• Journal of Microbiology and Biotechnology
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• v.32 no.8
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• pp.1041-1046
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• 2022

Nucleoside deoxyribosyltransferase (NDT) is an enzyme that replaces the purine or pyrimidine base of 2'-deoxyribonucleoside. This enzyme is generally used in the nucleotide salvage pathway in vivo and synthesizes many nucleoside analogs in vitro for various biotechnological purposes. Since NDT is known to exhibit relatively low reactivity toward nucleoside analogs such as 2'-fluoro-2'-deoxynucleoside, it is necessary to develop an enhanced NDT mutant enzyme suitable for nucleoside analogs. In this study, molecular evolution strategy via error-prone PCR was performed with ndt gene derived from Lactobacillus leichmannii as a template to obtain an engineered NDT with higher substrate specificity to 2FDU (2'-fluoro-2'-deoxyuridine). A mutant library of 214 ndt genes with different sequences was obtained and performed for the conversion of 2FDU to 2FDA (2'-fluoro-2'-deoxyadenosine). The E. coli containing a mutant NDT, named NDT^L59Q, showed 1.7-fold (at 40℃) and 4.4-fold (at 50℃) higher 2FDU-to-2FDA conversions compared to the NDT^WT, respectively. Subsequently, both NDT^WT and NDT^L59Q enzymes were over-expressed and purified using a His-tag system in E. coli. Characterization and enzyme kinetics revealed that the NDT^L59Q mutant enzyme containing a single point mutation of leucine to glutamine at the 59^th position exhibited superior thermal stability with enhanced substrate specificity to 2FDU.

• 한국생물공학회:학술대회논문집
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• 2000.04a
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• pp.189-192
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• 2000

The objective of this study was to investigate the effect of high content of light-absorbing pigments on overall photosynthetic efficiency in high density microalgal cultures. The light harvesting complex II (LHC II) regulating gene of Rhodobacter sphaeroides, photosynthetic purple bacterium, was removed to construct a mutant strain that had less pigment content. The mutant and wild type strains were cultured under various light intensity by adjusting the distance from the light source. The productivity of the mutant strain was higher at high light intensity (over 118 ${\mu}E/m^2/s$) compared with one of the wild type , and was lower at low light intensity (34 ${\mu}E/m^2/s$). Especially, the concentration of LHC II mutant strain was 56% higher at 118 ${\mu}E/m^2/s$. The reduction of per cell pigment contents in the mutant strain lessened the degree of the mutual shading and thus enhanced the overall photosynthetic efficiency.

• The Plant Pathology Journal
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• v.30 no.2
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• pp.215-219
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• 2014

The GacS/GacA two component system regulates various traits related to the biocontrol potential of plant-associated pseudomonads. The role of the sensor kinase, GacS, differs between strains in regulation of motility. In this study, we determined how a gacS mutation changed cell morphology and motility in Pseudomonas chlororaphis O6. The gacS mutant cells were elongated in stationary-phase compared to the wild type and the complemented gacS mutant, but cells did not differ in length in logarithmic phase. The gacS mutant had a two-fold increase in the number of flagella compared with the wild type strain; flagella number was restored to that of the wild type in the complemented gacS mutant. The more highly flagellated gacS mutant cells had greater swimming motilities than that of the wild type strain. Enhanced flagella formation in the gacS mutant correlated with increased expression of three genes, fleQ, fliQ and flhF, involved in flagellar formation. Expression of these genes in the complemented gacS mutant was similar to that of the wild type. These findings show that this root-colonizing pseudomonad adjusts flagella formation and cell morphology in stationary-phase using GacS as a major regulator.

• 한국생물공학회:학술대회논문집
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• 2003.10a
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• pp.434-437
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• 2003

A new bacterial strain, was isolated from activated sludge, identified and named P. aeruginosa EMS1. The new strain produced surface-active rhamnolipids by batch cultivation in mineral salts medium with waste flying oils. The mutant strain KH7, designated P. aeruginosa EMS1, derived by random mutagenesis with N-methyl-N-nitro-N-nitrosogoanidine treatment producing high levels of the biosurfactants was selected by an ion-pair plate assay. The mutant strain KH7 showed 4-5 times more hydrocarbon emulsification as compared to the parent when grown on waste frying oils and various hydrocarbons. Furthermore, P. aeruginosa EMS1 and mutant strain KH7 was also able to use whey as a co-substrate for growth and biosurfactant production. As results of this study, mutant strain KH7 is a very efficient biosurfactant producer, and its culture conditions are relatively inexpensive and economical. Rhamnolipid is synthesized by the rhlAB-encoded rhamnosyltransferase. To be convinced of these genes, we performed PCR based on P. aeruginosa PAO1 whole-genome database. rhl gene cluster nucleotide and amino acid sequences were compared for both parent and mutant. Comparison of nucleotide sequence of rhlAB, there were usually terminal's codons exchange.

• Research in Plant Disease
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• v.24 no.2
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• pp.162-169
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• 2018

The GacS/GacA system in the root colonizer Pseudomonas chlororaphis O6 is a key regulatory system of many traits relevant to the plant probiotic nature of this bacterium. The work in this paper elucidates proteins using proteomics approach in P. chlororaphis O6 under the control of the cytoplasmic regulatory protein, GacA. A gacA mutant of P. chlororaphis O6 showed loss in production of phenazines, acyl homoserine lactones, hydrogen cyanide, and protease, changes that were associated with reduced in vitro antifungal activity against plant fungal pathogens. Production of iron-chelating siderophore was significantly enhanced in the gacA mutant, also paralleling changes in a gacS mutant. However, proteomic analysis revealed proteins (13 downregulated and 7 upregulated proteins in the mutant compared to parental strain) under GacA control that were not apparent by a proteomic study of a gacS mutant. The putative identity of the downregulated proteins suggested that a gacA mutant would have altered transport potentials. Notable would be a predicted loss of type-VI secretion and PEP-dependent transport. Study of mutants of these GacA-regulated proteins will indicate further the features required for probiotic potential in this rhizobacterium.

• Korean Journal of Environmental Biology
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• v.23 no.4
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• pp.405-408
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• 2005

To induce the enhanced mutants of dinitroaniline herbicide pendimethalin degrading bacterium, Bacillus sp. MS202 was irradiated with gamma radiation at the dose of $LD_{99}$ (3.35 kGy). Three enhanced mutants (MS202m7, MS202m14, MS202m18) were isolated from the candidates by the generation - isolation method. Clear zone formation and the GC analysis confirmed that the degrading activity of each enhanced mutant (MS202m7, MS202m14, MS202m18), the formation of pendimethalin metabolite, increased by $11\%,\;45\%,\;and\;32\%$ than a wild type, respectively. It suggested that these mutants induced by gamma radiation could be useful for the application of pesticide degradation.

• KSBB Journal
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• v.19 no.4
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• pp.331-334
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• 2004

In vivo characterization of FadB homologous enzymes including PaaG, YdbU and YgfG for medium-chain-length (MCL) polyhydroxyalkanoate (PHA) biosynthesis was carried out in fadB mutant Escherichia coli. Previously, it was reported that amplification of FadB homologous enzymes such as PaaG and YdbU in fadB mutant E. coli resulted in enhanced biosynthesis of MCL-PHA by greater than two fold compared with control strain. In this study, we constructed paaG fadB double mutant E. coli WB114 and ydbU fadB double mutant E. coli WB115 to investigate the roles of PaaG and YdbU in biosynthesis of MCL-PHA. Inactivation of paaG and ydbU genes in fadB mutant E. coli harboring Pseudomonas sp. 61-3 phaC2 gene reduced the MCL-PHA production to 0.16 and 0.16 PHA g/L, respectively from 2 g/L of sodium decanoate, which are much lower than 0.43 PHA g/L obtained with fadB mutant E. coli WB101 harboring the phaC2 gene. Also, we identified new FadB homologous enzyme YgfG, and examined its roles by overexpression of ygfG and construction of ygfG fadB double mutant E. coli WB113.

• Korean Journal of Microbiology
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• v.45 no.3
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• pp.268-274
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• 2009

A photosynthetic cyanobacterium Arthrospira platensis, well known for health food supplement, was studied as a target species for atmospheric $CO_2$ removal as well as biomass production. Although the biomass of A. platensis was massively produced in many countries, the recovery cost of its biomass is still high. The purpose of this study was to develop the A. platensis mutant strains which have enhanced growth rate and floatation activity to reduce the recovery cost. A. platensis KCTC AG20590 was treated with 0.24% ethyl methanesulfonate (EMS) for 20 min at room temperature. The mutant strain A. platensis M20CJK3 was finally selected by its morphological and physiological features. The morphology of the mutant A. platensis M20CJK3 was changed from loose-coiled form to tight-coiled form showing short pitch. The growth and $CO_2$ uptake rate of A. platensis M20CJK3 were improved about 15% and 17% compared with A. platensis KCTC AG20590, respectively. The floatation activity of A. platensis M20CJK3 was enhanced in 2-fold compared with that of A. platensis KCTC AG20590. Soluble proteins extracted from two strains were analyzed by two dimensional electrophoresis (2-DE) and MALDI-TOF MS/MS. Among 15 protein spots induced in 2-DE analysis, two spots were the proteins related to photosynthesis and electron transfer system of the other cyanobacteria. As a consequence, it seems that the tight-coiled mutant A. platensis M20CJK3 has an advantage of high growth rate and floatation activity which are beneficial for the mass cultivation and recovery.

• Journal of Radiation Industry
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• v.4 no.1
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• pp.65-71
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• 2010

Two chitinase producing strains CHI2 and CHI4 were isolated from soybean rhizosphere soil. Both the strains belonged to Lysobacter enzymogenes as indicated by 16S rDNA sequence analysis. Though strain CHI2 and CHI4 produced extracellular chitinase, they differ in their chitinolytic activity. CHI4 produced approximately three times the higher amounts of enzyme than that of CHI2 under specified conditions. CHI2 produced $535.67U\;l^{-1}$ of chitinase after 48 h incubation with a specific activity of $3.91U\;mg^{-1}$ of protein while strain CHI4 produced $1584.13U\;l^{-1}$ of chitinase with a specific activity of $10.88U\;mg^{-1}$ protein. SDS-PAGE analysis indicated that the molecular weight of chitinase enzyme was approximately 45 kDa. A faint band with a molecular weight of 55 kDa reveals the possibility for the presence of another kind of chitin binding protein. Mutant library was developed by exposing the isolates to gamma rays at their $LD_{99}$ value (0.23 kGy). Totally, 11 mutants of CHI2 and CHI4 are reported to have enhanced chitinase activity. Several leaky mutant clones with decreased enzyme activity and a defective mutant (CHI2-M16) with complete loss of chitinase activity were also identified. CHI4-M18, CHI4-M8 and CHI4-M29 showed 78.8, 41.5, and 31.9% increased chitinase activity over wild type CHI4.

• Microbiology and Biotechnology Letters
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• v.47 no.4
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• pp.581-584
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• 2019

In this study, the carbon monoxide (CO)-fermenting acetogen, Clostridium sp. AWRP was subjected to chemical mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) to generate a CO-resistant mutant. Among the 26 colonies obtained, the highest alcohol production was observed in one isolate, named C1. Compared to the wild-type strain, the C1 strain exhibited 1.5- and 3.4-fold higher CO consumption rate and alcohol selectivity, respectively. The total CO consumption of strain C1 could be further enhanced by increasing the content of metal ions, such as nickel and iron. The highest ethanol titer (5.7 g/l) was achieved by 5-fold increase in the iron concentration.