Role of the Bacillus methanolicus citrate synthase II gene, citY, in regulating the secretion of glutamate in L-lysine-secreting mutants.
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 degrees 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 degrees 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.
Brautaset
Trygve
T
Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway.
Williams
Mark D
MD
Dillingham
Richard D
RD
Kaufmann
Christine
C
Bennaars
Assumpta
A
Crabbe
Edward
E
Flickinger
Michael C
MC
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D019149:Bioreactors; D001709:Biotechnology / Q000379:methods; D002950:Citrate (si)-Synthase / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D003470:Culture Media; D015964:Gene Expression Regulation, Bacterial; D018698:Glutamic Acid / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism*; D000432:Methanol / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D003470:Culture Media; D018698:Glutamic Acid; D002950:Citrate (si)-Synthase; D008239:Lysine; D000432:Methanol