• Current Research on Agriculture and Life Sciences
• /
• v.31 no.1
• /
• pp.45-50
• /
• 2013

Mutagenesis is used to study gene function and obtain new genetic resources for plant breeding. Soybean is an important oil crop in the world. Thus, to find new genetic resources, a mutation population was developed from the soybean cultivar Pungsannamul using 0.3% ethyl methane sulfonate. The variation of fatty acids was then evaluated among 892 M4 generation mutant lines selected from 3,774 mutant lines. While the wild type Pungsannamul showed 11.6, 3.4, 23.8, 53.3, and 7.8% for palmitic, stearic, oleic, linoleic and linolenic acid, respectively. the fatty acid variations in the mutant lines ranged from 7.4 to 19.7%, 2.2 to 13.0%, 14.7 to 49.0%, 31.8 to 63.9%, and 3.9 to 15.9% with an average of 10.8, 3.8, 25.3, 52.0, and 8.1% for palmitic, stearic, oleic, linoleic and linolenic acid, respectively. Thus, two mutation lines with higher plamitic acid, PE1542 (17.1%) and PE3058 (17.0%), one line with lower stearic acid, PE2166 (1.9%), one line with higher stearic acid, PE977 (12.7%), two lines with higher oleic acid, PE450 (44.4%) and PE2742 (47.7%), and two lines with lower linolenic acid, PE594 (4.6%) and PE1690 (3.7%), were selected from this study. The newly selected fatty acid variants will be good genetic sources for gene function analyses and breeding soybean varieties with altered fatty acids for various industrial and human food applications.

• Journal of Life Science
• /
• v.27 no.10
• /
• pp.1137-1144
• /
• 2017

The microalgae Chlorella vulgaris has been considered an important alternative resource for biodiesel production. However, its industrial-scale production has been constrained by the low productivity of the biomass and lipid. To overcome this problem, we isolated and characterized a potentially economical oleaginous strain of C. vulgaris via the random mutagenesis technique using UV irradiation. Two types of mass production systems were compared for their yield of biomass and lipid content. Among the several putatively oleaginous strains that were isolated, the particular mutant strain designated as UBM1-10 in the laboratory showed an approximately 1.5-fold higher cell yield and lipid content than those from the wild type. Based on these results, UBM1-10 was selected and cultivated under outdoor conditions using two different types of reactors, a tubular-type photobioreactor (TBPR) and an open pond-type reactor (OPR). The results indicated that the mutant strain cultivated in the TBPR showed more than 5 times higher cell concentrations ($2.6g\;l^{-1}$) as compared to that from the strain cultured in the OPR ($0.5g\;l^{-1}$). After the mass cultivation, the cells of UBM1-10 and the parental strain were further investigated for crude lipid content and composition. The results indicate a 3-fold higher crude lipid content from UBM1-10 (0.3%, w/w) as compared to that from the parent strain (0.1% w/w). Therefore, this study demonstrated that the economic potential of C. vulgaris as a biodiesel production resource can be increased with the use of a photoreactor type as well as the strategic mutant isolation technique.

• Journal of Microbiology and Biotechnology
• /
• v.27 no.10
• /
• pp.1783-1789
• /
• 2017

Thermostability is an important property of xylanase because high temperature is required for its applications, such as wood pulp bleaching, baking, and animal feedstuff processing. In this study, XynB from Thermobacillus composti, a moderately thermophilic gram-negative bacterium, was modified via site-directed mutagenesis (based on its 3D structure) to obtain thermostable xylanase, and the properties of this enzyme were analyzed. Results revealed that the half-life of xylanase at $65^{\circ}C$ increased from 10 to 50 min after a disulfide bridge was introduced between the ${\alpha}$-helix and its adjacent ${\beta}$-sheet at S98 and N145. Further mutation at the side of A153E named XynB-CE in the C-terminal of this ${\alpha}$-helix enhanced the half-life of xylanase for 60 min at $65^{\circ}C$. Therefore, the mutant may be utilized for industrial applications.

• Applied Chemistry for Engineering
• /
• v.26 no.5
• /
• pp.569-574
• /
• 2015

Arthrospira platensis has high lipid and pigment (such as chlorophyll and carotenoid) contents and thus evaluated as an important resource in functional food production. The cell growth rate and pigment concentration of EM24 increased by approximately 1.2-fold than those of the wild-type strain (WT). Fluorescence intensity levels in EM24, which were quantified with a lipid triolein standard curve, also increased by approximately 1.5-fold than those in WT (62.9 mg/Lvs. 38.9 mg/L). The analysis of fatty acid profiles indicated that the gamma-linoleic acid level in EM24 increased by 1.5-fold than that in WT.

• International Journal of Industrial Entomology and Biomaterials
• /
• v.10 no.2
• /
• pp.119-123
• /
• 2005

Benomyl-resistant mutants of entomopathogenic fungus, Metarhizium anisopliae were isolated and their physiological characteristics were investigated. These militants were obtained spontaneously or by UV irradiation in benomyl-treated media. Four spontaneous (S-2, S-11, S-18, S-19) and four UV-induced (UV-4, UV-5, UV-19, UV-24) mutants, which grow stably and normally were selected. No significant differences in conidia or hyphal shape, conidia viability, mycelial biomass, or virulent to the diamondback moth were observed between the wild type and their mutants. But differently from the mycelial growth of other benomyl-resistant mutants which was slower than that of the wild type on a modified Czapek-Dox, SDAY, $4\%$ chitin, or $1\%$ skim milk medium, that in the spontaneous mutants, S-18 and S-19, did not show any difference from the wild type. Especially, S-18 and S-19 grew well at benomyl concentrations up to 50 times or higher than that which inhibits wild type proliferation. These results suggested that S-18 and S-19 could potentially be used with the fungicide, benomyl.

• Journal of Microbiology and Biotechnology
• /
• v.25 no.12
• /
• pp.1977-1988
• /
• 2015

β-1,3-glucanosyltransferases play essential roles in cell wall biosynthesis in yeast. Kluyveromyces lactis has six putative β-1,3-glucanosyltransferase genes. KlGAS1-1 and KlGAS1-2 are homologs of Saccharomyces cerevisiae gene GAS1. RT-qPCR indicated the transcription level of KlGAS1-1 was significantly reduced while heterologous protein (thermostable xylanase B) secretion was enhanced during medium optimization. To evaluate if these two events were related, and to improve xylanase B secretion in K. lactis, we constructed KlGAS1-1 and KlGAS1-2 single deletion strains and double deletion strain, respectively. KlGAS1-1 gene deletion resulted in the highest xylanase B activity among the three mutants. Only the double deletion strain showed morphology similar to that of the GAS1 deletion mutant in S. cerevisiae. The two single deletion strains differed in terms of cell wall thickness and xylanase B secretion. Transcription levels of β-1,3-glucanosyltransferase genes and genes related to protein secretion and transport were assayed. The β-1,3-glucanosyltransferase genes displayed transcription complementation in the cell wall synthesis process. KlGAS1-1 and KlGAS1-2 affected transcription levels of secretion- and transport-related genes. Differences in protein secretion ratio among the three deletion strains were associated with changes of transcription levels of secretion- and transport-related genes. Our findings indicate that KlGAS1-1 deletion is an effective tool for enhancing industrial-scale heterologous protein secretion in K. lactis.

• Biotechnology and Bioprocess Engineering:BBE
• /
• v.10 no.6
• /
• pp.522-527
• /
• 2005

Various processes which produce L-lactic acid using ammonia-tolerant mutant strain, Rhizopus sp. MK-96-1196, in a 3L airlift bioreactor were evaluated. When the fed-batch culture was carried out by keeping the glucose concentration at 30g/L, more than 140 g/L of L-lactic acid was produced with a product yield of 83%. In the case of the batch culture with 200g/L of initial glucose concentration, 121g/L of L-lactic acid was obtained but the low product yield based on the amount of glucose consumed. In the case of a continuous culture, 1.5g/L/h of the volumetric productivity with a product yield of 71% was achieved at dilution rate of $0.024\;h^{-1}$. Basis on these results three processes were evaluated by simple variable cost estimation including carbon source, steam, and waste treatment costs. The total variable costs of the fed-batch and continuous cultures were 88% and 140%, respectively, compared to that of batch culture. The fed-batch culture with high L-lactic acid concentration and high product yield decreased variable costs, and was the best-suited for the industrial production of L-lactic acid.

• Journal of Microbiology and Biotechnology
• /
• v.22 no.5
• /
• pp.659-667
• /
• 2012

2,3-Butanediol (2,3-BDO) is an organic compound with a wide range of industrial applications. Although Escherichia coli is often used for the production of organic compounds, the wild-type E. coli does not contain two essential genes in the 2,3-BDO biosynthesis pathway, and cannot ferment 2,3-BDO. Therefore, a 2,3-BDO biosynthesis mutant strain of Escherichia coli was constructed and cultured. To determine the optimum culture factors for 2,3-BDO production, experiments were conducted under different culture environments ranging from strongly acidic to neutral pH. The extracellular metabolite profiles were obtained using high-performance liquid chromatography (HPLC), and the intracellular metabolite profiles were analyzed by ultra-performance liquid chromatography and quadruple time-of-flight mass spectrometry (UPLC/Q-TOF-MS). Metabolic flux analysis (MFA) was used to integrate these profiles. The metabolite profiles showed that 2,3-BDO production favors an acidic environment (pH 5), whereas cell mass favors a neutral environment. Furthermore, when the pH of the culture fell below 5, both the cell growth and 2,3-BDO production were inhibited.

• International Journal of Industrial Entomology and Biomaterials
• /
• v.39 no.1
• /
• pp.14-21
• /
• 2019

Genome editing by CRISPR/Cas9, a third-generation gene scissor in molecular breeding at the genome level, is attracting much attention as one of the breeding techniques of the future. In this study, genetic and phenotypic analysis was used to examine the responsiveness of the Bakokjam variety of the silkworm Bombyx mori to molecular breeding using CRISPR/Cas9 in editing the white egg 2 (w-2) gene. The nucleotide sequence of the w-2 gene was analyzed and three different guide RNAs (gRNA) were prepared. The synthesized gRNA was combined with Cas9 protein and then analyzed by T7 endonuclease I after introduction into the Bm-N silkworm cell line. To edit the silkworm gene, W1N and W2P gRNA and Cas9 complexes were microinjected into silkworm embryos. Based on the results of microinjection, the hatching rate was 16-24% and the incidence of mutation was 33-37%. The gene mutation was verified in the heterozygous F1 generation, but no phenotypic change was observed. In F2 homozygotes generated by F1 self-crosses, a mutant phenotype was observed. These results suggest that silkworm molecular breeding using the CRISPR/Cas9 system is possible and will be a very effective way to shorten the time required than the traditional breeding process.

• Applied Chemistry for Engineering
• /
• v.32 no.1
• /
• pp.10-14
• /
• 2021

mCherry is one of the well-understood red fluorescent proteins which has a similar tertiary structure as GFPs, but pH resistant due to the lack of hydrogen bond network. Whereas mCherry-I202T showed far-red fluorescence and also pH sensitive property because of the additional hydrogen bond formed by substituting Ile of 202 amino acid sequence on mCherry with Thr. In order to verify the pH sensitive characteristic of mCherry-I202T owing to the extension of hydrogen bond, UV-vis spectrum was measured over the range of acidic to basic pH. We also demonstrate further possibilities of applying mCherry-I202T as a pH sensor.