• Journal of Microbiology and Biotechnology
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• v.23 no.5
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• pp.674-680
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• 2013

Yeast Dekkera/Brettanomyces bruxellensis is probably the most common contaminant in wineries and ethanol production processes. The considerable economic losses caused by this yeast, but also its ability to produce and tolerate high ethanol concentrations, make it an attractive subject for research with potential for industrial applications. Unfortunately, efforts to understand the biology of D. bruxellensis and facilitate its broader use in industry are hampered by the lack of adequate procedures for delivery of exogenous DNA into this organism. Here we describe the development of transformation protocols (spheroplast transformation, LiAc/PEG method, and electroporation) and report the first genetic transformation of yeast D. bruxellensis. A linear heterologous DNA fragment carrying the kanMX4 sequence was used for transformation, which allowed transformants to be selected on plates containing geneticin. We found the spheroplast transformation method using 1M sorbitol as osmotic stabilizer to be inappropriate because sorbitol strikingly decreases the plating efficiency of both D. bruxellensis spheroplast and intact cells. However, we managed to modify the LiAc/PEG transformation method and electroporation to accommodate D. bruxellensis transformation, achieving efficiencies of 0.6-16 and 10-20 transformants/${\mu}g$ DNA, respectively. The stability of the transformants ranged from 93.6% to 100%. All putative transformants were analyzed by Southern blot using the kanMX4 sequence as a hybridization probe, which confirmed that the transforming DNA fragment had integrated into the genome. The results of the molecular analysis were consistent with the expected illegitimate integration of a heterologous transforming fragment.

• Plant Biotechnology Reports
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• v.5 no.3
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• pp.245-254
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• 2011

This study describes an efficient protocol for Agrobacterium tumefaciens-mediated transformation of two subgroups of genotype AAA bananas (Musa acuminata cv. Pei Chiao and Musa acuminata cv. Gros Michel). Instead of using suspension cells, cauliflower-like bud clumps, also known as multiple bud clumps (MBC), were induced from sucker buds on MS medium containing $N^6$-Benzylaminopurine (BA), Thidiazuron (TDZ), and Paclobutrazol (PP333). Bud slices were co-cultivated with A. tumefaciens C58C1 or EHA105 that carry a plasmid containing Arabidopsis root-type ferredoxin gene (Atfd3) and a plant ferredoxin-like protein (pflp) gene, respectively. These two strains showed differences in transformation efficiency. The EHA105 strain was more sensitive in Pei Chiao, 51.3% bud slices were pflp-transformed, and 12.6% slices were Atfd3-transformed. Gros Michel was susceptible to C58C1 and the transformation efficiency is 4.4% for pflp and 13.1% for Atfd3. Additionally, gene integration of the putative pflp was confirmed by Southern blot. Resulting from the pathogen inoculation assay, we found that the pflp transgenic banana exhibited resistance to Fusarium oxysporum f. sp. cubense tropical race 4. This protocol is highly advantageous to banana cultivars that have difficulties in setting up suspension cultures for the purpose of quality improvement through genetic transformation. In addition, this protocol would save at least 6 months in obtaining explants for transformation and reduce labor for weekly subculture in embryogenic cell suspension culture systems.

• Natural Product Sciences
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• v.23 no.4
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• pp.306-309
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• 2017

Microbial transformation of $({\pm})$-6-(1,1-dimethylallyl)naringenin (6-DMAN, 1) and $({\pm})$-5-(O-prenyl) naringenin-4',7-diacetate (5-O-PN, 2) was performed by using fungi. Scale-up fermentation studies with Mucor hiemalis, Cunninghamella elegans var. elegans, and Penicillium chrysogenum led to the isolation of five microbial metabolites. Chemical structures of the metabolites were determined by spectral analyses as $({\pm})$-8-prenylnaringenin (3), (2S)-5,4'-dihydroxy-7,8-[(R)-2-(1-hydroxy-1-methylethyl)-2,3-dihydrofurano]flavanone (4), $({\pm})$-5-(O-prenyl)naringenin-4'-acetate (5), $({\pm})$-naringenin-4'-acetate (6), and $({\pm})$-naringenin (7), of which 5 was identified as a new compound.

• Natural Product Sciences
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• v.26 no.4
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• pp.283-288
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• 2020

Microbial transformation of a potent progestin, norethisterone (17��-hydroxy-19-nor-17α-pregn-4-en-20-yn-3-one) (1) was carried out by using two filamentous fungi Aspergillus niger and Penicillium citrinum. Biotransformation of 1 with A. niger yielded a hydroxylated transformed product 10��,17��-diydroxy-19-nor-17α-pregn-4-en-20-yn-3-one (2) whereas 11��,17��-diydroxy-19-nor-17α-pregn-4-en-20-yn-3-one (3) was obtained through microbial transformation of 1 by P. citrinum. It is the first report of their production from 1 by using A. niger and P. citrinum with complete 1H- and 13C-NMR assignment. The structures of both metabolites were characterized by various spectroscopic techniques and reported data.

• Bulletin of the Korean Chemical Society
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• v.35 no.9
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• pp.2870-2872
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• 2014

• Journal of Microbiology and Biotechnology
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• v.20 no.1
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• pp.39-44
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• 2010

Acidithiobacillus caldus is an acidophilic, chemolithotrophic bacterium that plays an important role in bioleaching. Gene transformation into A. caldus is difficult, and only the conjugation method was reported successful, which was a relatively sophisticated method. In this research, electrotransformation of A. caldus species was achieved for the first time using A. caldus Y-3 and plasmid pJRD215. Transformants were confirmed by colony PCR specific to the str gene on pJRD215, and the recovery of the plasmid from the presumptive transformants. Optimizations were made and the transformation efficiency was increased from 0.8 to $3.6{\times}10^4$ transformants/${\mu}g$ plasmid DNA. The developed electrotransformation method was convenient in introducing foreign genes into A. caldus.

• Natural Product Sciences
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• v.5 no.3
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• pp.151-153
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• 1999

(+)-Hernandulcin is a sweet bisabolane-type sesquiterpene first isolated from Lippia dulcis Trev. (Verbenaceae). This oily compound is 1000-1500 times sweeter than sucrose but with poor solubility in water. Microbial transformation was employed to improve its water solubility, and a variety of microorganisms were screened for their ability to convert (+)-hernandulcin to more polar metabolites. Scale-up fermentation with Glomerella cinguiata, a fungal strain, has resulted in the isolation of a more polar metabolite (2).

• Natural Product Sciences
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• v.14 no.4
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• pp.269-273
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• 2008

Microbial transformation of isoxanthohumol (1), a prenylated flavanone from hops, has resulted in the production of a pair of glucosylated derivatives. The structures of these compounds were elucidated to be (2S)-5-methoxy-8-prenylnaringenin 7-O-${\beta}$-D-glucopyranoside (2) and (2R)-5-methoxy-8-prenylnaringenin 7-O-${\beta}$-Dglucopyranoside (3) based on the spectroscopic analyses.

• Korean Journal of Microbiology
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• v.42 no.1
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• pp.59-61
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• 2006

Genetic transformation of a mushroom-forming fungus Coprinellus congregatus to antibiotic resistance gene had been successfully carried out by electroporation to oidia instead of protoplasts. Since there was no protoplast generation step which required not only cell wall degrading enzymes but many skillful procedures, commercial herbicide (basta) could be used without any difficulty with simple procedure. The transformation yield was 10-20 $transformants/{\mu}g$ DNA, and the transformants were very stable even after 10 consecutive transfers through the non-selective medium.

• Proceedings of the Zoological Society Korea Conference
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• 1997.10b
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• pp.273.1-273
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• 1997

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