• Biotechnology and Bioprocess Engineering:BBE
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• v.7 no.3
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• pp.121-129
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• 2002

Existing methods for optimization of sequences by random mutagenesis generate libraries with a small number of mostly deleterious mutations, resulting in libraries containing a large fraction of non-functional clones that explore only a small part of sequence space. Large numbers of clones need to be screened to find the rare mutants with improvements. Library display formats are useful to screen very large libraries but impose screening limitations that limit the value of this approach for most commercial applications. By contrast, in both classical breeding and in DNA shuffling, natural diversity is permutated by homologous recombination, generating libraries of very high quality, from which improved clones can be identified with a small number of complex screens. Given that this small number of screens can be performed under the conditions of actual use of the product, commercially relevant improvements can be reliably obtained.

• Microbiology and Biotechnology Letters
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• v.30 no.4
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• pp.298-304
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• 2002

A family shuffling associating PCR-based and in vitro recombination and expression in Escherichia coli cdd mutant was carried out. Two cdd genes encoding cytidine deaminases (CDase) from thermophilic Bacillus caldolyticus and B. stearothermophilus were shuffled. Around 150 viable mutant colonies screened on AB minimal medium without uracil by E. coli cdd complementation were selected for cytidine deaminase assay and 4 candidates (SH1067, SH1077, SH1086, and SH1118) were chosen for the detailed study. The nucleotide sequence analyses of 4 selected mutants revealed that they have several point mutations and recombinations. Surprisingly, the SH 1067 showed 770 fold more specific CDase activity at $80^{\circ}C$ than that of T101 from parental B. stearothermophilus.

• Journal of Microbiology and Biotechnology
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• v.32 no.6
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• pp.800-807
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• 2022

Four aprE genes encoding alkaline serine proteases from B. subtilis strains were used as template genes for family gene shuffling. Shuffled genes obtained by DNase I digestion followed by consecutive primerless and regular PCR reactions were ligated with pHY300PLK, an E. coli-Bacillus shuttle vector. The ligation mixture was introduced into B. subtilis WB600 and one transformant (FSM4) showed higher fibrinolytic activity. DNA sequencing confirmed that the shuffled gene (aprEFSM4) consisted of DNA mostly originated from either aprEJS2 or aprE176 in addition to some DNA from either aprE3-5 or aprESJ4. Mature AprEFSM4 (275 amino acids) was different from mature AprEJS2 in 4 amino acids and mature AprE176 in 2 amino acids. aprEFSM4 was overexpressed in E. coli BL21 (DE3) by using pET26b(+) and recombinant AprEFSM4 was purified. The optimal temperature and pH of AprEFSM4 were similar to those of parental enzymes. However, AprEFM4 showed better thermostability and fibrinogen hydrolytic activity than the parental enzymes. The results indicated that DNA shuffling could be used to improve fibrinolytic enzymes from Bacillus sp. for industrial applications.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2001.06a
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• pp.47-57
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• 2001

• Korean Journal of Microbiology
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• v.39 no.2
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• pp.76-82
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• 2003

To prepare evolved PHB depolymerase with increased activity for PHB or P(3HB-co-3HV) compared to the activity of the original PHB depolymerase from Alcaligenes faecalis T1, random mutation of the cloned PHB depolymerase gene was performed by using a DNA shuffling method. A library of mutated PHB depolymerase genes from A. faecalis T1 was fused to the ice nucleation protein (INP) gene from Pseudomonas syringae in pJHCl 1 and approximately 7,000 transformants were isolated. Using M9 minimal medium containing PHB or P(3HB-co-3HV) as the carbon source, mutants showing alteration in PHB depolymerase activity were selected from the transformants. The PHB depolymease activity of the transformants was confirmed by the formation of halo around colony and the turbidity decrease tests using culture supermatants. The catalytic activity of PHB depolymerase of the best mutant II-4 for PHB or P(3HB-co-13 mol% 3HV) was approximately 1.8-fold and 3.2-fold, respectively, higher than that of the original PHB depolymerase. DNA sequence analysis revealed that three amino acid residues (Ala209Val, Leu258Phe, and Asp263Thr) were substituted in II-4. From the mutational analysis, it was presumed that the substitution of amino acids near catalytic triad to more hydrophobic amino acids enhance the catalytic activity of PHB depolymerase from A. faecalis T1.

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.135-135
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• 2005

• BMB Reports
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• v.43 no.8
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• pp.567-572
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• 2010

In this study, we cloned the ERF-B3 subfamily transcription factor gene BnaERF-B3-hy15 from Brassica napus L. Huyou15. This 600 bp gene encodes a 199 amino acid classic ethylene responsive factor (ERF), which shown no binding or very weak binding GCC box-binding activity by the yeast one-hybrid assay. We used gene shuffling and the yeast one-hybrid system to obtain three mutated sequences that can bind to the GCC box. Sequence analysis indicated that two residues, Gly156 in the AP2 domain and Phe62 at the N-terminal domain were mutated to arginine and serine, respectively. Changes of Gly156 to arginine and Phe62 to serine increased the GCC-binding activity of BnaERF-B3-hy15 and the alter of Gly156 to arginine changed the AP2-domain structure of BnaERF-B3-hy15.

• Journal of Microbiology
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• v.35 no.3
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• pp.228-233
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• 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.

• Journal of Microbiology and Biotechnology
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• v.10 no.6
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• pp.764-769
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• 2000

The lipase gene(lip) from Bacillus stearothermophilus was recombined in vitro by utilizing the DNA shuffling technique. After four rounds of shuffling, transformation, and screening based on the initial rate of clear zone formation on a tricaprylin plate, a clone (M10) was isolated, the cell extract of which showed about 2.8-fold increased lipase activity. The DNA sequence of the mutant lipase gene (m10) showed 3 base changes, resulting in two cryptic mutations and one amino acid substitution: S113($AGC{\rightarrow}AGT$), L252 ($TTG{\rightarrow}TTA$), and G275E ($GGA{\rightarrow}GAA$). SDS-PAGE analysis revealed that the increased enzyme activity observed in M10 was partly caused by high expression of the m10 lipase gene. The amount of the expressed G275E lipase was estimated to comprise as much as 41% of the total soluble proteins of the cell. The maximum velocity ($V_{max}$) of the purified mutant enzyme for the hydrolysis of olive oil was measured to be 3,200 U/mg, which was 10% higher than that of the parental (WT) lipase (2,900 U/mg). Its optimum temperature for the hydrolysis of olive oil was $68^{\circ}C$ and it showed a typical $Ca^{2+}$-dependent thermostability, properties fo which were the same as those of the WT lipase. However, the mutant enzyme exhibited a high enantiospecificity towards (S)-naproxen compared with the WT lipase. In addition, it showed increased hydrolytic activity towards triolein, tricaprin, tricaprylin, and tricaproin.

• Journal of Life Science
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• v.29 no.3
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• pp.376-381
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• 2019

To construct useful yeast strain for bioethanol production, we improved yeast harboring various phenotypes by using yeast protoplast fusion method. In this study, S. cerevisiae BYK-F11 strain which have ethanol tolerance, thermotolerance and ${\beta}-glucanase$ activity and P. $stipitis{\Delta}ura$ strain which has xylose metabolism pathway were fused by genome shuffling. P. $stipitis{\Delta}ura$ strain was constructed for protoplast fusion by URA3 gene disruption, resulting in uracil auxotroph. By protoplast fusion, several fused cells were selected and BYKPS-F8 strain (fused cell) showing both karyotypes from two parent strains (S. cerevisiae BYK-F11 and P. $stipitis{\Delta}ura$ strain) among 22 fused cells was finally selected. Sequentially, various phenotypes such as ${\beta}-glucanase$ activity, xylose utility, ethanol tolerance, thermotolerance and ethanol productivity were analyzed. The BYKPS-F8 strain obtained ${\beta}-glucanase$ activity from BYK-F11 strain and 1.2 fold increased xylose utility from P. $stipitis{\Delta}ura$ strain. Also, the BYKPS-F8 strain showed thermotolerance at $40^{\circ}C$ and increased ethanol tolerance in medium containing 8% ethanol. In this fused cell, 7.5 g/l ethanol from 20 g/l xylose was produced and the multiple phenotypes were stably remained during long term cultivation (260 hr). It was proved that novel biological system (yeast strains) is easily and efficiently bred by protoplast fusion among yeasts having different genus.