Protein can be provided by cultivating various microbes, which contain more than 30% protein content by cell dry weight. This study compared intracellular protein concentrations of various wild-type yeasts from different sources to select the best yeast strain with high protein concentration. Among them, Saccharomyces cerevisiae KCCM 34709, used for molasses fermentation, exhibited 4.1-fold higher protein concentration than a laboratory yeast strain, S. cerevisiae D452-2. In this study, an approach consisting of random mutagenesis coupled with the Bradford protein assay-based screening method was applied to enhance the S. cerevisiae KCCM 34709 protein content. Among 1,000 mutants, the #180 mutant strain produced 5,041±519 mg/L total amino acid in 48 h, which was 31% higher than the parental S. cerevisiae KCCM 34709 strain. These results demonstrate that the #180 mutant strain can be an attractive cell factory for animal-free protein production.

The cry gene from Bacillus thuringiensis (Bt), encoding the Cry protein, has been recently introduced into crops to generate transgenic plants that are resistant to pest insects. In this study, through the 3D structure prediction and accompanying mutagenesis study for the Mod-Cry1Ac, 7 and 16 amino acid residues from domain I and II, respectively, responsible for its insecticidal activity against larvae of Spodoptera exigua and Ostrinia furnacalis were identified. We used site-directed mutagenesis to improve the insecticidal activity of Mod-Cry1Ac, resulted 31 mutant cry genes. These mutant cry genes were expressed, as a polyhedrin fusion form, using a baculovirus expression system. The expressed proteins were 95 kDa and SDS-PAGE analysis of the recombinant polyhedra revealed that expressed Cry proteins was occluded into polyhedra and activated stably to 65 kDa by trypsin. When the insecticidal activities of these mutant Cry proteins against to larvae of P. xylostella, S. exigua and O. furnacalis were assayed, they showed higher or similar insecticidal activity compared to those of Cry1Ac and Cry1C. Especially, Mutant-N16 is considered to have the potential for the efficacious biological insecticide since it showed the highest insecticidal activity.

Bt crystal proteins, encoded by cry genes, are a group of insecticidal proteins unique in the Gram-positive and spore-forming bacterium, Bacillus thuringiensis. These cry genes are widely applied as one of the most successful candidates for constructing transgenic crops resistant to pest insects. In our previous report, we found Cry1-5 had high insecticidal activity against Spodoptera larvae although its amino acid sequences showed high similarity (95.6%) to those of Cry1Ac which had low activity. In comparison with Cry1Ac, Cry1-5 had 12 different residues on domain I and II. In order to convert these residues to Cry1-5 randomly, 10 mutagenic primers were designed. Through multi site-directed mutagenesis, we mutated the modified cry1Ac gene by plant codon usage in pOBI-Modcry1Ac based on cry1-5 and constructed 63 mutant cry genes. Among them, 10 mutant cry genes on domain II were selected and their recombinant proteins were expressed by baculovirus expression system. From bioassay results to P. xylostella and S. exigua, we found some mutants have high insecticidal activities to be applicable to transgenic crops.

Bt crystal proteins, encoded by cry genes, are a group of insecticidal proteins unique in the Gram-positive and spore-forming bacterium, Bacillus thuringiensis. These cry genes are widely applied as one of the most successful candidates for constructing transgenic plants resistant to pest insects. In our previous report, we found Cry1-5 had high insecticidal activity against Spodoptera larvae although its amino acid sequences showed high similarity (97.9%) to those of Cry1Ab which had low activity. In comparison with Cry1Ac, Cry1-5 had 12 different residues in domain Ⅰ and domain Ⅱ, and we focused on domain Ⅰand domain Ⅱ regions and designed 10 mutagenic primers to change 12 residues. Through multi site-directed mutagenesis, we mutated the modified cry1Ac gene by plant codon usage in pOBⅠ-Mod-cry1Ac based on cry1-5 and constructed 63 various mutant cry genes. In the further study, we will express those mutant proteins as a fusion form with polyhedrin using baculovirus expression system and subsequently do bioassay to Spodoptera larvae.

Human eryhropoietin (EPO) is acidic glycoprotein hormone that plays key role in hematopoiesis by facilitating differentiation of erythrocyte and formation of hemoglobin (Hb) and is used for the treatment of anemia. Human EPO is consist of 166 amino acids which is modified by three N-glycosylations (24, 38, 83) and single O-glycosylation (126). N-glycosylation is reported to be related to the cellular secretion and activity of EPO. In this study, we examined effects of mutagenesis in glycosylation site of recombinat hEPO for the cellular secretion during production from cultured CHO cell. We produced rhEpo which was cloned by PCR from human liver cDNA (TaKaRa) in cultured CHO cell. Using supernatant of the culture, ELISA assay and western analysis were performed. To estimate biological activity, 20IU of rhuEpo was subcutaneously injected into four ICR mice. After 8 days, HCT level was increased average 13 per cent, RBC was increased ca. 2106//. In disease model Rat (anemia c-kit, WSRC-WS/WS), HCT was increased ca. 12%, RBC was increased ca. 1.6106//. These results suggests that rhEpo we produced has biological activity. To remove glycosylation site by substituting 24, 38, 83, and 126th asparagine (or serine) with glutamic acid, overlapping -extension site-directed mutagenesis was performed. To add novel glycosylation sites, 69, 105th leucine was mutated to asparagine. Mutant EPO construct was transfected into CHO cell. Supernatant of the cell culture was analyzed using ELISA assay with monoclonal anti-EPO antibody (Medac, Germany). Since, several reports for mutagenesis of glycosylation sites showed case-by-case results, we examined both transient expression and stable expression. Addition of novel glycosylation sites resulted no secretion while deletion mutants had little effect except some double deletion mutants (24/83 and 38/83) and triple mutant. We suggest that not single but combination of glycosyl group affect secretion of EPO.

Promoters for milk proteins have been used far producing transgenic animals due to their temporal and spatial expression patterns. -casein, a calcium-sensitive casein, is a major milk protein that corresponds ca. 30 per cent of total milk protein. Expression of -casein is controlled by lactogenic hormones such as prolactin (PRL), composite response elements (CoREs) and transcription factors. CoREs are clusters of transcription factor binding sites containing both positive and negative regulatory elements. -casein gene promoter contains various regions (CoREs) for gene transcription. We analyzed the promoter region by mutagenesis using exonuclease III and linker-scanning. Transcription control elements usually are positioned in 5'-flanking region of the gene. However, in some cases, these elements are located in other regions such as intron 1. The nucleotide sequences of -casein promote. region has been reported (E12614). However, the properties of the promoter is not yet clear. In this study, we plan to investigate the properties of cis-regulating elements of porcine -casein by mutation analysis and expression analysis using dual-luciferase repoter assay system.

Zinc finger nucleases (ZFNs) have been used for targeted mutagenesis in eukaryotic cells. Custom-designed ZFNs can induce double-strand breaks (DSBs) at a specific locus. Our custom ZFN dimer was designed 3-finger of left and 4-finger of right with 2 kb size using 2A. A Ti-plasmid vector, pTA7002 containing the target site of SSS4A gene for a ZFN pair, that was shown to be active in yeast, was integrated in the rice genome. This promising technique for genome engineering was induced into 4 exon region of SSS4A gene in rice genome using Agrobacterium-mediated transformation. The SSS4A full-length cDNA was 5,070 bp consisting of a 318 bp 5′-untranslated region (UTR), a complete ORF of 2,928 bp encoding a polypeptide of 975 amino acids and a 3′-UTR of 1,824 bp. The vector is based on glucocorticoid receptor inducible gene expression system. Thus, SSS4A::ZFN expression was tightly controlled and the phenotype in low concentrations 10uM of the glucocorticoid hormone dexamethasone (DEX). In plant cells, transient ZFN expression is achieved by direct gene transfer into the target cells. For an alternative, ZFN delivery and production of mutant plants using a tobacco transient expression system for indirect transient delivery of ZFNs into a variety of tissues and cells of plants. ZFN activity was determined by PCR and sequence analysis of the target site. ZFN induced plants were obtained in up to 2% of the PCR products, consisting of deletions ranging between 1and 100 bp and insertions ranging between 1 and 10 bp. Our results describe an alternative to direct gene transfer for ZFN delivery and for the production of mutated rice.

Rapeseed (Brassica napus L.) oil with high oleic acid content is of great interest for both food and non-food uses. The ‘Tamla’ variety, characterized by oleic acid content of approximately 69%, was treated with 1% ethyl methane sulfonate (EMS) to induce mutations in the fatty acid biosynthesis pathway. M1 plants were selfed and subsequent generations (M2, M3, and M4 mutants) were analyzed to identify mutants having increased levels of oleic acid. M2 mutants showed oleic acid content ranging from 13.5% to 76.9% with some mutants (TR-458 and TR-544) having up to 74.7% and 76.9% oleic acid, which was an increase of nearly 5% and 7%, respectively, compared to untreated cv ‘Tamla’. We selected two M3 mutants with >75% oleic acid content. One mutant (TR-458-2) had increased oleic acid (75.9%) and decreased linoleic acid (12.5%) and linolenic acid (4.4%) contents. The other (TR-544-1) showed increased oleic acid content (75.7%) and decreased linoleic acid (13.5%) and linolenic acid (3.3%) contents. The accumulation or reduction of oleic acid content in the selected M4 mutants was also accompanied by a simultaneous decrease or increase in linoleic and linolenic acid contents. The high-oleic lines could be utilized further in breeding programs for improvement of rapeseed oil quality.

Two-component regulatory system (TCS) is the dominant mechanism that controls almost physiological processes of bacteria, such as nutrition assimilation, cell motility, chemotaxis, biofilm formation, quorum sensing and virulence. The intracellular informing process by a typical TCS accompany transfer of a phosphoryl group from His of a histidine kinase (HK) to Asp of a response regulator (RR). In Xanthomonas oryzae pv. oryzae (Xoo) genome, the TCS genes comprise approximately 3% of the nucleotide sequences with 58 response regulators (RRs), 32 orthodox histidine kinase (HKs) and 13 hybrid histidine kinase (HyHKs). However, there is not much understanding of RRs in Xoo except the reported RRs in Xanthomonas spp. including RpfC-RpfG, RavS-RavR, HrpG, VgrS-VgrR (also named ColS-ColR), VemR, RaxH-RaxR, and PhoQ-PhoP. Although a genome-scale mutagenesis and phenotypic characterization of TCSs were studied in Xanthomonas campestris pv. campestris ATCC 33913, there is not any genome-scale research of TCSs in Xanthomonas oryzae pv. oryzae. We have mutagenized 52 predicted RR genes in Xoo PXO99A by marker-exchange mutagenesis method and characterized the phenotype of mutants to identify RR genes involving in pathogenicity of Xoo and understand how Xoo TCSs work in given conditions. Ours investigation with the RR knock-out mutant strains have identified four novel RR genesthat are likely involved in virulence of Xoo. We have studied with these genes in molecular level to elucidate the mechanism for Xoo pathogenicity.

VitE (tocotrienols and tocopherols) are micronutrients with antioxidant properties synthesized by photosynthetic bacteria and plants that play important roles in animal and human nutrition. A new mutant line, T1001-1, was isolated from in vitro mutagenized population by ionizing radiation and shown to have increased VitE contents. The total VitE content was 26% increased in the T1001-1 mutant seeds compare with cv. Dongan (wild-type). In addition, we showed that the mutant confers retarded seedling growth during the early seedling growth stage in rice. To study the molecular mechanism of VitE biosynthesis, we used the rice microarray to identify genes that are upor down-regulated in T1001-1 mutant. In addition, we identified differentially regulated pathway using MapMan analysis, which provides deep insight into changes in transcript and metabolites. Our results enhanced the transcription of genes involved in starch and lipid metabolism in T1001-1 mutant. To identify the molecular mechanisms of the events involving transcription factors in tocopherol accumulation, we compared the expression patterns of transcription factors. The AP2-EREBP, WRKY, C2H2 transcription factor were up-regulated, whereas the MYB family was down-regulated in T1001-1 mutant. Our results demonstrate change of important transcript in high level of VitE accumulating rice mutant.

The world is facing a serious food and energy crisis. Plant mutation breeding has played an important role in overcoming this crisis and maintaining world stability. New techniques are required to achieve faster and more effective breeding. At RIKEN, we have developed a unique technology for mutation induction by using heavy-ion beams from particle accelerators at the RI Beam Factory. This development was achieved through an efficient synergistic link between agricultural science and accelerator physics. The use of ion beams for mutagenesis has a number of advantages: the approach has low exposure levels and high survival rates with high mutation rates, and it creates a wide variety of different mutations. Because heavy-ion beams provide a very high amount of energy, even a single ion is enough to significantly damage a gene – in fact, the beams have enough energy to break the double strand of the DNA. The technique is also very useful in producing mutants that lack just a single gene; multiple propagation technology can be used to convert these mutants into new cultivars. Examples of such breeds include ‘Safinia Rose’ (petunia), ‘Temari Bright Pink’ (vervena) and ‘Olivia Pure White’ (dianthus). The development period for producing new varieties is only 2 years. Over last decade, molecular biology has made great advancements through technological innovation. We use high-throughput DNA sequencing techniques such as next-generation sequence instruments and microarray technologies for analysis of gene mutations. Mutants have become more and more useful and important in modern genetic studies, enabling the discovery of genes that control important traits, and revealing the functions and mechanisms underlying their operations. The discovery of genes using mutants may lead to the emergence of a new field in biology, ‘Mutagenomics’.