Corynebacterium glutamicum possesses β-N-acetylglucosaminidase

Abstract

BackgroundIn Gram-positive Corynebacterium glutamicum and other members of the suborder Corynebacterianeae, which includes mycobacteria, cell elongation and peptidoglycan biosynthesis is mainly due to polar growth. C. glutamicum lacks an uptake system for the peptidoglycan constituent N-acetylglucosamine (GlcNAc), but is able to catabolize GlcNAc-6-phosphate. Due to its importance in white biotechnology and in order to ensure more sustainable processes based on non-food renewables and to reduce feedstock costs, C. glutamicum strains have previously been engineered to produce amino acids from GlcNAc. GlcNAc also is a constituent of chitin, but it is unknown if C. glutamicum possesses chitinolytic enzymes.ResultsChitin was shown here not to be growth substrate for C. glutamicum. However, its genome encodes a putative N-acetylglucosaminidase. The nagA2 gene product was active as β-N-acetylglucosaminidase with 0.27 mM 4-nitrophenyl N,N’-diacetyl-β-D-chitobioside as substrate supporting half-maximal activity. NagA2 was secreted into the culture medium when overproduced with TAT and Sec dependent signal peptides, while it remained cytoplasmic when overproduced without signal peptide. Heterologous expression of exochitinase gene chiB from Serratia marcescens resulted in chitinolytic activity and ChiB secretion was enhanced when a signal peptide from C. glutamicum was used. Colloidal chitin did not support growth of a strain secreting exochitinase ChiB and β-N-acetylglucosaminidase NagA2.ConclusionsC. glutamicum possesses β-N-acetylglucosaminidase. In the wild type, β-N-acetylglucosaminidase activity was too low to be detected. However, overproduction of the enzyme fused to TAT or Sec signal peptides led to secretion of active β-N-acetylglucosaminidase. The finding that concomitant secretion of endogenous NagA2 and exochitinase ChiB from S. marcescens did not entail growth with colloidal chitin as sole or combined carbon source, may indicate the requirement for higher or additional enzyme activities such as processive chitinase or endochitinase activities.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0795-3) contains supplementary material, which is available to authorized users.

Production of Food and Feed Additives From Non-food-competing Feedstocks: Valorizing N-acetylmuramic Acid for Amino Acid and Carotenoid Fermentation With Corynebacterium glutamicum

“…Corynebacterium glutamicum , which has been engineered to utilize GlcN and GlcNAc ( Matano et al, 2014 , 2016 ), cannot utilize MurNAc since no growth was observed in minimal medium with 25 mM MurNAc and 25 ± 0.1 mM MurNAc remained in the growth medium after 25 h of incubation ( Figure 2A ). As expected, the C. glutamicum genome lacks genes encoding a MurNAc PTS and MurNAc-6-phosphate etherase for uptake and conversion of MurNAc to GlcNAc-6-phosphate, an endogenous intermediate of C. glutamicum metabolism.…”

“…The mutated variation of murP was called murP opt . The genes of murP, crr and murQ were amplified via PCR from genomic DNA of E. coli K-12, while nagE from C. glycinophilum , was amplified from pVWEx1_ nagE ( Matano et al, 2016 ). The primers used in this study (see Supplementary Table S1 ) were obtained from Metabion international AG, Planegg.…”

“…Corynebacterium glutamicum is able to take up GlcN ( Figure 1 ) by using its glucose specific PTS PtsG ( Arndt and Eikmanns, 2008 ; Uhde et al, 2013 ). Efficient growth with GlcN required high levels of the endogenous NagB, e.g., in the absence of the repressor protein NanR ( Matano et al, 2016 ). By contrast, high levels of NagA and NagB were not sufficient to support growth with GlcNAc unless nagE from C. glycinophilum was expressed heterologously ( Matano et al, 2014 ).…”

Fermentative Production of l-2-Hydroxyglutarate by Engineered Corynebacterium glutamicum via Pathway Extension of l-Lysine Biosynthesis

“…C. glutamicum has been engineered to produce amino acids from pure and technical grade glycerol (Rittmann et al, 2008 ; Meiswinkel et al, 2013b ). Access to nitrogenous sidestreams from the fishery industry such as glucosamine or N -acetyl-glucosamine is important if the target product is containing nitrogen atoms (Matano et al, 2014 , 2016 ). For products lacking nitrogen atoms, access to lignocellulosics is pivotal (Gopinath et al, 2011 ; Buschke et al, 2013 ; Meiswinkel et al, 2013a ; Hadiati et al, 2014 ; Matsuura et al, 2019 ).…”

Recent Progress on Chemical Production From Non-food Renewable Feedstocks Using Corynebacterium glutamicum

“…Engineering microbes to utilize abundant polysaccharides from crustacean exoskeletons in order to produce chemicals has attracted extensive attention from researchers. Generally, the metabolic pathway of N-acetylglucosamine consists of a specific transport system and two enzyme actions, catalyzed by N-acetylglucosamine-6-phosphatedeacetylase (encoded by nagA) and glucosamine-6P deaminase (encoded by nagB), converting N-acetylglucosamine to fructose-6-phosphate that enters the glycolytic pathway (Matano et al, 2016). In C. glutamicum, the absence of a specific uptake system prevents the utilization of extracellular N-acetylglucosamine, requiring heterogeneous expression of nagE from Corynebacterium glycinophilum; this expression enables prompt transportation and feasible assimilation of N-acetylglucosamine.…”