• Journal of Microbiology and Biotechnology
• /
• v.33 no.4
• /
• pp.552-558
• /
• 2023

Levulinic acid (LA) is a valuable chemical used in fuel additives, fragrances, and polymers. In this study, we proposed possible biosynthetic pathways for LA production from lignin and poly(ethylene terephthalate). We also created a genetically encoded biosensor responsive to LA, which can be used for screening and evolving the LA biosynthesis pathway genes, by employing an LvaR transcriptional regulator of Pseudomonas putida KT2440 to express a fluorescent reporter gene. The LvaR regulator senses LA as a cognate ligand. The LA biosensor was first examined in an Escherichia coli strain and was found to be non-functional. When the host of the LA biosensor was switched from E. coli to P. putida KT2440, the LA biosensor showed a linear correlation between fluorescence intensity and LA concentration in the range of 0.156-10 mM LA. In addition, we determined that 0.156 mM LA was the limit of LA detection in P. putida KT2440 harboring an LA-responsive biosensor. The maximal fluorescence increase was 12.3-fold in the presence of 10 mM LA compared to that in the absence of LA. The individual cell responses to LA concentrations reflected the population-averaged responses, which enabled high-throughput screening of enzymes and metabolic pathways involved in LA biosynthesis and sustainable production of LA in engineered microbes.

• Journal of Microbiology and Biotechnology
• /
• v.30 no.9
• /
• pp.1430-1435
• /
• 2020

Bacterial cellulose (BC) has outstanding physical and chemical properties, including high crystallinity, moisture retention, and tensile strength. Currently, the major producer of BC is Komagataeibacter xylinus. However, due to limited tools of expression, this host is difficult to engineer metabolically to improve BC productivity. In this study, a regulated expression system for K. xylinus with synthetic ribosome binding site (RBS) was developed and used to engineer a BC biosynthesis pathway. A synthetic RBS library was constructed using green fluorescent protein (GFP) as a reporter, and three synthetic RBSs (R4, R15, and R6) with different strengths were successfully isolated by fluorescence-activated cell sorting (FACS). Using synthetic RBS, we optimized the expression of three homologous genes responsible for BC production, pgm, galU, and ndp, and thereby greatly increased it under both static and shaking culture conditions. The final titer of BC under static and shaking conditions was 5.28 and 3.67 g/l, respectively. Our findings demonstrate that reinforced metabolic flux towards BC through quantitative gene expression represents a practical strategy for the improvement of BC productivity.

• Journal of Ginseng Research
• /
• v.14 no.2
• /
• pp.200-206
• /
• 1990

The effects of ginseng saponins, SRbl and G-Rc on the rat liver LDH A-gene transcriptional activity was investigated during prereplicative phase of rat liver after partial hepatectomy. Changes in LDH A-mRNA levels in regenerating rat liver after intraperitoneal administrations of G-Rbl or 'G-Rc were tested by slot blot hybridization methods. The results showed that G-Rbl (1 mg/100g B.W) and G-Rc (1 mg/100g B.W) caused marked increases of LDH A-mRNA contents by respectively 1.9- and 1.5-fold in rat liver at 5-hours after partial hepatectomy Dose dependent elect of G-Rbl and G-Rc (1-25 mg/ 100g B.W) on the LDH A-mRNA levels on regenerating rat liver were also analyzed. The maximal increases of liver LDH A-mRNA levels were observed with the doses of 1 mg for G-Rbl and 5 mg for G-Rc. However, when the administration doses of G-Rbl and G-Rc were increased to 20 mg, G-Rbl caused a marked decrease of LDH A-mRNA level to 61% of those in sham-operated rat liver. In contrast, G-Rc slightly decreased the liver LDH A-mRNA contents by 30% as compared to those of the maximum value but still maintained 22% higher LDH A-mRNA levels then those of sham-operated rate liver. On the basis of these experimental results, we conclude that ginseng saponin, G-Rbl and G-Rc have stimulatory effect at the lower concentration (1 mg/ 100g B.W) and inhibitory effect at the higher concentration (20 mg/ 100g B.W) on the LDH A-gene transcription during regeneration of rat liver. Additionally we also investigated the stimulatory effects of ginsenosides on the protein and DNA sinthetic activities in hepatocyte primary cell cultures isolated from regenerating rat liver. Both of G-Rc and -Re increased the synthetic rates of hepatocytes proteins and DNA at the administration doses of 50 us and 100 $\mu\textrm{g}$/3 ml/dish respectively representing 1.3-1.6 fold increases. From these results we postulate that G-Rc and -Re may have a mitogen ehincer activity for the hepatocyte proliferation during rat liver regeneration period.

• Asian-Australasian Journal of Animal Sciences
• /
• v.21 no.5
• /
• pp.628-632
• /
• 2008

Egg production traits are economically important both for egg-laying and broiler lines of chicken. In sheep, the Q249R mutation in BMPR-IB is associated with ovulation rate. The present study cloned a partial chicken BMPR-IB fragment which contained the corresponding ovine Q249R mutation, including partial exon 6 and exon 7 and full-length intron 6. Five nucleotide changes were identified by alignment of the fragment amplified from Jining Bairi and Zang chickens. Among these nucleotide substitutions, the C/T transition at the base position of 35 and the A/G transition at the base position of 287 were found to be highly polymorphic, and named as SNPs C35T and A287G, respectively. For the SNP C35T, 331 hens of a synthetic broiler line were genotyped by a PCR-SSCP approach and allele C was found to be dominant. For the SNP A287G, 604 birds from the synthetic broiler line, a commercial egg-laying line, as well as three Chinese indigenous chicken breeds were genotyped by a PCR-RFLP technique. The associations of these two SNPs with egg production traits in the broiler line were analyzed. The results indicated that both the C35T and the A287G SNPs were not associated with egg production at 33wks and from 33wks to 42 wks (p>0.1), whereas the SNP A287G was associated with egg production from 47 to 56 wks (p<0.05). The dominance genetic effects on this latter trait and on egg production from 33 to 42 wks were significant (p<0.05).

• Asian-Australasian Journal of Animal Sciences
• /
• v.12 no.1
• /
• pp.9-14
• /
• 1999

Factors affecting gene gun-mediated expression of the human erythropoietin gene were investigated in primary cultured oviduct cells from laying hens. The human erythropoietin gene was transfected by a gene gun method at $1.25{\mu}g$ per dish, and cultured in a synthetic serum-free medium for 72 hrs. The concentration of human erythropoietin mRNA was determined by RNA : RNA solution hybridization. In experiment 1, the effect of changing the shooting pressure of DNA-coated microparticles with nitrogen gas was tested at 20 and $60kgf/cm^2$. The results showed that the erythropoietin mRNA concentration was significantly higher at 60 than $20kgf/cm^2$. In experiment 2, the effects of supplementing the medium with fetal calf serum at 10%, and raising the shooting pressure from 60 to $80kgf/cm^2$ on the cell number and erythropoietin gene expression were examined. Although supplementation with fetal calf serum significantly increased the cell numbes compared with no supplemented controls (p < 0.05), erythropoietin mRNA concentration per $10^3$ cells was not affected. Raising the shooting pressure from 60 to $80kgf/cm^2$ did not affect either of the parameters, In experiment 3, the effect of supplementing ascorbate 2-phosphate at 0.5 mM was tested. The results indicated that the ascorbate supplementation significantly increased the cell number (p < 0.05), and tended to increase erythropoietin mRNA concentration (p < 0.1). Thus, for human erythropoietin gene expression by using the gene gun method, shooting pressure with nitrogen gas should be sufficient at $60kgf/cm^2$ and supplementation with ascorbate phosphate would be useful to enhance not only the cell proliferation but also erythropoietin gene expression.

• Journal of Microbiology
• /
• v.45 no.2
• /
• pp.139-145
• /
• 2007

The Bombyx mori parvo-like virus (China isolate) DNA polymerase (BmDNV-3 dnapol) gene has been tentatively identified based on the presence of conserved motifs. In the present study, we perform a transcriptional analysis of the BmDNV-3 dnapol gene using the total RNA isolated from BmDNV-3 infected silkworm at different times. Northern blot analysis with a BmDNV-3 dnapol-specific riboprobe showed a major transcript of 3.3 kb. 5'-RACE revealed that the major transcription start point was located 20 nucleotides downstream of the TATA box. In a temporal expression analysis using differential RT-PCR, BmDNV-3 dnapol transcript was detected at low levels at 6 h.p.i., increased from 6 to 36 h.p.i., and remained fairly constant thereafter. Analysis of the predicted DNA polymerase sequence using neighborjoining and protein parsimony algorithms indicated that the predicted 1115-residue polypeptide contained five motifs associated with DNA polymerases synthetic activities and three additional motifs associated with polymerases possessing 3' to 5' exonuclease activity. The molecular phylogenetic analysis of this gene supported the placement of Bombyx mori parvo-like virus in a separate virus family.

• Bulletin of the Korean Chemical Society
• /
• v.34 no.9
• /
• pp.2589-2593
• /
• 2013

Gene therapy using nonviral gene delivery carriers has focused on the development and modification of synthetic carriers such as liposomes and polymers. Most polymers that are commercially used are taking advantage of their polycationic character which allows not only strong ligand-DNA affinity but also competent cell penetration. Despite the relatively high transfection efficiencies, high cytotoxicity is continuously pointed out as one of the major shortcomings of polycationic polymers such as PEI. Studies on the utilization of peptides have therefore been carried out recently to overcome these problems. For these reasons, the human transcription factor Hph-1, which is currently known as a protein transduction domain (PTD), was investigated in this study to evaluate its potential as a gene delivery carrier. Although its transfection efficiency was about 10-fold lower than PEI, it displayed almost no cytotoxicity even at concentrations as high as $100{\mu}M$. Hph-1 was oxidatively polymerized to yield poly-Hph-1. The cell viability of poly-Hph-1 transfected U87MG and NIH-3T3 cells was almost as high as the control (untreated) groups, and the transfection efficiency was about 10-fold higher than PEI. This study serves as a preliminary evaluation of Hph-1 and encourages further investigation.

• BMB Reports
• /
• v.43 no.7
• /
• pp.451-460
• /
• 2010

Starting with the first publication of lacZ gene fusion in 1980, reporter genes have just entered their fourth decade. Initial studies relied on the simple fusion of a promoter or gene with a particular reporter gene of interest. Such constructs were then used to determine the promoter activity under specific conditions or within a given cell or organ. Although this protocol was, and still is, very effective, current research shows a paradigm shift has occurred in the use of reporter systems. With the advent of innovative cloning and synthetic biology techniques and microfluidic/nanodroplet systems, reporter genes and their proteins are now finding themselves used in increasingly intricate and novel applications. For example, researchers have used fluorescent proteins to study biofilm formation and discovered that microchannels develop within the biofilm. Furthermore, there has recently been a "fusion" of art and science; through the construction of genetic circuits and regulatory systems, researchers are using bacteria to "paint" pictures based upon external stimuli. As such, this review will discuss the past and current trends in reporter gene applications as well as some exciting potential applications and models that are being developed based upon these remarkable proteins.

• Microbiology and Biotechnology Letters
• /
• v.20 no.3
• /
• pp.280-288
• /
• 1992

Molecular cloning of metalloprotease gene from Serratia marcescens ATCC 21074 into Escherichia coli JM109 was carried out. Chromosomal DNA of S. marcescens was completely digested with Hind111 and southern hybridization with a synthetic oligonucleotide probe revealed that a 50 KD metalloprotease gene was contained in 4.0 Kb chromosomal DNA fragment, 4.0 Kb chromosomal DNA fragments eluted from agarose gel were ligated with pUC19 and transformed into E. coli JM109. Nine positive clones were obtained from about $1\times 10^3$ transformants by colony hybridization. Their recombinant plasmids, pSPl and pSP2 have same chromosomal DNA fragments in pUC19 in opposite-orientations. When cloned metalloprotease gene was expressed in E. coli, about 52 KD precursor protein of metalloprotease was detected by western blot analysis from E. coli harboring a recombinant plasmid pSP2. Plasmid pSP2 showed no protease activities in E. coli but overproduced the active metalloprotease in S. rnarcescens ATCC 27117.

• Molecules and Cells
• /
• v.38 no.12
• /
• pp.1105-1110
• /
• 2015

Phleichrome, a pigment produced by the phytopathogenic fungus Cladosporium phlei, is a fungal perylenequinone whose photodynamic activity has been studied intensively. To determine the biological function of phleichrome and to engineer a strain with enhanced production of phleichrome, we identified the gene responsible for the synthesis of phleichrome. Structural comparison of phleichrome with other fungal perylenequinones suggested that phleichrome is synthesized via polyketide pathway. We recently identified four different polyketide synthase (PKS) genes encompassing three major clades of fungal PKSs that differ with respect to reducing conditions for the polyketide product. Based on in silico analysis of cloned genes, we hypothesized that the non-reducing PKS gene, Cppks1, is involved in phleichrome biosynthesis. Increased accumulation of Cppks1 transcript was observed in response to supplementation with the application of synthetic inducer cyclo-(${_L}-Pro-{_L}-Phe$). In addition, heterologous expression of the Cppks1 gene in Cryphonectria parasitica resulted in the production of phleichrome. These results provide convincing evidence that the Cppks1 gene is responsible for the biosynthesis of phleichrome.