A New Way to Control Microbial Metabolism Revealed
Posté le 30/6/2020 à 14:29 - 0 Commentaires - poster un commentaire - Lien
Microorganisms can be engineered to produce a variety of useful compounds, including plastics, biofuels and pharmaceuticals. However, in many cases, bacteria need to compete in the metabolic pathways of “self-sustaining” and “synthetic products”.To help optimize the ability of cells to produce desired compounds and maintain their own growth, chemical engineers at MIT have designed a method that can induce bacteria to switch between different metabolic pathways at different times. These switches are “programmed” into the bacterial genome and are triggered or closed depending on changes in population density without manual intervention.Kristala Prather, professor of chemistry and the author of the article, said: “We hope this will allow more precise regulation of bacterial metabolismto obtain higher productivity, but at the same time minimize the number of interventions.” This conversion allows researchers to increase the microbial yield of two different products by tenfold.
Christina Dinh, a graduate student at MIT, is the lead author of the paper, which was recently published in PNAS.
To enable microbes to synthesize compounds that are not normally produced, engineers inserted genes for enzymes involved in metabolic pathways. In some cases, the intermediates produced during these reactions are also part of the metabolic pathways already present in the cell. When cells transfer these intermediates to routes other than the engineering route, the total yield of the final product is reduced.
By using a concept called dynamic metabolic engineering, Prather has previously constructed switches that can help cells maintain a balance between their own metabolic needs and the pathways that produce the desired products. Her idea was to program the cells to automatically switch between pathways without any intervention by personnel operating the reactive fermenter.
The MIT team engineered their E. coli cells to secrete a quorum-sensing molecule called AHL. When the concentration of AHL reaches a certain level, the cell turns off an enzyme that translocates gluconeic acid into one of the cell's own metabolic pathways. This allows the cells to grow and divide normally until the population is large enough to begin producing large quantities of the desired products.
In the new PNAS paper, Prather and Dinh set out to design multiple switching points into their units, thereby giving them a greater degree of control over the production process. For this purpose, they used two quorum-sensing systems from two different bacteria. They integrated these systems into E. coli and engineered them to produce a compound called grapefruit, a flavonoid naturally present in citrus fruits with multiple beneficial effects on health.
Using these quorum sensing systems, the researchers designed two switching points into the cell. A switch was designed to prevent bacteria from transferring the bombesin called malonyl-CoA into the cell's own metabolic pathway. At another transition point, the researchers delayed the production of enzymes in their engineered pathway to avoid accumulation of excessive amounts that in turn inhibited the synthesis of grapefruit.
The researchers created hundreds of E. coli variants to execute these two switches at different population densities, thus allowing them to identify which was the most productive. The best performing strains showed a ten-fold increase in bombesin production compared to strains without built-in these control switches.
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Morphology and Crystal Structure of SARS-CoV-2
Posté le 29/6/2020 à 13:05 - 0 Commentaires - poster un commentaire - Lien
The 2019 new coronavirus (2019-nCoV) is a kind of spherical, protruding surface, which looks like a crown under the electron microscope. The viral gene is a continuous linear single-stranded RNA with a diameter of 75-160nm. 2019-nCoV is the seventh member of the Coronaviridae family that has been discovered to infect humans. The other 6 members are: HCoV 229E, HCoV NL63, HCoV OC43, HCoV HKU1, SARS-CoV and MERS-CoV. 2019-nCoV belongs to β-coronavirus as well as SARS-CoV and MERS-CoV.
On February 12, 2020, the International Committee on Taxonomy of Viruses (ICTV) issued a statement officially renaming the 2019-nCoV to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It is also recognized that this virus is the sister virus of SARS coronavirus, which indicates that the new coronavirus is a close relative of SARS coronavirus (SARS-CoV) from a taxonomic point of view.
On February 11, 2020, the world The World Health Organization (WHO) announced that it will name the new coronavirus pneumonia "COVID-19": the letter CO stands for "corona", the letter VI stands for "virus", and the letter D stands for "disease" "(Disease), the number 19 indicates that the disease was discovered in 2019.
Morphology of SARS-CoV-2
Negative staining was used to observe the virus under an electron microscope. Viruses are generally spherical and some are polymorphic, with a diameter between 75-160nm. The edges of the virus particles have protrusions similar to the corona, about 9-12nm, and look like a crown. Extracellular free virus particles and inclusion bodies filled with virus particles in the cytoplasmic membrane sac were found in ultrathin sections of human airway epithelium. This morphology was consistent with the Coronaviridae.
Crystal structure of SARS-CoV-2
On January 16, 2020, the high-rate crystal structure of the new coronavirus 3CL hydrolase determined by the Rao Zihe/Yang Haitao research group of Shanghai University of Science and Technology was announced to the public.
3CL hydrolase (Mpro) is encoded by ORF1 (located at nsp5), located in the central region of the replicase gene, and is a key protein in the replication of new coronavirus RNA.
Mechanism of 3CL hydrolase action
The active form of the 3CL hydrolase molecule is a dimer formed by two homologous monomers, which are composed of the N-terminal heptapeptide SGFRKMA (N-finger), three domains (Domain I, II, III) and the connecting structure loop of domain II and domain III.
Domains I and II are β-sheets, and the enzyme active center is located in the gap between domains I and II. The interaction between the two monomer domains III to further stabilize the dimer structure of 3CL hydrolase. The N-terminal heptapeptides of the two monomers are inserted into the grooves of the domain II of each other, and the molecular conformation of the active center of the 3CL hydrolase is stably maintained by hydrogen bonding and salt bonding. The mature 3CL hydrolase can catalyze and hydrolyze 11 conserved sites of the replicator precursor polyprotein downstream of it, producing 13 end products of hydrolysis, as well as multiple intermediate products. These include the two most conserved regions of replicase, RNA polymerase and RNA helicase, which are necessary for viral RNA replication.
If the function of 3CL hydrolase is inhibited, the replication of viruses can be blocked with a high probability, indicating the direction for the development of antiviral drugs.
Atomic diagram of SARS-CoV-2
A new study published in the journal Science by the research team of the University of Texas at Austin and the National Institutes of Health on February 19, 2020 pointed out that they created the first 3D atom of the new coronavirus to attach and infect parts of human cells Scale structure diagram, which is a key step in developing vaccines and treatment methods.
Mechanism of SARS-CoV-2 invading human body
The new coronavirus uses highly glycosylated homotrimeric S protein to enter the host cell. The S protein undergoes many structural rearrangements to fuse the virus into the cell membrane of the host cell. This process involves the binding of the viral S1 subunit to the host cell receptor, triggering trimer instability, which in turn causes the S1 subunit to fall off the S2 subunit to form a highly stable post-fusion structure.
In order to access the host cell receptor, the receptor binding domain (RBD) in the S1 subunit undergoes a hinge-like conformational movement to hide or expose key sites for receptor binding. In this process, there are two states of S1: the "down" structure represents the receptor unbound state, and the "up" structure represents the receptor bindable state, but at the same time the "up" structure is relatively unstable.
Based on the published genome sequence, the research team carried out in vitro protein purification by affinity chromatography and gel exclusion chromatography, and then used cold electron microscopy technology to initially screen the new coronavirus S protein images showing high particle density. After 3D reconstruction, a spike protein (S protein) trimer structure with a resolution of 3.5 Å was finally obtained.
The research team has also sent the spine protein molecular structure map to the international database, waiting for publication, hoping that other teams will use their results to develop a vaccine as soon as possible.