The production of recombinant proteins in Escherichia coli is often challenged by cytoplasmic expression due to proteolytic degradation and inclusion body formation. Extracellular expression can overcome these problems by simplifying downstream processing and improving protein yields. This study aims to compare the efficiency of two Bacillus subtilis chitosanase signal peptides in mediating extracellular secretion in E. coli. We identified a naturally occurring mutant signal peptide (mCsn2-SP) from B. subtilis CH2 chitosanase (CH2CSN), which is characterized by a deletion of six amino acids in the N-region relative to the signal peptide (Csn1-SP) from B. subtilis CH1 chitosanase (CH1CSN). The CH1CSN and CH2CSN genes were cloned into the pET-11a vector and protein secretion was evaluated in E. coli BL21(DE3) host cells. Expression was induced with 0.1 mM and 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 30 °C for one and three days. CH2CSN showed higher secretion levels compared to CH1CSN under all experimental conditions, especially with 0.1 mM IPTG induction for 3 days, which resulted in a 2.37-fold increase in secretion. Furthermore, it was demonstrated that mCsn2-SP is capable of secreting human Cu,Zn-superoxide dismutase (hSOD) in E. coli BL21(DE3) and successfully translocating it to the periplasmic region.
This study represents the inaugural investigation into the utilisation of a naturally modified signal peptide, thereby corroborating the assertion that signal peptide deletion variants can influence protein secretion efficiency.
Furthermore, the findings substantiate the proposition that such variants can serve as a viable alternative for the secretion of heterologous proteins in E.
coli.
Md Rezaul Karim, Safia Iqbal, Shahnawaz Mohammad, Jong-Hoon Kim, Li Ling, Changbao Chen, Abdus Samad, Md Anwarul Haque, Deok-Chun Yang, Yeon Ju Kim, Dong Uk Yang
J. Microbiol. 2024;62(10):919-927. Published online October 8, 2024
In recent years, kidney cancer has become one of the most serious medical issues.
Kidney cancer is treated with a variety of active compounds that trigger genes that cause cancer. We identified in our earlier research that isoquercitrin (IQ) can activate PIK3CA, IGF1R, and PTGS2. However, it has a very low bioavailability because of its lower solubility in water. So, we utilized sub-merge fermentation technology with two well-known probiotics, Lactobacillus acidophilus and Bacillus subtilis, as a microbial source and mulberry fruit extract as a substrate, which has a high IQ level to improve IQ yield. Furthermore, we compared the total phenolic, flavonoid, and antioxidant contents of fermented and non-fermented samples, and we found that the fermented samples had greater levels than non-fermented sample. In addition, the high-performance liquid chromatography (HPLC) results showed that the fermented mulberry fruit extract from B. subtilis and L. acidophilus showed higher IQ values (190.73 ± 0.004 μg/ml and 220.54 ± 0.007 μg/ml, respectively), compared to the non-fermented samples, which had IQ values (80.12 ± 0.002 μg/ml). Additionally, at 62.5 µg/ml doses of each sample, a normal kidney cell line (HEK 293) showed higher cell viability for fermented and non-fermented samples. Conversely, at the same doses, the fermented samples of L. acidophilus and B. subtilis in a kidney cancer cell line (A498) showed an inhibition of cell growth around 36% and 31%, respectively. Finally, we performed RT and qRT PCR assay, and we found a significant reduction in the expression of the PTGS2, PIK3CA, and IGF1R genes. We therefore can conclude that the fermented samples have a higher concentration of isoquercitrin, and also can inhibit the expression of the genes PTGS2, PIK3CA, and IGF1R, which in turn regulates kidney cancer and inflammation.
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Candida albicans is the primary etiological agent associated with candidiasis in humans. Unrestricted growth of C. albicans can progress to systemic infections in the worst situation. This study investigates the antifungal activity of Hydroxychloroquine (HCQ) and mode of action against C. albicans. HCQ inhibited the planktonic growth and yeast to hyphal form morphogenesis of C. albicans significantly at 0.5 mg/ml concentration. The minimum inhibitory concentrations (MIC(50)) of HCQ for C. albicans adhesion and biofilm formation on the polystyrene surface was at 2 mg/ml and 4 mg/ml respectively. Various methods, such as scanning electron microscopy, exploration of the ergosterol biosynthesis pathway, cell cycle analysis, and assessment of S oxygen species (ROS) generation, were employed to investigate HCQ exerting its antifungal effects. HCQ was observed to reduce ergosterol levels in the cell membranes of C. albicans in a dose-dependent manner. Furthermore, HCQ treatment caused a substantial arrest of the C. albicans cell cycle at the G0/G1 phase, which impeded normal cell growth. Gene expression analysis revealed upregulation of SOD2, SOD1, and CAT1 genes after HCQ treatment, while genes like HWP1, RAS1, TEC1, and CDC 35 were downregulated. The study also assessed the in vivo efficacy of HCQ in a mice model, revealing a reduction in the pathogenicity of C. albicans after HCQ treatment. These results indicate that HCQ holds for the development of novel antifungal therapies.
Glycyrrhizic acid, glycyrrhetinic acid, and their oxo, ester, lactone, and other derivatives, are known for their anti-inflammatory,
anti-oxidant, and hypoglycemic pharmacological activities. In this study, chryseno[2,1-c]oxepin-12-carboxylic acid
(MG) was first biosynthesized from glycyrrhizic acid through sequential hydrolysis, oxidation, and esterification using
Aspergillus terreus TMZ05-2, providing a novel in vitro biosynthetic pathway for glycyrrhizic acid derivatives. Assessing
the influence of fermentation conditions and variation of strains during culture under stress-induction strategies enhanced
the final molar yield to 88.3% (5 g/L glycyrrhizic acid). CCK8 assays showed no cytotoxicity and good cell proliferation,
and anti-inflammatory experiments demonstrated strong inhibition of NO release (36.3%, low-dose MG vs. model), transcriptional
downregulation of classical effective cellular factors tumor necrosis factor-α (TNF-α; 72.2%, low-dose MG vs.
model), interleukin-6 (IL-6; 58.3%, low-dose MG vs. model) and interleukin-1β (IL-1β; 76.4%, low-dose MG vs. model),
and decreased abundance of P-IKK-α, P-IKB-α, and P-P65 proteins, thereby alleviating inflammatory responses through
the NF-κB pathway in LPS-induced RAW264.7 cells. The findings provide a reference for the biosynthesis of lactone compounds
from medicinal plants.
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Efficient directional biosynthesis of isoquercitrin from quercetin by
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CD-2 and its anti-inflammatory activity
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The disposal of sewage in significant quantities poses a health hazard to aquatic ecosystems. These effluents can contain
a wide range of pathogens, making faecal contamination a leading source of waterborne diseases around the world. Yet
monitoring bacteria or viruses in aquatic environments is time consuming and expensive. The standard indicators of faecal
pollution all have limitations, including difficulty in determining the source due to lack of host specificity, poor connection
with the presence of non-bacterial pathogens, or low environmental persistence. Innovative monitoring techniques are sorely
needed to provide more accurate and targeted solutions. Viruses are a promising alternative to faecal indicator bacteria for
monitoring, as they are more persistent in ambient water, more abundant in faeces, and are extremely host-specific. Given
the range of viruses found in diverse contexts, it is not easy to find one “ideal” viral indicator of faecal pollution; however,
several are of interest. In parallel, the ongoing development of molecular techniques coupled with metagenomics and bioinformatics
should enable improved ways to detect faecal contamination using viruses. This review examines the evolution
of faecal contamination monitoring with the following aims (i) to identify the characteristics of the main viral indicators of
faecal contamination, including human enteric viruses, bacteriophages, CRESS and plant viruses, (ii) to assess how these
have been used to monitor water pollution in recent years, (iii) to evaluate the reliability of recent detection methods of such
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Our recent genome-based study indicated that Mycobacterium paragordonae (Mpg) has evolved to become more adapted to
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castellanii was also proved. Here, we sought to investigate potential use of Mpg for antimycobacterial drug screening
systems. Our data showed that Mpg is more susceptible to various antibiotics compared to the close species M. marinum
(Mmar) and M. gordonae, further supporting its intracellular lifestyle in environments, which would explain its protection
from environmental insults. In addition, we developed two bacterial whole-cell-based drug screening systems using a
recombinant Mpg stain harboring a luciferase reporter vector (rMpg-LuxG13): one for direct application to rMpg-LuxG13
and the other for drug screening via the interaction of rMpg-LuxG13 with A. castellanii. Direct application to rMpg-LuxG13
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IC50) values of rifampin, isoniazid, clarithromycin, and ciprofloxacin against
Mpg compared to Mmar. Application of drug screening system via the interaction of rMpg-LuxG13 with A. castellanii also
exhibited lower IC50
values for rifampin against Mpg compared to Mmar. In conclusion, our data indicate that Mpg is more
susceptible to various antibiotics than other strains. In addition, our data also demonstrate the feasibility of two whole cellbased
drug screening systems using rMpg-LuxG13 strain for the discovery of novel anti-mycobacterial drugs.
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Candida species cause the most prevalent fungal illness, candidiasis.
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produced. In this study, the biological activity of 40 coumarin
derivatives was used to create a three-dimensional quantitative
structure activity relationship (3D-QSAR) model. The
training and test minimum inhibitory concentration values
of C. albicans active compounds were split, and a regression
model based on statistical data was established. This model
served as a foundation for the creation of coumarin derivative
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A novel bacterium designated RR4-40T was isolated from a
biofilter of seawater recirculating aquaculture system in Busan,
South Korea. Cells are strictly aerobic, Gram-negative, irregular
short rod, non-motile, and oxidase- and catalase-negative.
Growth was observed at 15–30°C, 0.5–6% NaCl (w/v),
and pH 5.0–9.5. The strain grew optimally at 28°C, 3% salinity
(w/v), and pH 8.5. The phylogenetic analysis based on
16S rRNA gene sequences showed that strain RR4-40T was
most closely related to Marinirhabdus gelatinilytica NH83T
(94.16% of 16S rRNA gene similarity) and formed a cluster
with genera within the family Flavobacteriaceae. The values
of the average nucleotide identity (ANI), digital DNA-DNA
hybridization (dDDH), and average amino acid identity (AAI)
between genomes of strain RR4-40T and M. gelatinilytica
NH83T were 72.91, 18.2, and 76.84%, respectively, and the
values against the strains in the other genera were lower than
those. The major fatty acids were iso-C15:0 (31.34%), iso-C17:0
3-OH (13.65%), iso-C16:0 3-OH (10.61%), and iso-C15:1 G
(10.38%). The polar lipids comprised phosphatidylglycerol,
diphosphatidylglycerol, aminophospholipid, aminolipid, glycolipid,
and sphingolipid. The major respiratory quinone was
menaquinone-6 (MK-6) and the DNA G + C content of strain
RR4-40T was 37.4 mol%. According to the polyphasic analysis,
strain RR4-40T is considered to represent a novel genus within
the family Flavobacteriaceae, for which the name Rasiella
rasia gen. nov, sp. nov. is proposed. The type strain is RR4-40T
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stresses. The antioxidant sulforaphane (SFN) has been
reported to exhibit anticancer activity and inhibit the growth
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exhibited bactericidal activity in a time and dose-dependent
manner. The anti-tubercular activity of SFN was significantly
correlated with bacterial reactive oxygen species (ROS) levels.
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on intracellular bacteria in a dose-dependent manner,
mediated by increasing intracellular mitochondrial ROS
levels and decreasing cytoplasmic ROS levels. Taken together,
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proliferation of bacteriophages. The Salmonella-infecting
phage SPN3US contains a putative N-acetyltransferase, which
is widely found in bacteriophages. However, due to low sequence
similarity to the N-acetyltransferases from bacteria
and eukaryotic cells, the structure and function of phage-encoded
acetyltransferases are mainly unknown. This study
determines the crystal structure of the putative N-acetyltransferase
of SPN3US in complex with acetyl-CoA. The crystal
structure showed a novel homodimeric arrangement stabilized
by exchanging the C-terminal α-helix within the dimer.
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specificity. Further studies are required to reveal the biochemical
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efficiency and accuracy is becoming more important. In this
review, we summarize the computational workflows used in
the research field of antibiotic resistome based on genome or
metagenome sequencing. We introduce workflows, software
tools, and data resources that have been successfully employed
in this rapidly developing field. The workflow described in
this review can be used to list the known antibiotic resistance
genes from genomes and metagenomes, quantitatively profile
them, and investigate the epidemiological and evolutionary
contexts behind their emergence and transmission. We also
discuss how novel antibiotic resistance genes can be discovered
and how the association between the resistome and
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There are presently no studies on the genes for sexual development
of Aspergillus fumigatus in situ using mating culture,
primarily because of challenging experimental conditions
that require a significantly long period of induction and produce
developmentally heterogenous culture, harboring very
few sexual organs. In order to overcome these challenges, we
developed an efficient and convenient procedure called ‘vegetative
mass mating (VeM)’ for study at a molecular level.
The VeM method enabled production of a developmentally
homogenous A. fumigatus culture, harboring many sexual
organs in a plate within a short period of two weeks. Feasibility
of the use of VeM for functional study of genes during
A. fumigatus sexual development was evaluated by analyzing
the transcription pattern of genes involved in pheromone signal
transduction and regulation of sexual development. Here,
we present for the first time, an in situ expression pattern of
sexual genes during the mating process, induced by the VeM method , which will enable and promote the sexual development
study of A. fumigatus at the molecular level.
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Translation initiation factor eIF4E forms eIF4E-eIF4G complex
at the 5’ cap of mRNA. This interaction can be inhibited
by the family of 4E-binding proteins (4E-BP). In yeast
Saccharomyces cerevisiae, two 4E-BPs, Caf20 and Eap1, compete
with eIF4G for binding to eIF4E via the shared conserved
interaction motif. In order to investigate the roles of Caf20
in gene-specific translational regulation and the formation
of mRNA granules (P-bodies), we introduced substitution
mutations, caf20-Y4A or caf20-L9A, in the eIF4E-binding
motif for CAF20. Overexpression of the wild-type CAF20
showed an increased protein level of Ste12 transcription factor
as well as highly developed P-body formation. However,
4E-binding site mutations of CAF20 led to a reduced number
of P-body foci and decreased levels of Ste12 protein. The
phenotypes of the caf20 deletion mutation were also analyzed,
and we suggest that Caf20 plays a critical role in Ste12 protein
expression and in the control of P-body formation.
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In the present study, a new strain, Bacillus cereus ZY12, producing
phospholipase D (PLD) was identified. The expression
of PLD in this strain was found to be induced by its substrate,
phosphatidylcholine (PC), and completely silenced by
other carbon sources, such as glucose, fructose, and maltose,
which are generally used in microbial growth cultures, thus
presenting a unique expression pattern different from other
PLD-producing microorganisms. This study is the first to
report on the ability of B. cereus to produce PLD, and successfully
clone its PLD-coding gene and identify its function,
extending the knowledge on PLD distribution and evolution
in microorganisms.
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Construction of a Super-Folder Fluorescent Protein-Guided Secretory Expression System for the Production of Phospholipase D in Bacillus subtilis Haiyang Zhang, Xuehan Li, Qi Liu, Jianan Sun, Francesco Secundo, Xiangzhao Mao Journal of Agricultural and Food Chemistry.2021; 69(24): 6842. CrossRef
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Cyclomaltodextrinases (CDases) belong to Glycoside Hydrolases
(GH) family 13, which show versatile hydrolyzing
and/or transglycosylation activity against cyclodextrin (CD),
starch, and pullulan. Especially, some CDases have been reported
to hydrolyze acarbose, a potent α-glucosidase inhibitor,
and transfer the resulting acarviosine-glucose to various
acceptors. In this study, a novel CDase (LPCD) gene was
cloned from Lactobacillus plantarum WCFS1, which encodes
574 amino acids (64.6 kDa) and shares less than 44% of identities
with the known CDase-family enzymes. Recombinant
LPCD with C-terminal six-histidines was produced and purified
from Escherichia coli. It showed the highest activity
on β-CD at 45°C and pH 5.0, respectively. Gel permeation
chromatography analysis revealed that LPCD exists as a dodecameric
form (~826 kDa). Its hydrolyzing activity on β-
CD is almost same as that on starch, whereas it can hardly
attack pullulan. Most interestingly, LPCD catalyzed the unique
modes of action in acarbose hydrolysis to produce maltose
and acarviosine, as well as to glucose and acarviosineglucose.
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Genetic and enzymatic characterization of Amy13E from
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reclassifies it as a cyclodextrinase also capable of α-diglucoside degradation
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