Objective To establish a mouse lethal model of influenza B virus , which will facilitate the study on the mechanism of pathogenesis , transmission of influenza B virus , development of new vaccines and drugs against influenza B virus.Methods We obtained a mouse adaptive B/Lee/1940 virus by continuously passaging it in mice for 5 cycles.The P5 virus was propagated in MDCK cells , which was used for infecting mice .The body mass and survival rate of mice were monitored during the following 14 days after infection.At the same time,the 8 gene segments (PB2, PB1, PA, HA, NA/NB, NP, M, and NS) of P0 and P5 virus were sequenced and analyzed .Results and Conclusion Virus was detected in the lungs of mice in each generation in the process of virus passaging .The body mass of mice infected with the deadly mouse adaptive virus changed dramatically .The mortality of mice was 100%, and virus was detected in mouse lungs . Sequence analysis results indicated that the amino acid mutations occurred in PB 2 and NP.A series of experiments indicated that we had established a mouse lethal model of influenza B virus .

Coenzyme Q10 (CoQ10) is a lipophilic antioxidant that improves human immunity, delays senility and enhances the vitality of the human body and has wide applications in pharmaceutical and cosmetic industries. Microbial fermentation is a sustainable way to produce CoQ10, and attracts increased interest. In this work, the native CoQ8 synthetic pathway of Escherichia coli was replaced by the CoQ10 synthetic pathway through integrating decaprenyl diphosphate synthase gene (dps) from Rhodobacter sphaeroides into chromosome of E. coli ATCC 8739, followed by deletion of the native octaprenyl diphosphate synthase gene (ispB). The resulting strain GD-14 produced 0.68 mg/L CoQ10 with a yield of 0.54 mg/g DCW. Modulation of dxs and idi genes of the MEP pathway and ubiCA genes in combination led to 2.46-fold increase of CoQ10 production (from 0.54 to 1.87 mg/g DCW). Recruiting glucose facilitator protein of Zymomonas mobilis to replace the native phosphoenolpyruvate: carbohydrate phosphotransferase systems (PTS) further led to a 16% increase of CoQ10 yield. Finally, fed-batch fermentation of the best strain GD-51 was performed, which produced 433 mg/L CoQ10 with a yield of 11.7 mg/g DCW. To the best of our knowledge, this was the highest CoQ10 titer and yield obtained for engineered E. coli.
Alkyl and Aryl Transferases ; genetics ; Bacterial Proteins ; genetics ; Batch Cell Culture Techniques ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Gene Deletion ; Industrial Microbiology ; Metabolic Engineering ; Rhodobacter sphaeroides ; enzymology ; genetics ; Ubiquinone ; analogs & derivatives ; biosynthesis ; Zymomonas ; genetics

β-carotene belongs to carotenoids family, widely applied in pharmaceuticals, neutraceuticals, cosmetics and food industries. In this study, three key genes (dxs, idi, and crt operon) within β-carotene synthetic pathway in recombinant Escherichia coli strain CAR005 were modulated with RBS Library to improve β-carotene production. There were 7%, 11% and 17% increase of β-carotene yield respectively after modulating dxs, idi and crt operon genes with RBS Library, demonstrating that modulating gene expression with regulatory parts libraries would have more opportunities to obtain optimal production of target compound. Combined modulation of crt operon, dxs and idi genes led to 35% increase of β-carotene yield compared to parent strain CAR005. The optimal gene expression strength identified in single gene modulation would not be the optimal strength when used in combined modulation. Our study provides a new strategy for improving production of target compound through modulation of gene expression.
Escherichia coli ; metabolism ; Gene Expression ; Gene Library ; Operon ; beta Carotene ; biosynthesis

A N-acetylneuraminate lyase gene (shnal) from Staphylococcus hominis was cloned into pET-28a and expressed in Escherichia coli BL21 (DE3) host cells. The recombinant enzyme was purified and characterized. It is a homotetrameric enzyme with the optimum pH at 8.0 for the cleavage direction and the optimum pH and temperature were 7.5 and 45 degrees C for the synthetic direction. The activity of ShNAL is stable when incubated at 45 degrees C for 2 h but decreased rapidly over 50 degrees C. ShNAL showed high stability in a wide range pH from 5.0 to 10.0 with the residual activity being > 70% when the enzyme was incubated in different buffers at 4 degrees C for 24 h. Its K(m) towards N-acetylneuraminic acid, pyruvate and ManNAc were (4.0 +/- 0.2) mmol/L, (35.1 +/- 3.2) mmol/L and (131.7 +/- 12.1) mmol/L, respectively. The k(cat)/K(m) value of Neu5Ac, ManNAc, and Pyr for ShNAL were 1.9 L/(mmol x s), 0.08 L/(mmol x s) and 0.08 L/(mmol x s), respectively.
Bacterial Proteins ; genetics ; metabolism ; Cloning, Molecular ; Enzyme Stability ; Escherichia coli ; genetics ; metabolism ; Hydrogen-Ion Concentration ; Oxo-Acid-Lyases ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Staphylococcus hominis ; enzymology ; Temperature

Xylulose as a metabolic intermediate is the precursor of rare sugars, and its unique pattern of biological activity plays an important role in the fields of food, health, medicine and so on. The aim of this study was to design a new pathway for xylulose synthesis from formaldehyde, which is one of the most simple and basic organic substrate. The pathway was comprised of 3 steps: (1) formaldehyde was converted to glycolaldehyde by benzoylformate decarboxylase mutant BFD-M3 (from Pseudomonas putida); (2) formaldehyde and glycolaldehyde were converted to dihydroxyacetone by BFD-M3 as well; (3) glycolaldehyde and dihydroxyacetone were converted to xylulose by transaldolase mutant TalB-F178Y (from Escherichia coli). By adding formaldehyde (5 g/L), BFD-M3 and TalB-F178Y in one pot, xylulose was produced at a conversion rate of 0.4%. Through optimizing the concentration of formaldehyde, the conversion rate of xylulose was increased to 4.6% (20 g/L formaldehyde), which is 11.5 folds higher than the initial value. In order to further improve the xylulose conversion rate, we employed Scaffold Self-Assembly technique to co-immobilize BFD-M3 and TalB-F178Y. Finally, the xylulose conversion rate reached 14.02%. This study provides a new scheme for the biosynthesis of rare sugars.

Higher alcohols are one of the main by-products of Saccharomyces cerevisiae in brewing. High concentration of higher alcohols in alcoholic beverages easily causes headache, thirst and other symptoms after drinking. It is also the main reason for chronic drunkenness and difficulty in sobering up after intoxication. The main objective of this review is to present an overview of the flavor characteristics and metabolic pathways of higher alcohols as well as the application of mutagenesis breeding techniques in the regulation of higher alcohol metabolism in S. cerevisiae. In particular, we review the application of metabolic engineering technology in genetic modification of amino transferase, α-keto acid metabolism, acetate metabolism and carbon-nitrogen metabolism. Moreover, key challenges and future perspectives of realizing optimization of higher alcohols metabolism are discussed. This review is intended to provide a comprehensive understanding of metabolic regulation system of higher alcohols in S. cerevisiae and to provide insights into the rational development of the excellent industrial S. cerevisiae strains producing higher alcohols.
Alcoholic Beverages ; Alcohols/analysis* ; Fermentation ; Saccharomyces cerevisiae/metabolism* ; Saccharomyces cerevisiae Proteins/metabolism*

As a new beer fermentation technology, high temperature and high gravity fermentation has brought many benefits to brewery industry, but there are also a series of problems such as the decrease of yeast flocculation ability at the end of fermentation and the high concentration of higher alcohols. To increase yeast flocculation ability and reduce the production of higher alcohols in high temperature and high gravity fermentation of beer, BAT2 was replaced by the FLO5 expression cassette to obtain the mutant strain S6-BF2. Real-time quantitative PCR showed that the relative transcriptional level of FLO5 in S6-BF2 improved 17.8 times compared with that in S6. The flocculation ability of mutant S6-BF2 heightened by 63% compared to that of the original strain S6, and the concentration of higher alcohols decreased from 175.58 mg/L to 159.58 mg/L in high temperature and high gravity fermentation of beer. Moreover, the activity of mitochondrial branched-chain amino acid transferase was repressed, resulting in the production of higher alcohols of 142.13 mg/L, reduced by 18.4% compared to that of the original strain S6, meanwhile, the flocculation ability of mutant S6-BF2B1 kept unchanged compared to the mutant S6-BF2. The determination result of flavor compounds showed that the higher alcohols/ester ratio in beer was reasonable. This research has suggested an effective strategy for enhancing yeast flocculation ability and decreasing production of higher alcohols in high-temperature and high-gravity brewing.
Beer ; Fermentation ; Hypergravity ; Saccharomyces cerevisiae ; Saccharomyces cerevisiae Proteins ; Temperature ; Transaminases

Limonene (C₁₀H₁₆) and bisabolene (C₁₅H₂₄) are both naturally occurring terpenes in plants. Depending on the number of C₅ units, limonene and bisabolene are recognized as representative monoterpenes and sesquiterpenes, respectively. Limonene and bisabolene are important pharmaceutical and nutraceutical products used in the prevention and treatment of cancer and many other diseases. In addition, they can be used as starting materials to produce a range of commercially valuable products, such as pharmaceuticals, nutraceuticals, cosmetics, and biofuels. The low abundance or yield of limonene and bisabolene in plants renders their isolation from plant sources non-economically viable. Isolation of limonene and bisabolene from plants also suffers from low efficiency and often requires harsh reaction conditions, prolonged reaction times, and expensive equipment cost. Recently, the rapid developments in metabolic engineering of microbes provide a promising alternative route for producing these plant natural products. Therefore, producing limonene and bisabolene by engineering microbial cells into microbial factories is becoming an attractive alternative approach that can overcome the bottlenecks, making it more sustainable, environmentally friendly and economically competitive. Here, we reviewed the status of metabolic engineering of microbes that produce limonene and bisabolene including microbial hosts, key enzymes, metabolic pathways and engineering of limonene/bisabolene biosynthesis. Furthermore, key challenges and future perspectives were discussed.

In recent years, adaptive laboratory evolution (ALE) has emerged as a powerful tool for basic research in microbiology (e.g., molecular mechanisms of microbial evolution) and efforts on evolutionary engineering of microbial strains (e.g., accelerated evolution of industrial strains by bringing beneficial mutations). The ongoing rapid development of next-generation sequencing platforms has provided novel insights into growth kinetics and metabolism of microbes, and thus led to great advances of this technique. In this review, we summarize recent advances in the applications of long-term and short-term ALE techniques mainly for microbial strain engineering, and different modes of ALE are also introduced. Furthermore, we discuss the current limitations of ALE and potential solutions. We believe that the information reviewed here will make a significant contribution to further advancement of ALE.
High-Throughput Nucleotide Sequencing ; Laboratories ; Mutation

Yarrowia lipolytica is a non-conventional yeast with unique physiological and metabolic characteristics. It is suitable for production of various products due to its natural ability to utilize a variety of inexpensive carbon sources, excellent tolerance to low pH, and strong ability to secrete metabolites. Currently, Y. lipolytica has been demonstrated to produce a wide range of carboxylic acids with high efficiency. This article summarized the progress in engineering Y. lipolytica to produce various carboxylic acids by using metabolic engineering and synthetic biology approaches. The current bottlenecks and solutions for high-level production of carboxylic acids by engineered Y. lipolytica were also discussed, with the aim to provide useful information for relevant studies in this field.
Carboxylic Acids/metabolism* ; Metabolic Engineering ; Synthetic Biology ; Yarrowia/metabolism*