Objective:To express fusion antimicrobial peptide in Pichia pastoris SMD1168 and determine its in vitro bioactivity.Methods:The constructed fusion anti-peptide RHKJT gene fragment was cloned from the recombinant pVAX1-RHKJT vector previously constructed in laboratory. The RHKJT gene fragment was inserted into plasmid pGAPZaA, then verified by PCR and sequenced to construct recombinant pGAPZα-RHKJT vector. The linearized pGAPZα-RHKJT was electroporated into Pichia pastoris SMD1168 to obtain the recombinant yeast SMDpG-RHKJT. The recombinant Pichia pastoris SMDpG-RHKJT was fermented and confirmed by PCR and RT-PCR. Fermentation supernatants were collected for in vitro bioactivity assay.Results:The recombinant yeast SMDpG-RHKJT was successfully obtained, and the recombinant yeast’s fermentation supernatant had significant inhibition effects on Escherichia coli, Salmonella, Staphylococcus aureus and Streptococcus pneumonia.Conclusion:The recombinant antimicrobial peptide expressed by recombinant yeast has marked antibacterial activity and would facilitate the development of novel antibacterial additive feed later.
The genus Trichoderma comprises a large number of strains found in multitude of ecological niches. Some of them have been developed as biocontrol agents against plant diseases, which can antagonize plant pathogens and facilitate the growth of plants at the same time, showing an important value for biocontrol in agriculture. Carbon catabolite repression (CCR) is a wide-domain regulatory circuit, allowing the preferred assimilation of carbon sources of high nutritional value over others. CCR is mediated by the Cys2His2 type transcription factor CreA/CREI which has been cloned from numerous filamentous fungi. The latest studies have found that CRE1, the carbon catabolite repressor, regulates cell growth and metabolic procedures of Trichoderma spp., which would be helpful for strains’ antagonism against various pathogens and their own survival at different habitats.On basis of wide type strain T.atroviride 23, T23 and its cre1 knock out mutant strain T23 Δcre1 kept in laboratory, the phenotypic assays and function analysis of both strains have been carried out on different mediums to draw the following conclusions. Firstly, the cre1 gene in Trichoderma is essential for the growth and sporulation of T.atroviride. When cre1 is knocked out, the growth rate of T23 Δcre1 slows down, and its sporulation apparently lags behind that of T23 with the number of spores reduced an order of magnitude; Furthermore, the response of T23 Δcre1 to carbon sources and nitrogen sources are diffirent from those of T23. The results further indicate cre1-mediated regulation of growth and formation of sporulation depends on specific culture. Secondly, how CRE1 regulates the expression of some cell-wall degraditon enzymes, such as chitnase, β-1,3-glucanase, protease and cellulase, is detected. Results of enzyme activity assay on plate and qRT-PCR show that CRE1 represses the expression of the chitianse and β-1,3-glucanase obviously. In line with that, transcription activity of two enzyme activity encoding genes, chit42 and glucanase, respectively increase by 2 times and 4 times in T23Δcre1 compared with in T23. Enzyme activity comparison of cellulase and protease conducted on plate also indicates a higher activity level in T23Δcre1 rather than in T23. Finally, the regulation of CRE1 on the expression of several non-ribosomal peptides synthases (NRPSs) and polyketide synthases (PKSs), which are found in charge of the synthesis of most valuable secondary metabolies for biocontrol, are deterimined. According to results of qRT-PCR, CRE1 plays a complicate role in the regulation on the transcription of NRPSs and PKSs. Among the three choosen NRPS encoding genes, the knock out of cre1 results in an increase of 8.72-fold expression of NRPS2, while NRPS1 expression is reduced to 1/2 and expression of NRPS3 is not significantly changed. Also, expression of PKS3 coding conidia pigment formation in T23Δcre1 increases by 3 times compared with that in T23,and PKS1 expression is detected being inhibited in T23. Thus, CRE1 indirectly affects the synthesis and secretion of Trichoderma secondary metabolites.In conclusion, carbon catabolite repressor CRE1 regulates mycelia growth, secondary metabolites biosynthesis, which is exactly of great importance in biocontrol procedure of T.atroviride. Those findings are pretty valuable for controlling synthesis and secretion of metabolites of T.atroviride and further to improve its biocontrol effects.
Glycosyltransferase Alg11, which is an important protein in N-glycosylation pathway, transfers the mannose moiety from GDP-Man to DPGn2M3 (Dolichyl-pyrophosphate-GlcNAc2Mannose3), forming DPGn2M4 and DPGn2M5 LLO (lipid-linked oligosaccharide) precursors. The structural analysis of Saccharomyces cerevisiae Alg11 showed the prediction of a hydrophobic N-terminal transmembrane domain. Thus, truncated Alg11 lacking the first 44 amino acid was designed and successfully overexpressed in Escherichia coli. The induction time and inducer concentration were optimized and the recombinant protein Alg1145-548 was purified. After the transferase activity assay, reaction mixture was applied to the liquid chromatography tandem mass spectrometry (LC-MS), which showed Alg1145-548 was capable to generate PPGn2M5 (Phytanyl-pyrophosphate-GlcNAc2Mannose5) from substrate PPGn2M3. Structural analysis of Gn2M5 showed the newly formed two glycosidic bonds could be cleaved by α-1,2 mannosidase, meaning the two mannose moieties were attached to Gn2M3 by α-1,2 linkages. Substrate specificity assay indicated the recombinant Alg11 specifically recognized PPGn2M3 rather than other LLOs, such as PPGn2 and PPGn2M1. Additionally, oligosaccharide Gn2M3 was not elongated by Alg1145-548, suggesting the lipid chain in the substrate PPGn2M3 was critical for the recognition. The achievement of active Alg11 provides an effective tool for producing Gn2M5, as well as for the further investigation of kinetic and mechanistic features of related mannosyltransferases.
Cholesterol oxidase, which catalyzes the reaction of cholesterol to cholest-4-en-3-one, is widely used in clinical and food processing industry. Cholesterol oxidase from Pimelobacter simplex (PsCO4) was transformed into E.coli BL21(DE3), Rosetta(DE3), and C41(DE3). The bacteria were induced at different temperatures (15℃, 25℃, 37℃), and different IPTG concentrations (0.01mmol/L, 0.1mmol/L, 0.5mmol/L). The results showed that PsCO4 was expressed greatly in supernatant of Rosetta(DE3) (0.63mg/ml), at the conditions of 0.1mmol/L IPTG and 15℃. The optimum temperature and pH of heterologous expressed PsCO4 was 30℃ and 7.5. The product of cholesterol catalyzed by PsCO4 was identified by TLC and GC-MS. The substrate specificity of PsCO4 towards cholesterol, β-sitosterol, stigmasterol and pregnenolone was determined. The Km/kcat value of cholesterol (0.08s -1·μM -1) is higher than β-sitosterol (0.04s -1·μM -1), stigmasterol (0.005s -1·μM -1) and pregnenolone (0.02s -1·μM -1).
Objective: To study the effects of pH on the production cyclic β-1,2-glucan, and analyze the structure of cyclic glucans under pH control. Methods:Rhizobium radiobacter ATCC 1333 was used to analyze the effects of pH on fermentation. The cyclic glucans were separated, purified and purified, then analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-MS), monosaccharide composition analysis, electrospray ionization mass spectrometry(ESI-MS), fourier transform infrared spectrometer(FTIR) and nuclear magnetic resonance spectroscopy(NMR). Results: The two stage pH strategy was developed in which pH was controlled at 7.0 for the cell growth and then shift to 5.5. The results showed that biomass was increased by 102% and the cyclic glucan concentration increased by 52%, and the fermentation color was controlled at the background level. Also, the glucans from Rhizobium radiobacter ATCC 1333 were cyclic glucans consisting of glucose by β-1,2 linkages with a degree of polymerization ranging from 17~22, mainly 19. Conclusion: The pH controlled at 7.0 has no effect on the structure of cyclic β-1,2-glucans, and provides a theoretical basis for the fermentation optimization and reliable source of cyclic β-1,2-glucans.
Objective: Construct YOD1 gene knockout mice based on CRISPR/Cas9 technology. Methods: Design and synthesize single-guide RNA (sgRNA) according to the YOD1 sequence in Genbank. Cas9 and sgRNA are transcribed to RNA in vitro, these RNA are then microinjected into zygotes of mice. The genotype is analyzed by PCR and sequencing. After YOD1 heterozygotes self-crossing and analysis of genotype of live offspring at weaning, wild type(WT)and knockout genotype(KO)littermates of YOD1 gene are verified. It is recorded that quantity and ratio of each genotype of live offspring of YOD1 heterozygotes self-crossing. And it is evaluated whether the ratio is in agreement with Mendel’s law of segregation. Protein lysates are made from main organs of the WT and KO littermates. And western blotting is used to assay the expression of YOD1 protein of these tissues. Meanwhile, size and weight of main organs and tissues of KO and WT mice are compared. Then analyze pathological phenotype of liver by H.E. staining. The glucose tolerance test (GTT) are carried out on the male mice of 6 months old. Results: According to PCR analysis and sequencing results, it is chose that mouse with deletion mutation and frameshift mutation in exon 2 of YOD1 gene to breed. After YOD1 heterozygotes self-crossing, WT and KO littermates are generated. According to statistics results, it is in agreement with Mendel’s law of segregation that the ratio of live offspring. Therefore, it is suggested that YOD1 KO mice birth normally without embryonic lethality. Western blotting results show that the expression of YOD1 in main organs is knocked-out significantly. Liver of YOD1 KO mouse is smaller in size than of WT littermate. There is no significant pathological phenotype in liver of YOD1 KO mice. YOD1 KO mice have general glycemic control in a GTT as compared to the control mice. Conclusions: YOD1 gene knockout mice are constructed successfully on CRISPR/Cas9 technology. And YOD1 KO mice birth and live normally without embryonic lethality. Compared to the control mice, livers of YOD1 KO mice are smaller in size and YOD1 KO mice have general glycemic control.
Objective:To establish a mouse model that can trace the differentiation of CD133 positive neural stem cells.Methods:The CD133-Cre-ERT2 transgenic mice were mated with Rosa26-CAG-LSL-ZsGreen transgenic mice to produce CD133-CreER;CAG-ZsGreen double transgenic in C57B16 mice.Result:The immunohistochemistry and laser scanning confocal imaging analysis showed that Tamoxifen-Inducible Cre/loxP recombination, as depicted by green cells, existed in lateral ventricle SVZ, the ventricular zone of third ventricle and the fourth ventricle in adult mice, and the green fluorescence overlaps with CD133+ red fluorescence in these regions.Conclusion:CD133+ is the sign of the resting nerve stem cell in the ventricle membrane, so it can trace the cell differentiation lineages of neural stem cells by analyzing ZsGreen positive cells in CD133-CreER;CAG-ZsGreen mice. The mouse model for in vivo lineage tracing of endogenous CD133+ cell was established successfully, which will be helpful to explore the activation, proliferation, migration and differentiation of CD133+ neural stem cells in the brain.
Five different kinds of basal media were screened in batch mode to find out the optimal basal medium for cell growth of Sp2/0. The peak viable cell density in batch mode was 13.12×10 6cells/ml, and the culture duration was 7 days. Various cell culture conditions including different shaking speeds, concentrations of carbon dioxide, glutamine replaced by GlutaMAX TM, addition of trace element and different culture temperatures were studied in batch mode as well. Few different cell growth were found with these conditions except for culture temperatures, which leaded to different peak viable cell concentrations, different viabilites and then different culture durations. Fourteen kinds of combinations of feed media were screened in fed-batch mode. The peak viable cell densities were up to 30×10 6cells/ml, the culture duration were around 9 days, and the highest daily Mab-A production was 27.20mg/L. Batch re-feed mode was used in the third part of the study. The peak viable cell density was 50.42×10 6cells/ml, the culture duration was 14 days, and the highest daily Mab-A production (141.10mg/L) was 5.19 fold greater than in fed-batch mode. These studies suggest batch re-feed mode is the optimal scale-down mode for cell growth and Mab-A production of Sp2/0 cells.
It is well known that glucose and fatty acids are key metabolic substrates for pancreatic β-cells. Pancreatic β-cells secrete insulin by glucose stimulating to keep blood glucose levels within a homeostatic range. When insulin resistance occurs, β-cells metabolic flexibility has become the first victim of pancreatic β-cells dysfunction. Compensation to decompensation of pancreatic β-cells is the key for the development of obesity insulin resistance to type 2 diabetes. Nuclear transcription factor FoxO1 (forkhead box O1) belongs to the Fox family, which is widely expressed in pancreatic β-cells. FoxO1 is a key regulator of the insulin-signaling pathway, and is reported to play important roles in pancreatic β-cells differentiation, proliferation, apoptosis and stress resistance. In view of the key role of FoxO1 in the maintenance of pancreatic β-cells function, an overview focused on the role of FoxO1 in the impaired metabolic flexibility and decompensation progress of pancreatic beta cells is provided.
Widely conserved iron-storage protein plays crucial role in ferric ion metabolism maintaining iron homeostasis, resisting oxidative stress and eliminating other toxic effects of excessive metal ions. With gaining the knowledge from structural and molecular studies, iron-storage protein complex in which iron cluster formed from absorbing free iron has been successfully applied in fields of biomedical engineering, nanomaterial, and biomolecule imaging. Herein, the recent progresses in studying the catalytic mechanism of iron cluster formation are briefly introduced. And the cutting-edge bioengineering applications in which iron-storage protein were engineered as a versatile molecular scaffold for presenting special chemicals, or natural magnetic particle for constructing remote control molecular machine are reviewed, although related researches has arisen academic argument.
As important components of cell membrane,bacterial lipoproteins play critical roles in many physiological process of bacteria, such as nutrient acquisition, environmental sensing, maintaining cell envelope and cell wall stability and electron transfer. With the continuous development and improvement of biotechnology, more and more lipoprotein as well as their functions have been discovered. This paper reviews the research progress of lipoprotein functions, biosynthesis and applications in Gram-positive bacteria. The influence of Lgt and LspA, key enzymes in lipoprotein biosynthesis, on physiological activity of Gram-positive bacteria is also addressed. Finally, the prospect and suggestion are provided for the future research on Gram-positive bacteria lipoproteins.
The EU has been at the forefront of the world in risk prevention,Its legal regulation of genetically modified organisms has always pursued the precautionary principle.Under the guidance of this principle,the EU has set up a very stringent legal framework for the regulation of GMOs and applies the principle to all aspects of the regulation of GMOs.Limited by the abstractness,fuzziness and other defects of the precautionary principle,the principle in EU regulation of GMO has causes some problems such as:member states abused protective measures and EU international trade frictions intensified,which has made the application of the precautionary principle in dilemma.The EU is trying to get out of the dilemma by law amendment and Court of Justice of the European Union’s judicial interpretation,and make the precautionary principle more effective.Our country has a similar understanding and application of the precautionary principle as the EU, and the existing legal framework is too harsh on the regulation of GMOs.Drawing on the experience of other countries and based on the needs of the local community,we should,at present,fully understand the precautionary principle and re-examine the relationship between science and law and appropriately relax the legal supervision over GMOs.
In order to study the maturity of cellulose pretreatment technology, the maturity of main pretreatment technologies such as steam explosion method, acid treatment method, alkali treatment method and biological method was studied based on multi-source literature data using Fisher-Pry model. The results show that the steam explosion method, acid pretreatment method, and alkali pretreatment method have developed rapidly since 2005 and are currently at a mature stage. However, due to the fact that these methods have inhibitory products, hydrolysis corrosion and high cost, it is difficult to make further breakthroughs. The biological method is still in a period of rapid growth and has great prospects. After the Fisher-Pry curve fitting, it is expected that the biological method will reach maturity around 2043 based on Fisher-Pry model.
