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Role of the Bacillus methanolicus Citrate Synthase II Gene, citY, in Regulating the Secretion of Glutamate in L-Lysine-Secreting Mutants

Trygve Brautaset,¹ Mark D. Williams,² Richard D. Dillingham,² Christine Kaufmann,² Assumpta Bennaars,³ Edward Crabbe,^2, and Michael C. Flickinger^2,3*

Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway,¹ Biotechnology Institute,² Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, St. Paul, Minnesota 55108³

Received 4 December 2002/ Accepted 8 April 2003

The thermotolerant, restrictive methylotroph Bacillus methanolicus MGA3 (ATCC 53907) can secrete 55 g of glutamate per liter (maximum yield, 0.36 g/g) at 50°C with methanol as a carbon source and a source of ammonia in fed-batch bioreactors. A homoserine dehydrogenase mutant, 13A52-8A66, secreting up to 35 g of L-lysine per liter in fed-batch fermentations had minimal 2-oxoglutarate dehydrogenase activity [7.3 nmol min^-1 (mg of protein)^-1], threefold-increased pyruvate carboxylase activity [535 nmol min^-1 (mg of protein)^-1], and elevated citrate synthase (CS) activity [292 nmol min^-1 (mg of protein)^-1] and simultaneously secreted glutamate (20 to 30 g per liter) and L-lysine. The flow of carbon from oxaloacetate is split between transamination to aspartate and formation of citrate. To investigate the regulation of this branch point, the B. methanolicus gene citY encoding a CSII protein with activity at 50°C was cloned from 13A52-8A66 into a CS-deficient Escherichia coli K2-1-4 strain. A citY-deficient B. methanolicus mutant, NCS-L-7, was also isolated from the parent strain of 13A52-8A66 by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis, followed by selection with monofluoroacetate disks on glutamate plates. Characterization of these strains confirmed that citY in strain 13A52-8A66 was not altered and that B. methanolicus possessed several forms of CS. Analysis of citY cloned from NCS-L-7 showed that the reduced CS activity resulted from a frameshift mutation. The level of glutamate secreted by NCS-L-7 was reduced sevenfold and the ratio of L-lysine to glutamate secreted was increased 4.5-fold compared to the wild type in fed-batch cultures with glutamate feeding. This indicates that glutamate secretion in L-lysine-overproducing mutants can be altered in favor of increased L-lysine secretion by regulating in vivo CS activity.

Present address: Surrey, British Columbia, Canada V3R 4C5.

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