Creatinase (creatine amidinohydrolase), an important medical enzyme, has been used for clinical diagnosis of renal function because of its high substrate specificity. Recently, we successfully cloned a NaN3-resistant creatinase encoding gene from Arthrobacter nicotianae. By optimizing the cultivation process, we realized its high-level expression in Escherichia coli. In this addendum, production of this NaN3-resistant creatinase in E. coli and future research were further discussed.
arthrobacter nicotianae; creatinase; escherichia coli; NaN3-resistance
The identification of core genes involved in the biosynthesis of saxitoxin (STX) offers a great opportunity to detect toxic algae associated with paralytic shellfish toxins (PST). In the Yellow Sea (YS) in China, both toxic and nontoxic Alexandrium species are present, which makes it a difficult issue to specifically monitor PST-producing toxic algae. In this study, a quantitative PCR (qPCR) assay targeting sxtA4, a domain in the sxt gene cluster that encodes a unique enzyme involved in STX biosynthesis, was applied to analyze samples collected from the YS in spring of 2012. The abundance of two toxic species within the Alexandrium tamarense species complex, i.e., A. fundyense and A. pacificum, was also determined with TaqMan-based qPCR assays, and PSTs in net-concentrated phytoplankton samples were analyzed with high-performance liquid chromatography coupled with a fluorescence detector. It was found that the distribution of the sxtA4 gene in the YS was consistent with the toxic algae and PSTs, and the quantitation results of sxtA4 correlated well with the abundance of the two toxic species (r = 0.857). These results suggested that the two toxic species were major PST producers during the sampling season and that sxtA-based qPCR is a promising method to detect toxic algae associated with PSTs in the YS. The correlation between PST levels and sxtA-based qPCR results, however, was less significant (r = 0.552), implying that sxtA-based qPCR is not accurate enough to reflect the toxicity of PST-producing toxic algae. The combination of an sxtA-based qPCR assay and chemical means might be a promising method for monitoring toxic algal blooms.
N-terminal sequences play crucial roles in regulating expression, translation, activation and enzymatic properties of proteins. To reduce cell toxicity of intracellular trypsin and increase secretory expression, we developed a novel auto-catalyzed strategy to produce recombinant trypsin by engineering the N-terminus of mature Streptomyces griseus trypsin (SGT). The engineered N-terminal peptide of SGT was composed of the thioredoxin, glycine-serine linker, His6-tag and the partial bovine trypsinogen pro-peptide (DDDDK). Furthermore, we constructed a variant TLEI with insertion of the artificial peptide at N-terminus and site-directed mutagenesis of the autolysis residue R145. In fed-batch fermentation, the production of extracellular trypsin activity was significantly improved to 47.4 ± 1.2 U·ml−1 (amidase activity, 8532 ± 142.2 U·ml−1 BAEE activity) with a productivity of 0.49 U·ml−1·h−1, which was 329% greater than that of parent strain Pichia pastoris GS115-SGT. This work has significant potential to be scaled-up for microbial production of SGT. In addition, the N-terminal peptide engineering strategy can be extended to improve heterologous expression of other toxic enzymes.
Owing to our limited understanding of the relationship between sequence and function and the interaction between intracellular pathways and regulatory systems, the rational design of enzyme-coding genes and de novo assembly of a brand-new artificial genome for a desired functionality or phenotype are difficult to achieve. As an alternative approach, directed evolution has been widely used to engineer genomes and enzyme-coding genes. In particular, significant developments toward DNA synthesis, DNA assembly (in vitro or in vivo), recombination-mediated genetic engineering, and high-throughput screening techniques in the field of synthetic biology have been matured and widely adopted, enabling rapid semi-rational genome engineering to generate variants with desired properties. In this commentary, these novel tools and their corresponding applications in the directed evolution of genomes and enzymes are discussed. Moreover, the strategies for genome engineering and rapid in vitro enzyme evolution are also proposed.
directed evolution; DNA assembly; enzyme; genome engineering; metabolic engineering; recombineering; synthetic biology
Puccinia striiformis f. sp. tritici (Pst) is the obligate biotrophic fungus responsible for stripe rust wheat. In this study, we developed and characterized 20 polymorphic microsatellite markers from the genomic sequence of an isolate of Chinese Pst race CY32. Polymorphism at each simple sequence repeat (SSR) locus was determined using 32 Pst isolates from 7 countries. The number of alleles varied from 2 to 7 across isolates, and the observed and expected heterozygosities ranged from 0.33 to 0.97 (mean 0.62) and 0.23 to 0.73 (mean 0.51), respectively. As expected the genomic SSR markers were more polymorphic than the expressed sequence tag (EST)-SSR markers developed previously. These markers will be more useful for population genetics and molecular genetics studies in Pst.
Microsatellites; Simple sequence repeat; Stripe rust
AIM: To compare two different laparotomy methods for modeling rabbit VX2 hepatocarcinoma.
METHODS: Thirty New Zealand rabbits were randomly divided into two groups: A and B. Group A was assigned a traditional laparotomy method (embedding tumor fragments directly into the liver with tweezers). Group B was subjected to an improved laparotomy method (injection of tumor fragments into the liver through a 15 G syringe needle). The operation time, incision length, incision infection rate, and mortality rate were compared between the two groups after laparotomy. Magnetic resonance imaging (MRI) was performed to evaluate tumor formation rates and the characteristics of the tumors 2 wk after laparotomy.
RESULTS: The mean operation times for the two groups (Group A vs Group B) were 23.2 ± 3.4 min vs 17.5 ± 2.9 min (P < 0.05); the incision length was 3.3 ± 0.5 cm vs 2.4 ± 0.6 cm (P < 0.05); and the mortality rate after 2 wk was 26.7% vs 0% (P < 0.05); all of these outcomes were significantly different between the two groups. The incision infection rates in the two groups were 6.7% vs 0% (P > 0.05), which were not significantly different. MRI performed after 2 weeks showed that the tumor formation rates in the two groups were 90.9% vs 93.3% (P > 0.05). These rates were not significantly different between the two groups. The celiac implantation rate and abdominal wall metastasis rate in the two groups were 36.4% vs 13.3% (P < 0.05) and 27.2% vs 6.7% (P < 0.05), respectively, which were significantly different between the two groups.
CONCLUSION: The tumor formation rates were not significantly different between the two methods for modeling rabbit VX2 hepatocarcinoma. However, the improved method is recommended because it has certain advantages.
Rabbit VX2 hepatocarcinoma; Laparotomy; Modeling; Magnetic resonance imaging
Several studies have shown that CNS provides the regulation of gastric functions. Recent evidence indicated that the activation of melanocortin 4 receptors (MC4R) in brain nuclei played an important role in modulating gastric activity. This study was designed to assess whether MC4R signaling existed in autonomic circuitry modulated the activity of stomach by a virally mediated transsynaptic tracing study. Pseudorabies virus (PRV)-614 was injected into the ventral stomach wall in adult male MC4R-green fluorescent protein (GFP) transgenic mice (n = 5). After a survival time of 5 days, the mice were assigned to humanely sacrifice, and spinal cords and caudal brainstem were removed and sectioned, and processed for PRV-614 visualization. Neurons involved in the efferent control of the stomach were identified following visualization of PRV-614 retrograde tracing. The neurochemical phenotype of MC4R-GFP-positive neurons was identified using fluorescence immunocytochemical labeling. PRV-614/MC4R-GFP dual labeled neurons were detected in spinal IML and the dorsal motor nucleus of the vagus nerve (DMV). Our findings support the hypothesis that MC4R signaling in autonomic circuitry may participate in the modulation of gastric activity by the melanocortinergic-sympathetic pathway or melanocortinergic-parasympathetic pathway.
Stomach; Melanocortin-4 receptor; spinal cord; autonomic nervous system; pseudorabies virus; transsynaptic tracing
5-Aminolevulinic acid (ALA), the committed intermediate of the heme biosynthesis pathway, shows significant promise for cancer treatment. Here, we identified that in addition to hemA and hemL, hemB, hemD, hemF, hemG and hemH are also the major regulatory targets of the heme biosynthesis pathway. Interestingly, up-regulation of hemD and hemF benefited ALA accumulation whereas overexpression of hemB, hemG and hemH diminished ALA accumulation. Accordingly, by combinatorial overexpression of the hemA, hemL, hemD and hemF with different copy-number plasmids, the titer of ALA was improved to 3.25 g l−1. Furthermore, in combination with transcriptional and enzymatic analysis, we demonstrated that ALA dehydratase (HemB) encoded by hemB is feedback inhibited by the downstream intermediate protoporphyrinogen IX. This work has great potential to be scaled-up for microbial production of ALA and provides new important insights into the regulatory mechanism of the heme biosynthesis pathway.
Vitamin B12 is a fascinating molecule which acts as a co-factor in the metabolism of many organisms, especially affecting DNA synthesis and regulation, fatty acid synthesis and energy production. The synthesis of vitamin B12 is limited to a few of bacteria and archaea. Therefore, industrial microbial fermentation is used to meet annual demands worldwide of vitamin B12 and as an alternative method to the chemical synthesis which requires at least 60 steps that is uneconomical. Bacillus megaterium is one of vitamin B12 producers and an ideal host for many biotechnology applications and being one of the best tools for the industrial production of several enzymes. Therefore, a two-step optimization strategy was established to produce high yield of vitamin B12 by B. megaterium through the provision of the production requirements and the suitable conditions for the biosynthesis of vitamin B12.
We achieved the optimum conditions for the fermentation process of B. megaterium to produce high yield of vitamin B12 in a practical way based on statistical design and analysis which allowed vitamin B12 production to increase up to 759-fold (204.46 μg/l) as compared with control without parameters (0.26 μg/L). High performance liquid chromatography coupled to variable wavelength detector and mass spectrometry has been used to identify vitamin B12 forms and confirm the results.
We developed the fermentation process of B. megaterium to enhance the production of vitamin B12 by providing the required supplements for the synthesis of vitamin B12 (CoCl2, δ-aminolevulinic acid (ALA) and 5,6-dimethylbenzimidazole (DMB)) and dividing the fermentation process into three stages. In addition, the optimum incubation times of the three fermentation stages were investigated and performed with reducing number of experimental and evaluated multiple parameters and their interactions by using statistical experimental design and analysis. All of these strategies has proven successful in enhancing the production of vitamin B12 up to 204.46 μg/l and demonstrated that B. megaterium could be a good candidate for the industrial production of vitamin B12.
Vitamin B12; Bacillus megaterium; δ-Aminolevulinate (ALA); 5; 6-dimethylbenzimidazole (DMB)
Gastric cancer (GC) is one of the leading causes of cancer death in the world. The role of histone deacetylase 4 (HDAC4) in specific cell and tissue types has been identified. However, its biological roles in the development of gastric cancer remain largely unexplored. Quantitative real time PCR (qRT-PCR) and western blot were used to analyze the expression of HDAC4 in the clinical samples. siRNA and overexpression of HDAC4 and siRNA p21 were used to study functional effects in a proliferation, a colony formation, a adenosine 5′-triphosphate (ATP) assay and reactive oxygen species(ROS) generation, cell cycle, cell apoptosis rates, and autophagy assays. HDAC4 was up-regulated in gastric cancer tissues and several gastric cancer cell lines. The proliferation, colony formation ability and ATP level were enhanced in HDAC4 overexpression SGC-7901 cells, but inhibited in HDAC4 knockdown SGC-7901 cells. HDAC4 knockdown led to G0/G1 phase cell arrest and caused apoptosis and ROS increase. Moreover, HDAC4 was found to inhibit p21 expression in gastric cancer SGC-7901 cells. p21 knockdown dramatically attenuated cell proliferation inhibition, cell cycle arrest, cell apoptosis promotion and autophagy up-regulation in HDAC4-siRNA SGC-7901 cells. We demonstrated that HDAC4 promotes gastric cancer cell progression mediated through the repression of p21. Our results provide an experimental basis for understanding the pro-tumor mechanism of HDAC4 as treatment for gastric cancer.
Hyaluronidases (HAases), particularly leech HAases, have attracted intense attention due to their broad applications in medical treatments and great potential for the enzymatic production of hyaluronan oligosaccharides. However, little is known about this third interesting family of HAases. Here, we applied the random amplification of cDNA ends polymerase chain reaction (RACE-PCR) approach to identify the first leech HAase-encoding gene. By combining protein engineering and high-density culture, we achieved high-level production (8.42 × 105 U ml−1) in the yeast Pichia pastoris secretory expression system. Compared with the commercial bovine testicular HAase, the recombinant leech HAase exhibited superior enzymatic properties. Furthermore, analysis of the hydrolytic process suggested that this novel enzyme adopts a nonprocessive endolytic mode, yielding a narrow-spectrum of specific HA oligosaccharides with different incubation times. Large-scale production of this novel leech HAase will not only greatly promote medical applications but also facilitate the enzymatic production of specific HA oligosaccharides.
Applying in silico simulations and in vitro experiments, the amino acid proline was proved to have a profound influence on Streptomyces griseus trypsinogen, and the hydrogen bond between H57 and D102 was found to be crucial for trypsin activity. By introducing an artificial propeptide, IVEF, the titer of trypsin was increased 6.71-fold.
The organic molecule in the title monohydrate, C6N6O5·H2O, presents an almost planar configuration, the greatest deviation from the least-squares plane through the atoms being 0.061 (1) Å for the O atom within the seven-membered ring. Each water H atom is bifurcated, one forming two O—H⋯N hydrogen bonds and the other forming O—H⋯N,O hydrogen bonds. The result of the hydrogen bonding is the formation of supramolecular layers with a zigzag topology that stack along .
Diagnostic information for psychiatric research often depends on both clinical interviews and medical records. Although discrepancies between these two sources are well known, there have been few studies into the degree and origins of inconsistencies.
We compared data from structured interviews and medical records on 1,970 Han Chinese women with recurrent DSM-IV major depression (MD). Correlations were high for age at onset of MD (0.93) and number of episodes (0.70), intermediate for family history (+0.62) and duration of longest episode (+0.43) and variable but generally more modest for individual depressive symptoms (mean kappa = 0.32). Four factors were identified for twelve symptoms from medical records and the same four factors emerged from analysis of structured interviews. Factor congruencies were high but the correlation of factors between interviews and records were modest (i.e. +0.2 to +0.4).
Structured interviews and medical records are highly concordant for age of onset, and the number and length of episodes, but agree more modestly for individual symptoms and symptom factors. The modesty of these correlations probably arises from multiple factors including i) inconsistency in the definition of the worst episode, ii) inaccuracies in self-report and iii) difficulties in coding medical records where symptoms were recorded solely for clinical purposes.
Poly-β-hydroxybutyrate (PHB) mobilization in bacteria has been proposed as a mechanism that can benefit their host for survival under stress conditions. Here we reported for the first time that a stress-induced system enabled E. coli, a non-PHB producer, to mobilize PHB in vivo by mimicking natural PHB accumulation bacteria.
The successful expression of PHB biosynthesis and PHB depolymerase genes in E. coli was confirmed by PHB production and 3-hydroxybutyrate secretion. Starvation experiment demonstrated that the complete PHB mobilization system in E. coli served as an intracellular energy and carbon storage system, which increased the survival rate of the host when carbon resources were limited. Stress tolerance experiment indicated that E. coli strains with PHB production and mobilization system exhibited an enhanced stress resistance capability.
This engineered E. coli with PHB mobilization has a potential biotechnological application as immobilized cell factories for biocatalysis and biotransformation.
Rice dwarf virus (RDV) is a member of the genus Phytoreovirus, which is composed of viruses with segmented double-stranded RNA genomes. Proteins that support the intercellular movement of these viruses in the host have not been identified. Microprojectile bombardment was used to determine which open reading frames (ORFs) support intercellular movement of a heterologous virus. A plasmid containing an infectious clone of Potato virus X (PVX) defective in cell-to-cell movement and expressing either β-glucuronidase or green fluorescent protein (GFP) was used for cobombardment with plasmids containing ORFs from RDV gene segments S1 through S12 onto leaves of Nicotiana benthamiana. Cell-to-cell movement of the movement-defective PVX was restored by cobombardment with a plasmid containing S6. In the absence of S6, no other gene segment supported movement. Identical results were obtained with Nicotiana tabacum, a host that allows fewer viruses to infect and spread within its tissue. S6 supported the cell-to-cell movement of the movement-defective PVX in sink and source leaves of N. benthamiana. A mutant S6 lacking the translation start codon did not complement the cell-to-cell movement of the movement-defective PVX. An S6 protein product (Pns6)-enhanced GFP fusion was observed near or within cell walls of epidermal cells from N. tabacum. By immunocytochemistry, unfused Pns6 was localized to plasmodesmata in rice leaves infected with RDV. S6 thus encodes a protein with characteristics identical to those of other viral proteins required for the cell-to-cell movement of their genome and therefore is likely required for the cell-to-cell movement of RDV.