Xueliang Li , Juntao Qian , Jin Liu , Jun Fang , Jian Chen
2020, 36(11):2241-2249. DOI: 10.13345/j.cjb.200162 CSTR: 32114.14.j.cjb.200162
Abstract:Micro- and mini-bioreactors are characterized by their miniature working volume and comprehensive monitoring of process data, e.g., biomass, pH, dissolved oxygen, and fluorescence that are on par with conventional bench-top systems. The technical advancements of micro- and mini-bioreactors are supported by single-use material and micro-manufacturing, non-invasive optical sensors, automation such as industrial robotics and the integration of design of experiment software with data acquisition and process control. Owing to the miniature scales, micro-bioreactors typically feature lower turbulence intensity and energy dissipation rate, resulting in different mass transfer, mixing and shear conditions as compared to industrial scale equipment. Mini-bioreactors, nevertheless, are closer to large vessels. Micro- and mini-bioreactors are used mostly in screening and process development nowadays, owing to their combined high throughput and richness of data. They are also the hardware that will enable “precision medicine” in the near future.
Xiaomei Zhang , Yujie Gao , Ling Yang , Yi Yang , Ping Zheng , Jinbin Sun , Jinsong Shi , Zhenghong Xu
2020, 36(11):2250-2259. DOI: 10.13345/j.cjb.200154 CSTR: 32114.14.j.cjb.200154
Abstract:Amino acids are important compounds with a wide range of applications in the food, medicine and chemical industries. Corynebacterium glutamicum is a powerful workhorse commonly used in industrial amino acid production, with the scale of more than one million tons. In addition to its efficient anabolism, the effective exporters also ensure the high amino acid production by C. glutamicum. In this review, the research progress of amino acid exporter of C. glutamicum is summarized, to provide the foundation for further improving amino acid production by C. glutamicum via metabolic engineering.
Xutao Zhang , Lefan Ma , Kunpeng Zhou , Hongbing Li , Faxing Qin , Wufei Xie , Yingsheng Xu
2020, 36(11):2260-2276. DOI: 10.13345/j.cjb.200153 CSTR: 32114.14.j.cjb.200153
Abstract:Dissolving pulp consists of high purity cellulose and is widely used to as raw materials for the production of regenerated cellulose fiber, cellulose ester and cellulose ether. The characteristic of dissolving pulp affects greatly the production and processing performance of subsequent products. The α-cellulose content, hemicellulose content, pulp viscosity, ash, transition metal ion content, fiber morphology, molecular weight distribution of cellulose and the reactivity are the important properties. Because of its green, mild and high efficiency, the application of enzymes in improving the properties of dissolving pulp has a promising application prospect and has been researched significantly. In this review, the main properties of dissolving pulp are presented first, followed by a recommendation of the enzymes to improve these properties. The application and current research of cellulase and xylanase in improving the properties of dissolving pulp are emphasized. The main problems and the future research areas in improving the properties of dissolving pulp by enzymes are revealed. Finally, the technology prospects in this field are proposed.
Ruolin Hu , Chao Yuan , Yi Niu , Qinglin Tang , Dayong Wei , Zhimin Wang
2020, 36(11):2277-2286. DOI: 10.13345/j.cjb.200125 CSTR: 32114.14.j.cjb.200125
Abstract:MYB transcription factor is one of the largest transcription families and involved in plant growth and development, stress response, product metabolism and other processes. It regulates the development of plant flowers, especially anther development, a key role in the reproduction of plant progeny. Here, we discuss the regulatory effects of MYB transcription factors on the development of anther, including tapetum development, anther dehiscence, pollen development, carbohydrates and hormone pathways. We provide a reference for the further study of the regulation mechanism and network of plant anther development.
Lulu Huang , Yufeng Gu , Cuirong Wu , Junhong Huang , Guyue Cheng
2020, 36(11):2287-2297. DOI: 10.13345/j.cjb.200144 CSTR: 32114.14.j.cjb.200144
Abstract:Overuse of antibiotics in medical care and animal husbandry has led to the development of bacterial antimicrobial resistance, causing increasingly more health concern. In addition to genetic mutations and the formation of resistance, the various stresses bacteria encountered in the natural environment trigger their stress responses, which not only protect them from these stresses, but also change their tolerance to antimicrobials. The emergence of antimicrobial tolerance will inevitably affect the physiological metabolism of bacteria. However, bacteria can restore their sensitivity to drugs by regulating their own metabolism. This article reviews recent studies on the relationship between bacterial stress responses or the physiological metabolism and antimicrobial tolerance, intending to take more effective measures to control the occurrence and spread of antimicrobial resistance.
Zhenghao Fu , Meihua Su , Xiaoping Liu , Yunyu Chen
2020, 36(11):2298-2312. DOI: 10.13345/j.cjb.200132 CSTR: 32114.14.j.cjb.200132
Abstract:Polo-like kinase 1 (Plk1) is widely regarded as one of the most promising targets for cancer therapy due to its essential role in cell division and tumor cell survival. At present, most Plk1 inhibitors have been developed based on kinase domain, some of which are in clinical trial. However, inhibitors targeting kinase domain face off-target effect and drug resistance owing to the conserved nature and the frequent mutations in the ATP-binding pocket. In addition to a highly conserved kinase domain, Plk1 also contains a unique Polo-Box domain (PBD), which is essential for Plk1’s subcellular localization and mitotic functions. Inhibitors targeting Plk1 PBD show stronger selectivity and less drug resistance for cancer therapy. Therefore, Plk1 PBD is an attractive target for the development of anti-cancer agents. In this review, we will summarize the up-to date drug discovery for targeting Plk1 PBD, including the molecular structure and cellular functions of Plk1 PBD. Small-molecule inhibitors targeting Plk1 PBD not only provide an opportunity to specifically inhibit Plk1 activity for cancer treatment, but also unveil novel biological basis regarding the molecular recognition of Plk1 and its substrates.
Beining Xu , Yinkun Liu , Yu’an Xie
2020, 36(11):2313-2326. DOI: 10.13345/j.cjb.200129 CSTR: 32114.14.j.cjb.200129
Abstract:Glycosylation is one of the common post-translational modifications of proteins to regulate the ability of tumor invasion, metastasis and tumor heterogeneity by interacting with glycan-binding proteins such as lectins and antibodies. Glycan microarray can be constructed by chemical synthesis, chemical-enzyme synthesis or natural glycan releasing. Glycan microarray is an essential analytical tool to discover the interaction between glycan and its binding proteins. Here we summarize the standard techniques to construct glycan microarray for the application in cancer vaccine, monoclonal antibody and diagnostic markers.
2020, 36(11):2327-2333. DOI: 10.13345/j.cjb.200147 CSTR: 32114.14.j.cjb.200147
Abstract:Therapeutic antibody drugs have achieved great success in clinical practice. However, their efficacy and safety still need to be improved. At the same time, excessive concentration of drug targets will cause problems such as repeated development and waste of resources. Therefore, pharmaceutical companies need to explore differentiated discovery strategies when researching antibody drugs in order to survive and develop in the fierce market competition. In this paper, the differential development strategy of therapeutic antibody drugs is discussed from the aspects of drug sources and formats, drug target selection, drug mechanism and differential drug characteristics.
Shihong Tian , Xiang Wang , Hongmei Li , Li Bai , Hong Liu , Xibin Zhang , Qingli Dong
2020, 36(11):2334-2344. DOI: 10.13345/j.cjb.200158 CSTR: 32114.14.j.cjb.200158
Abstract:Strain variability is one of the most important factors to influence the accuracy of foodborne pathogens risk assessment, such as Listeria monocytogenes, Salmonella spp. Strain-to-strain variation is defined as the inherent differences among identically treated strains of the same microbial species. The differences cannot be eliminated by changing test methods or improving test protocols. This review addresses presently related studies of strain variability. Based on the effect of strain variability on the outcome of risk assessment, we summarize sources of variabilities in food chain, strain phenotypic variabilities and the methods to integrate strain variability in growth and inactivation into predictive modelling, and indicate the inadequacies in the study of strain variability. We suggest further study the mechanism of strain variability, expand the comparison of variability among different sources, and integrate the variability of gene expression, protein and cell metabolism into the predictive modelling.
Debin Zhang , Yao Luo , Wenjin Chen
2020, 36(11):2345-2356. DOI: 10.13345/j.cjb.190557 CSTR: 32114.14.j.cjb.190557
Abstract:As the breakthrough in gene editing, represented by CRISPR/Cas9, gene manipulations now are more maneuverable, economically feasible and time saving. It is possible for China to catch an overtaking in researching and industrializing of downside sections (especially the application of plant gene editing), also the incubation of professional companies in gene editing fields. For this consideration, it is necessary and urgent to find the key demands and potential application for gene editing in China. Questionnaire and statistic analysis were carried out to find the key demands and the most potential application fields of the development for gene editing. Firstly, an ordered multi-classification Logistic regression model was established following with dependent variable analysis. Eight out of 24 questionnaires questions in 4 categories were regarded as independent variables with significance test. Then, regression model based on ordered multi-classification logistic method was established to analyze the specific impact of different options on the development of gene editing. The results showed that most researchers in the field of gene editing take the view that development of potential competitive advantages lies in the field of plant science. The results also showed that major gene editing experts believe more attention should be paid on how to carry out technology industrialization while focusing on basic technology development, as well as the development of potential competitive advantages of gene editing technology in plant field. To promote the development of gene editing in China, not only the participation of scientific research institution was needed, but also the synergy of various forces both universities and governments. It is urgent both properly guiding public opinion on gene editing and establishing a national safety standard system. At the same time, the key point of technology risk avoidance should be put on biological weapons and bioterrorism, gene editing related infectious disease, and the potential risk of species genetic change on the ecological environment, etc.
Yuanfang Fu , Wei He , Pu Sun , Lin Yang , Huifang Bao , Yimei Cao , Xingwen Bai , Pinghua Li , Dong Li , Yingli Chen , Lei Liu , Zengjun Lu , Zaixin Liu
2020, 36(11):2357-2366. DOI: 10.13345/j.cjb.200149 CSTR: 32114.14.j.cjb.200149
Abstract:Antigenic purity is important for quality control of the foot-and-mouth (FMD) whole virus inactivated vaccine. The recommended method for evaluation the antigenic purity of FMD vaccine is to check the serum conversion to non-structural protein (NSP) 3AB antibody after 2 to 3 times inoculation of animals with inactivated vaccine. In this study, we developed a quantitative ELISA to detect the amount of residual 3AB in vaccine antigen, to provide a reference to evaluate the antigenic purity of FMD vaccine. Monoclonal antibody (Mab) of NSP 3A and HRP-conjugated Mab of NSP 3B were used to establish a sandwich ELISA to quantify the NSP 3AB in vaccine antigen of FMD. Purified NSP 3AB expressed in Escherichia coli was serially diluted and detected to draw the standard curve. The detectable limit was determined to be the lowest concentration of standard where the ratio of its OD value to OD blank well was not less than 2.0. Results: The OD value was linearly corelated with the concentration of 3AB protein within the range between 4.7 and 600 ng/mL. The correlation coefficient R2 is greater than 0.99, and the lowest detectable limit is 4.7 ng/mL. The amount of 3AB protein in non-purified inactivated virus antigen was detected between 9.3 and 200 ng/mL depending on the 12 different virus strains, whereas the amount of 3AB in purified virus antigen was below the lowest detectable limit. The amount of 3AB in 9 batches of commercial FMD vaccine antigens was between 9.0 and 74 ng/mL, whereas it was below the detectable limit in other 24 batches of commercial vaccine antigens. Conclusion: the sandwich ELISA established in this study is specific and sensitive to detect the content of 3AB protein in vaccine antigen of FMD, which will be a useful method for evaluation of the antigenic purity and quality control of FMD inactivated vaccine.
Yanan Huo , Fengli Wu , Guotian Song , Ran Tu , Wujiu Chen , Erbing Hua , Qinhong Wang
2020, 36(11):2367-2376. DOI: 10.13345/j.cjb.200122 CSTR: 32114.14.j.cjb.200122
Abstract:p-coumaric acid is an important natural phenolic compound with a variety of pharmacological activities, and also a precursor for the biosynthesis of many natural compounds. It is widely used in foods, cosmetics and medicines. Compared with the chemical synthesis and plant extraction, microbial production of p-coumaric acid has many advantages, such as energy saving and emission reduction. However, the yield of p-coumaric acid by microbial synthesis is too low to meet the requirements of large-scale industrial production. Here, to further improve p-coumaric acid production, the directed evolution of tyrosine ammonia lyase (TAL) encoded by Rhodotorula glutinis tal gene was conducted, and a high-throughput screening method was established to screen the mutant library for improve the property of TAL. A mutant with a doubled TAL catalytic activity was screened from about 10,000 colonies of the mutant library. There were three mutational amino acid sites in this TAL, namely S9Y, A11N, and E518A. It was further verified by a single point saturation mutation. When S9 was mutated to Y, I or N, or A11 was mutated to N, T or Y, the catalytic activity of TAL increased by more than 1-fold. Through combinatorial mutation of three types of mutations at the S9 and A11, the TAL catalytic activity of S9Y/A11N or S9N/A11Y mutants were significantly higher than that of other mutants. Then, the plasmid containing S9N/A11Y mutant was transformed into CP032, a tyrosine-producing E. coli strain. The engineered strain produced 394.2 mg/L p-coumaric acid, which is 2.2-fold higher than that of the control strain, via shake flask fermentation at 48 h. This work provides a new insight for the biosynthesis study of p-coumaric acid.
Miaomiao Wang , Huimin Yu , Xin He , Yanmei Li , Huaiyu Yang
2020, 36(11):2377-2386. DOI: 10.13345/j.cjb.200156 CSTR: 32114.14.j.cjb.200156
Abstract:Surfactin has great potential applications in enhancing oil recovery, agriculture, pharmaceuticals, foods and beverages, and cosmetics due to its extraordinary surface activity, biodegradability, anti-bacterial activity and biocompatibility. Enhancing surfactin production by engineering surfactin-producer and optimizing culture conditions is the key of its industrial production and subsequent applications. In this study, the effect of fatty acid synthesis pathway on surfactin synthesis was investigated, and Bacillus subtilis THBS-2 and THBS-8 with high surfactin titer were constructed by overexpressing key genes involved in the fatty acid synthesis pathway. To optimize culture condition, the amount and adding time of isopropyl-beta-D-thiogalactopyranoside (IPTG) and amino acids were studied, and a two-stage culture method was obtained: IPTG (final concentration: 1.25 mmol/L) and leucine (final concentration: 5 g/L) were added at 3 h, leucine (final concentration 5 g/L) and condensed culture medium (5 mL) were added at 24 h. Applying this strategy, the surfactin titer of B. subtilis THBS-2 reached to 24 g/L in shake flask at 48 h and up to 34 g/L after 68 h fermentation in a 30-L fermentor. The results provide basis for large-scale production and broad application of surfactin.
Fengli Wu , Yanxia Liang , Yuanyuan Zhang , Yanan Huo , Qinhong Wang
2020, 36(11):2387-2397. DOI: 10.13345/j.cjb.200157 CSTR: 32114.14.j.cjb.200157
Abstract:Recently, fast-growing Vibrio natriegens, as the great potential chassis, has shown a wide application in synthetic biology. Genome editing is an indispensable tool for genetic modification in synthetic biology. However, genome editing tools with high efficiency and fidelity are still to be developed for V. natriegens synthetic biology. To deal with this problem, the physiological characteristics of 6 V. natriegens strains were evaluated, and CICC 10908 strain with fast and stable growth was selected as the host strain for genome editing study. Then, the natural transformation system of V. natriegens was established and optimized. The efficiencies of optimized natural transformation that integrates antibiotic resistance marker cat-sacB or KanR onto the chromosome of V. natriegens could reach 4×10–5 and 4×10–4, respectively. Based on the optimized natural transformation, a double-selection cassette was used to achieve seamless genome editing with high efficiency and fidelity. The positive rates of four different types of genetic manipulation, including gene deletion, complementation, insertion and substitution, were 93.8%, 100%, 95.7% and 100%, respectively. Finally, transformation and elimination of the recombinant plasmid could be easily achieved in V. natriegens. This work provides a seamless genome editing system with high efficiency and fidelity for V. natriegens synthetic biology.
Yuanping Xiang , Yuntong Huang , Hongjun He , Qijiang Xu
2020, 36(11):2398-2412. DOI: 10.13345/j.cjb.200422 CSTR: 32114.14.j.cjb.200422
Abstract:The E class MADS-box genes SEPALLATA (SEP)-like play critical roles in angiosperm reproductive growth, especially in floral organ differentiation. To analyze the sequence characteristics and spatio-temporal expression patterns of E-function MADS-box SEP-like genes during kale (Brassica oleracea L. var. acephala) flower development, BroaSEP1/2/3 (GenBank No. KC967957, KC967958, KC967960) homologues, three kale SEP MADS-box gene, were isolated from the kale variety ‘Fourteen Line’ using Rapid amplification of cDNA ends (RACE). Sequence and phylogenetic analysis indicated that these three SEP genes had a high degree of identity with SEP1, SEP2, SEP3 from Brassica oleracea var. oleracea, Brassica rapa, Raphanus sativus and Brassica napus, respectively. Alignment of the predicted amino acid sequences from these genes, along with previously published subfamily members, demonstrated that these genes comprise four regions of the typical MIKC-type MADS-box proteins: the MADS domain, intervening (I) domain and keratin-like (K) domain, and the C-terminal domain SEPⅠ and SEPⅡ motif. The longest open reading frame deduced from the cDNA sequences of BroaSEP1, BroaSEP2, and BroaSEP3 appeared to be 801 bp, 759 bp, 753 bp in length, respectively, which encoded proteins of 266, 252, and 250 amino acids respectively. Expression analyses using semi-quantitative RT-PCR and quantitative real-time PCR indicate that BroaSEP1/2/3 are specifically expressed in floral buds of kale during flower development process. The expression levels?of the three genes are very different at different developmental stages, also in wild type, mutant flower with increased petals, and mutant flower with decreased petals. These?different patterns of gene expression maybe cause the flowers to increase or decrease the petal number.
Yun Ma , Tingting Li , Riyue Feng , Guijie Guo , Qidong Pan , Jianning Li , Jilong Chen
2020, 36(11):2413-2423. DOI: 10.13345/j.cjb.200015 CSTR: 32114.14.j.cjb.200015
Abstract:Human acute leukemia (AL) is a clonal malignancy with abnormal hematopoietic stem cells. Clinically, AL is very difficult to cure due to its sudden onset and short course of disease progression. Previous studies have shown that eukaryotic initiation factor 4B (eIF4B) plays a critical role in the development of chronic leukemia. However, the involvement of eIF4B in human acute leukemia is still largely unknown. Therefore, we studied eIF4B function and its regulatory mechanism in human acute leukemia. We found that phosphorylation levels of eIF4B in acute leukemia cells were significantly reduced in response to treatment with either LY294002 (PI3K inhibitor), AKTi (AKT inhibitor) or SMI-4A (Pim inhibitor). Co-treatment with inhibitors targeting JAK/STAT5/Pim and PI3K/AKT/mTOR signaling dramatically promoted apoptosis of acute leukemia cells by downregulating eIF4B phosphorylation. Furthermore, in vitro and in vivo functional experiments showed that eIF4B played an important anti-apoptosis role in the acute leukemia cells by regulating the expression of anti-apoptotic proteins Bcl-2 and Bcl-XL. In contrast, silencing eIF4B inhibited the growth of acute leukemia cells as engrafted tumors in nude mice. Taken together, our results indicate the synergistic role of JAK/STAT5/Pim and PI3K/AKT/mTOR signaling pathways in regulating eIF4B phosphorylation in acute leukemia, and highlight eIF4B as a candidate therapeutic target for treatment of acute leukemia.
Xiaoqin Shu , Xiaomei Hu , Jian Zheng , Jie Li , Juan Zhang
2020, 36(11):2424-2434. DOI: 10.13345/j.cjb.200135 CSTR: 32114.14.j.cjb.200135
Abstract:This study intends to obtain recombinant proteins of ALT1 and ALT2 isozymes by using genetic recombination technology. Monoclonal antibodies ALT1 and ALT2 with high specificity and high activity were prepared and screened (ALT1 monoclonal antibody has been successfully prepared and published). The localization, distribution and expression of ALT1 and ALT2 isozymes in human tissues were discussed. The ALT2 genes were amplified from human liver cancer cell (HepG2) by RT-PCR method. The mature ALT2 gene was subcloned into the pET32a-ALT2 prokaryotic expression vector. Its ligation product was transformed into BL21(DE3) competent cells, and transformed into competent cells to express ALT2 proteins induced by IPTG. The recombinant proteins of ALT2 were purified by nickel column (Ni+) affinity chromatography. Balb/c mice were immunized with recombinant proteins of ALT2. Positive serum mouse spleen cells and myeloma cells SP2/0 were selected for cell fusion. The positive cell lines were selected by indirect ELISA and subcloned by limited dilution method. Affinity chromatography was used to purify ALT2 antibodies. The expression and distribution of ALT2 in human normal tissues were detected by RT-PCR and Western blotting. Results show that the expression of ALT isoenzyme in tissues was almost the same at gene mRNA level and protein level. ALT1 is highly expressed in liver, kidney and skeletal muscle, and moderately expressed in gastrointestinal smooth muscle. ALT2 is highly expressed in fat, skeletal muscle and myocardium, and is poorly expressed in gastrointestinal smooth muscle. Immunohistochemical studies show that ALT1 is highly expressed in hepatocytes, renal medullary tubules and muscle fibers, ALT2 is highly expressed in adipocytes and myocardial cells, and ALT1 and ALT2 in gastrointestinal tissues are mainly expressed in mucosa of upper intestinal wall region. The results showed that the isoenzymes ALT1 and ALT2 were mainly expressed in the mucosa of the upper part of the intestinal wall. It is widely distributed in the tissues, providing theoretical basis for understanding the mechanism of ALT activity increase under different pathological conditions.
Hongli Liu , Yan Shen , Wenwen Gao , Haichuan Yu , Shoumin Xi , Guomin Shen
2020, 36(11):2435-2442. DOI: 10.13345/j.cjb.200128 CSTR: 32114.14.j.cjb.200128
Abstract:In recent years, mass spectrometry has been widely used to study membrane protein structure and function. However, the application of mass spectrometry to study integral membrane protein is limited because there are many hydrophobic amino acids in the trans-membrane domain of integral membrane protein to cause low sequence coverage detected by LC-MS/MS. Therefore, we used vitamin K epoxide reductase (VKORC1), a human integral membrane protein, as a model to optimize the digestion conditions of chymotrypsin, and developed an in-gel digestion method of chymotrypsin to improve sequence coverage of membrane protein by mass spectrometry. By exploring the effects of calcium concentration, pH value and buffer system on the percentage of sequence coverage, number of total detected and types of unique peptide, and the size of unique peptide, sequence coverage and peptide diversity could be considered under condition of Tris-HCl buffer with 5–10 mmol/L calcium ion concentration and pH value 8.0–8.5. This method could make the sequence coverage of membrane protein to reach more than 80%. It could be widely used in the study of membrane protein structure and function, identification of interaction site between membrane proteins, and identification of binding site between membrane protein and small molecular drug.
2020, 36(11):2443-2450. DOI: 10.13345/j.cjb.200146 CSTR: 32114.14.j.cjb.200146
Abstract:To establish a method for identifying protein epitopes recognized by therapeutic monoclonal antibodies, the programmed death receptor-1 (PD-1) was selected as the target protein. Based on the alanine scanning strategy, a rapid expression method of antigen mutants combining site-directed mutagenesis with mammalian cell expression system was established, the conditions for eukaryotic expression element amplification and cell transfection expression were established. 150 PD-1 protein mutants were co-expressed, and the binding ability of these mutants to anti-PD-1 antibody Pembrolizumab was identified. The epitopes of Pembrolizumab were determined based on the binding ability of protein mutants to antibodies and combined with protein structure analysis, which was highly consistent with the reported crystal structure-based epitopes, indicating that this method is simple and accurate and can be used for epitope mapping of therapeutic monoclonal antibodies.
Xiaohe Li , Jian Li , Wen Hou , Shen Zheng , Xiangdong Zhu
2020, 36(11):2451-2458. DOI: 10.13345/j.cjb.200160 CSTR: 32114.14.j.cjb.200160
Abstract:To screen strains with antibacterial and antitumor activity, pregnenolone was used as the sole carbon source for screening bacteria from soil. Based on bacteriostatic activity assay, Pseudomonas aeruginosa HBD-12 was found to be effectively inhibiting the growth of Escherichia coli, Bacillus thuringiensis, Penicillium digitatum and Penicillium italicum, and its fermentation broth was separated and purified using column chromatography. Then, structure of the obtained monomeric compounds was analyzed by spectrum analysis, and their antitumor activity was measured using HTRF kinase detection kit. The isolated monomeric compounds 1-hydroxy-9,10-phenanthroline and 3-hydroxy-9,10-dihydrophenanthroline had significant antitumor activity. At 20 μg/mL, 1-hydroxy-9,10-phenanthroline and 3-hydroxy-9,10-dihydrophenanthroline inhibited 78.39±2.29% and 60.34±8.35% Aurora kinase A, respectively. Therefore, the secondary metabolites of Pseudomonas aeruginosa HBD-12 have the potential to develop antibacterial and antitumor drugs.
Ying Wang , Wei Zhang , Yixuan Li , Fang Jiang , Tingting Huang , Xin Yu , Hongwei Zhu , Xingxiao Zhang
2020, 36(11):2459-2466. DOI: 10.13345/j.cjb.200171 CSTR: 32114.14.j.cjb.200171
Abstract:Salmonella enterica serovar Enteritidis (SE) is one of the most important zoonotic pathogens that cause enteritis and systemic infection in animals and human. Understanding invasive capacities of SE isolates is of vital importance to elucidate pathogenesis of Salmonella infection. To improve the throughput capacity and repeatability of classical gentamicin protection assay (GPA), a modified PGA was developed by taking high-throughput advantage of 96-well cell plates and multichannel pipettes. In addition, drop plate technique rather than spread plate method was applied in the modified GPA protocol for bacterial enumeration. The modified GPA protocol was evaluated by phenotyping intracellular replication of a high virulent and a low virulent SE isolates, JL228 and LN248, in a phagocytic cell line RAW264.7. The protocol was then applied in invasive phenotype determination of 16 SE strains to non-phagocytes (HT-29) and the intracellular replication of 43 SE strains to phagocytes (RAW264.7). Significant lower intra-group and inter-group coefficient of variations of the modified GPA was observed, implying good repeatability and reproducibility over traditional protocol. Further, replication phenotypes were also correlated with those from direct observation by confocal microscopy. Collectively, the improved GPA protocol had advantages of high throughput capacity, good repeatability and reliability, it was also noticed that the protocol also represented a fast and labor-saving alternative scheme for the invasive phenotype determination of Salmonella Enteritidis, and providing reliable phenotype profiles for Salmonella-host interplay interpretation.
Xueqing Xie , Yuqi Tian , Jinghuan Tian , Wenyan Ning , Chunmei Wang
2020, 36(11):2467-2477. DOI: 10.13345/j.cjb.200173 CSTR: 32114.14.j.cjb.200173
Abstract:The low expression rate of exogenous genes in cyanobacteria is one of the bottlenecks of cyanobacteria genetic engineering. The T7 RNA polymerase expression system has achieved the efficient expression of exogenous genes in Escherichia coli. Cyanobacteria and E. coli are both Gram-negative bacteria with high genetic homology. The construction of T7 RNA polymerase expression system in cyanobacteria may improve the expression of foreign genes. In order to construct the T7 RNA polymerase expression system in Anabaena sp. PCC 7120, methods such as overlapping extension PCR and digestion-ligation technique were used to construct a site-specific integration vector pEASY-T1-F1-TacT7RNAPCmR-F2 and a shuttle expression vector pRL-T7-hG-CSF. The site-specific integration vector is capable of expressing T7 RNA polymerase, and the shuttle expression vector expresses hG-CSF driven by the T7 promoter. Then we introduced the site-specific integration vector into the wild type cyanobacteria by electroporation and transferred the shuttle expression vector into the site-integrated transgenic cyanobacteria by triparental conjugative transfer. In the end, we identified the presence of foreign genes in cyanobacteria by PCR, tested the transcription level of foreign genes in cyanobacteria by RT-PCR, and detected the protein expression of foreign genes in cyanobacteria by Western blotting. The two vectors were successfully constructed, the T7 RNA polymerase gene and hG-CSF gene were transferred into cyanobacteria well, and both genes were also expressed in cyanobacteria. In summary, the T7 RNA polymerase expression system was successfully constructed in cyanobacteria, and the expression rate of hG-CSF gene was doubled than the traditional cyanobacteria expression systems. This expression system will provide a better tool for the application of cyanobacteria genetic engineering and will promote the development of cyanobacteria as a chassis cell in the fields of synthetic biology in the future.
Ting Zhang , Qing He , Zijun Xu , Feiya Suo , Chengwu Zhang , Qiang Hu
2020, 36(11):2478-2493. DOI: 10.13345/j.cjb.200264 CSTR: 32114.14.j.cjb.200264
Abstract:Filamentous microalga Tribonema sp. has the advantages of highly resistance to zooplankton-predation, easy harvesting, and high cellular lipid content, in particular large amounts of palmitoleic acid (PA) and eicosapentaenoic acid (EPA). Therefore, Tribonema sp. is considered as a promising biomass feedstock to produce biodiesel and high-value products. In this work, we studied the effect of different concentrations of nitrogen (NaNO3: 255–3 060 mg/L), phosphorus (K2HPO4: 4–240 mg/L), iron ((NH4)3FeC12H10O14: 0.6–12 mg/L) and magnesium (MgSO4: 7.5–450 mg/L) on the biomass, lipid content, and fatty acid composition of Tribonema sp. FACHB-1786, aiming at enhancing cell lipid productivity. The growth of Tribonema sp. had a positive correlation with the concentration of magnesium, and the maximum biomass of Tribonema sp. (under the condition of 450 mg/L MgSO4) was 8.09 g/L, much greater than those reported in previous studies using the same and other Tribonema species under autotrophic conditions. Different nitrogen concentrations exerted no significant effect on algal growth (P > 0.05), but a higher nitrogen concentration resulted in a greater amount of lipid in the cells. The maximum volumetric productivities of total lipids (319. 6 mg/(L·d)), palmitoleic acid (135.7 mg/(L·d)), and eicosapentaenoic acid (24.2 mg/(L·d)) of Tribonema sp. were obtained when the concentrations of NaNO3, K2HPO4, (NH4)3FeC12H10O14, and MgSO4 were 765 mg/L, 80 mg/L, 6 mg/L, and 75 mg/L, respectively. This study will provide a reference for substrate optimization for Tribonema sp. growth and lipid production.
Gang Li , Xiaomei Hu , Qiwen Hu
2020, 36(11):2494-2500. DOI: 10.13345/j.cjb.200120 CSTR: 32114.14.j.cjb.200120
Abstract:The Chinese national college students’ life science competition has been held for three times, with good organization, large scale and high participation degree. The competition plays an important role in promoting life science education and research. This paper reports the form and status of the competition, statistically analyses the registration data and competition results by region and year, based on the previous three competitions. By combining new changes and understanding in the field of life science, we also indicate prospects on how to better promote the competition.
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