l-lysine production by Bacillus methanolicus: Genome-based mutational analysis and l-lysine secretion engineering

Abstract

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 lysE^MGA3. Inspection of the wild type B. methanolicus strain PB1 genome sequence identified two homologous putative l-lysine transporter genes, lysE^PB1 and lysE2^PB1. The biological role of these putative l-lysine transporter genes, together with the heterologous l-lysine exporter gene lysE^Cg from Corynebacterium glutamicum, were therefore investigated. Our results demonstrated that the titer of secreted l-lysine in B. methanolicus was significantly increased by overexpression of lysE^Cg while overexpression of lysE^MGA3, lysE^PB1 and lysE2^PB1 had no measurable effect.

Keywords: Methylotrophy; Mutational analysis; Thermotolerant bacilli; l-lysine secretion.