.com.

Applied and Environmental Microbiology, July 2000, p. 2811-2816, Vol. 66, No. 7
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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