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Applied and Environmental Microbiology, July 2009, p. 4240-4247, Vol. 75, No. 13
0099-2240/09/$08.00+0     doi:10.1128/AEM.00640-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Screening of Thermotolerant Gluconobacter Strains for Production of 5-Keto-D-Gluconic Acid and Disruption of Flavin Adenine Dinucleotide-Containing D-Gluconate Dehydrogenase{triangledown}

Ittipon Saichana,1 Duangtip Moonmangmee,3 Osao Adachi,1 Kazunobu Matsushita,1 and Hirohide Toyama2*

Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan,1 Department of Bioscience and Biotechnology, Faculty of Agriculture, University of the Ryukyus, Okinawa 903-0213, Japan,2 Department of Microbiology, Faculty of Science, King Mongkut's University of Technology Thonburi, Bangkok 10140, Thailand3

Received 18 March 2009/ Accepted 24 April 2009

We isolated thermotolerant Gluconobacter strains that are able to produce 5-keto-D-gluconic acid (5KGA) at 37°C, a temperature at which regular mesophilic 5KGA-producing strains showed much less growth and 5KGA production. The thermotolerant strains produced 2KGA as the major product at both 30 and 37°C. The amount of ketogluconates produced at 37°C was slightly less than the amount produced at 30°C. To improve the yield of 5KGA in these strains, we disrupted flavin adenine dinucleotide-gluconate dehydrogenase (FAD-GADH), which is responsible for 2KGA production. Genes for FAD-GADH were cloned by using inverse PCR and an in vitro cloning strategy. The sequences obtained for three thermotolerant strains were identical and showed high levels of identity to the FAD-GADH sequence reported for the genome of Gluconobacter oxydans 621 H. A kanamycin resistance gene cassette was used to disrupt the FAD-GADH genes in the thermotolerant strains. The mutant strains produced 5KGA exclusively, and the final yields were over 90% at 30°C and 50% at 37°C. We found that the activity of pyrroloquinoline quinone (PQQ)-dependent glycerol dehydrogenase, which is responsible for 5KGA production, increased in response to addition of PQQ and CaCl2 in vitro when cells were grown at 37°C. Addition of 5 mM CaCl2 to the culture medium of the mutant strains increased 5KGA production to the point where over 90% of the initial substrate was converted. The thermotolerant Gluconobacter strains that we isolated in this study provide a promising new option for industrial 5KGA production.

* Corresponding author. Mailing address: Department of Bioscience and Biotechnology, Faculty of Agriculture, University of the Ryukyus, Okinawa 903-0213, Japan. Phone and fax: 81-98-895-8805. E-mail: toyama{at}agr.u-ryukyu.ac.jp

{triangledown} Published ahead of print on 1 May 2009.

Applied and Environmental Microbiology, July 2009, p. 4240-4247, Vol. 75, No. 13
0099-2240/09/$08.00+0     doi:10.1128/AEM.00640-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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