• Microbiology and Biotechnology Letters
• /
• v.47 no.1
• /
• pp.78-86
• /
• 2019

Recombinant Corynebacterium glutamicum producing N-acetylglucosamine (GlcNAc) was constructed by metabolic engineering. To construct a basal strain producing GlcNAc, the genes nagA, nagB, and nanE encoding N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, and N-acetylmannosamine-6-phosphate epimerase, respectively, were sequentially deleted from C. glutamicum ATCC 13032, yielding strain KG208. In addition, the genes glmS and gna1 encoding glucosamine-6-phosphate synthase and glucosamine-6-phosphate N-acetyltransferase, which originated from C. glutamicum and Saccharomyces cerevisiae, respectively, were expressed in several expression vectors. Among several combinations of glmS and gna1 expression, recombinant cells expressing glmS and gna1 under control of the ilvC promoter produced 1.77 g/l of GlcNAc and 0.63 g/l of glucosamine in flask cultures.

• Journal of Applied Biological Chemistry
• /
• v.58 no.3
• /
• pp.227-232
• /
• 2015

A microarray study has been employed to understand changes of gene expression in E. coli KD43162 resistant to ampicillin, ampicillin-sulbactam, piperacillin, piperacillin-tazobactam, cefazolin, cefepime, aztreonam, imipenem, meropenem, gentamicin, tobramycin, ciprofloxacin, levofloxacin, moxifloxacin, fosfomycin, and trimethoprim-sulfamethoxazole except for amikacin using disk diffusion assay. Using Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and MALDI-TOF MS analyses, 36 kDa of outer membrane proteins (OMPs) was found to be deleted in the multidrug resistant E. coli KD 43162. Microarray analysis was used to determine up- and down-regulated genes in relation to multidrug resistant E. coli KD43162. Among the up-regulated genes, these genes were corresponded to express the proteins as penicillin-binding proteins (PBPs), tartronate semialdehyde reductase, ethanolamine utilization protein, shikimate kinase I, allantoinase, predicted SAM-dependent methyltransferase, L-glutamine: D-fructose-6-phosphate aminotransferase (GFAT), phospho-glucosamine mutase, predicted N-acetylmannosamine kinase, and predicted N-acetylmannosamine-6-P epimerase. Up-regulation of PBPs, one of primary target sites of antibiotics, might be responsible for the multidrug resistance in E. coli with increasing amount of target sites. Up-regulation of GFAT enzyme may be related to the up-regulation of PBPs because GFAT produces N-acetylglucosamine, a precursor of peptidoglycans. One of GFAT inhibitors, azaserine, showed a potent inhibition on the growth of E. coli KD43162. In conclusion, up-regulation of PBPs and GFATs with the loss of 36 kDa OMP refers the multidrug resistance in E. coli KD 43162.

• Microbiology and Biotechnology Letters
• /
• v.32 no.2
• /
• pp.142-148
• /
• 2004

Erythropoietin is a main regulator of human erythropoiesis. Recombinant human erythropoietin (rhEPO) is one of the glycoproteins produced in animal cells, and it has oligo saccharides chains which comprise about 40％ of its molecular mass. Because the content of sialic acid can extend circulatory lifetime, the high degree of sialylation is often a desirable feature of therapeutic glycoproteins. In this study, the sialylation of rhEPO produced by chinese hamster ovary cell culture was maximized by supplementing the culture medium with N-acetylm-annosamine (ManNAc), a direct intracellular precursor for sialic acid synthesis and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (NeuAc2en), a sialidase inhibitor. Feeding of 20 mM ManNAc/0.5 mM NeuAc2en into culture medium increased the sialic acid content by nearly tenfold compared with unsupplemented medium. This effect was achieved without affecting the cell growth or product yield. Six erythropoietin fractions differing in sialic acid content, ranging from 11∼15％ of EPO, were identified from chinese hamster ovary cell-derived rhEPO by mono Q column chromatography. It was found that, at 20 mM ManNAc/0.5 mM NeuAc2en feeding, productivity of hyper-glycosylated EPO increased up to 50％, compared with the unsupplemented medium.

• Journal of Life Science
• /
• v.24 no.3
• /
• pp.311-317
• /
• 2014

Distal myopathy with rimmed vacuoles (DMRV) or hereditary inclusion body myopathy 2 is an autosomal recessive muscular disorder characterized by early adult-onset weakness of distal muscles and rimmed vacuoles in muscle biopsy. Mutations in the UDP-N-acetylglucosamine 2-epimerase/N-ace-tylmannosamine kinase (GNE) gene are associated with the development of DMRV. In this study, whole exome sequencing (WES) revealed compound heterozygous GNE mutations of p.Asp176Val and p.Val572Leu in a patient with distal limb weakness. Three hundred healthy controls did not show these mutations. All other variants found in distal myopathy-relevant genes were polymorphic. These findings confirmed that the patient had DMRV. This work underscores the usefulness of WES in improving the molecular diagnosis of myopathy.

• Journal of Microbiology and Biotechnology
• /
• v.18 no.6
• /
• pp.1024-1032
• /
• 2008

Efficient expression of the Salmonella Typhimurium tdc ABCDEG operon involved in the degradation of L-serine and L-threonine requires TdcA, the transcriptional activator of the tdc operon. We found that the tdcA gene was transiently activated when the bacterial growth condition was changed from aerobic to anaerobic, but this was not observed if Salmonella was grown anaerobically from the beginning of the culture. Expression kinetics of six tdc genes after anaerobic shock demonstrated by a real-time PCR assay showed that the tdc CDEG genes were not induced in the tdcA mutant but tdcB maintained its inducibility by anaerobic shock even in the absence of tdcA, suggesting that an additional unknown transcriptional regulation may be working for the tdcB expression. We also investigated the effects of nucleoid-associated proteins by primer extension analysis and found that H-NS repressed tdcA under anaerobic shock conditions, and fis mutation delayed the peak expression time of the tdc operon. DNA microarray analysis of genes regulated by TdcA revealed that the genes involved in N-acetylmannosamine, maltose, and propanediol utilization were significantly induced in a tdcA mutant. These findings suggest that Tdc enzymes may playa pivotal role in energy metabolism under a sudden change of oxygen tension.

• Journal of Microbiology and Biotechnology
• /
• v.31 no.1
• /
• pp.115-122
• /
• 2021

Phenol-soluble modulins (PSMs) are responsible for regulating biofilm formation, persister cell formation, pmtR expression, host cell lysis, and anti-bacterial effects. To determine the effect of psm deletion on methicillin-resistant Staphylococcus aureus, we investigated psm deletion mutants including Δpsmα, Δpsmβ, and Δpsmαβ. These mutants exhibited increased β-lactam antibiotic resistance to ampicillin and oxacillin that was shown to be caused by increased N-acetylmannosamine kinase (nanK) mRNA expression, which regulates persister cell formation, leading to changes in the pattern of phospholipid fatty acids resulting in increased anteiso-C15:0, and increased membrane hydrophobicity with the deletion of PSMs. When synthetic PSMs were applied to Δpsmα and Δpsmβ mutants, treatment of Δpsmα with PSMα1-4 and Δpsmβ with PSMβ1-2 restored the sensitivity to oxacillin and slightly reduced the biofilm formation. Addition of a single fragment showed that α1, α2, α3, and β2 had an inhibiting effect on biofilms in Δpsmα; however, β1 showed an enhancing effect on biofilms in Δpsmβ. This study demonstrates a possible reason for the increased antibiotic resistance in psm mutants and the effect of PSMs on biofilm formation.