• Mycobiology
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• v.47 no.1
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• pp.59-65
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• 2019

Agrobacterium tumefaciens-mediated transformation (ATMT), as a simple and versatile method, achieves successful transformation in the yeast-like cells (YLCs) of Tremella fuciformis with lower efficiency. Establishment of a more efficient transformation system of YLCs is important for functional genomics research and biotechnological application. In this study, an enzymolysis-assisted ATMT method was developed. The degradation degree of YLCs depends on the concentration and digestion time of Lywallzyme. Lower concentration (${\leq}0.1%$) of Lywallzyme was capable of formation of limited wounds on the surface of YLCs and has less influence on their growth. In addition, there is no significant difference of YLCs growth among groups treated with 0.1% Lywallzyme for different time. The binary vector pGEH under the control of T. fuciformis glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter was utilized to transform the enzymolytic wounded YLCs with different concentrations and digestion time. The results of PCR, Southern blot, quantitative real-time PCR (qRT-PCR) and fluorescence microscopy revealed that the T-DNA was integrated into the YLCs genome, suggesting an efficient enzymolysis-assisted ATMT method of YLCs was established. The highest transformation frequency reached 1200 transformants per $10^6$ YLCs by 0.05% (w/v) Lywallzyme digestion for 15 min, and the transformants were genetically stable. Compared with the mechanical wounding methods, enzymolytic wounding is thought to be a tender, safer and more effective method.

• KSBB Journal
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• v.10 no.1
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• pp.38-45
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• 1995

The expression of Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpdA) and trpC promoters in A. oryzae were compared using E. coli lacZ gents fusions. The specific activities of the expressed E. coli $\beta$-galactosidase in A. oryzae transformants containing the A. nidulans gpdA promoter were around 2,000 units per ug of protein. The specific activities of transformants containing the A. nidulans trpC promoter were very low, ranging from 10.5 to 52.3 units per ug of protein. These results showed that the expression of the A. nidulans gpdA promoter in A. oryzae was approximately 70 times greater than the A. nidulans trpC promoter. In western blot analysis, immunoreactive bands of a imlilar molecular weight as the E. coli $\beta$-galactosidase were observed in A. oryzae carrying the gpdA-lacZ fusion and to a lesser intensity in those carrying the tvpC-lacZ fusion. Southern analysis showed that the higher expression of the gpdA-lacZ fusion as compared to the trpC-lacZ fusion was not due a greater number of integrated plasmids.

• Journal of Marine Bioscience and Biotechnology
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• v.16 no.1
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• pp.45-54
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• 2024

Microalgae have great potential in the biomedical and pharmaceutical industries as a new type of bioreactor that can produce proteins for specific purposes, including recombinant proteins, pharmaceuticals, and industrial enzymes. Despite the production advantages and importance of microalgae-based expression systems, studies on secretion efficiency are limited. In this study, for stable expression and efficient secretion of the heterologous protein (human SCF and human INFγ) in Chlorella vulgaris, we constructed SP:hSCF:His and SP:hINFγ:His plant binary vectors using the signal peptide (SP) of Chlamydomonas reinhardtii, and we obtained stable transformants through the effective agrobacterium-mediated transformation of these vectors. Transformants with accurately inserted hSCF and hINFγ demonstrated stably increased mRNA and protein expression using RT-PCR and western blotting under the same culture conditions. Following the analysis of the proteins secreted into the culture medium using ELISA, it was confirmed that hINFγ was effectively produced in the transformed C. vulgaris culture medium. The overall findings indicate that the combination of heterologous protein and SP may be crucial for ensuring the expression and secretion of recombinant proteins in Chlorella culture systems.

• Research in Plant Disease
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• v.19 no.4
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• pp.254-258
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• 2013

Fusaric acid (FA) is a mycotoxin produced by Fusarium species. Its toxicity is relatively low but often associated with other mycotoxins, thus enhancing total toxicity. To date, biosynthetic genes or enzymes for FA have not been identified in F. oxysporum. In order to explore the genetic element(s) for FA biosynthesis, restriction enzyme mediated integration (REMI) procedure as an insertional mutagenesis was employed using FA producing-F. oxysporum strains. Genetic transformation of two F. oxysporum strains by REMI yielded more than 7,100 transformants with efficiency of average 3.2 transformants/${\mu}g$ DNA. To develop a screening system using phytotoxicity of FA, eleven various grains and vegetable seeds were tested for germination in cultures containing FA: Kimchi cabbage seed was selected as the most sensitive host. Screening for FA non-producer of F. oxysporum was done by growing each fungal REMI transformant in Czapek-Dox broth for 3 weeks at $25^{\circ}C$ then observing if the Kimchi cabbage seeds germinated in the culture filtrate. Of more than 5,000 REMI transformants screened, fifty-three made the seeds germinated, indicating that they produced little or fewer FA. Among them, twenty-six were analyzed for FA production by HPLC and two turned out to produce less than 1% of FA produced by a wild type strain. Sequencing of genomic DNA regions (252 bp) flanking the vector insertion site revealed an uncharacterized genomic region homologous (93%) to the F. fujikuroi genome. Further study is necessary to determine if the vector insertion sites in FA-deficient mutants are associated with FA production.

• Journal of Microbiology
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• v.37 no.2
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• pp.51-58
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• 1999

Using yeast, Saccharomyces cerevisiae, and the integrate vector system, we have isolated and characterized an autonomously replicating sequence (ARS) from Aspergillus nidulans. The DNA fragment, designated ANR1, is 5.0 kb in size and maintained free from the chromosome in S. cerevisiae. The YIplac211-ANR1 recombinant plasmid, which consists of sequences derived from the yeast integrative vector YIplac211 and 5.0 kb ANR1 fragment, showed a 104-fold enhancement in transformation efficiency over that found for YIplac211, and was easily recovered from the transformed yeast. Genetic analysis of transformants showed that YIplac21-ANR1 could be over 96% cured when cultured over 20 generations in complete medium and thus suggests that this sequence is mitotically unstable. In A. nidulans, recombinant plasmid PILJ16-4.5 which carries the 4.5 kb EcoRI fragment of ANR1 showed a 170-fold enhancement in transformation efficiency compared to that of the integrative vector PILJ16.

• Journal of Microbiology and Biotechnology
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• v.18 no.4
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• pp.754-758
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• 2008

Agrobacterium tumefaciens-mediated transformation (ATMT) was successfully applied to Monascus ruber. The optimum cocultivation time was 84 h with an efficiency of 900 to 1,000 transformants when $1{\times}10^6$ spores were used with the same volume of bacteria. The stability of transform ants was over 98% after five generations. When M. ruber was transformed with A. tumefaciens YL-63 containing the green fluorescent protein gene (egfp), the green fluorescent signal was observed throughout hyphae, confirming expression of the gene. This efficient transformation and expression system of M. ruber by ATMT will facilitate the study of this fungus at a molecular genetic level.

• Food Science and Biotechnology
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• v.17 no.2
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• pp.438-441
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• 2008

A novel lactobacilli replicon from plasmids of Lactobacillus reuteri KCTC 3678 was isolated. Eight L. reuteri strains from Korean Collection for Type Cultures (KCTC) and Korea Food Research Institute (KFRI) were screened for cryptic plasmids and most strains harbored 1 or 2 plasm ids. Particularly, L. reuteri KCTC 3678 contained 6 plasm ids which all were used for screening of lactobacilli replicon. EcoRI digests of the plasmid DNA prep from L. reuteri KCTC 3678 were ligated with pUC19 and the recombinant DNAs were serially named from pLR1 to pLR7. A cat (chloramphenicol acetyltransferase; $Cm^r$) gene originated from pC194 was introduced into pLR1-7, resulting in pLR1cat-pLR7cat, respectively. The recombinant plasmids were introduced into L. reuteri KCTC 3679, and only transformants harboring pLR5cat were obtained, indicating that the insert in pLR5 functioned as a lactobacilli replicon.

• 한국생물공학회:학술대회논문집
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• 2000.04a
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• pp.195-198
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• 2000

Production of eight avermectin components was improved in Streptomyces avermitilis wild type strain (ATCC31267) and high producing mutant strain (ATCC31780) when transformed with a foreign regulatory gene, afsR2 of Streptomyces lividans. Wild type and the high producing strain of S. avermitilis transformed with multiple copies of afsR2 improved total avermectin productions by 2.3 fold and 1.5 fold, respectively. In both of wild type and the high producing transformants carrying afsR2, glycerol was proved to be the best carbon source for the stimulation of avermectin production.

• KSBB Journal
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• v.6 no.3
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• pp.223-230
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• 1991

Using the shuttle vector pECCGl between Escherichia coli and Corynebacterium glutamicum and C. glutamicum strain JS231 grown in the medium supplemented with penicillin-G, which inhibits the formation of cross-links in the peptidoglycan of bacterial cell wall, various parameters involved in electroporation system including resistance, electric field strength, capacitance, DNA concentration, and cell density were investigated independently and optimized for the high efficiency transformation of coryneform bacteria. Using cells grown with 0.3U/ml of penicillin-G and harvested at A600 of 0.7-0.8, transformation efficiencies of 107-l08 transformants/$\mu\textrm{g}$ of DNA with Corynebcctertum glutamicum strain JS231 and wild type ATCC13032 were achieved under conditions of 12.5kV/cm of electric field strength, 400 ohms of resistance, $25\mu$F of capacitance, 3$\times$108 cells per transformation(1.2$\times$1010 cells/ml) and 100ng of plasmid DNA per transformation.

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.467-470
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• 2003

The endoxylanase (642 bp; 213 amino acids) and ${\beta}-xylosidase$ (1,602 bp; 533 amino acids) genes from Bacillus sp. were amplified by PCR and separately inserted downstream of the yeast ADH1 promoters, resulting in the pAEDX-1 and pAEX plasmid. When the yeast transformants, S. cerevisiae SEY2102 harboring pAEDX-1 or pAEX, were grown on YPD medium, the total activities of the enzymes reached about 9.8 unit/mL for endoxylanase and 2.9 unit/mL for ${\beta}-xylosidase$. When the three kinds of xylan from oat spelts, birch wood, and corncob were hydrolyzed by treatment of recombinant endoxylanase and ${\beta}-xylosidase$, it was found that xylose, xylobiose and xylotriose were produced and xylose was the major product after 12 h reaction. In addition, with the higher amount of enzymes, the more amount of xylose was produced.