• Journal of Microbiology and Biotechnology
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• v.25 no.7
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• pp.988-998
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• 2015

The filamentous fungus Aspergillus oryzae is a well-known expression host used to express homologous and heterologous proteins in a number of industrial applications. To facilitate higher yields of proteins of interest, we constructed the pAsOP vector to express heterologous proteins in A. oryzae. pAsOP carries a selectable marker, pyrG, derived from Aspergillus nidulans, and a strong promoter and a terminator of the amyB gene derived from A. oryzae. pAsOP transformed A. oryzae efficiently via the PEG-CaCl₂-mediated transformation method. As proof of concept, green fluorescent protein (GFP) was successfully expressed in A. oryzae transformed by pAsOP-GFP. Additionally, we identified a novel fungal α-amylase (PcAmy) gene from Penicillium sp. and cloned the gene into the vector. After transformation by pAsOPPcAmy, the α-amylase PcAmy from Penicillium sp. was successfully expressed in a heterologous host system for the first time. The α-amylase activity in the A. oryzae transformant was increased by 62.3% compared with the untransformed A. oryzae control. The PcAmy protein produced in the system had an optimum pH of 5.0 and optimum temperature of 30^oC. As a cold-adapted enzyme, PcAmy shows potential value in industrial applications because of its high catalytic activity at low temperature. Furthermore, the expression vector reported in this study provides promising utility for further scientific research and biotechnological applications.

• Journal of Microbiology and Biotechnology
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• v.23 no.5
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• pp.674-680
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• 2013

Yeast Dekkera/Brettanomyces bruxellensis is probably the most common contaminant in wineries and ethanol production processes. The considerable economic losses caused by this yeast, but also its ability to produce and tolerate high ethanol concentrations, make it an attractive subject for research with potential for industrial applications. Unfortunately, efforts to understand the biology of D. bruxellensis and facilitate its broader use in industry are hampered by the lack of adequate procedures for delivery of exogenous DNA into this organism. Here we describe the development of transformation protocols (spheroplast transformation, LiAc/PEG method, and electroporation) and report the first genetic transformation of yeast D. bruxellensis. A linear heterologous DNA fragment carrying the kanMX4 sequence was used for transformation, which allowed transformants to be selected on plates containing geneticin. We found the spheroplast transformation method using 1M sorbitol as osmotic stabilizer to be inappropriate because sorbitol strikingly decreases the plating efficiency of both D. bruxellensis spheroplast and intact cells. However, we managed to modify the LiAc/PEG transformation method and electroporation to accommodate D. bruxellensis transformation, achieving efficiencies of 0.6-16 and 10-20 transformants/${\mu}g$ DNA, respectively. The stability of the transformants ranged from 93.6% to 100%. All putative transformants were analyzed by Southern blot using the kanMX4 sequence as a hybridization probe, which confirmed that the transforming DNA fragment had integrated into the genome. The results of the molecular analysis were consistent with the expected illegitimate integration of a heterologous transforming fragment.

• Proceedings of the Microbiological Society of Korea Conference
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• 2000.10a
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• pp.85-91
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• 2000

Aspergillus oryzae is a filamentous fungus classified in the group Aspergillaceae Ascomycetes. It is an important microorganism for industrial production of enzymes and fermented food productions. It secrets large quantities of proteins or enzymes into the culture medium which makes this organism appealing for the production of heterologous proteins. Recently Electric field-mediated transformation method, electroporation, has been applied to fungal transformation. In this study, fungal transformation was carried out by bypassing the protoplast isolation step, decreasing the culturing time and non-protoplast transformation for the increment of transformation efficiency. Transformants were obtained with electroporation in optimal condition 2,500 voltage, 1,540 ohm and 0.50 capacitance. More than 1,000 transform ants were obtained with 6-10 hrs cultured mycelia without enzyme treatment, called non-protoplast transformation.

• Microbiology and Biotechnology Letters
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• v.16 no.6
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• pp.489-493
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• 1988

To obtain a new yeast strain that is able to efficiently produce ethanol from starch, the glucoamylase gene of Saccharomyces diastaticus was transformed into S. cerevisiae without a cloning vector. The competent cells of S. cerevisiae, induced by the treatment of Li$_2$SO$_4$, were transformed with the partial BamHI-digests of chromosomal DNA of S. diastaticus, and the transformants were selected by their abilities to utilize and ferment starch. The transformants, which appeared at a frequency of 8.5$\times$10$^{-7}$, were able to withstand up to 800 ppm of copper sulfate like the recipient and retained the phenotypic expression of the recipient with the exception of the acquisition of STA gene and MAL gene, as regards fermentation of carbohydrates. The enzymatic properties of glucoamylases produced by transformants were very similar to those produced by S. diastaticus as based on optimium pH and temperature.

• Journal of Microbiology and Biotechnology
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• v.16 no.6
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• pp.952-960
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• 2006

As a unicellular green alga that possesses many of the metabolic pathways present in higher plants, Chlorelia offers many advantages for expression of heterologous proteins. Since strong and constitutive promoters are necessary for efficient expression in heterologous expression systems, the development of such promoters for use in the Chlorella system was the aim of this study. Proteins encoded by the early genes of algal viruses are expressed before viral replication, probably by the host transcriptional machinery, and the promoters of these genes might be useful for heterologous expression in Chlorella. In this study, putative promoter regions of DNA polymerase, ATP-dependent DNA ligase, and chitinase genes were amplified from eight Korean Chlorella virus isolates by using primer sets designed based on the sequence of the genome of PBCV-1, the prototype of the Phycodnaviridae. These putative promoter regions were found to contain several cis-acting elements for transcription factors, including the TATA, CAAT, NTBBF1, GATA, and CCAAT boxes. The amplified promoter regions were placed into Chlorella transformation vectors containing a green fluorescence protein (GFP) reporter gene and the Sh ble gene for phleomycin resistance. C. vulgaris protoplasts were transformed and then selected with phleomycin. The GFP fluorescence intensities of cells transformed with chitinase, DNA polymerase, and DNA ligase gene promoter-GFP fusion constructs were 101.5, 100.8, and 95.8%, respectively, of that of CaMV 35S-GFP-transformed Chlorella cells. These results demonstrate that these viral promoters are active in transformed Chlorella.

• Mycobiology
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• v.47 no.3
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• pp.301-307
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• 2019

The $11{\alpha}$-hydroxylation of $16{\alpha}$, 17-epoxyprogesterone (EP) catalyzed by Rhizopus nigricans is crucial for the steroid industry. However, lower conversion rate of the biohydroxylation restricts its potential industrial application. The $11{\alpha}$-steroid hydroxylase CYP509C12 from R. oryzae were reported to play a crucial role in the $11{\alpha}$-hydroxylation in recombinant fission yeast. In the present study, the CYP509C12 of R. oryzae (RoCYP) was introduced into R. nigricans using the liposome-mediated mycelial transformation. Heterologous expression of RoCYP resulted in increased fungal growth and improved intracellular reactive oxygen species content in R. nigricans. The $H_2O_2$ levels in RoCYP transformants were approximately 2-folder that of the R. nigricans wild type (RnWT) strain, with the superoxide dismutase activities increased approximately 45% and catalase activities decreased approximately 68%. Furthermore, the $11{\alpha}$-hydroxylation rates of EP in RoCYP transformants (C4, C6 and C9) were 39.7%, 38.3% and 38.7%, which were 12.1%, 8.2% and 9.4% higher than the rate of the RnWT strain, respectively. This paper investigated the effect of heterologous expression of RoCYP in R. nigricans, providing an effective genetic method to construct the engineered strains for steroid industry.

• Mycobiology
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• v.49 no.2
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• pp.95-104
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• 2021

Species of the genus Aspergillus have a variety of effects on humans and have been considered industrial cell factories due to their prominent ability for manufacturing several products such as heterologous proteins, secondary metabolites, and organic acids. Scientists are trying to improve fungal strains and re-design metabolic processes through advanced genetic manipulation techniques and gene delivery systems to enhance their industrial efficiency and utility. In this review, we describe the current status of the genetic manipulation techniques and transformation methods for species of the genus Aspergillus. The host strains, selective markers, and experimental materials required for the genetic manipulation and fungal transformation are described in detail. Furthermore, the advantages and disadvantages of these techniques are described.

• Journal of Microbiology and Biotechnology
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• v.22 no.9
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• pp.1279-1287
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• 2012

We established an efficient transformation method for thermophile Geobacillus kaustophilus HTA426 using conjugative transfer from Escherichia coli of host-mimicking plasmids that imitate DNA methylation of strain HTA426 to circumvent its DNA restriction barriers. Two conjugative plasmids, pSTE33T and pUCG18T, capable of shuttling between E. coli and Geobacillus spp., were constructed. The plasmids were first introduced into E. coli BR408, which expressed one inherent DNA methylase gene (dam) and two heterologous methylase genes from strain HTA426 (GK1380-GK1381 and GK0343-GK0344). The plasmids were then directly transferred from E. coli cells to strain HTA426 by conjugative transfer using pUB307 or pRK2013 as a helper plasmid. pUCG18T was introduced very efficiently (transfer efficiency, $10^{-5}-10^{-3}\;recipient^{-1}$). pSTE33T showed lower efficiency ($10^{-7}-10^{-6}\;recipient^{-1}$) but had a high copy number and high segregational stability. Methylase genes in the donor substantially affected the transfer efficiency, demonstrating that the host-mimicking strategy contributes to efficient transformation. The transformation method, along with the two distinguishing plasmids, increases the potential of G. kaustophilus HTA426 as a thermophilic host to be used in various applications and as a model for biological studies of this genus. Our results also demonstrate that conjugative transfer is a promising approach for introducing exogenous DNA into thermophiles.

• Journal of Plant Biotechnology
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• v.46 no.4
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• pp.323-330
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• 2019

The high expression level of industrial and metabolically important proteins in plants can be achieved by plastid transformation. The CaIA vector, a Capsicum-specific vector harboring aadA (spectinomycin resistance), is a selectable marker controlled by the PsbA promoter, and the terminator is flanked by the trnA and trnI regions of the inverted repeat (IR) region of the plastid. The CaIA vector can introduce foreign genes into the IR region of the plastid genome. The biolistic method was used for chloroplast transformation in Scoparia dulcis with leaf explants followed by antibiotic selection on regeneration medium. Transplastomes were successfully screened, and the transformation efficiency of 3 transgenic lines from 25 bombarded leaf explants was determined. Transplastomic lines were evaluated by PCR and Southern blotting for the confirmation of aadA insertion and its integration into the chloroplast genome. Seeds collected from transplastomes were analyzed on spectinomycin medium with wild types to determine genetic stability. The increased chloroplast transformation efficiency (3 transplastomic lines from 25 bombarded explants) would be useful for expressing therapeutically and industrially important genes in Scoparia dulcis L.

• Journal of Plant Biotechnology
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• v.5 no.1
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• pp.19-23
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• 2003

The Ac (activator) which is one of the well-characterized transposable elements from maize was examined for its transposition possibility to the heterologous plant (P.nigra x maximowiczii) genome via Agrobacterium tumefacience (LBA4404) mediated transformation system. A number of transgenic plants were successfully recovered after 30 weeks by amount reduction from 50 to 15 g/$m\ell$ kanamycin for in vitro selection to minimize phytotoxic effects and to increase callus growth and regeneration efficiency. Among transgenic plants, 62 out of 106 transgenic poplars (58.5%) showed abnormal phenotypes such as severe serrated leaves and light leaf coloration. Indigo staining with X-gluc proved indirectly the restoration of Gus enzyme function and the presence of Ac in poplar genome by PCR. Southern analysis indicated the transposition and existence of Ac element in poplar genomes. In this research, an Agrobacterium-mediated transformation system in poplar species was developed and identified that Ac derived from maize can be excised and trans posed into other poplar genomes.