• Journal of Microbiology and Biotechnology
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• v.31 no.9
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• pp.1305-1310
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• 2021

Shikimate is a key high-demand metabolite for synthesizing valuable antiviral drugs, such as the anti-influenza drug, oseltamivir (Tamiflu). Microbial-based strategies for shikimate production have been developed to overcome the unstable and expensive supply of shikimate derived from traditional plant extraction processes. In this study, a microbial cell factory using Corynebacterium glutamicum was designed to overproduce shikimate in a fed-batch culture system. First, the shikimate kinase gene (aroK) responsible for converting shikimate to the next step was disrupted to facilitate the accumulation of shikimate. Several genes encoding the shikimate bypass route, such as dehydroshikimate dehydratase (QsuB), pyruvate kinase (Pyk1), and quinate/shikimate dehydrogenase (QsuD), were disrupted sequentially. An artificial operon containing several shikimate pathway genes, including aroE, aroB, aroF, and aroG were overexpressed to maximize the glucose uptake and intermediate flux. The rationally designed shikimate-overproducing C. glutamicum strain grown in an optimized medium produced approximately 37.3 g/l of shikimate in 7-L fed-batch fermentation. Overall, rational cell factory design and culture process optimization for the microbial-based production of shikimate will play a key role in complementing traditional plant-derived shikimate production processes.

• Journal of Life Science
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• v.22 no.8
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• pp.1009-1017
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• 2012

The orf282 gene of Rhodobacter sphaeroides is located between the ccoNOQP operon encoding $cbb_3$ terminal oxidase and the fnrL gene encoding an anaerobic activator, FnrL. Its function remains unknown. In an attempt to reveal the function of the orf282 gene, we disrupted the gene by deleting a portion of the orf282 gene and constructed an orf282-knockout mutant. Two FnrL binding sites were found to be located upstream of orf282, and it was demonstrated that orf282 is positively regulated by FnrL. The orf282 gene is not involved in the regulation of spectral complex formation. The $cbb_3$ oxidase activity detected in the orf282 mutant was comparable to that in the wild-type sample, indicating that the orf282 gene is not involved in the regulation of the ccoNOQP operon and the biosynthesis of the cbb3 cytochrome c oxidase. The elevated promoter activity of the nifH and nifA genes, which are the structural genes of nitrogenase and its regulator, respectively, in the orf282 mutant, suggests that the orf282 gene product acts as a negative effector for nifH and nifA expression.

• Korean Journal of Microbiology
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• v.41 no.1
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• pp.1-7
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• 2005

Methylophaga aminosulfidovorans SK1 (KCTC 10323 BP) can utilize trimethylamine as a sole carbon, nitrogen, and energy source. The bacterial flavin-containing monooxygenase (bFMO) gene was identified in the strain and the recombinant enzyme expressed in E. coli oxidized trimethylamine. To study the function and regulation of the bfmo, over 8,000 nucleotide sequences of the neighboring regions including the bfmo were determined. Three open reading frames proceeding to the bfmo gene encoded analogues to highly conserved nitrate/nitrite sensing two-component system regulators and a methyl accepting protein. Two small open reading frames just downstream of the bfmo gene showed no similar proteins of known functions but the sequences were conserved among other bacteria. Reverse transcription-polymerase chain reaction analysis showed that the six putative genes consisted of three transcription units. The three regulatory genes located upstream of the bfmo gene formed two separate transcription units. The bfmo and the two downstream genes were transcribed from a single promoter.

• Korean Journal of Microbiology
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• v.30 no.5
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• pp.371-376
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• 1992

Plasmid pSL4 of plasmid pKM 101 mutant have high protection effects and mutagenecity for UV and methyl methanesulfonate, The mucA gene and a pan of mucE gene of pKM 101 and pSL4 were sucloned onto lacZ' fusion vector pMC874 and the hybrid plasmids pBH31 and pBH30 were selected. These plsmids were intrduced into $recA^{+}lexA^{-}$, $recA^{-}와lexA^{+}$ strains and determined the activity of $\beta$-galactosidase for UV. In $recA^{+}lexA^{+}$ strain.$\beta$-galactosidase activity of pBH30 included mue region of pSL4 was higher thall pBH31 inclued muc region of pKM 10 I and the tf-galactosidase of two plasmids was not induced in reeA and leeA mutants with or without UV illumination. Without UV illumination. the .$\beta$-galactosidasc of pBH30 was expressed a little higher level than that of pBH3L We suggest that the functional difference of pKM 10l and pSL4 are due to the variety of mue regulatory region. Also. a plasmid pBH 100 earring umuC' -lacZ' gene fusion was constructed in vitro to study the regulation of the umu operon. It was shown that the umu operon is induced by UV and is regulated by the reeA and lexA genes.

• Korean Chemical Engineering Research
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• v.48 no.2
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• pp.147-156
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• 2010

There are four key factors for gas-phase biofilters; biocatalysts(microorganisms), packing materials, design/operating techniques, and diagnosis/management techniques. Biofilter performance is significantly affected by microbial community structures as well as loading conditions. The microbial studies on biofilters are mostly performed on basis of culture-dependent methods. Recently, advanced methods have been proposed to characterize the microbial community structure in environmental samples. In this study, the physiological, biochemical and molecular methods for profiling microbial communities are reviewed, and their applicability to biofilters is discussed. Community-level physiological profile is based on the utilization capability of carbon substrate by heterotrophic community in environmental samples. Phospholipid fatty acid analysis method is based on the variability of fatty acids present in cell membranes of different microorganisms. Molecular methods using DNA directly extracted from environmental samples can be divided into "partial community DNA analysis" and "whole community DNA analysis" approaches. The former approaches consist in the analysis of PCR-amplified sequence, the genes of ribosomal operon are the most commonly used sequences. These methods include PCR fragment cloning and genetic fingerprinting such as denaturing gradient gel electrophoresis, terminal-restriction fragment length polymorphism, ribosomal intergenic spacer analysis, and random amplified polymorphic DNA. The whole community DNA analysis methods are total genomic cross-DNA hybridization, thermal denaturation and reassociation of whole extracted DNA and extracted whole DNA fractionation using density gradient.

• Journal of Microbiology and Biotechnology
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• v.8 no.5
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• pp.509-516
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• 1998

A plasmid vector, pXGPRMATG-lac-Tgx, was developed for overproduction of $\beta$-galactosidase in Escherichia coli using the genetic components of groEx, a heat-shock gene cloned from symbiotic X-bacteria in Amoeba proteus. The vector is composed of intragenic promoters P3 and P4 of groEx, the structural gene of lac operon, transcription tenninator signals of lac and groEx, and ColEl and amp'of pBluescript SKII. The optimized host, E. coli DH5$\alpha$, transfonned with the vector constitutively produced 117,310-171,961 Miller units of $\beta$-galactosidase per mg protein in crude extract. The amount of enzyme in crude extract was 53% of total water-soluble proteins. About 43% of the enzyme could be purified to a specific activity of 322,249 Miller units/mg protein after two-fold purification, using two cycles of precipitation with ammonium sulfate and one step of gel filtration. Thus, the expression system developed in this study presents a low-cost and simple method for purifying overproduced $\beta$-galactosidase in E. coli.

• Journal of Microbiology and Biotechnology
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• v.25 no.5
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• pp.696-703
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• 2015

A gamma-aminobutyric acid (GABA)-producing microorganism was isolated from jeot-gal (anchovy), a Korean fermented seafood. The isolate, A156, produced GABA profusely when incubated in MRS broth with monosodium glutamate (3% (w/v)) at 37℃ for 48 h. A156 was identified as Lactobacillus sakei by 16S rRNA gene sequencing. The GABA conversion yield was 86% as determined by GABase enzyme assay. The gadB gene encoding glutamate decarboxylase (GAD) was cloned by PCR. gadC encoding a glutamate/GABA antiporter was located immediately upstream of gadB. The operon structure of gadCB was confirmed by RT-PCR. gadB was overexpressed in Escherichia coli BL21(DE3) and recombinant GAD was purified. The purified GAD was 54.4 kDa in size by SDS-PAGE. Maximum GAD activity was observed at pH 5.0 and 55℃ and the activity was dependent on pyridoxal 5'-phosphate. The K_m and V_max of GAD were 0.045 mM and 0.011 mM/min, respectively, when glutamate was used as the substrate.

• Biotechnology and Bioprocess Engineering:BBE
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• v.9 no.4
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• pp.252-255
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• 2004

A pfl ldhA double mutant Escherichia coli strain NZN 111 was used to produce succinic acid by overexpressing the E. coli malic enzyme gene (sfcA). This strain, however, produced a large amount of malic acid as well as succinic acid. After the analyses of the metabolic pathways, the fumB gene encoding the anaerobic fumarase of E. coli was co-amplified to solve the problem of malic acid accumulation. A plasmid, pTrcMLFu, was constructed, which contains an artificial operon (sfcA-fumB) under the control of the inducible trc promoter. From the batch culture of recombinant E. coli NZN 111 harboring pTrcMLFu, 7 g/L of succinic acid was produced from 20 g/L of glucose, with no accumulation of malic acid. From the metabolic flux analysis the strain was found under reducing power limiting conditions by severe reorientation of metabolic fluxes.

• Biomedical Science Letters
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• v.10 no.2
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• pp.153-161
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• 2004

Antibiotic resistance is often associated with the production of inner membrane proteins (for example, AcrAB/TolC efflux pump) that are capable to extrude antibiotics, detergents, dyes and organic solvents. In order to evaluate the unknown MarB function of Escherichia coli, especially focused on the function of OmpF porin, several mutants were construted by T4GT7 transduction. MarA plays a major roles in mar (multiple antibiotic resistance) phenotype with AcrAB/TolC efflux pump in E. coli K-12. Futhermore, MarA decreases OmpF porin expression via micF antisense RNA. Expression of acrAB is increased in strains containing mutation in marR, and in those carrying multicopy plasmid expressing marA. MarB protein of E. coli K-12 showed its activity at OmpF porin & TolC protein as target molecule. Some paper reported MarB positively regulates OmpF function. MarA shows mar phenotype, and MarB along with MarA show decreased MIC through OmpF function. By this experiment, MarB could decrease MIC through the OmpF porin & TolC protein as target.

• The Plant Pathology Journal
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• v.18 no.2
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• pp.57-62
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• 2002

In vivo expression technology (IVET) has been developed to study bacterial gene expression in Salmonella typhimurium during host infection. The expression of selected genes by IVET has been elevated in vivo but not in vitro. The selected genes turned out to be important for bacterial virulence and/or pathogenicity. IVET depends on a synthetic operon with a promoterless transcriptional fusion between a selection marker gene and a reporter gene. The IVET approach has been successfully adapted in other bacterial pathogens and plant-associated bacteria using different selection markers. Pseudomonas putida suppresses citrus root rot caused by Phytophthora parasitica and enhances citrus seedling growth. The WET strategy was adapted based on a transcriptional fusion, pyrBC'-lacZ, in P. putida to study the bacterial traits important far biocontrol activities. Several genes appeared to be induced on P. parasitica hyphae and were found to be related with metabolism and regulation of gene expression. It is likely that the biocontrol strain took a metabolic advantage from the plant pathogenic fungus and then suppressed citrus root rot effectively. The result was parallel with those from the adaptation of IVET in P. fluorescens, a plant growth promoting rhizobacteria (PGPR). Interestingly, genes encoding components for type III secretion system have been identified as rhizosphere-induced genes in the PGPR strain. The type III secretion system may play a certain role during interaction with its counterpart plants. Application of IVET has been demonstrated in a wide range of bacteria. It is an important strategy to genetically understand complicated bacterial traits in the environment.