l-lysine production by Bacillus methanolicus: Genome-based mutational analysis and l-lysine secretion engineering.
Bacillus methanolicus is a methylotrophic bacterium with an increasing interest in academic research and for biotechnological applications. This bacterium was previously applied for methanol-based production of l-glutamate, l-lysine and the five-carbon diamine cadaverine by wild type, classical mutant and recombinant strains. The genomes of two different l-lysine secreting B. methanolicus classical mutant strains, NOA2#13A52-8A66 and M168-20, were sequenced. We focused on mutational mapping in genes present in l-lysine and other relevant amino acid biosynthetic pathways, as well as in the primary cell metabolism important for precursor supply. In addition to mutations in the aspartate pathway genes dapG, lysA and hom-1, new mutational target genes like alr, proA, proB1, leuC, odhA and pdhD were identified. Surprisingly, no mutations were found in the putative l-lysine transporter gene lysEMGA3. Inspection of the wild type B. methanolicus strain PB1 genome sequence identified two homologous putative l-lysine transporter genes, lysEPB1and lysE2PB1. The biological role of these putative l-lysine transporter genes, together with the heterologous l-lysine exporter gene lysECgfrom Corynebacterium glutamicum, were therefore investigated. Our results demonstrated that the titer of secreted l-lysine in B. methanolicus was significantly increased by overexpression of lysECgwhile overexpression of lysEMGA3, lysEPB1and lysE2PB1had no measurable effect.
SINTEF Materials and Chemistry, Department of Biotechnology and Nanomedicine, Trondheim, Norway.
Ingemar
I
Nærdal
SINTEF Materials and Chemistry, Department of Biotechnology and Nanomedicine, Trondheim, Norway.
Roman
R
Netzer
Chair of Genetics of Prokaryotes, Faculty of Biology & CeBiTec, Bielefeld University, Bielefeld, Germany.
Marta
M
Irla
SINTEF Materials and Chemistry, Department of Biotechnology and Nanomedicine, Trondheim, Norway.
Anne
A
Krog
SINTEF Materials and Chemistry, Department of Biotechnology and Nanomedicine, Trondheim, Norway.
Tonje Marita Bjerkan
TMB
Heggeset
Chair of Genetics of Prokaryotes, Faculty of Biology & CeBiTec, Bielefeld University, Bielefeld, Germany.
Volker F
VF
Wendisch
NTNU, Norwegian University of Science and Technology, Department of Biotechnology, Trondheim, Norway. Electronic address: trygve.brautaset@ntnu.no.
Trygve
T
Brautaset
D001407:Bacillus / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics*; D053898:Biosynthetic Pathways; D003198:Computer Simulation; D004252:DNA Mutational Analysis / Q000379:methods*; D016680:Genome, Bacterial; D008239:Lysine / Q000096:biosynthesis* / Q000235:genetics; D017354:Point Mutation; D017422:Sequence Analysis, DNA / Q000379:methods
D001426:Bacterial Proteins; D008239:Lysine
Methylotrophy; Mutational analysis; Thermotolerant bacilli; l-lysine secretion