Roles of maltodextrin and glycogen phosphorylases in maltose utilization and glycogen metabolism in Corynebacterium glutamicum

Abstract

Corynebacterium glutamicum transiently accumulates large amounts of glycogen, when cultivated on glucose and other sugars as a source of carbon and energy. Apart from the debranching enzyme GlgX, which is required for the formation of maltodextrins from glycogen, a-glucan phosphorylases were assumed to be involved in glycogen degradation, forming a-glucose 1-phosphate from glycogen and from maltodextrins. We show here that C. glutamicum in fact possesses two a-glucan phosphorylases, which act as a glycogen phosphorylase (GlgP) and as a maltodextrin phosphorylase (MalP). By chromosomal inactivation and subsequent analysis of the mutant, cg1479 was identified as the malP gene. The deletion mutant C. glutamicum DmalP completely lacked MalP activity and showed reduced intracellular glycogen degradation, confirming the proposed pathway for glycogen degradation in C. glutamicum via GlgP, GlgX and MalP. Surprisingly, the DmalP mutant showed impaired growth, reduced viability and altered cell morphology on maltose and accumulated much higher concentrations of glycogen and maltodextrins than the wild-type during growth on this substrate, suggesting an additional role of MalP in maltose metabolism of C. glutamicum. Further assessment of enzyme activities revealed the presence of 4-a-glucanotransferase (MalQ), glucokinase (Glk) and a-phosphoglucomutase (a-Pgm), and the absence of maltose hydrolase, maltose phosphorylase and b-Pgm, all three known to be involved in maltose utilization by Gram-positive bacteria. Based on these findings, we conclude that C. glutamicum metabolizes maltose via a pathway involving maltodextrin and glucose formation by MalQ, glucose phosphorylation by Glk and maltodextrin degradation via the reactions of MalP and a-Pgm, a pathway hitherto known to be present in Gram-negative rather than in Gram-positive bacteria. INTRODUCTIONCorynebacterium glutamicum is a Gram-positive soil bacterium employed in the production of various amino acids (Eggeling & Bott, 2005), and serves as a model organism for the mycolic acid-containing suborder Corynebacterianeae, which includes pathogenic species such as Corynebacterium diphtheriae, Mycobacterium tuberculosis and Mycobacterium leprae. The organism grows on a variety of mono-and disaccharides, alcohols and organic acids as single or combined sources of carbon and energy (Liebl, 2005). and transiently forms intracellular glycogen when cultivated on sugar substrates (Tzvetkov et al., 2003;. We recently showed that C. glutamicum forms maltodextrins in the course of glycogen degradation , and thus we speculate that glycogen is degraded in C. glutamicum by a pathway which proceeds similarly or identically to the one present in Escherichia coli. This pathway involves the interplay of six enzymes (Fig. 1). Glycogen phosphorylase (GlgP) cleaves a-1,4-glycosidic bonds at the non-reducing ends of glycogen and forms glucose 1-phosphate and phosphorylase-limited dextrins (pl-dextrins) (Dauvillee et al., 2005;Alonso-Casajus et al., 2006). Cleavage of the a-1,6-glycosidic bon...

Phosphotransferase System-Independent Glucose Utilization in Corynebacterium glutamicum by Inositol Permeases and Glucokinases

“…The ATP-dependent glucokinase encoded by glk has not been described to be involved in growth with glucose; however, a glk mutant showed growth perturbations in maltose media (43), which also was observed for a ppgK mutant (36). During the utilization of maltose or maltodextrins, glucose is generated intracellularly (63), thus necessitating glucose phosphorylation by Glk or PPGK. When iolT1 or iolT2 is overexpressed in the PTS-deficient mutant ⌬hpr, glucose phosphorylation becomes rate limiting because growth rates in glucose medium comparable to those of the wild type were observed only if ppgK was overexpressed (Table 4).…”

Transcription of malP is subject to phosphotransferase system-dependent regulation in Corynebacterium glutamicum

“…In contrast to the situation in E. coli, in which the activity of the ABC transporter for maltose is controlled by the PTS (Bao & Duong, 2013;Blüschke et al, 2006;Böhm et al, 2002;Chen et al, 2013;Saier & Roseman, 1976a, b), maltose uptake was not negatively influenced in PTSdeficient C. glutamicum strains. However, we found in C. glutamicum that transcription of malP, which encodes an a-glucan phosphorylase required for maltose utilization (Seibold et al, 2009), depends on PTS activity. Besides maltose utilization, the malP-encoded a-glucan phosphorylase contributes in C. glutamicum to glycogen degradation.…”

“…In cultivations with sugars, C. glutamicum transiently accumulates large amounts of glycogen in the early exponential growth phases, which are degraded in the course of the late exponential growth phase just before the initially provided substrate is consumed . Besides slowed down growth on maltose, the deletion of malP in C. glutamicum causes a delayed degradation of glycogen in cultivations on glucose (Seibold et al, 2009). The recently identified control of C. glutamicum MalP by competitive inhibition by the glycogen synthesis intermediate ADP-glucose (a typical feature of glycogen phosphorylases) also pointed out a prominent role of this enzyme in the control of the glycogen content (Clermont et al, 2015).…”

“…Activities of MalP, Pgm, glucokinase, pyruvate kinase (Pyk) and MalQ were assayed in a final volume of 1 ml by spectrophotometric measurement of the variation in the NADP(H) concentration at 340 nm at 30 uC as described elsewhere (Clermont et al, 2015;Netzer et al, 2004;Seibold et al, 2009;.…”

“…As depicted in Fig. 1(a), maltose is then metabolized by the concomitant action of amylomaltase MalQ, maltodextrin phosphorylase MalP, the glucokinases Glk and Ppgk, and phosophoglucomutase Pgm (Clermont et al, 2015;Lindner et al, 2010;Seibold et al, 2009;; genes for maltose utilization are not clustered within the C. glutamicum genome ( Fig. 1b; Kalinowski et al, 2003).…”