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.
Highlights
In 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 ATCC13032 possesses a GH3 Nacetylglucosaminidase BLAST alignments indicate that nagA2 encodes a β Nacetylglucosaminidase belonging to the family 3 of glycosidases, which is predicted to be secreted
The residues involved in the catalytic mechanism are boxed in light grey: while in β-glucosidases, a glutamate at a C-terminal region is responsible for the acid/base catalytic mechanism, N-acetylglucosaminidases are characterized by the D (S/T)H motif containing the Asp-His catalytic dyad [40]
Summary
In 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 is a model bacterium for white biotechnology since it is used for the production of amino acids and derived products [1] This bacterium can use a variety of carbon sources for growth and production, e.g. sugars (glucose, fructose, sucrose, Matano et al BMC Microbiology (2016) 16:177 glucosamine (GlcN) and N-acetyl-glucosamine (GlcNAc) have been developed [22, 23]. GlcN and GlcNAc represent interesting feedstocks for biotechnological applications since they can be produced by acid hydrolysis of chitin, one of the major waste components generated from shellfish industry [24, 25]. The amino sugar fraction of shellfish waste could represent an example of a sustainable, renewable feedstock for industrial fermentations, in particular for nitrogenous compounds such as amino acids and diamines
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