Bacillus methanolicus pyruvate carboxylase and homoserine dehydrogenase I and II and their roles for L-lysine production from methanol at 50 degrees C.
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.
Brautaset
Trygve
T
Department of Biotechnology, SINTEF Materials and Chemistry, 7465, Trondheim, Norway. trygve.brautaset@sintef.no
Jakobsen
Øyvind M
ØM
Degnes
Kristin F
KF
Netzer
Roman
R
Naerdal
Ingemar
I
Krog
Anne
A
Dillingham
Rick
R
Flickinger
Michael C
MC
Ellingsen
Trond E
TE
D001407:Bacillus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D005285:Fermentation; D018698:Glutamic Acid / Q000378:metabolism; D006715:Homoserine Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D008239:Lysine / Q000096:biosynthesis*; D000432:Methanol / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011766:Pyruvate Carboxylase / Q000235:genetics / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism
D001426:Bacterial Proteins; D013912:Threonine; D018698:Glutamic Acid; D008715:Methionine; D006715:Homoserine Dehydrogenase; D011766:Pyruvate Carboxylase; D008239:Lysine; D000432:Methanol