1. Bacillus methanolicus pyruvate carboxylase and homoserine dehydrogenase I and II and their roles for L-lysine production from methanol at 50 degrees C.
- Author
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Brautaset T, Jakobsen ØM, Degnes KF, Netzer R, Naerdal I, Krog A, Dillingham R, Flickinger MC, and Ellingsen TE
- Subjects
- Bacillus genetics, Bacillus metabolism, Bacterial Proteins genetics, Cloning, Molecular, Fermentation, Glutamic Acid metabolism, Homoserine Dehydrogenase genetics, Hot Temperature, Methionine metabolism, Molecular Sequence Data, Mutation, Pyruvate Carboxylase genetics, Threonine metabolism, Bacillus enzymology, Bacterial Proteins metabolism, Homoserine Dehydrogenase metabolism, Lysine biosynthesis, Methanol metabolism, Pyruvate Carboxylase metabolism
- Abstract
We here present the pyc gene encoding pyruvate carboxylase (PC), and the hom-1 and hom-2 genes encoding two active homoserine dehydrogenase (HD) proteins, in methylotrophic Bacillus methanolicus MGA3. In general, both PC and HD are regarded as key targets for improving bacterial L-lysine production; PC plays a role in precursor oxaloacetate (OAA) supply while HD controls an important branch point in the L-lysine biosynthetic pathway. The hom-1 and hom-2 genes were strongly repressed by L-threonine and L-methionine, respectively. Wild-type MGA3 cells secreted 0.4 g/l L-lysine and 59 g/l L-glutamate under optimised fed batch methanol fermentation. The hom-1 mutant M168-20 constructed herein secreted 11 g/l L-lysine and 69 g/l of L-glutamate, while a sixfold higher L-lysine overproduction (65 g/l) of the previously constructed classical B. methanolicus mutant NOA2#13A52-8A66 was accompanied with reduced L-glutamate production (28 g/l) and threefold elevated pyc transcription level. Overproduction of PC and its mutant enzyme P455S in M168-20 had no positive effect on the volumetric L-lysine yield and the L-lysine yield on methanol, and caused significantly reduced volumetric L-glutamate yield and L: -glutamate yield on methanol. Our results demonstrated that hom-1 represents one key target for achieving L-lysine overproduction, PC activity plays an important role in controlling L-glutamate production from methanol, and that OAA precursor supply is not a major bottleneck for L-lysine overproduction by B. methanolicus.
- Published
- 2010
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