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Abstract:Given the rapid development of technologies in biochemical engineering and genetic engineering, biological capture, conversion and utilization of greenhouse gases(carbon dioxide and methane) into value-added products have been progressed rapidly. The efficiency of electron transfer and energy supply are essential for microbial carbon fixation. In this review, the concepts of direct and indirect electron transfer chains in methanotrophic and chemoautotrophic microbes were introduced firstly. Subsequently, the strategies of supplying light and electrical energy as well as their effects on metabolic flux, synthetic pathway and energy supply efficiency during microbial carbon fixation were discussed. Finally, solutions and application prospects to address the key technical challenges of microbial carbon fixation were discussed.

Abstract:Microplastics(MPs) have been detected in many ecosystems, such as the ocean, land and the atmosphere. A large number of MPs in urban sewage are trapped in the activated sludge by sewage treatment plants, but tens of thousands of MPs ‘escape' the treatment and are discharged into the nature.Meanwhile, most of the MPs are transferred into the activated sludge during sewage treatment, and the sludge will be further used in agriculture, leading to secondary pollution of the MPs. Through literature research, we summarized the sources, distribution and hazards of MPs in the environment, the treatment of MPs with activated sludge, and the treatment methods of residual MPs in activated sludge, and summed up the potentials of biotechnology and synthetic biology in the genetic modification of key bacteria in activated sludge to endow them with MPs-degrading ability. The conclusion is expected to serve as a reference for optimizing the biodegradation of MPs in wastewater treatment plants.

Abstract:Recently, drug resistance resulted from the extensive abuse and over-use of antibiotics has posed a great threat to human health. Scholars have conducted numerous studies on the impacts of antibiotic resistant bacteria and antibiotic resistance genes(ARGs) in different types of environments.Aerosol is not only a potential reservoir for ARGs, but also an important route for transmission of ARGs in the environment. However, a systematic summary of its sources, transmission, human exposure, and health risks is lacking. This review focused on four types of typical sites for aerosol research: human functional living places, farms, urban wastewater treatment plants, and hospitals. The sources,transmission routes, human exposure, and health risks of ARGs in the aerosol of these four typical sites were reviewed. This article also provides a reference for prevention and control of ARGs in aerosols.

Abstract:In order to adapt to different environmental stresses including heat stress, oxidative stress,osmotic pressure stress and ultraviolet stress in the process of growing and infecting agricultural products,fungi have developed a set of high osmolarity glycerol mitogen-activated protein kinase(HOG-MAPK)pathway to alleviate the environmental stresses. This pathway plays an important role in the growth,development, mycotoxin production and pathogenicity of fungi. There are two branches in the HOG-MAPK pathway, among which the SLN1 branch is more sensitive to osmotic stress than another branch(SHO1 branch), and is able to respond to high osmotic pressure and high salt concentration. The SHO1 branch is involved in a variety of signal transduction in response to oxidative stress and thermal stress. This paper reviews the functions of key genes sln1, sho1, ste11, ssk2, pbs2 and hog1 in the HOG-MAPK pathway of phytopathogenic fungi in responses to different environmental stresses such as osmotic stress and oxidative stress. We show that the HOG-MAPK pathway can respond to a variety of environmental signals and is involved in regulating the growth of phytopathogenic fungi such as Aspergillus flavus and Aspergillus ochraceus, and the production of mycotoxins such as aflatoxins and ochratoxins. Understanding the mechanism of how HOG-MAPK pathway regulates mycotoxins' formation under different environmental stresses that provides a theoretical basis and guidance for the prevention and control of mycotoxins in agricultural products such as food and feed.

Abstract:The CRISPR(clustered regularly interspaced short palindromic repeats)-Cas(CRISPR-associated proteins) system is an efficient and precise gene editing tool. The development of this technology has promoted genome editing into a rapid development stage. The most widely used Cas9 protein is SpyCas9 from Streptococcus pyogenes. As a “gene scissors”, the SpyCas9 protein is widely used in eukaryotes including mammals and plants. However, the application of this protein in some lactic acid bacteria(LAB) is still hampered by many factors. It has been identified that LAB genomes possess many types of CRISPR system and are rich in uncharacterized Cas proteins. Genome editing of LAB is possible by repurposing the endogenous CRISPR-Cas systems in LAB combined with exogenous single guide RNA(sgRNA) and homologous recombination template. This method employs its endogenous CRISPR-Cas system for gene editing, which has the advantages including easiness for transformation due to the relatively small targeting vector, and no concern about the toxicity of heterologous Cas9 to host cells. Compared to CRISPR-SpyCas9, the endogenous CRISPR-Cas system is more suitable for genome editing of LAB, and it may become the main genome editing tool for some LAB in the future.This article summarizes the advances in this field.

Abstract:Synthetic biology has brought enormous impacts to the advancement of agriculture, energy,manufacturing and medicine by redesigning existing natural systems or creating biological elements,modules and systems to endow life with new functions. The precise, quantitative and efficient regulation of biological elements, modules and systems is critical for controlling living systems. Bacterial small regulatory RNAs are a class of functional molecules with a length of 50–300 nt and usually do not translate into proteins. They play an irreplaceable role in environmental stress response, metabolic adaptation and bacterial virulence control. In recent years, synthetic small regulatory RNAs have been designed based on natural small RNAs and were used to effectively inhibit or activate target genes and even pathways. Synthetic small regulatory RNAs have broad application advantages in synthetic biology because of their small size, high flexibility, programmability, easy design and little metabolic burden on host cells. To better understand the synthetic small regulatory RNAs and promote its application, this review summarized several synthetic small RNA systems and their representative applications in synthetic biology. Finally, future optimization directions were prospected.

Abstract:Due to the large amount of greenhouse gas emissions and the high dependence on fossil fuels,the sustainable development of aviation industry has attracted worldwide attention. Bio-jet fuel is considered to be a promising alternative to traditional aviation fuel. This article summarizes the representative technological route, development status, opportunities and challenges faced by the development of bio-jet fuel industry. So far, several bio-jet fuel production technologies have been certified by the American Society for Testing and Materials(ASTM). Hydroprocessed esters and fatty acids is currently the most mature process that can be fully commercialized. Considering economic characteristics and technology maturity, Fischer-Tropsch is promising in near term.

Abstract:Lignin is the only natural aromatic polymer and the second most abundant lignocellulosic resource in nature after cellulose, and its derived high-value products can be used in a variety of fields. An efficient, highly value-added and high-quality production of lignin is critical for lignocellulose biorefinery,but the complex and variable structure of lignin macromolecules, poor activity of reactions, and redundant functional groups make it difficult to prepare polymeric materials with stable properties. With the increasing research on lignin modification, the application of lignin composite hydrogels has also received great attention. In this paper, the preparation of lignin composite hydrogels based on the basic structural composition and reaction properties of lignin was briefly outlined. The current applications of lignin composite hydrogels, including biosensors, controlled release materials, environmentally responsive materials, adsorbent materials, electrode materials, and other materials, were summarized. Moreover,future perspectives of lignin-based composite hydrogels were prospected.

Abstract:Protein cross-linking plays important roles in food, chemical, medicine and other fields.Enzyme-catalyzed protein cross-linking is an efficient and economically viable alternative to physical and chemical cross-linking. However, detailed analysis of enzyme-catalyzed protein cross-linking at molecular level is still lacking. This review summarized the mechanisms of enzyme-catalyzed protein cross-linking,its effects on protein structure, and its applications in food, chemical and pharmaceutical fields.

Abstract:A bio-electrochemical system can promote the interaction between microorganism and electrode and consequently change cellular metabolism. To investigate the metabolic performance of Zymomonas mobilis in the bio-electrochemical system, we applied an H-type bio-electrochemical reactor to control Z. mobilis fermentation under 3 V. Compared with the control group without applied voltage,the glycerol in the anode chamber increased by 24%, while the glucose consumption in the cathode chamber increased by 16%, and the ethanol and succinic acid concentration increased by 13% and 8%,respectively. Transcriptomic analysis revealed that the pathways related to organic acid metabolism,redox balance, and electron transfer played roles in metabolic changes. Three significantly differentially expressed genes, ZMO1060(superoxide dismutase), ZMO0401(diguanylate cyclase), and ZMO1819(nitrogen fixation protein), were selected to verify their functions in the bio-electrochemical system.Overexpression of ZMO1060 and ZMO1819 improved the electrochemical activity of Z. mobilis. This study provides insights into the microbial metabolism regulated by the bio-electrochemical system.

Abstract:L-ascorbic acid 2-glucoside(AA-2G) is a derivative of L-ascorbic acid(L-AA). Compared with L-AA, it has good stability and is easily decomposed by enzyme in the human body. α-Glucosidase(AG) was the first enzyme found capable of producing AA-2G. However, researches on this enzyme is still in infancy. We took AG derived from Aspergillus niger(AAG), Japanese rice(JrAG) and Rattus rattus(RAG), and compared their specific enzymatic activity and transglycosidation rate, with the aim to improve the synthesis of AA-2G by the transglycosidation of AG. The genes encoding these three different AG were cloned and expressed in engineered yeast. The conditions for the transglycosidation reaction of these three enzymes were optimized and the transglycosidation efficiency and yield of AA-2G under the optimized conditions were compared. The specific activity of AAG reached 1.0 U/mg,while the yield of AA-2G reached 153.1 mg/L with a transglycosidation rate of 0.5%. The specific activity of RAG reached 0.4 U/mg, while the yield of AA-2G reached 861.0 mg/L with a transglycosidation rate of 2.5%. JrAG showed the highest specific activity and transglycosidation rate.The enzyme specific activity of JrAG reached 1.9 U/mg, while the yield of AA-2G reached 2 577.2 mg/L with a transglycosidation rate of 7.6%, much higher than that of the other two glucosidases. JrAG may thus have potential to improve the synthesis of AA-2G.

Abstract:Natamycin is a natural, broad spectrum and highly efficient antifungal compound that belongs to polyene macrolide antibiotics. It has been used in prevention of food fungal contamination and treatment of clinical fungal infection. The extracellular transport efficiency of natamycin may be an important factor hampering the yield of natamycin produced by Streptomyces gilvosporeus. The extracellular transporter SgnA/B of natamycin was analyzed by bioinformatics tools and molecular docking techniques. This ATP-binding cassette transporter, consisted of SgnA and SgnB, is a heterodimers with inward-facing conformation. The difference between the natamycin combining efficiency of the two drug-binding cavities in SgnA/B is favorable for natamycin extracellular transport.sgnA/B gene was overexpressed in S. gilvosporeus F607 and the effects of sgnA/B gene overexpression on natamycin synthesis and extracellular transport were analyzed. In F-EX strain, the extracellular/intracellular ratio of natamycin in logarithmic synthesis stage was increased, and the total fermentation yield at 120 h was increased by 12.5% and reached to 7.38 g/L. Moreover, transcriptome sequencing analysis showed that sgnA/B gene overexpression affected the expression of genes involved in the metabolism of various amino acids, propionate, glucose, C5-branched dibasic acid and TCA cycle.This research demonstrated that the enhanced extracellular transport increased the synthesis of natamycin by S. gilvosporeus, and S. gilvosporeus F-EX showed good potential for the industrial production of natamycin.

Abstract:D-mannitol is widely used in the pharmaceutical and medical industries as an important precursor of antitumor drugs and immune stimulants. However, the cost of the current enzymatic process for D-mannitol synthesis is high, thus not suitable for commercialization. To address this issue, an efficient mannitol dehydrogenase LpGDH used for the conversion and a glucose dehydrogenase BaGDH used for NADH regeneration were screened, respectively. These two enzymes were co-expressed in Escherichia coli BL21(DE3) to construct a two-enzyme cascade catalytic reaction for the efficient synthesis of D-mannitol, with a conversion rate of 59.7% from D-fructose achieved. The regeneration of cofactor NADH was enhanced by increasing the copy number of Bagdh, and a recombinant strain E. coli BL21/pETDuet-Lpmdh-Bagdh-Bagdh was constructed to address the imbalance between cofactor amount and key enzyme expression level in the two-enzyme cascade catalytic reaction. An optimized whole cell transformation process was conducted under 30 ℃, initial pH 6.5, cell mass(OD₆₀₀) 30,100 g/L D-fructose substrate and an equivalent molar concentration of glucose. The highest yield of D-mannitol was 81.9 g/L with a molar conversion rate of 81.9% in 5 L fermenter under the optimal conversion conditions. This study provides a green and efficient biotransformation method for future large-scale production of D-mannitol, which is also of great importance for the production of other sugar alcohols.

Abstract:Malonic acid is an important dicarboxylic acid, which can be widely used in the fields of chemical industry, medicine and food. In this study, a recombinant Escherichia coli strain BL21(TPP)was constructed to synthesize malonate through overexpressing six genes of ppc, aspC, panD, pa0132,yneI and pyc. Under shake flask fermentation conditons, strain BL21(TPP) produced 0.61 g/L malonic acid. In a 5 L fermentor, the production of malonic acid reached 3.32 g/L by using an intermittent feeding strategy. Next, a recombinant strain BL21(SCR) was constructed by fusional expression of ppc and aspC, as well as pa0132 and yneI, respectively. As a result, the production of malonic acid increased to 0.83 g/L at the shake flask level, which was a 36% increase over the starting strain BL21(TPP).Finally, the highest malonate production reached 5.61 g/L in a 5 L fermentor, which was a 69% increase over the starting strain BL21(TPP). Production of malonic acid by metabolically engineered E. coli provides a basis for further optimization, and may also serve as a reference for the biosynthesis of other dicarboxylic acids.

Abstract:Biliverdin is an important cellular antioxidant. Traditionally, biliverdin is produced by chemical oxidation of bilirubin, which is a complex process and the final product is of low purity. Here we report an efficient, green and safe process for biotechnological production of biliverdin. A heme oxygenase(HO) gene from Clostridium tetani was screened, and a recombinant strain Escherichia coli BL21/pETDuet-hoCt with the ability of transforming heme into biliverdin was constructed. A biliverdin yield of 32.9 mg/L from 100 mg/L substrate was achieved under pH 7.0 and 35 ℃. In order to improve the supply of reducing power, an NADPH regeneration system using glutamate dehydrogenase(GdhA)was constructed, resulting in a recombinant strain E. coli BL21/pETDuet-gdhAEc-hoCt which was capable of producing 71.5 mg/L biliverdin. Moreover, through introduction of a membrane surface display system, a recombinant strain E. coli BL21/pETDuet-gdhAEc-blc/hoCt was constructed to shorten the transformation time, and the production of biliverdin was further increased to 76.3 mg/L, this is the highest titer of biosynthesized biliverdin reported to date, and the research may thus facilitate the green production of biliverdin.

Abstract:Chondroitin sulfate(CS) is a linear polysaccharide, which is widely used in medical, health care and other fields. Compared with the traditional animal tissue extraction method, microbial synthesis of CS has the advantages of controllability and easiness of scaling-up. In order to achieve an efficient synthesis of chondroitin sulfate A(CSA), we constructed a recombinant Pichia pastoris GS115 strain capable of synthesizing chondroitin(Ch) from glycerol by introducing the Ch synthase coding genes kfoC, kfoA and UDP-glucose dehydrogenase coding gene tuaD into the P. pastoris chromosome. The titer of Ch reached 2.6 g/L in fed-batch cultures upon optimizing the synthesis pathway of Ch. After further expressing the chondroitin-4-O-sulfotransferase(C4ST), we developed a one-pot biosynthesis system for CSA production by directly adding 3'-adenosine-5'-phosphoryl sulfate and C4ST into the high-pressure homogenized recombinant P. pastoris cells. Eventually, controllable synthesis of 0-40%CSA with different sulfation degrees were achieved by optimizing the catalytic conditions. The one-pot biosynthesis system constructed here is easy to operate and easy to scale up for industrial production of CSA. The idea of the present study may also facilitate the biosynthesis of other glycosaminoglycan, for instance, heparin.

Abstract:Promoters with different sensitivity and response intensity are useful tools in gene expression regulation and metabolic engineering. Maltose induced promoter P_glvc was engineered to obtain promoters with different induced expression intensities. A promoter P_glvc mutant library was built by error-prone PCR, and screened by a growth-associated method using tetracycline resistance as an indicator. A library of promoter mutants with different sensitivity and intensity was obtained, and the maltose-induced response threshold range of promoter mutants(MT2, MT3, MT4, MT6) was extended from 0–3 g/L to 0–15 g/L. Among them, the highest induced expression intensity(MT8) was about 3.15 times higher than that of the original promoter for eGFP expression, which would be useful for its application in metabolic engineering and synthetic biology.

Abstract:Biodegradation of antibiotic pollutants by microorganisms has received widespread attention, to which the identification of microorganisms capable of efficiently degrading antibiotics is a key. In this study, a strain DM-1 with high degradation capability was successfully isolated from monensin-contaminated chicken manure by using monensin as the sole carbon source. The strain was further identified basing on morphological, physiological and biochemical characteristics and 16S rRNA gene sequence-based phylogenetic analysis. The degradation efficiency of DM-1 for monensin was determined by HPLC post-column derivatization, and then the degradation conditions of DM-1 were optimized. DM-1 was identified as a strain of Acinetobacter and named as Acinetobacter baumannii DM-1. The optimal conditions for monensin degradation by strain DM-1 were pH 7.0, 30 ℃, and initial monensin concentration of 50 mg/L. The strain DM-1 degraded more than 87.51% of monensin at an initial concentration of 10 mg/L in 28 days, while only a slight decrease in monensin concentration was observed in the control without monensin-degrading strain. This study indicates that the strain DM-1 has a promising application prospect in the bioremediation of monensin-contaminated environment.

Abstract:To achieve an efficient preparation of lactoferrin N-lobe, we optimized the fermentation process for a recombinant Bacillus subtilis pMA0911-D60Y/Y92D producing lactoferrin N-lobe. The IOD of the lactoferrin N-lobe reached 68.03% under the optimized cultural conditions, that is using glucose and tryptone as the best carbon and nitrogen source, respectively, and conduct the fermentation under pH 7.0, 28 ℃, for 25.5 h. An optimized fermentation process was obtained through fermentation optimization on a 10 L fermenter. That is, culturing the recombinant strain at 30 ℃, pH 7.5 within 0-7 h, and switching to induction at 28 ℃, pH 7.5 within 7-25 h for production of lactoferrin N-lobe,using an agitation speed of 300 r/min throughout the fermentation. After the fermentation, the cells were collected and disrupted, followed by purification of the lactoferrin N-lobe to homogeneity by using HisTrap HP-affinity and a Superdex^TM 200(10/300 GL)-affinity chromatography. The purified lactoferrin N-lobe proteins with over 94% purity were obtained. One liter culture of recombinant B. subtilis pMA0911-D60Y/Y92D produced 23.5 mg of pure protein. This study may facilitate the fermentative production of the recombinant lactoferrin N-lobe.

Abstract:Synthetic biology, a course with a sound theoretical system and a wide application range, plays a role in the cultivation of innovative talents in the field of bioengineering. To this end, we have set up a synthetic biology course in our university. First, according to the concept of imparting basic knowledge,highlighting innovative practice, and keeping up with cutting-edge progress, we assembled a high-level teaching team for synthetic biology. The team constantly adjusted and optimized the course contents and achieved a novel and reasonable course system. Second, we introduced frontier cases of synthetic biology reported in high-level journals, as well as breaking news in this field in classroom teaching, which enriched the teaching contents and aroused students' interest. Third, taking these cases as the breakthrough point, we guided students to in-depth discussions through the learning-centered teaching mode to improve students' abilities of critical thinking and theoretical innovation. In summary, the course has achieved good teaching outcomes and improved the cultivation of innovative talents. Therefore, we share our work with peer teachers, aiming to give new insights into the teaching reform of synthetic biology and other related courses.

Abstract:Microbes are involved in every aspect of human life. Microbiology is a mandatory subject at the undergraduate level covering majors including life sciences, pharmacy, medicine, agriculture,forestry and food. Along with internationalization and development of the first-class disciplines,teaching microbiology courses in English is highly valued. Here we discuss how to conduct curriculum reform of microbiology teaching in English, and what are the advantages and challenges when teaching in English. The teaching system can be advanced by enhancing interdisciplinary communication so as to promote study and research for students and teachers. We take this practical exploration as an example to communicate with relevant teachers.

Abstract:Talent training is the core of future national competition. “Strengthening Basic Disciplines Program” is an important initiative exploring the training mode for top-notch creative talents in basic disciplines, and an important measure to meet the needs for fostering future talents. The students enrolled in the “Strengthening Basic Disciplines Program” often have excellent grades and strong motivation,which puts forward new requirements and new goals for the talents training mode. Among them, the teaching reform under the background of “Strengthening Basic Disciplines Program” is imperative. The science, technology, engineering, arts, mathematics(STEAM) education concept is an interdisciplinary comprehensive educational philosophy that happens to coincide with the philosophy of the “Strengthening Basic Disciplines Program”. In view of this, the School of Life Sciences of Tianjin University has explored and practiced the reform of biology experimental teaching based on the objective of fostering biology talents in the context of “Strengthening Basic Disciplines Plan” and the STEAM education concept, by taking the course of comprehensive biology design as an example.

Abstract:Improving students' comprehensive quality and developing their core literacy are the training objectives of high school subject curriculums, which puts forward new requirements for teachers' professionalism and core literacy. In order to adapt to the high school curriculum reform and the new college entrance examination reform, the Ministry of Education approved the development of high-quality, integrated and master-level high school teachers training pilot program. The aim of this program was to foster a group of dual-disciplinary integrated high school teachers who are suitable for teaching, enjoying teaching and skillful in teaching. How to foster the dual-disciplinary core literacy of normal students through subject curriculum is one of the challenges faced by the pilot program. Following the training objectives of the pilot program “Chemistry and Biological Sciences”, we proposed to integrate the dual-disciplinary literacy into four aspects: material concept, scientific thinking, inquiry practice,attitude and responsibility. This was proposed based on analyzing the core literacy of the disciplines of chemistry and biology, and aimed to promote teachers and normal students' understanding and practice of dual-discipline core literacy. With biochemistry course as an example, we further explored and practiced on how to foster the dual-disciplinary core literacy of normal students, aiming to provide reference for the reform of other courses included in the dual-disciplinary integrated programs.