Cellulosic ethanol, a low-carbon, clean and green energy, has broad application prospects. It can be mixed with traditional petroleum-based liquid fuels. The production of cellulosic ethanol goes through processes such as lignocellulose pretreatment, saccharification and Saccharomyces cerevisiae fermentation. However, the pretreatment process will produce many by-products, which significantly inhibit the growth rate and fermentation performance of yeast. Therefore, constructing inhibitor-tolerant yeast chassis cells can contribute to improving the production efficiency of cellulosic ethanol and reduce production costs. A review is conducted on the design and construction of inhibitor-tolerant yeast chassis cells, the mechanism of inhibitors, the methods of strengthening tolerant chassis cells, and means for mining inhibitor-tolerant genes. Finally, the latest progress in SCRaMbLE to improve yeast tolerance is discussed.
Sesquiterpenes, which belong to terpenes and have strong fragrance and excellent biological activity, can be used in the synthesis of flavors, biofuels and pharmaceuticals. At present, the common methods of obtaining sesquiterpenes in industry are chemical synthesis and plant extraction. Due to the inevitable problems of low yield, high cost and large pollution in common methods, researchers begin to pay attention to the research of microbial synthesis of sesquiterpenes and used metabolic engineering, enzyme engineering and methods of synthetic biology to construct microbial cell factories of Saccharomyces cerevisiae, which can produce various sesquiterpenes. The sesquiterpene synthesis pathway has been introduced and analyzed in the paper. Focusing on the accumulation of the acetyl-CoA, the improvement and modification of the mevalonate pathway, and the inhibition of competitive pathways, specific strategies and relative examples about the improvement and modification of the sesquiterpene synthesis pathway are reviewed. The research progress about the characterization and mutation of sesquiterpene synthases in recent years is summarized. Finally, the prospect and suggestions are proposed to improve the efficiency of sesquiterpene synthesis in Saccharomyces cerevisiae.
Saccharomyces cerevisiae is widely applied in fields like food, brewing, chemical industry and medicine. Based on constructed production line or demonstrated production line for Saccharomyces cerevisiae, the paper reviews the development process and essential technologies of yeast biomass manufacturing industry; the application of yeast biomass in wine brewing, functional food and food additives manufacturing field is introduced; the product manufacturing plan of yeast cell factory is summarized and the industrial development of products like bulk chemicals made by yeast cell factory, refined chemicals and biological fuels are introduced; all these provide reference to biological manufacturing from laboratory to industrial production.
Clostridium butyricum is an obligate anaerobic bacterium, which can be modified by multiple genetic manipulation methods such as multi-gene overexpression, homologous recombination, homologous recombination based on non-replicating plasmids and non-replicating plasmids synthesis direction. Butyric acid is one of the fermentation products of Clostridium butyricum. It has a wide range of uses. It is used as a feed additive to improve animal resistance and reduce the use of antibiotics.The yield of butyric acid in the fermentation of Clostridium butyricum is still low, which is not conducive to industrial production. It is necessary to optimize the way of producing butyric acid by Clostridium butyricum through metabolic engineering.This article reviews the research progress of Clostridium butyricum’s main metabolic pathways, genetic manipulation systems, and the optimization of butyric acid synthesis pathways. On this basis, the thoughts and ideas for further transformation of Clostridium butyricum were prospected.
Xanthan gum is an extracellular polysaccharide produced by genus Xanthomonas. It is widely used in food, petroleum, agriculture and other industries because of its superior rheology and stability. At present, the synthetic pathway of xanthan gum has been clarified, and its research mainly focuses on how to affect its synthesis and modification through molecular regulation to meet the needs of different industries.By introducing the primary and secondary structure, rheology, stability and biosynthetic pathway of xanthan gum, this paper summarizes the research progress on the molecular regulation of xanthan gum biosynthesis in Xanthomonas sp. The main conclusions are as follows: the existing research on molecular regulation focuses on the regulation of key genes in each stage of xanthan gum synthesis pathway, signal molecules and other factors; in the synthesis stage of xanthan gum precursor, xanthan gum production can be regulated by changing the expression of related genes involved in the conversion of glucose to phosphate sugar, the conversion of phosphate sugar precursor to nucleoside diphosphate, utilization and transport of intracellular carbohydrate; in the assembly and secretion stage of xanthan gum, the synthesis of xanthan gum can be regulated by regulating the structural proteins and promoter regulators of gum gene cluster; the regulation of signal molecule level in c-di-GMP signal network system and quorum sensing (QS) system can affect the synthesis and secretion of xanthan gum; other factors can also regulate the synthesis of xanthan gum, including genes related to lipopolysaccharide modified O-antigen, genes related to protein/metal transport and secretion, substrate competition pathways of peptidoglycan and polyhydroxyalkanoate (PHA), and hemoglobin genes. In the future, we will further explore new regulatory factors for xanthan gum biosynthesis and reveal the molecular regulatory mechanism.
Objective: To investigate the effects of UPF1 on the proliferation, migration and invasion of human breast cancer cells MDA-MB-231 and its possible mechanism. Methods: The role of UPF1 in breast cancer and the expression level of UPF1 in pan-cancer and breast cancer was evaluated by bioinformatics analysis. Breast cancer cells MDA-MB-231 and MCF-7 were transfected with siRNA. The experiment was divided into two groups: control group (transfected with siRNA negative control) and treatment group (transfected with siUPF1). The mRNA and protein levels of UPF1, MMP9, EMT-related makers were measured by qRT-PCR and Western blot; the proliferation of MDA-MB-231 and MCF-7 cells was detected by CCK-8 assay; the lateral migration ability was studied by wound healing assay; the longitudinal migration and invasion abilities were evaluated by Transwell migration and invasion assays. Results: Bioinformatics analysis showed that UPF1 was highly expressed in breast cancer and associated with immune cell infiltration processes and positively correlated with tumor suppressor genes. UPF1 was also highly expressed in breast cancer cells. After knockdown of UPF1, the mRNA and protein expression levels of UPF1 in MDA-MB-231 and MCF-7 breast cancer cells were significantly decreased (P<0.05, P<0.05). Furthermore, the proliferation, migration and invasion abilities of MDA-MB-231 and MCF-7 cells were significantly enhanced. The mRNA and protein levels of MMP9 and Vimentin were increased, but E-cadherin decreased. Conclusion: UPF1 is highly expressed but plays a cancer-inhibiting role in breast cancer. UPF1 may inhibit the proliferation, migration and invasion of breast cancer MDA-MB-231 and MCF-7 cells by inhibiting the EMT signaling pathway.
Objective: To explore the effects of miR-324-3p on prostate cancer (PCa) cell ferroptosis and its underlying mechanism. Methods: qRT-PCR was performed to detect the expression of miR-324-3p and glutathione peroxidase 4 (GPX4) mRNA in PCa tissues, matched adjacent tissues, and cell lines. Western blot was employed to examine the protein level of GPX4 in PCa cell lines and normal prostate epithelial cells. CCK-8 assay was used to evaluate cell proliferation. The levels of glutathione (GSH), lipid oxidation, and reactive oxygen species (ROS) were determined using GSH detection assay kit, lipid peroxidation MDA assay kit and DCFH-DA fluorescent probe assay. Dual-luciferase reporter gene was applied to verify the interaction of miR-324-3p and GPX4. Results: The expression of miR-324-3p in PCa tissues was lower than that in the corresponding paracancerous tissues, and the expression of miR-324-3p was downregulated in PCa cell lines compared with normal prostate epithelial cells. GPX4 was highly-expressed in PCa tissues and cell lines in comparison with the corresponding paracancerous tissues and normal prostate epithelial cells. Dual-luciferase reporter results showed that miR-324-3p was directly targeted to negatively regulate GPX4. Overexpression of miR-324-3p significantly inhibited PCa cell proliferation, reduced GSH production and enhanced the levels of lipid oxidation and ROS; while treatment with the ferroptosis inhibitor Fer-1 or GPX4 reversed the promotion effect of miR-324-3p on ferroptosis in PCa cells. Conclusion: miR-324-3p promoted ferroptosis in PCa cells by targeting negative regulation of GPX4 expression and thus exerted anticancer effects on PCa.
Objective: To construct the eukaryotic expression vector of NGFP-TERT and TPP1-CGFP genes, then verify whether TPP1-CGFP protein can be recruited to the telomere region, and observe the interaction between TERT and TPP1 through the spontaneous reconstruction of GFP protein. Methods: Using the corresponding plasmid as a template, the coding sequences of CDS regions of NGFP, CGFP and TPP1 were amplified by polymerase chain reaction (PCR) technology. NGFP was inserted into the pCDH-Flag-TERT vector by restriction digestion and recombination, and TPP1-CGFP was inserted into the pCDH-Myc-POT1-v5tag vector. After bacterial liquid PCR, vector enzyme digestion and sequencing verification, it was transfected into 293T cells, and its expression was detected by Western blot. Immunofluorescence and telomere Fish were used to verify whether TPP1-CGFP can be recruited to the telomere region. Plasmid co-transformation verified that it can be reassembled into GFP protein. Results: The results of double enzyme digestion showed that the NGFP-TERT and TPP1-CGFP vectors were successfully constructed; the plasmid was extracted and transfected into 293T cells, and Western blot showed that the gene protein was successfully expressed. Immunofluorescence and telomere Fish showed that TPP1-CGFP can be recruited to the telomere region, and plasmid co-transformation can reassemble the split proteins into GFP. Conclusion: The eukaryotic expression vector was successfully constructed and proved that TPP1-CGFP can be recruited to the telomere region, and the split GFP protein can be reassembled by the interaction of connexins and emit green fluorescence. This paper provides a visual cell model for the study of telomerase and TPP1 protein and the interaction of telomeres.
Canine interferon-α(CaIFN) was expressed in Pichia pastoris in the previous studies, which had high biological activity and relatively low yield. Therefore, increasing the yield of recombinant protein is the key task to promote the application of CaIFN. Yeast transformants containing 1, 2, 4, 6, and 8 copies of CaIFN gene were generated, and the yield of KM-6CaIFN was the highest, which was 200.6% higher than that of KM-1CaIFN. 8 molecular chaperones were co-expressed with CaIFN, and the Hac protein could increase the target protein yield of KM-6CaIFN and KM-8CaIFN by 32.1% and 113.1%, respectively. More copies of CaIFN were integrated into KM-8CaIFN-Hac strain. SDS-PAGE analysis showed that the yield of KM-12CaIFN-Hac was 1.33 times that of KM-8CaIFN-Hac, and 5.61 times that of KM-1CaIFN, respectively. By comparing protein bands with serially dilution BSA, the yield of KM-12CaIFN-Hac was estimated to be about 581 mg/L, which is the highest value in P. pastoris reported so far.
7-aminocephalosporanic acid (7-ACA) is an important intermediate for synthesis of cephalosporin antibiotics, which is produced by enzymatic conversion of cephalosporin C using cephalosporin C acylase in industry. However, during the reaction process, there is a major impurity 3-deacetyl-7-aminocephalosporanic acid (D-7-ACA) generated from the degradation of cephalosporin C or 7-ACA by cephalosporin C acetyl esterase encoded by the aes gene of Escherichia coli. In order to obtain high-quality 7-ACA and reduce downstream refining costs, it is necessary to prevent the formation of D-7-ACA. Therefore, the corresponding gRNA and donor DNA fragments were designed and the gene aes was knocked out from the chromosome of E. coli BL21(DE3) to generate the engineer E. coli BL21(DE3)△aes using the pTargetF/pCas knockout system. Then, the plasmid of pET30-CPCacy was constructed by inserting the gene CPCacy encoding cephalosporin C acylase into the backbone of pET30(a). The cell lysis supernatants of recombinant strains expressing the cephalosporin C acylase plasmids, including E. coli BL21(DE3)/pET30-CPCacy and E. coli BL21(DE3)△aes/pET30-CPCacy, were applied to the production of the 7-ACA. During the process of cephalosporin C bioconversion, the cephalosporin C utilization efficiency, the yield of 7-ACA and impurity D-7-ACA by each engineered strain were compared. The cephalosporin C conversion rate was 98.8% in E. coli BL21(DE3)△aes/pET30-CPCacy and 98.5% in the original strain, respectively. At the same time, the yield of 7-ACA was 80.7% while that of the original strain was 80.2%,and the yield of impurity D-7-ACA was only 0.1% which was a quarter of the original strain. This work would lay a foundation for the further production of high-quality 7-ACA.
The classic Wnt/β-catenin signaling pathway is involved in regulating various biological functions including stem cell self-renewal, cell proliferation, differentiation, apoptosis, early embryonic development and tissue regeneration, and it is closely related to the occurrence and development of cancer. In addition, this signaling pathway plays an important role in the development and differentiation of thymic T cells, and affects many aspects of the anti-tumor immune effect. Abnormally activated Wnt/β-catenin signaling pathway can induce the formation of malignant tumors and mediate tumor immune escape. This review elaborated on the correlation of Wnt/β-catenin signaling pathway with cancer occurrence and development as well as anti-tumor immunotherapy, and discussed the research progress of drugs targeting Wnt/β-catenin signaling pathway, and the challenges and limitations of its clinical application.
Compared with 2D cell model and animal model, organoids can better reproduce the key structural and functional characteristics of the source organs, which have been widely studied and applied in the biomedical field. Organoids-on-a-chip combines organoid culture chamber, microfluidics and other functional units, which can not only be designed according to researchers' cognition of target organs, but also simulate the complexity of target organs. With the characteristics of high throughput and high sensitivity, it can control and detect the changes of the microenvironment in which organoids are located. This review summarizes the units and applications of organoids-on-a-chip in medical research, including construction of biological models and disease models, drug research and development, and immune evaluation, and discusses the shortcomings of organoids-on-a-chip in current research and application and proposes directions for future research. The aim is to provide a powerful strategy for the study of disease or biological development mechanism and preclinical research.
Low temperature can slow down biochemical reaction and extend the life of biological materials. In order to avoid the cryoinjury caused by the ice crystal during traditional isobaric (atmospheric pressure) freezing process, Dr. Rubinsky developed two new biopreservation technologies at isochoric (constant volume) conditions. One technology is isochoric freezing, during which part of liquid is frozen and the formed ice expands to generate hydrostatic pressure inside the rigid isochoric chamber and biomatters can be stored at subfreezing temperatures in supercooled phase without any internal ice formation damage. The other technology is isochoric supercooling, which can also preserve organisms in supercooling state with a higher stability in a rigid isochoric chamber, resulting in no damage from ice crystals. In this paper, the principle, application and research progress of isochoric freezing and isochoric supercooling are reviewed, and the possible future research direction of cryopreservation under isochoric conditions is prospected.
High-throughput omics technology provided detailed data for studying life system components. Through the interaction of components among the genome, transcriptome, proteome, and metabolome, it promoted the construction of the genome scale of metabolic network model (GSMM). GSMM, as a commonly used tool in systems biology, allows the complex life process of cells to be studied as a whole system, so it shows more holistic thinking contrary to traditional reductionism. Flux balance analysis (FBA), as the mainstream method of GSMM, is usually difficult to obtain the unique optimal solution due to enough constraints. Thermodynamics is closely related to biological metabolism, so in addition to introducing multiple omics data into the GSMM as additional constraints, adding thermodynamic constraints has also become an effective way to reduce the solution space further. This paper first reviews the method of introducing thermodynamic constraints into the GSMM and the advantages and disadvantages of the methods themselves, and then summarizes the methods and tools for obtaining relevant thermodynamic parameters. Finally, this review introduces the metabolic network model integrating multi-omics and thermodynamic constraints and discusses the practical application of the model based on thermodynamic principle constraints, and puts forward a prospect on how to apply thermodynamic constraints to improve the accuracy of the GSMM.
Bacteriophages are widely distributed in nature. They are a kind of viruses that infect only bacteria. Phages multiply rapidly and have very high specificity for host selection, and are less likely to cause host bacteria to develop resistance when they are used as an antibiotic replacement therapy for bacterial infections. Because it is strictly selected by parasitism, its proliferation process is affected not only by environmental factors (pH, ionic strength, temperature), but also by the host metabolic level. Among the external environmental factors, temperature has an important influence on phage activity, stability, preservation and evolution. In this paper, the research progress of phage in temperature adaptation mechanism was summarized. In addition, the research on phage adaptation evolution under temperatures stress was classified further, and thus will provide help for research in this field.
Edible fungi polysaccharides have attracted much attention because of their biological activities such as antioxidant, immune regulation, anti-tumor, and hypoglycemic and hypolipidemic effect. The structure of edible fungi polysaccharides affects their biological activityies and has the characteristics of (1→3), (1→4), and (1→6) and mixed glycosidic bonds β-D-glucan is one of the structural characteristics of highly active edible fungi polysaccharides. It shows different functions, such as improving the activity of antioxidant enzymes, promoting the secretion of anticancer factors, and stimulating the proliferation of spleen and thymocytes. Different extraction and purification methods such as acid-base, ultrasonic and microwave, Sevag method, resin method and affinity chromatography will affect the yield of edible fungi polysaccharides, change their structure and affect their biological activities. The extraction and purification methods of edible fungi polysaccharides and their effects on structure and activity, the composition, structure and structure-activity relationship of edible fungi polysaccharides, as well as the functions, structural characteristics and biological activities of edible fungi polysaccharides in antioxidant, antitumor, immune regulation, hypoglycemic and hypolipidemic aspects were described in detail. The molecular mechanism of edible fungi polysaccharides formation, modification of polysaccharide active sites, polysaccharide metabolic kinetics and other future research directions were proposed.
Carbon neutralization means that the carbon dioxide emissions produced are offset, leading to carbon dioxide “zero emissions” within a certain period, through afforestation, energy conservation, and emission reduction. Microalgae is generally a term for the microorganism that contains chlorophyll a and can carry out photosynthesis. It has the characteristic property of being carbon neutral. It can efficiently fix carbon dioxide through the CO2 concentration mechanism (CCM) by photosynthesis and fix organic carbon through heterotrophic assimilation. The organic carbon recycling is coupled with microalgae cultivation by using nutrients in wastewater from sewage sludge, agriculture, or food industry. The new alternative and clean energy originated from microalgae includes biodiesel, alcohol-based fuel, hydrogen, and hydrocarbons. The biomass can be converted into biofuels, biomaterials, and biofertilizers to replace fossil fuels, plastics, and fertilizers. Biotechnology applications through the full life cycle include species selection, multi-omics regulation, cultivation in photobioreactors, harvesting, extraction, and purification of microalgae biomass or products. Herein, the application and research status of microalgae biotechnology in the field of carbon neutralization are reviewed based on the carbon balance between intake and output. The value, significance, existing problems, and improvement direction of microalgae biotechnology are discussed. The low efficiency and high energy consumption of cultivation, harvesting, and extraction process are the main cause of carbon footprint. The screening of suitable microalgal strains, regulating of metabolic pathways, optimizing of culture conditions and bioreactors, and optimizing of downstream processing (such as harvesting, extraction, and purification) with the purpose of increasing the efficiency of carbon sequestration and utilization efficiency of light energy are expected to reduce costs and improve carbon footprint. More applications of microalgae in carbon neutral fields can be realized by targeting key nodes to improve the carbon footprint.
Silicon nanowire (SiNW) is one of the one-dimensional nanomaterials, and has been emerged as the promising sensing materials due to large surface area to volume ratio and high stability. The research of SiNW in sensing field has received wide attention. With the development of SiNW synthesis techniques and modification methods, biosensors based on silicon nanowires are now introduced into many research areas, including the detection of metal ions, early protein biomarkers and drug screening. On the other hand, high biocompatibility and commercial feasibility of silicon nanowires provide the potential to dynamic and real-time monitoring of single cell. Meanwhile, the research on silicon-based biosensors reveals different mechanisms like electrical, optical methods. Sensitivity, specificity and stability are important indicators of sensors to measure the performance during the detection of biochemical substances. Stable chemical properties of silicon nanowires provide an ideal platform for the fabrication of sensors. However, the surface modification remains as one of the challenges when biosensors based on silicon nanowires are used for different application scenarios. Depending on different requirements for sensing, silicon-based biosensors with different sensing mechanisms have been proposed. In terms of biosensors based on electrical signals, SiNW field-effect transistor (SiNW-FET) has been widely studied, which achieves ultra-sensitive detection of target substances by detecting the output electrical signal of sensor through the conductivity change of nanowire induced by surface charge density. Among biosensors based on optical signals, SiNW-based fluorescent sensor achieves detection by measuring the change of fluorescence intensity or wavelength, enabling fast and convenient detection. In this paper, the applications of silicon nanowires in biosensors are summarized, and the sensing mechanisms of SiNW-FET and SiNW-based fluorescent sensor are discussed. Finally, the future research and development of silicon nanowires in biosensors are prospected.
Base mutation, gene re-arrangement or horizontal gene transfer are the fundamental mechanisms involved in bacterial evolution. During adaptive evolution, they are mainly affected by biological and abiological factors. Among them, heavy metal ion stress is also one of the main reasons during bacterial adaptive evolution, and it drives bacteria to adaptively strengthen the metabolic pathways related to metal input and/or transformation. On the other hand, excessive metal ion also can induce metal accumulation and efflux. Under heavy metal stress, heavy metal resistance (HMR) gene and enzyme protein play an important role involved in mechanisms of bacterial adaptive evolution. The mechanisms include the adaptation of isolation, the regulatory adaptation of metal regulatory protein and the adaptation of enzyme detoxification. The current research status and progress were summarized in this paper. Heavy metal ions have polluted the environment and threatened human health and ecosystem stability. Therefore, to elucidate the molecular mechanisms of adaptive evolution of bacteria under heavy metal stress, this paper not only enriches the content of bacterial evolutionism, but also provides a theoretical basis for the biological remediation of environmental pollution by heavy metal ions.
Based on the data of domestic patent transfer/license database, this paper systematically combs and analyzes the transformation of biomedical patent achievements in China. The research shows that although the conversion rate of biomedical patents in China is low at present, and the transformation in some fields lags behind obviously, after the revision of the Law of Promoting the Transformation of Scientific and Technological Achievements in 2015, all parties actively explore a new mode of transformation of achievements, and the transformation technology trend is basically consistent with the development trend of biomedical industry, indicating that the policy effectively drives the transformation work. It is suggested that relevant departments continue to improve policies, establish a transformation ecosystem, and play a greater role in the transformation of biomedical patent achievements in epidemic prevention and control.
