• Journal of fish pathology
• /
• v.24 no.3
• /
• pp.297-305
• /
• 2011

A trap identification system for isolating functional sequences to allow the constitutive expression of foreign protein from Edwardsiella tarda was developed. Using the green fluorescent protein (GFP) reporter-based trap system, various functional sequences to drive heterologous expression of the GFP were selectable in Escherichia coli host. However from the bioinformatic sequence analysis, all the segments predicted as regulatory regions were not native promoters actually existing upstream of endogenous E. tarda genes. Instead, a number of non-authentic sequences, possibly resulted from the random shuffling and/or intermolecular ligation were also proven to be able to display a potent GFP expression in the recombinant E. coli. Further analysis with selected clones showed that both authentic and non-authentic sequences could function in as a constitutive promoter, leading quite a consistent and stable GFP expression after repetitive subcultures. Microscopic examination also confirmed the uniform pattern of GFP expression in every host bacterium. Semi-quantitative assay of GFP showed that there was no clear relationship between expression levels and organizational features of the promoters trapped. Functional promoter-like elements achieved in the present study could be a good starting material for multivalent genetic engineering of E. tarda in order to produce recombinant vaccines in a cost-effective fashion.

• Journal of Microbiology and Biotechnology
• /
• v.31 no.5
• /
• pp.740-746
• /
• 2021

Efficient cellulolytic enzyme production is important for the development of lignocellulose-degrading enzyme mixtures. However, purification of cellulases from their native hosts is time- and labor-consuming. In this study, a constitutive expression system was developed in Penicillium oxalicum for the secreted production of proteins. Using a constitutive polyubiquitin gene promoter and cultivating with glucose as the sole carbon source, nine cellulolytic enzymes of different origins with relatively high purity were produced within 48 h. When supplemented to a commercial cellulase preparation, cellobiohydrolase I from P. funiculosum and cellobiohydrolase II from Talaromyces verruculosus showed remarkable enhancing effects on the hydrolysis of steam-exploded corn stover. Additionally, a synergistic effect was observed for these two cellobiohydrolases during the hydrolysis. Taken together, the constitutive expression system provides a convenient tool for the production of cellulolytic enzymes, which is expected to be useful in the development of highly efficient lignocellulose-degrading enzyme mixtures.

• Plant Biotechnology Reports
• /
• v.3 no.4
• /
• pp.325-331
• /
• 2009

To efficiently express a gene of interest in transgenic plants, the choice of promoter is a crucial factor as it directly affects the expression of the transgene that will yield the desired phenotype. The Arabidopsis ${\beta}-carotene$ hydroxylase 1 gene (AtBch1) shows constitutive and ubiquitous expression and was thus selected as one of best candidates for constitutive promoter analysis by both in silico northern blotting and semi-quantitative RT-PCR analysis. To investigate AtBch1 promoter activity, the 1,981-bp 5'-upstream region of this gene was fused with ${\beta}-glucuronidase$ (GUS) and transformed into Arabidopsis. Through the molecular characterization of transgenic leaf tissues, the AtBch1 promoter generated strong activity that drives 1.8- and 2-fold higher GUS expression than the cauliflower mosaic virus 35S (35S) promoter at the transcriptional and translational levels, respectively. Furthermore, the GUS enzyme activity driven by the AtBch1 promoter was 2.8-fold higher than that produced by the 35S promoter. By histochemical GUS staining, the ubiquitous expression of the AtBch1 promoter was observed in all tissues of Arabidopsis. Semi-quantitative RT-PCR analysis with different tissues further showed that this promoter serves as a strong constitutive driver of transgene expression in dicot plants.

• Microbiology and Biotechnology Letters
• /
• v.35 no.3
• /
• pp.184-190
• /
• 2007

Approximately 2.0 kb of the promoter region of the Pichia pastoris phosphoglycerate kinase gene (PGK1) was reduced to a 266 bp fragment and this minimized portion was used for construction of a new episomal constitutive expression vector in P. pastoris. As an approach to developing a constitutive expression vector in P. pastoris, the GAP promoter region of the Pichia expression vector pGAPZB was replaced with sequentially deleted PGK1 promoter fragments fused to a beta-galactosidase gene. When a lacZ gene was used as a reporter gene, PGK1 promoter strength was lower than that of the constitutive GAP promoter but it was higher than TEF1. We report here the development of the pPGKZ-E vector as a new episomal expression vector for heterologous gene expression by removing non-essential regions of the PGK1 promoter. This broadens the choice of episomal expression vectors for controlled constitutive expression in P. pastoris.

• Korean Chemical Engineering Research
• /
• v.58 no.2
• /
• pp.280-285
• /
• 2020

We have developed a constitutive display system of the Pseudomonas fluorescens SIK W1 TliA lipase on the cell surface of Escherichia coli using E. coli outer membrane protein C (OmpC) as an anchoring motif, which is an economical compared to induced system. For the constitutive expression of truncated OmpC-TliA fusion proteins, gntT104 promoter was employed. Cell growth was not affected by over expression of fusion protein during entire culture time, suggesting cell lysis was not a problem. The localization of truncated OmpC-TliA fusion protein on the cell surface was confirmed by immunofluorescence microscopy and measuring whole cell lipase activity. Constitutively displayed lipase was very stable, retaining activity enantioselectivity throughout the five repeated reactions. These results suggest that OmpC from E. coli be a useful anchoring motif for displaying enzymes on the cell surface without any inducers, and this stable surface display system can be employed for a broad range of biotechnological applications.

• Microbiology and Biotechnology Letters
• /
• v.47 no.3
• /
• pp.337-342
• /
• 2019

We published that bacterial heme was over-produced in a recombinant Corynebacterium glutamicum expressing 5-aminolevulinic acid synthase ($hemA^+$) under control of a constitutive promoter ($P_{180}$) and the heme-producing C. glutamicum had commercial potentials; as an iron feed additive for swine and as a preservative for lactic acid bacteria. To enhance the heme production, the $hemA^+$ gene was expressed under controls of various promoters in the recombinant C. glutamicum. The $hemA^+$ expression by $P_{gapA}$ (a constitutive glycolytic promoter of glyceraldehyde-3-phosphate dehydrogenase) led 75% increase of heme production while the expression by $P_{H36}$ (a constitutive, very strong synthetic promoter) resulted in 50% decrease compared with the control ($hemA^+$ expression by $P_{180}$ constitutive promoter). The $hemA^+$ expression by a late log-phase activating $P_{sod}$ (an oxidative-stress responding promoter of superoxide dismutase) led 50% greater heme production than the control. The $hemA^+$ expression led by a heat-shock responding chaperone promoter ($P_{dnaK}$) resulted in 121% increase of heme production at the optimized heat-shock conditions. The promoter strength and induction phase are discussed based on the results for the heme production at an industrial scale.

• Proceedings of the Botanical Society of Korea Conference
• /
• 1996.07a
• /
• pp.4-9
• /
• 1996

The majority of plants sense environmental signals, such as day length or temperature, to select their transition timing from vegetative growth t flowering. Here, we report the identification of a regulatory gene, OsMADS1, that controls the photoperiod sensitivity of flowering time. Constitutive expression of OsMADS1 in a long-day flowering plant, Nicotiana sylvestris, resulted in flowering in both short-day long-day conditions. Similarly, ectopic expression of the gene in a short-day flowering plant, N. tabacum cv. Maryland Mammoth, also induced flowering regardless of the day length. The transition time was dependent on the level of the OsMADS1 transcript in transgenic plants. These suggest that OsMADS1 is a key regulatory factor that determines the transition from shoot apex to floral meristem and that it can be used for controlling flowering time in a variety of plant species.

• Journal of Life Science
• /
• v.14 no.3
• /
• pp.391-395
• /
• 2004

To overproduce the cyclodextrin glucanotransferase(CGTase) of Bacillus stearothermophilus NO2 in B. subtilis, the pJH-CGTl plasmid (8.14 kb) was constructed, in which the ORF of CGTase gene could be transcribed by strong constitutive promoter, P$\_$JH/. To overproduce CGTase from a recombinant B. subtilis, the effect of media on the cell growth and expression level of CGTase were investigated with various media (LB, 2${\times}$LB, 5% molasses+2% CSL, CS, LBG) in the flask culture. Among them, [5% molasses+2% CSL] medium revealed the maximum expression level of CGTase with 1.8 unit/$m\ell$ at 9 hr culture. In the batch culture on [10% molasses+5% corn steep liquor] medium the expression level of CGTase, the secretion efficiency, and plasmid stability were about 4.2 unit/$m\ell$, 90% and 90%, respectively, at 30 hr culture. The cell growth and expression level in the fermenter culture with the industrial molasses medium were increased by 2-folds over the flask culture.

• Journal of Microbiology and Biotechnology
• /
• v.18 no.10
• /
• pp.1634-1640
• /
• 2008

This study was focused on the screening of valuable genetic resources, such as promoters from metagenome, and describes a promoter trapping system with a bidirectional probe concept, which can select promoters or operons from various biological resources including metagenomic DNA. A pair of reporters, GFP and DsRed, facing the opposite direction without promoters, is an effective system that can function regardless of the direction of inserted promoters. The feasibility of this system was tested for the isolation of constitutively expressed promoters in E. coli from a soil metagenome, resulting in a potential pool of various promoters for practical application. The analyses of structural organization of the trapped genes demonstrated that constitutively expressible promoters in E. coli were broadly distributed within the metagenome, and suggested that some promoters were useful for the construction of expression vectors. Based on these observations, three constitutive promoters were employed in the expression vector system and their potentials for practical application were evaluated in terms of expression level, protein solubility, and effects on host growth.

• Radiation Oncology Journal
• /
• v.24 no.2
• /
• pp.128-137
• /
• 2006

Purpose: We investigated whether a relationship exists between tumor control dose 50 ($TCD_{50}$) or tumor growth delay (TGD) and radiation induced apoptosis (RIA) in syngeneic murine tumors. Also we investigated the biological markers that can predict radiosensitivity in murine tumor system through analysis of relationship between $TCD_{50}$, TGD, RIA and constitutive expression levels of the genetic products regulating RIA. Materials and Methods: Syngeneic murine tumors such as ovarian adenocarcinoma, mammary carcinoma, squamous cell carcinoma, fibrosarcoma, hepatocarcinoma were used In this study. C3H/HeJ mice were bred and maintained in our specific pathogen free mouse colony and were $8{\sim}12$ weeks old when used for the experiments. The tumors, growing in the right hind legs of mice, were analyzed for $TCD_{50}$, TGD, and RIA at 8 mm in diameter. The tumors were also analyzed for the constitutive expression levels of $p53,\;p21^{WAF1/CIP1},\;BAX,\;Bcl-2,\;Bcl-X_L,\;Bcl-X_S$, and p34. Correlation analysis was peformed whether the level of RIA were correlated with $TCD_{50}$ or TGD, and the constitutive expression levels of genetic products regulating RIA were correlated with $TCD_{50}$, TGD, RIA. Results: The level of RIA showed a significant positive correlation (R=0.922, p=0.026) with TGD, and showed a trend to correlation (R=-0.848), marginally significant correlation with $TCD_{50}$ (p=0.070). It indicates that tumors that respond to radiation with high percentage of apoptosis were more radiosensitive. The constitutive expression levels of $p21^{WAF1/CIP1}$ and 34 showed a significant correlation either with $TCD_{50}$ (R=0.893, p=0.041 and R=0.904, p=0.035) or with TGD (R=-0.922, p=0.026 and R=-0.890 p=0.043). The tumors with high constitutive expression levels of $p21^{WAF1/CIP1}$ or p34 were less radiosensitive than those with low expression. Conclusion: Radiosensitivity may be predicted with the level of RIA in murine tumors. The constitutive expression levels of $p21^{WAF1/CIP1}$ or p34 can be used as biological markers which predict the radiosensitivity.