Yunsoo Yeo

Characterization of a gene encoding cellulase from uncultured soil bacteria.

To detect cellulases encoded by uncultured microorganisms, we constructed metagenomic libraries from Korean soil DNAs. Screenings of the libraries revealed a clone pCM2 that uses carboxymethyl cellulose (CMC) as a sole carbon source. Further analysis of the insert showed two consecutive ORFs (celM2 and xynM2) encoding proteins of 226 and 662 amino acids, respectively. A multiple sequence analysis with the deduced amino acid sequences of celM2 showed 36% sequence identity with cellulase from the Synechococcus sp., while xynM2 had 59% identity to endo-1,4-beta-xylanase A from Cellulomonas pachnodae. The highest enzymatic CMC hydrolysis was observable at pH 4.0 and 45 degrees C with recombinant CelM2 protein. Although the enzyme CelM2 additionally hydrolyzed avicel and xylan, no substrate hydrolysis was observed on oligosaccharides such as cellobiose, pNP-beta-cellobioside, pNP-beta-glucoside, and pNP-beta-xyloside. These results showed that CelM2 is a novel endo-type cellulase.

Unintended polar metabolite profiling of carotenoid-biofortified transgenic rice reveals substantial equivalence to its non-transgenic counterpart

Substantial equivalence is a critical concept for biosafety assessment of genetically modified (GM) crops. To investigate substantial equivalence among carotenoid-biofortified GM rice and five conventional rice cultivars having common white (three) and red (two) grain colors, profiles of 52 polar metabolites were analyzed using gas chromatography time-of-flight mass spectrometry. The results were compared to evaluate the differences among GM and non-GM rice cultivars using principal components analysis. The GM rice is more comparable to its non-transgenic counterpart rice variety according to the closer co-separation than for other cultivars tested. This suggests that profiling of unintended polar metabolites could be a useful tool to reveal substantial equivalence of GM rice.

Metabolite profiling approach reveals the interface of primary and secondary metabolism in colored cauliflowers (Brassica oleracea L. ssp. botrytis).

In the present study, carotenoids, anthocyanins, and phenolic acids of cauliflowers ( Brassica oleracea L. ssp. botrytis) with various colored florets (white, yellow, green, and purple) were characterized to determine their phytochemical diversity. Additionally, 48 metabolites comprising amino acids, organic acids, sugars, and sugar alcohols were identified using gas chromatography-time-of-flight mass spectrometry (GC-TOFMS). Carotenoid content was considerably higher in green cauliflower; anthocyanins were detected only in purple cauliflower. Phenolic acids were higher in both green and purple cauliflower. Results of partial least-squares discriminant, Pearson correlation, and hierarchical clustering analyses showed that green cauliflower is distinct on the basis of the high levels of amino acids and clusters derived from common or closely related biochemical pathways. These results suggest that GC-TOFMS-based metabolite profiling, combined with chemometrics, is a useful tool for determining phenotypic variation and identifying metabolic networks connecting primary and secondary metabolism.

Identification of a novel 4-hydroxyphenylpyruvate dioxygenase from the soil metagenome.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a Fe(II)-dependent, non-heme oxygenase that converts 4-hydroxyphenylpyruvate to homogentisate. Essential cofactors, such as plastoquinone and tocopherol, are produced by HPPD-dependent anabolic pathways in plants. To isolate a novel hppd using culture-independent method, a cosmid metagenomic library was constructed from soil in Korea. Screening of Escherichia coli metagenomic libraries led to the identification of a positive clone, YS103B, producing dark brown pigment in Luria-Bertani medium supplemented with l-tyrosine. In vitro transposon mutagenesis of YS103B showed that the 1.3kb insert was sufficient to produce the hemolytic brown pigment. Sequence analysis of YS103B disclosed one open reading frame encoding a 41.4kDa protein with the well-conserved prokaryotic oxygenase motif of the HPPD family of enzymes. The HPPD-specific beta-triketone herbicide, sulcotrione, inhibited YS103B pigmentation. The recombinant protein expressed in E. coli generated homogentisic acid. Thus, we present the successful heterologous expression of a previously uncharacterized hppd gene from an uncultured soil bacterium.

Crystal structure of engineered β‐glucosidase from a soil metagenome

Crystal structure of engineered b-glucosidase from a soil metagenome Ki Hyun Nam,1y Soo-Jin Kim,2y Min-Young Kim, Jae Hee Kim, Yun-Soo Yeo, Chang-Muk Lee, Hong-Ki Jun, and Kwang Yeon Hwang* 1Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul, Korea 2Microbial Genetics Division, National Institute of Agricultural Biotechnology, Rural Development Administration, Suwon, Korea 3Department of Microbiology, Pusan National University, Pusan, Korea

Determination of phenolic acids in Korean rice (Oryza sativa L.) cultivars using gas chromatography-time-of-flight mass spectrometry

Total soluble phenolic acids (free and esterified forms) from 15 Korean rice cultivars (Oryza sativa L.) were characterized to determine the total soluble phenolic acid contents and the diversity among the phenolic acids. The individual phenolic acids were identified by GC coupled to time-of-flight MS (TOFMS) including tertbutyldimethylsilyl (TBDMS) derivatization. This method requires only 13 min for the GC-TOFMS run. Total soluble phenolic acid contents were higher in pigmented rice (168–368 μg/g) than nonpigmented rice (146–172 μg/g). Freeform phenolic acids accounted for less than 10% of the total soluble phenolic acids. The dominant compounds were ferulic and sinapic acid, which were detected as cisand trans-isomers. The content of salicylic acid in rice grain varied, ranging from 7.95 to 29.61 μg/g. The quantitative results of this study indicate that the black rice ‘Josengheugchalbyeo’ could facilitate development of phenolic-rich rice or food.

Identification and quantification of carotenoids in paprika fruits and cabbage, kale, and lettuce leaves

Twelve carotenoids were identified in Korean leafy vegetables and paprikas. by high-performance liquid chromatography, Carotenoid contents varied greatly, with red paprika having a higher antheraxanthin and capsanthin contents than other paprikas. Orange paprika had higher levels of zeaxanthin, β-cryptoxanthin, lutein, and α-carotene compared to those of other paprikas. The results of Pearson’s correlation analysis using quantitative data of carotenoids revealed that significant positive relationships were apparent between capsanthin and antheraxanthin (r=0.9870, p <0.0001), zeaxanthin and α-cryptoxanthin (r=0.9951, p <0.0001), as well as lutein and α-carotene (r=0.9612, p <0.0001). Because the correlations between carotenoids levels have provided valuable information regarding metabolic associations, this technique will contribute to identifying metabolic links for carotenoid biosynthesis.

Comparative Analysis of Phytochemicals and Polar Metabolites from Colored Sweet Potato (Ipomoea batatas L.) Tubers

We determined the phytochemical diversity, including carotenoids, flavonoids, anthocyanins, and phenolic acids, in sweet potatoes (Ipomoea batatas L.) with distinctive flesh colors (white, orange, and purple) and identified hydrophilic primary metabolites. Carotenoid content was considerably higher in orange-fleshed sweet potatoes, wherein β-carotene was the most plentiful, and anthocyanins were detected only in purple-fleshed sweet potatoes. The levels of phenolic acids and flavonoids were relatively higher in purple-fleshed sweet potatoes than those in the other two varieties. Forty-one primary and 18 secondary metabolite profiles were subjected to multivariate statistical analyses, which fully distinguished among the varieties and separated orange- and purple-fleshed sweet potatoes from white-fleshed sweet potatoes based on the high levels of sugars, sugar alcohols, and secondary metabolites. This is the first study to determine comprehensive metabolic differences among different color-fleshed sweet potatoes and provides useful information for genetic manipulation of sweet potatoes to influence primary and secondary metabolism.

Comparative Analysis of Nutritional Composition between the Disease-resistant Rice Variety OsCK1 and Conventional Comparators

The disease-resistant OsCK1 (Oryza sativa L. cv. Nakdong) was developed in Korea by inserting a choline kinase (CK1) gene into the rice genome. The key nutrients and anti-nutrients of OsCK1 were analyzed and compared with those of its non-transgenic counterpart and four commercial cultivars grown together at two different locations. The levels of proximates, amino acids, fatty acids, minerals, vitamins, trypsin inhibitors, and phytic acid in OsCK1 were comparable to those of its parent rice and the commercial rice varieties. The results of principal component analysis performed using quantification data from 47 nutritional components revealed differences among the rice samples according to their growing locations rather than by their genotypes. These results confirm that the nutritional quality of rice grains was not affected by the insertion of the CK1 gene and suggest that the quality is more affected by environmental factors such as growing conditions than by genetic factors.

Identification of the Oligotrophic Bacteria Strain 7F Biocontrolling Phytophthora Blight Disease of Red-pepper

A total of 10,753 oligotrophic bacteria were isolated from the cultivated soils of red-pepper infected by Phytophthora blight disease in various regions of Korea (Chungju, Anmyon, Taean, Andong, Eumsung and Goesan). Seven bacteria isolates among these collected resources were selected by the first screening of in vitro antagonistic assay against major several plant pathogenic fungi including Phytophthora capsici. Finally, strain 7F was selected by pot assay for a possible biological control agent against Phytophthora blight disease of pepper seedling in the greenhouse. Strain 7F was identified as Bacillus subtilis on the basis of its 16S rDNA sequence analysis and as standardized biochemical characteristics assay kits such as API20 NE. In the experiment of P. capsici zoospore infected red-pepper on the pot test, infection rate of red-pepper with nonetreatment to Phytophthora blight disease was 87%, while the rate was only 6% in the pot treated with strain 7F. This result indicated that the Bacillus subtilis strain 7F will be useful as a potential biocontrol agent for Phytophthora blight disease of red-pepper.