Sang-Hong Yoon

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.

Novel Metagenome-Derived Carboxylesterase That Hydrolyzes β-Lactam Antibiotics

ABSTRACT It has been proposed that family VIII carboxylesterases and class C β-lactamases are phylogenetically related; however, none of carboxylesterases has been reported to hydrolyze β-lactam antibiotics except nitrocefin, a nonclinical chromogenic substrate. Here, we describe the first example of a novel carboxylesterase derived from a metagenome that is able to cleave the amide bond of various β-lactam substrates and the ester bond of p-nitrophenyl esters. A clone with lipolytic activity was selected by functional screening of a metagenomic library using tributyrin agar plates. The sequence analysis of the clone revealed the presence of an open reading frame (estU1) encoding a polypeptide of 426 amino acids, retaining an S-X-X-K motif that is conserved in class C β-lactamases and family VIII carboxylesterases. The gene was overexpressed in Escherichia coli, and the purified recombinant protein (EstU1) was further characterized. EstU1 showed esterase activity toward various chromogenic p-nitrophenyl esters. In addition, it exhibited hydrolytic activity toward nitrocefin, leading us to investigate whether EstU1 could hydrolyze β-lactam antibiotics. EstU1 was able to hydrolyze first-generation β-lactam antibiotics, such as cephalosporins, cephaloridine, cephalothin, and cefazolin. In a kinetic study, EstU1 showed a similar range of substrate affinities for both p-nitrophenyl butyrate and first-generation cephalosporins while the turnover efficiency for the latter was much lower. Furthermore, site-directed mutagenesis studies revealed that the catalytic triad of EstU1 plays a crucial role in hydrolyzing both ester bonds of p-nitrophenyl esters and amide bonds of the β-lactam ring of antibiotics, implicating the predicted catalytic triad of EstU1 in both activities.

Methylobacterium iners sp. nov. and Methylobacterium aerolatum sp. nov., isolated from air samples in Korea

Two bacterial strains isolated from air samples, 5317S-33(T) and 5413S-11(T), were characterized by determining their phenotypic characteristics, cellular fatty acid profiles and phylogenetic positions based on 16S rRNA gene sequence analysis. 16S rRNA gene sequence analysis showed that these isolates belonged to the genus Methylobacterium. Strain 5317S-33(T) was most closely related to Methylobacterium adhaesivum AR27(T) (97.9% sequence similarity). Strain 5413S-11(T) was most closely related to Methylobacterium fujisawaense DSM 5686(T) (97.3% sequence similarity), Methylobacterium oryzae CBMB20(T) (97.1% similarity) and Methylobacterium radiotolerans JCM 2831(T) (97.0% similarity). Cells of both strains were strictly aerobic, Gram-negative, motile and rod-shaped. The major fatty acid was C(18:1)omega7c. The G+C contents of the genomic DNA were 68.0 mol% for strain 5317S-33(T) and 73.2 mol% for strain 5413S-11(T). According to DNA-DNA hybridization data, strain 5317S-33(T) showed a level of DNA-DNA relatedness of 33 % with M. adhaesivum DSM 17169(T), and strain 5413S-11(T) showed low levels of DNA-DNA relatedness (<35%) with M. fujisawaense DSM 5686(T), M. oryzae CBMB20(T) and M. radiotolerans DSM 1819(T). On the basis of this polyphasic analysis, it was concluded that strains 5317S-33(T) and 5413S-11(T) represent two novel species within the genus Methylobacterium, for which the names Methylobacterium iners sp. nov. (type strain 5317S-33(T) =KACC 11765(T) =DSM 19015(T)) and Methylobacterium aerolatum sp. nov. (type strain 5413S-11(T) =KACC 11766(T) =DSM 19013(T)) are proposed.

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.

Structural and functional analysis of a novel hormone-sensitive lipase from a metagenome library

Structural and functional analysis of a novel hormone-sensitive lipase from a metagenome library Ki Hyun Nam,1y Min-Young Kim,2y Soo-Jin Kim, Amit Priyadarshi, Suk-Tae Kwon, Bon-Sung Koo, Sang-Hong Yoon, and Kwang Yeon Hwang* 1Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea 2National Agrobiodiversity Center, National Academy of Agricultural Science, Rural Development Administration, Suwon 441-707, Korea 3 Biomedical Research Center, Korea Institute of Science and Technology, Seoul 136-791, Korea 4Department of Genetic Engineering, Sungkyunkwan University, Jangan-gu, Suwon 440-746, Korea 5 Fermentation and Food Processing Division, Department of Korean Food Research for Globalization, National Academy of Agricultural Science,

Aquitalea denitrificans sp. nov., isolated from a Korean wetland

A novel bacterium, designated strain 5YN1-3(T), was isolated from wetland peat collected from Yongneup, Korea. The bacterium was facultatively anaerobic, Gram-negative, yellow-coloured, rod-shaped, mesophilic and motile with one polar flagellum. The strain grew optimally at 30 degrees C, at pH 6.0-9.0 and with 0-1 % NaCl (w/v). 16S rRNA gene sequence analysis showed the highest similarity to the sequence from Aquitalea magnusonii TRO-001DR8(T), with 98.7 % sequence similarity. However, strain 5YN1-3(T) showed DNA-DNA relatedness of 43 % (40 % in a reciprocal experiment) with A. magnusonii LMG 23054(T). The strain contained summed feature 3 (iso-C(15 : 0) 2-OH and/or C(16 : 1)omega7c) and C(16 : 0) as major cellular fatty acids. On the basis of DNA-DNA relatedness and physiological and biochemical characterization, strain 5YN1-3(T) should be assigned to a novel species of the genus Aquitalea, for which the name Aquitalea denitrificans sp. nov. is proposed. The type strain is 5YN1-3(T) (=KACC 12729(T) =DSM 21300(T)).

Identification of a novel alkaline amylopullulanase from a gut metagenome of Hermetia illucens

A novel pullulanase gene, PulSS4, was identified from the gut microflora of Hermetia illucens by a function-based metagenome screening. The PulSS4 gene had an open reading frame of 4455 base pairs, and encoded a mature protein of 1484 amino acids, with a signal peptide sequence of 44 amino acids. The deduced amino acid sequence of PulSS4 gene showed 51% identity with that of the amylopullulanase of Amphibacillus xylanus, exhibiting no significant sequence homology to already known pullulanases. A conserved domain analysis revealed it to be a pullulanase type II with respective active sites at the N-terminal pullulanase and C-terminal amylase domain. PulSS4 was active in the temperature range of 10-50°C, with an optimum activity at 40°C. It was active in the pH range of 6.5-10.5, with optimum pH at 9.0, and retained more than 80% of its original activity in a broad pH range of 5-11 for 24h at 30°C. Also, PulSS4 was highly stable against many different chemical reagents, including 10% polar organic solvents and 1% non-ionic detergents. Overall, PulSS4 is expected to have the strong potential for application in biotechnological industries that require high activity at moderate temperature and alkaline conditions.

Burkholderia denitrificans sp. nov., isolated from the soil of Dokdo Island, Korea.

A novel, Gram-negative, bacterial strain KIS30-44T was identified from wet forest soil collected on the Korean island of Dokdo. Growth of the strain was observed at 15–30°C, pH 5–9, 0–3% NaCl, and 950 mM KNO3. KIS30-44T reduced nitrate to nitrogen gas. Analysis of the 16S rRNA gene sequence showed that KIS30-44T was phylogenetically related to Burkholderia sacchari, Burkholderia mimosarum, and Burkholderia oxyphila (98.1%, 98.0%, and 98.0% sequence similarity, respectively). The genomic G+C content was 63.5 mol%. KIS30-44T exhibited less than 52% DNA-DNA relatedness with the type strains of 9 closely related Burkholderia species. The major isoprenoid quinone was Q-8. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and two unknown aminolipids. The major fatty acids in KIS30-44T were C16:0, C18:1ω7c and summed feature 3 (iso-C15:0 2-OH and C16:1ω7c), and the strain contained half the amount of C17:0 cyclo found in the 9 closely related Burkholderia species. The results of these phenotypic, 16S rRNA gene sequence, DNA-DNA hybridization, and chemotaxonomic data indicate that KIS30-44T represents a novel species within the genus Burkholderia, for which the name Burkholderia denitrificans (Type strain KIS30-44T =KACC 12733T =DSM 24336T) is proposed.

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.

Expressed sequence tags (EST) analysis of a normalized full-length cDNA library from the pinewood nematode (Bursaphelenchus xylophilus)

The pinewood nematode ( Bursaphelenchus xylophilus ) infects pine trees and causes pine wilt disease. To clarify the functions and subcellular localization of B. xylophilus genes/proteins transcribed and predicted from mixed stages (egg, J1, J2, J3 J4 and adult), we prepared a normalized full-length B. xylophilus cDNA library and analyzed expressed sequence tags (ESTs) using the Pendant-Pro sequence analysis suite. Most cDNAs inserted into the library ranged from 0.9 to 1.8 kb (average 1.5 kb). The 1,902 ESTs from B. xylophilus consisted of 286 clusters and 1,273 singletons. EST sizes ranged from 9 to 743 bp with a mean of 336 bp. The predicted protein length from B. xylophilus ESTs revealed that most proteins ranged from 50 to 149 amino acids. Enzyme nomenclature (EC) numbers were classified into 133 (8.5%) of 1,559 contigs using UniProt database hits by the EC numbers method. Transmembrane regions of 1559 clusters were predicted using the TMpred algorithm. The 1,559 contigs with transmembrane regions were annotated; 481 (30.8%) contigs were assigned ‘above one’ domain and 1,078 (69.1%) were assigned ‘none.’ Additionally, taxonomy was classified for 672 (43.1%) of 1,559 contigs. Of the 1,559 contigs, 685 (43.9%) were assigned gene ontology terms using the gomerger method of contigs, including singletons. Thirty-one (31) contigs of predicted proteins grouped by BLASTP identity values had significant homology to genes expressed in subcellular structures (for example, mitochondrion, plasma membrane, endoplasmic reticulum, nucleus and golgi). B. xylophilus ESTs provide the foundation for research information on related plant parasite nematodes and contribute to finding an important novel parasite control strategy. Keywords: Pinewood nematode, Bursaphelenchus xylophilus, pine wilt disease, expressed sequence tag, Pendant-Pro Sequence Analysis Suite. African Journal of Biotechnology , Vol 13(31) 3332-3343