• Journal of Photoscience
• /
• 제7권3호
• /
• pp.103-108
• /
• 2000

We have evaluated a new mutagenesis strategy called random insertional mutagenesis with subtracted cDNA fragments. The cDNAs from long day Arabidopsis plants were subtracted by cDNAs from short day plants using PCR based cDNA subtraction. The subtracted cDNAs were inserted between 35S promoter and 3'-NOS terminator regardless of orientation. When the cDNA library was used for the random insertion into Arabidopsis genome by Agrobacterium-mediated transformation, approximately 15% of transformants showed abnormal development in leaf, floral organ, shoot apex. When 20 mutants were analyzed, 12 mutants showed single cDNA fragment insertion and 8 mutants showed more than 2 transgene insertions. Only two mutants among 12 mutants that have single cDNA insert showed consistent phenotype at T2 generation, suggesting the genetic instability of the mutants.

• Journal of Microbiology and Biotechnology
• /
• 제19권9호
• /
• pp.869-872
• /
• 2009

The cyclic undecapeptide cyclosporin A (CyA), one of the most valuable immunosuppressive drugs, is produced nonribosomally by a multifunctional cyclosporin synthetase enzyme complex by the filamentous fungus Tolypocladium niveum. To increase CyA productivity by wild-type T. niveum (ATCC 34921), random mutagenesis was first performed using an antifungal agar-plug colony assay (APCA) selection approach. This generated a mutant strain producing more than 9-fold greater CyA than the wild-type strain. Additionally, a foreign bacterial gene, Vitreoscilla hemoglobin gene (VHb), was transformed via protoplast regeneration and its transcription was confirmed by RT-PCR in the UV-irradiated mutant cell. This led to an additional 33.5% increase of CyA production. Although most protoplast-regenerated T. niveum transformants tend to lose CyA productivity, the optimized combination of random mutagenesis and protoplast transformation described here should be an efficient strategy to generate a commercially valuable, yet metabolite low-producing, fungal species, such as CyA-producing T. niveum.

• Journal of Microbiology and Biotechnology
• /
• 제20권12호
• /
• pp.1696-1701
• /
• 2010

The ${\alpha}$-amylases activity was improved by random mutagenesis and screening. A region comprising residues from the position 34-281 was randomly mutated in B. licheniformis ${\alpha}$-amylase (AmyL), and the library with mutations ranging from low, medium, and high frequencies was generated. The library was screened using an effective liquid-phase screening method to isolate mutants with an altered pH profile. The sequencing of improved variants indicated 2-5 amino acid changes. Among them, mutant TP8H5 showed an altered pH profile as compared with that of wild type. The sequencing of variant TP8H5 indicated 2 amino acid changes, Ile157Ser and Trp193Arg, which were located in the solvent accessible flexible loop region in domain B.

• Journal of Microbiology
• /
• 제37권3호
• /
• pp.154-158
• /
• 1999

The arginine residue at position 243 (Arg 243) of the yeast transcription factor, Gcn4p, is invariably conserved among bZIP transcription factors. Using site-directed oligonucleotide saturation mutagenesis involving two-step polymerase chain reaction (PCR) amplification, random mutations were successfully introduced at the codon of 243 in the basic domain of Gcn4p. This mutant library was transformed ito Gcn4p defective yeast strain and selected for the transcriptionally active colonies. All colonies which were transcriptionally active had arginines in the codon 243. In this study, the strand preference by Taq polymerase during mutagenesis was also tested. Oligonucleotides were specially designed to test whether or not the polymerase was preferred using the strand as a template. A population of randomly mutated products were cloned into an appropriate vector and characterized by DNA sequencing analysis. Saturation mutagenesis which was performed efficiently by this method revealed a strong bias in terms of strand preference of Taq polymerase by an approximate ratio of 3 to 1 in this study.

• Journal of Microbiology
• /
• 제35권4호
• /
• pp.300-308
• /
• 1997

The catechol 2,3-dioxygenase (C23O) encoded by the Pseudomonas putida xylE gene was over-produced in Escherichia coli and purified to homogeneity. The activity of the C23O required the reduced form of the Fe(II) ion since the enzyme was highly susceptible to inactivation with hydrogen perocide but reactivated with the addition of ferrous sulfate in conjunction with ascorbic acid. The C23O activity was abolished by treatment with the chemical reagents, diethyl-pyrocarbonate (DEPC), tetranitromethane (TNM), and 1-cyclohexy1-3-(2-morpholinoethyl) car-bodiimidemetho-ρ-toluenesulfontate (CMC), which are modifying reagents of histidine, tyrosine and glutamic acid, respectively. These results suggest that histidine, tyrosine and glutamic acid residues may be good active sites for the enzyme activity. These amino acid residues are conserved residues may be good active sites for the enzyme activity. These amino acid residues are conserved residues among several extradion dioxygenases and have the chemical potential to serveas ligands for Fe(II) coordination. Analysis of random point mutants in the C23O gene derived by PCR technique revealed that the mutated positions of two mutants, T179S and S211R, were located near the conserved His165 amd Hos217 residues, respectively. This finding indicates that these two positions, along with the conserved histidine residues, are specially effective regions for the enzyme function.

• Journal of Microbiology
• /
• 제35권3호
• /
• pp.228-233
• /
• 1997

The MTF1 gene of Saccharomyces cerevisiae encodes a 43 kDa MITOCHONDRIAL RNA polymerase specificity factor which recognizes mitochondrial promoters to initiate correct transcription. To better understand structure-function of the MTF1 gene as well as the transcription mechanism of mitochondrial RNA polymerase, two cold-sensitive alleles of the MTF1 mutation were isolated by plasmid shuffling method after PCR-based random mutagenesis of the MTF1 gene. The mutation sites were analyzed by nucleotide sequencing. These cs phenotype mtf1 mutants were respiration competent on the nonfermentible glycerol medium at the permissive temperature, but incompetent at 13.deg.C. The cs phenotype allele of the MTF1, yJH147, encoded an L146P replacement. The other cs allele, yJH148, contained K179E and K214M double replacements. Mutations in both alleles were in a region of Mtflp which is located between domains with amino acid sequence similarities to conserved regions 2 and 3 of bacterial s factors.

• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2003년도 생물공학의 동향(XII)
• /
• pp.445-449
• /
• 2003

As for the purpose, we first introduce an random mutation into wild-type gene to expand a mutation space, and then further recombine the mutant genes by staggered extension process PCR. As a result, we obtained the best clones 6-52 that showed a high activity and stability, from a round of error prone and staggered extension process PCR. The purified enzyme showed a similar pH stability to the wild-type enzyme and reveal a slightly high optimum pH at 12. In the optimum temperature, an identical dependency was also showed and a quite high stability in the thermal stability was obtained. Along with this, the enzyme was also stable at a reaction that supplement with a 15 % of ethanol as an additive. The addition of other solvents and surfactants did not improve the reaction and thus resulted in a similar profile to those of wild-type enzyme. The specific activity on the target compound rac-ketoprofen ethyl ester was calculated to be about 85, 000 unit, and the kinetic constants Km and Vmax were determined to be 0.2 mM and 90 mM/mg-protein/min respectively. The deduced amino acid alignment with the wild type enzyme revealed five mutations at L120P, I208V, T249A, D287H and T357A. Based on these observations, the site directed mutagenesis to delineate the mutagenic effect is under progress.

• KSBB Journal
• /
• 제21권2호
• /
• pp.90-95
• /
• 2006

라이코펜 생산성을 향상시키기 위하여 몇 가지 대장균 균주들을 대상으로 생산성 시험을 한 결과 MG1655 균주가 가장 우수하였다. 대장균 MG1655 균주의 염색체 DNA 내에 존재하는 특정 유전자의 기능이 상실되었을 때 라이코펜 생산성이 증가되는 변이체를 선발하기 위해 transposon 돌연변이를 수행하였으며, 5종의 우수 transposon 변이체를 얻었다. 특히, Tn4 변이체는 야생형 MG1655에 비해서 라이코펜 생산량은 6배, 균체 내 함량은 7배로 가장 높았다. Transposon 삽입에 의해 결손된 유전자를 파악하기 위해 inverse PCR을 통해 염기서열을 확인하였으며, 결손된 유전자는 rfaH, treB, B2436으로 규명되었다. 또한 특정 유전자 기능의 상실뿐만 아니라 기능의 강화나 변화 등에 의한 라이코펜 생산성이 증가된 변이체를 선발하기 위해 NTG 돌연변이를 수행하였으며, 4개의 우수한 NTG 변이체를 얻었다. 이들 중에 NTG4 변이체가 가장 높은 라이코펜 생산량을 보였다. 특히, NTG4 변이체는 발현유도물질인 arabinose의 소량(0.013mM) 첨가 시에 라이코펜 농도, 6 mg/L와 균체 내 함량 4 mg/g DCW로 최대의 라이코펜 생산성을 보였고, 이것은 야생형 MG1655에 비해서 각각 18배와 12배 높은 생산성이다.

• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2003년도 생물공학의 동향(XIII)
• /
• pp.434-437
• /
• 2003

A new bacterial strain, was isolated from activated sludge, identified and named P. aeruginosa EMS1. The new strain produced surface-active rhamnolipids by batch cultivation in mineral salts medium with waste flying oils. The mutant strain KH7, designated P. aeruginosa EMS1, derived by random mutagenesis with N-methyl-N-nitro-N-nitrosogoanidine treatment producing high levels of the biosurfactants was selected by an ion-pair plate assay. The mutant strain KH7 showed 4-5 times more hydrocarbon emulsification as compared to the parent when grown on waste frying oils and various hydrocarbons. Furthermore, P. aeruginosa EMS1 and mutant strain KH7 was also able to use whey as a co-substrate for growth and biosurfactant production. As results of this study, mutant strain KH7 is a very efficient biosurfactant producer, and its culture conditions are relatively inexpensive and economical. Rhamnolipid is synthesized by the rhlAB-encoded rhamnosyltransferase. To be convinced of these genes, we performed PCR based on P. aeruginosa PAO1 whole-genome database. rhl gene cluster nucleotide and amino acid sequences were compared for both parent and mutant. Comparison of nucleotide sequence of rhlAB, there were usually terminal's codons exchange.

• BMB Reports
• /
• 제36권4호
• /
• pp.344-348
• /
• 2003

Protein kinase CKII is composed of two catalytic ($\alpha$ or $\alpha$') subunits and two regulatory ($\beta$) subunits. The $CKII{\beta}$ subunit is thought to mediate the tetramer formation and interact with other target proteins. However, its physiological function remains obscure. In this study, point mutants of $CKII{\beta}$ that are defective for the L41 binding were isolated by using the reverse two-hybrid system. A sequence analysis of the point mutants revealed that Asp-26, Met-52, and Met-78 of $CKII{\beta}$ are critical for L41 binding; Asn-67 (and/or Lys-139) and Met-52 are important for $CKII{\beta}$ homodimerization. Two point mutants, R75 and R83, of $CKII{\beta}$ interacted with L5, topoisomerase $II{\beta}$, and CKBBP1/SAG, but not with the wild-type $CKII{\beta}$. This indicates that $CKII{\beta}$ homodimerization is not a prerequisite for its binding to target proteins. These $CKII{\beta}$ point mutants may be useful in exploring the biochemical physiological functions of $CKII{\beta}$.