• Plant Biotechnology Reports
• /
• 제5권3호
• /
• pp.245-254
• /
• 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.

• Journal of Microbiology and Biotechnology
• /
• 제29권1호
• /
• pp.44-54
• /
• 2019

Geldanamycin and its derivatives, inhibitors of heat shock protein 90, are considered potent anticancer drugs, although their biosynthetic pathways have not yet been fully elucidated. The key step of conversion of 4,5-dihydrogeldanamycin to geldanamycin was expected to catalyze by a P450 monooxygenase, Gel16. The adequate bioconversions by cytochrome P450 mostly rely upon its interaction with redox partners. Several ferredoxin and ferredoxin reductases are available in the genome of certain organisms, but only a few suitable partners can operate in full efficiency. In this study, we have expressed cytochrome P450 gel16 in Escherichia coli and performed an in vitro assay using 4,5-dihydrogeldanamycin as a substrate. We demonstrated that the in silico method can be applicable for the efficient mining of convenient endogenous redox partners (9 ferredoxins and 6 ferredoxin reductases) against CYP Gel16 from Streptomyces hygroscopicus. The distances for ligand FDX4-FDR6 were found to be $9.384{\AA}$. Similarly, the binding energy between Gel16-FDX4 and FDX4-FDR6 were -611.88 kcal/mol and -834.48 kcal/mol, respectively, suggesting the lowest distance and binding energy rather than other redox partners. These findings suggest that the best redox partners of Gel16 could be NADPH ${\rightarrow}$ FDR6 ${\rightarrow}$ FDX4 ${\rightarrow}$ Gel16.

• Journal of Microbiology and Biotechnology
• /
• 제2권4호
• /
• pp.248-254
• /
• 1992

The initial growth of Clostridium acetobutylicum was not inhibited by 1 atm of H$_2$ while H$_2$ reduced glucose consumption in a solventogenic culture of a phosphate limited 2-stage chemostat. Under 1 atm of H$_2$, a solventogenic culture consumed hydrogen, but an acidogenic culture produced hydrogen. H$_2$ consumption by the solventogenic culture was enhanced by the addition of 5 mM neutral red, an artificial electron carrier with a redox potential of -325 mV. Hydrogenase activity, measured in both directions of production and consumption, showed that activity coupled with methyl viologen is higher in an acidogenic culture than in a solventogenic culture, and that the two cultures have similar activities for methylene blue reduction. The solventogenic culture showed a higher activity coupled with neutral red than the acidogenic culture. From these results, it is hypothesized that hydrogen producing hydrogenase activity is high during the acidogenic phase, and decreases as solventogenesis starts, and that the solventogenic culture produces a second hydrogenase which uses an electron carrier other than ferredoxin. This hypothesis was supported by the fact that enzyme activities involved in electron flow can be coupled to neutral red, indepedent of ferredoxin, and that neutral red addition to the fermentation system increased butanol yield, with a decrease in production of less reduced fermentation products, and $H^2$.

• Archives of Pharmacal Research
• /
• 제10권3호
• /
• pp.193-196
• /
• 1987

Protein methylation occurs ubiquitously in nature and involves N-methylation of lysine, arginine, histidine, alanine, proline and glutamine, O-methylesterfication o dicarboxylic acids, and S-methylation of cysteine and methionine. In nature, methylated amino acids accur in highly specialized proteins such as histones, flagella proteins, myosin, actin, ribosomal proteins. hn RNA-bound protein, HMG-1 and HMG-2 protein, opsin, EF-Tu, EF-$1\alpha$, porcine heart citrate synthase, calmodulin, ferredoxin, $1\alpha$-amylase, heat shock protein, scleroderma antigen, nucleolar protein C23 and IF-3l.

• Journal of Microbiology
• /
• 제40권3호
• /
• pp.211-218
• /
• 2002

A 4.8-kb DNA fragment encoding the P-450 type hydroxylase and ferredoxin genes was cloned from Pseudonocardia autotrophica IFO 12743 that can convert vitamin D$\_$3/ into its hydroxylated active forms. In order to isolate the P-450 gene cluster in this organism, we designed PCR primers on the basis of the regions of an oxygen binding site and a heme ligand pocket that are general characteristics of the P-450 hydroxylase. Sequencing analysis of the BamHI fragment revealed the presence of four complete and one incomplete ORFs, named PauA, PauB, PauC, and PauD, respectively. As a result of computer-based analyses, PauA and PauB have homology with enoyl-CoA hydratase from several organisms and the positive regulators belonging to the tetR family, respectively. PauC and PauD show similarity with SuaB/C proteins and ferredoxins, respectively, which are composed of P-450 monooxygenase systems for metabolizing two sulfonylurea herbicides in Streptomyces griseolus PauC shows the highest similarity with another CytP-450$\_$Sca2/ protein that is responsible for production of a specific HMG-CoA reductase inhibitor, pravastatin, in S. carbophilus. Cultures of Steptomyces lividans transformant, containing the P-450 gene cluster on the pWHM3 plasmid, was unable to convert vitamin D$\_$3/ to its hydroxylated forms.

• 미생물학회지
• /
• 제39권3호
• /
• pp.123-127
• /
• 2003

Sphingomonas chungbuken교 DJ77은 phenanthrene을 단일 탄소원과 에너지원으로 이용하여 살아갈 수 있다. pUPXS는 phenanthrene과 naphthalene분해를 위해 필요한 2-hydroxychromene-2-carboxylate (HCCA) isomerase를 암호화하는phnS유전자를 포함한다. 본 논문에서는 phnS유전자가 포함된 3271 bp의 염기 서열을 결정하였다. 이 유전자는 ATG를 개시코돈으로 사용하며, TAA를 종결 코돈으로 사용하고 있다. 또한 개시 코돈의 5 bp앞쪽으로 GGAA라는 ribosomal binding site를 갖는다. phnS는 총 594 bP의 open reading frame을 포함하며,158개의 아미노산으로 구성되었다. phns를 구성하는 아미노산서열은 S. aromaticivorans F199의 유사서열과 87％의 유사성을 나타냈다. phns 유전자는 biphenyl dioxygenase를 구성하는 2,3-dihydroxybiphenyl 1,2-dioxygenase를 암호화하는 phnQ와 ferredoxin를 암호화 하는 phnR과 같은 operon을 이루며, 이들 유전자의 downstream에 위치하고 있다.

• Journal of Microbiology and Biotechnology
• /
• 제11권5호
• /
• pp.763-771
• /
• 2001

Biphenyl dioxygenase (BPDO), which catalyzes the first step in the bacterial degradation of biphenyl and polychlorinated biphenyls, was characterized in Pseudomonas sp. B4. The bphA locus containing the four structural genes encoding BPDO were cloned and sequenced. A regulatory gene as well as a putative regulatory sequence were identified upstream of this locus. A transposase-like gene was found within a 1-kb region further upstream, thereby suggesting that the bphA locus may be carried on a transposable element. The three components of the BPDO enzyme have been separately overexpressed and purified from E. coli. The ferredoxin and terminal dioxygenase components showed biochemical properties comparable to those of two previously characterized BPDOs, whereas the ferredoxin reductase exhibited an unusually high lability. The substrate selectivity of BPDO was examined in vivo using resting cell assays performed with mixtures of selected polychlorinated biphenyls. The results indicated that para-substituted congeners were the preferred substrates. In vitro studies were carried out on a BPDO complex where the reductase from strain B4 we replaced by the more stable isoform from Comamonas testosteroni B-356. The BPDO enzyme had a specific activity of $0.26{\pm}0.02 {\mu}mol {min^-1}{mg^-1}\;of\;ISP_{BPH}$ with biphenyl as the substrate. The 2,3-, 4,4'-, and 2,4,4'-chlorobiphenyls were converted to single dihydrodiols, while 2,4'-dichlorobiphenyl gave rise to two dihydrodiols. The current data also indicated that 2,4,4'-trichlorobiphenyl was a better substrate than the 4,4'-dichlorinated congener.

• Journal of Microbiology and Biotechnology
• /
• 제33권3호
• /
• pp.378-386
• /
• 2023

The CYP707A family genes encoding ABA 8'-hydroxylase catabolize abscisic acid (ABA), a plant stress hormone that plays an important role in stress condition, such as drought, heat, cold and salinity. Phaseic acid (PA) is a catabolic product of ABA. Recent studies have shown that PA is important for the physiological functions in plants. It is also a neuroprotective molecule that protects against ischemic brain injury in mice. To obtain enzymes for the PA production, four CaCYP707A genes (CaCYP707A1, CaCYP707A2, CaCYP707A3 and CaCYP707A4) were isolated from hot pepper. They were heterologously expressed in Escherichia coli. Among them, CaCYP707A2 showed significantly higher expression levels in both the membrane fraction and the soluble fraction. Preferred redox partners were investigated to improve the efficiency of CaCYP707A2's catalytic reaction, and NADPH-cytochrome P450 reductase (CPR) from hot pepper (CaCPR) was preferred over other redox partners (i.e., rat CPR and ferredoxin reductase/ferredoxin). The production of 8'-hydroxy ABA and PA by ABA hydroxylation activity was confirmed in CaCYP707A2 from both membrane and soluble fractions. Therefore, CaCYP707A2 is the first identified plant CYP protein that is expressed a soluble form in cytosolic fraction having stable activity. Taken together, we propose a new CYP707A protein with industrial applications for PA production without additional modifications in E. coli heterologous expression.

• Journal of Microbiology and Biotechnology
• /
• 제6권1호
• /
• pp.43-49
• /
• 1996

Modulation of the catabolic PEP-pathway of Anaerobiospirillum succiniciproducens was tried using some enzymatic inhibitors such as gases and chemicals in order to enhance succinic acid production. 10$\%$ CO increased the succinic acid/acetic acid (S/A) ratio but inhibited growth as well as production of succinic and acetic acid. Hydrogen gas also increased the S/A ratio and inhibited the synthesis of pyruvate: ferredoxin oxidoreductase when used in mixture with $CO_2$, Catabolic repression by acetic, lactic and formic acid was not recognized and other modulators such as glyoxylate, pyruvate derivatives, arsenic salt, phosphate and sulfate were shown not to be effective. Magesium carbonate was shown effective for repressing acetate production. Palmitic acid, myristic acid and phenylalanine did not affect acetate production but carprylic acid completely inhibited growth.

• Journal of Microbiology and Biotechnology
• /
• 제16권3호
• /
• pp.386-390
• /
• 2006

A green fluorescent protein-based Pseudomonas putida reporter was successfully constructed and shown to be capable of detecting oxidative stress. In this whole-cell reporter, the promoter of the paraquat-inducible ferredoxin-$NADP^+$ reductase (fpr) was fused to a promoterless gfp gene on a broad-host-range promoter probe vector. Pseudomonas putida KT2440 harboring this reporter plasmid exhibited an increased level of gfp expression in the presence of redox-cycling agents (paraquat and menadione), hydrogen peroxide, and potential environmental pollutant chemicals such as toluene, paint thinner, gasoline, and diesel. Induction of fpr in the presence of these chemicals was confirmed using Northern blot analysis.