A novelgndmutation leading to increased l-lysine production inCorynebacterium glutamicum

Abstract

Toward more efficient L-lysine production, we have been challenging genome-based strain breeding by the approach of assembling only relevant mutations in a single wild-type background. Following the creation of a new L-lysine producer Corynebacterium glutamicum AHP-3 that carried three useful mutations (lysC311, hom59, and pyc458) on the relevant downstream pathways, we shifted our target to the pentose phosphate pathway. Comparative genomic analysis for the pathway between a classically derived L-lysine producer and its parental wild-type identified several mutations. Among these mutations, a Ser-361-->Phe mutation in the 6-phosphogluconate dehydrogenase gene (gnd) was defined as a useful mutation for L-lysine production. Introduction of the gnd mutation into strain AHP-3 by allelic replacement led to approximately 15% increased L-lysine production. Enzymatic analysis revealed that the mutant enzyme was less sensitive than the wild-type enzyme to allosteric inhibition by intracellular metabolites, such as fructose 1,6-bisphosphate, D-glyceraldehyde 3-phosphate, phosphoribosyl pyrophosphate, ATP, and NADPH, which were known to inhibit this enzyme. Isotope-based metabolic flux analysis demonstrated that the gnd mutation resulted in 8% increased carbon flux through the pentose phosphate pathway during L-lysine production. These results indicate that the gnd mutation is responsible for diminished allosteric regulation and contributes to redirection of more carbon to the pentose phosphate pathway that was identified as the primary source for NADPH essential for L-lysine biosynthesis, thereby leading to improved product formation.

Analysis and Engineering of Metabolic Pathway Fluxes in Corynebacterium glutamicum

“…Different reactions are linked to the supply of NADPH. The major enzymes are glucose 6-phosphate dehydrogenase [28,29] and 6-phosphogluconate dehydrogenase [28,30] in the oxidative part of the PPP and the TCA cycle enzyme isocitrate dehydrogenase [31]. In selected cases, NADPH supply might involve malic enzyme [32,33].…”

“…Due to these various hurdles, classical mutagenesis and selection did not yield high levels of production, although efforts have been continuing now for almost four decades [6]. The development of recombinant DNA technologies, allowing targeted genetic modification, has initiated intensive research towards rational optimization of C. glutamicum, resulting in remarkable progress in production efficiency [30,135,136]. In the following, the advances in metabolic pathway engineering of C. glutamicum are highlighted.…”

“…In further successful examples, an increased PPP flux was achieved by modifying the regulatory properties of enzymes in the PPP, i.e., partially releasing glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase from allosteric inhibition. The substitution A243T in the zwf gene encoding for glucose 6-phosphate dehydrogenase [116,153] and the substitution S361F in the gnd gene encoding for 6-phosphogluconate dehydrogenase [30] both led to significantly increased lysine titer. The modifications cause an increase in the PPP flux which probably resulted from positively changed kinetics of the enzymes.…”

Regulation and Intensification Ways of Lysine Biosynthesis

“…Óсі гени корі-небактерій, що кодують öі ферìенти, ізольовано та секвеновано [6,7,8,17]. Для кожного гена визна÷ені його ìісöе і функöія у ìетаболі÷ноìу шляху та у÷асть у транспортуванні лізину [10,11,12] [13][14][15][16] .…”

A genome-based approach to create a minimally mutated Corynebacterium glutamicum strain for efficient l-lysine production

“…Reconstructing an L-lysine producer according to genome information Reconstruction of an L-lysine producer is carried out in the following way [8,9,[14][15][16].…”